Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

KERTAS KERJA POSTER
NO P-01 PENULIS/PEMBENTANG ROSHAIDA MUHAMMAD, AMINAH ABDULLAH, NORRAKIAH ABDULLAH SANI TENGKU FARIZAN IZZI CHE KU JUSOH, SURIAH ABDUL RAHMAN AND NORRAKIAH ABDULLAH SANI MD. JELAS HARON, SAIFUL ADLI MASDAN, MOHD ZOBIR HUSSEIN AND ZULKARNAIN ZAINAL A. M. YUSOF AND MOHAMMAD ADIL MOHD. ZOBIR HUSSEIN, LIM SHEAU WEN, ASMAH HAJI YAHAYA AND ZULKARNAIN ZAINAL COLLIN G. JOSEPH, HASNUL FAZLI MD. ZAIN DAN SITI FATIMAH DEK IBRAHIM BABA, NORMAH AWANG, SRIHANUM ADNAN, YANG FARINA ABDUL AZIZ DAN BOHARI M. YAMIN M.F. MOHAMAD BUKHORI, N. ABDUL RAHMAN, N. KHALID AND V. PILLAI MARCUS JOPONY AND TING TEO MING KAREN A.CROUSE, M. IBRAHIM.M. TAHIR AND THAHIRA BEGUM TAJUK KERTAS KERJA MINERAL AND HEAVY METAL CONTENTS IN BOTTLE DRINKING WATER DETERMINATION OF TOTAL, SOLUBLE AND INSOLUBLE DIETARY FIBERS IN BAKERY PRODUCTS KINETICS AND THERMODYNAMIC FOR SORPTION OF ARSENATE BY LANTHANUM-EXCHANGED ZEOLITE PHOSPHORIC ACID ACTIVATED CARBON AS BORDERLINE AND SOFT METAL IONS SCAVENGER LAMELLA STRUCTURE OF ZN-ALLAYERED DOUBLE HYDROXIDE AS MOLECULAR CONTAINERS FOR THE PREPARATION OF MICRO-MESOPOROUS CARBONS TREATMENT OF LANDFILL LEACHETES IN KAYU MADANG, SABAH:TEXTURAL AND CHEMICAL CHARACTERIZATION (PART 1) KOMPLEKS BIS(N,N-SEKBUTILPROPILDITIOKARBAMATO)KADMIU M(II) DAN BIS(N,NBUTILETANOLDITIOKARBAMATO)KADMI UM(II) DENGAN LIGAN 1,10-FENANTROLIN CARPAINE FROM CARICA PAPAYA L. VAR. EKSOTIKA I PHYSICOCHEMICAL CHARACTERISTICS AND PROFILES OF MUD VOLCANOES AND ITS SURROUNDINGS SYNTHESIS, CHARACTERISATION & BIOLOGICAL ACTIVITIES OF MIXEDLIGAND COPPER(II) COMPLEXES CONTAINING SACCHARIN AS ONE OF THE LIGANDS COMPARATIVE STUDY ON CLEANUP PROCEDURES FOR THE DETERMINATION OF ORGANOPHOPHORUS PESTICDES IN VEGETABLES THE INFLUENCE OF THE MEDIA SRB ON THE CORROSION OF ALLOY STEEL BY DESULFOVIBRIO SP. BACTERIA: AN ELECTROCHEMICAL STUDY

P-02

P-03

P-04

P-05

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P-07

P-08

P-09

P-10

P-11

ALVIN CHAI LIAN KUET AND LAU SENG

P-12

FATHUL KARIM SAHRANI, ZAHARAH IBRAHIM, MADZLAN AZIZ AND ADIBAH YAHYA

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

NO P-13

PENULIS/PEMBENTANG BADRI MUHAMMAD, KAREN A. CROUSE, NORAZLINALIZA SALIM, AND KIO SOO SAN CHUN FOONG CHAN AND RICHARD C.S. WONG

TAJUK KERTAS KERJA SYNTHESIS AND CHARACTERIZATION OF QUATERNARY AMMONIUM SALTS (QAS) FROM α-IODOKETONES A PRELIMINARY STUDY ON THE DISTRIBUTION OF SOME ORGANIC PESTICIDES RELATED TO AQUACULTURE ACTIVITIES IN THE MANJUNG STRAITS SYNTHESIS, CHARACTERIZATION AND ANTIMICROBIAL ACTIVITY OF BIS(NBENZYL-NISOPROPYLDITHIOCARBAMATE)ZINC(II) WITH THE BIDENTATE AMINES 2, 2’ BIPYRIDYL AND 1, 10-PHENANTHROLINE: CRYSTAL STRUCTURE OF ZN[S2CN(C7H7)(IC3H7)]2(BIPY) BINDING OF MESO-TETRA KIS (4-NMETHYL-PYRIDINIUM) PORPHYRIN AND ITS MN (III) AND CO (III) COMPLEXES WITH CALF THYMUS DNA: A THERMODYNAMIC APPROACH IN SILICO PROTEIN ENGINEERING : FUNDAMENTAL APPROACH OF BIOCHEMICAL MOLECULAR INTERACTION IN DESIGNING POTENTIAL PROTEIN COMPLEXES AS INDUSTRIAL BIOCATALYST MENENTUKAN KANDUNGAN PROTEIN LARUT DALAM SARONG TANGAN LATEK GETAH ASLI AN ASSESSMENT OF ABSORBED DOSE AND RADIATION HAZARD INDEX FROM NATURAL RADIOACTIVITY MEASURED IN JENGKA 15 OIL PALM AREA, PAHANG DARUL MAKMUR ANGGARAN KADAR PEMENDAPAN SEDIMEN DI PERAIRAN PANTAI TIMUR SEMENANJUNG MALAYSIA MENGGUNAKAN TEKNIK 210PB

P-14

P-15

NORMAH AWANG, IBRAHIM BABA, YANG FARINA ABD. AZIZ AND BOHARI M. YAMIN

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NASIRIAN

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P-18

MOHD BASYARUDDIN ABDUL RAHMAN, AHMAD HANIFF JAAFAR, MAHIRAN BASRI, RAJA NOR ZALIHA ABDUL RAHMAN, ABU BAKAR SALLEH AND HABIBAH ABDUL WAHAB SALIHAN BIN SIAIS, ABDUL AZIZ AWANG MASITAH ALIAS, ZAINI HAMZAH, AHMAD SAAT, MOHAMAT OMAR AND ABDUL KHALIK WOOD NORFAIZAL MOHAMED, ZAHARUDIN AHMUD, ABD. KADIR ISHAK, ZAL UYUN WAN MAHMOOD, YII MEI WO, JALAL SHARIB @ SARIP, KAMAROZAMAN ISHAK YII MEI WO AND ZAHARUDIN AHMAD

P-19

P-20

P-21

P-22

KARIMAH KASSIM, YONG SOON KONG, HADARIAH BAHRON, NOR HADIANI ISMAIL

P-23

MD. PAUZI ABDULLAH DAN LIM FANG YEE

VALIDATION OF RA-226 AND K-40 MEASUREMENT IN ENVIRONMENTAL SAMPLES USING GAMMA SPECTROMETRY SYSTEM INVESTIGATION ON NEW TRANSITION METAL COMPLEXES OF THE SCHIFF BASES DERIVED FROM SALICYLALDEHYDE AND 2HYDROXYACETOPHENONE HUBUNGAN PERMINTAAN KLORIN DENGAN KUALITI AIR MENTAH

ii

Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

NO P-24

PENULIS/PEMBENTANG OOI YONG YONG

TAJUK KERTAS KERJA PHYSICAL AND CHEMICAL PROPERTIES OF LIPID FRACTION FROM HEVEA BRASILIENSIS AND ELATERIOSPERMUM TAPOS KETOHYDRAZONE COMPLEXES AS POTENTIAL EMITTING MATERIAL BY OLED REMOVAL OF DYE BY IMMOBILISED PHOTOCATALYST LOADED ACTIVATED CARBON THE PRELIMINARY STUDY ON PHYTOCHEMICAL CONSTITUENT FROM QUASSIA INDICA

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HAZNITA ROSE BAHARI, MOHD. NORDIN GARIF AND MUSTAFFA SHAMSUDDIN ZULKARNAIN ZAINAL, CHANG SOOK KENG AND ABDUL HALIM ABDULLAH MOHD HUSSIN ZAIN, ZALILAWATI MAT RASHID, JURIFFAH ARIFFIN, HABSAH MOHAMAD, AND MOHD RAZALI SALAM LIM YING SIEW, LIM YING CHIN AND PAULINE MAH JIN WEE

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USWATUN HASANAH ZAIDAN, MOHD. BASYARUDDIN ABD. RAHMAN NOOR MONA BINTI MD YUNUS, MOHD. BASYARUDDIN BIN ABDUL RAHMAN, MAHIRAN BINTI BASRI, MOHD ZOBIR BIN HUSSEIN, ABU BAKAR BIN SALLEH AZMAHANI SULAIMAN, MAHIRAN BASRI, MOHD BASYARUDDIN ABD. RAHMAN, ABU BAKAR SALLEH, RAJA NOOR ZALIHA ABDUL RAHMAN AND SALMIAH AHMAD PHANG FEONG KUAN, ZAHARRUDIN AHMAD AND CHE ABD. RAHIM MOHAMED DEVAGI, K., CONNIE, A. J., AND SHABDIN, M. L MOHD. BASYARUDDIN ABD. RAHMAN, CHANG KOK KHAN

ARIMA AND INTEGRATED ARFIMA MODELS FOR FORECASTING AIR POLLUTION INDEX IN SHAH ALAM, SELANGOR SYNTHESIS OF PETROCHEMICAL-BASED ADIPATE ESTER USING IMMOBILISED LIPASES IMMOBILIZATION OF LIPASE FROM CANDIDA RUGOSA ONTO LAYERED DOUBLE HYDROXIDES FOR THE SYNTHESIS OF ESTER

THE EVALUATION OF COSMETIC AND PHARMACEUTICAL EMULSION AGING PROCESS USING CLASSICAL TECHNIQUE AND A NEW METHOD: FTIR FLUCTUATION ACTIVITIES OF 210PO IN WATER THE WATER COLUMN AT BAGAN LALANG, SELANGOR HEAVY METAL CONCENTRATIONS IN THE RAZOR CLAM (SOLEN SP) FROM MUARA TEBAS, SARAWAK ENGINEERING NOVEL METALLOPROTEIN: DESIGN OF METAL-LIGAND-BINDING SITES INTO LIPASE BY STRUCTURAL PREDICTION AND MOLECULAR MODELING SYNTHESIS AND CHARACTERIZATION OF DISCOTIC LIQUID CRYSTAL MOLECULE CONTAINING 1,3,5-TRIAZINE

P-34

P-35

NOOR HAFIZAH ABDUL HALIM, SIDIK SILONG, FAZILAH M. AYOP, MOHAMAD ZAKI AB. RAHMAN, WAN MD ZIN WAN YUNUS, MOHD M. MOKSIN

iii

Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

NO P-36

PENULIS/PEMBENTANG ABDUL HALIM ABDULLAH, LOO LI YIN AND NOOR HIDAYAH BT ABU SAMAH SHARINA SHAMSUDIN DAN JUMAT SALIMON NURASYIKIN ABDUL RAHMAN DAN JUMAT SALIMON ZAINAB RAMLI, DEWI JAMALIAH KAMSIAR DAN HASIDAH MOHD. ARSAT FAZILAH MAHMAD AYOP , SIDIK SILONG, MOHAMAD ZAKI AB. RAHMAN, WAN MD ZIN WAN YUNUS MOHD MAROOF MOKSIN, HAFIZAH HALIM M. IBRAHIM M. TAHIR, KAREN A. CROUSE, TAN SANG LOON, W. A. RAHMAN W.A. KADIR AND KHOO TENG JIN

TAJUK KERTAS KERJA PHYSICOCHEMICAL CHARACTERISTICS OF MODIFIED ACTIVATED CARBON CHARACTERISTICS OF AJI-AJI LIPID SERIOLA NIGROFASCIATA PHYSICO-CHEMICAL CHARACTERISTICS OF TERUBOK OIL (TENUALOSA TOLI). PERTUKARAN FASA ZEOLIT ASLI KE FASA ZEOLIT SINTETIK YANG DICIRIKAN OLEH XRD BAGI MENGHASILKAN BAHAN PENUKAR ION SYNTHESIS AND CHARACTERIZATION OF DISCOTIC LIQUID CRYSTAL MOLECULE CONTAINING 1,3,4-THIADIAZOLE MOIETIES SYNTHESIS, CHARACTERISATION AND BIOLOGICAL ACTIVITIES OF METAL COMPLEXES CONTAINING N'-(1-METHYLHEPTYLIDENE)HYDRAZINECARBODITHIOIC ACID BENZYL ESTER DILUTE-SOLUTION PROPERTIES OF BRANCHED POLYSTYRENE EXTRACTION OF CONDENSED TANNINS FROM MANGROVE BACK

P-37 P-38 P-39

P-40

P-41

P-42 P-43

P-44

MAIMUNAH SOKRO, ARBA’AT HASSAN AND HANITA OTHMAN MAIMUNAH SOKRO, LIEW SIEW HAR, LIEW HAN WOON, ARBA’AT HASSAN, HANITA OTHMAN MAIMUNAH SOKR, ARBA’AT HASSAN, HANITA OTHMAN ZILDAWARNI IRWAN, JAFARIAH JAAFAR AND RAHMALAN BIN AHAMAD

P-45

P-46

ASMAH HJ. YAHAYA, MOHD ZOBIR HUSSEIN, ZULKARNAIN ZAINAL AND ADILA MOHAMAD JAAFAR

P-47

ILYANA, S.I., MAMOT, S. AND NAZARUDDIN, R. HABSAH MOHAMAD, KHAMSAH SURYATI MOHD, NURHANA FAUJAN, NORHAYATI IBRAHIM, JASNIZAT SAIDIN, ABDUL MANAF ALI, MOHD NORDIN HJ

P-48

THE PERCEPTIONS ON ENVIRONMENTAL ISSUES OF SELECTED RESIDENTS IN KUALA PERLIS ON-LINE PRECONCENTRATION TECHNIQUE BY DYNAMIC PH JUNCTION FOR THE DETERMINATION OF ARSENIC SPECIES IN CAPILLARY ELECTROPHORESIS THE EFFECT OF MG/AL RATIO ON THE FORMATION OF 2,4DICHLOROPHENOXYACETATE-LAYERED DOUBLE HYDROXIDE-NANOCOMPOSITES SYNTHESISED BY CO-PRECIPITATION METHOD ENZYMATIC SYNTHESIS OF STRUCTURED LIPID FROM RAMBUTAN KERNEL STEARIN (RKFST) AND FLUIDIZED PALM OIL (FPO) TAXONOMIC IDENTIFICATION, IN-VITRO SCREENING FOR CYTOTOXIC ACTIVITY AND CHEMICAL CONSTITUENT OF A TOTAL OF 37 MARINE SPONGES COLLECTED FROM PULAU BIDONG AND

iv

Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

NO

P-49

P-50

P-51

P-52

PENULIS/PEMBENTANG LAJIS, FAIZAH SHAHAROM AND MOHAMAD HUSSIN ZAIN M.Z. ABDUL RAHMAN, A.A. MD. HAFIDZIN, N.K. ABU BAKAR, AND M.J. MAAH MOHD RAZIP ASARUDDIN, GABRIEL TONGA NOWEG, ENDELA TIPOT, JACK LIAM MOHD RAZIP ASARUDDIN, DEVAGI KANAKARAJU, JIBBY JINANG ONG, M.C., KAMARUZZAMAN, B.Y. AND AIRIZA, Z.

TAJUK KERTAS KERJA PULAU PERHENTIAN, TERENGGANU COPPER, LEAD AND ZINC IN ANADARA GRANOSA AND SEDIMENT FROM BERNAM RIVER ESTUARY, BAGAN DATOH, PERAK MEDICINAL PLANTS OF LOAGAN BUNUT NATIONAL PARK, SARAWAK STUDY ON ANTIDIABETIC PROPERTIES OF MICHELIA ALBA D.C ALUMINIUM (AL) AS A TRACER OF ANTHROPOGENIC INPUT OF HEAVY METALS IN THE SURFACE SEDIMENT AT KEMAMAN MANGROVE FOREST, MALAYSIA THIN FILMS FROM WATER-SOLUBLE CHITOSAN DERIVATIVES

P-53

P-54

P-55

ZANARIAH UJANG, SITI KASMARIZAWATY SUBOH, NORHANI NASIR, AHMAD HAZRI ABD. RASHID, MAZITA MOHD. DIAH, NOORRASYIDAH MOHD. SARMIN, ZULIANA AHMAD MD PAUZI ABDULLAH, KAMARRUDDDIN ASRI, MOHAMAD SALLEH RAMLI AND MAIMUNAH SULAIMAN @ WAHID JAMIL, B. T., KAMARUZZAMAN, B.Y., NOOR AZHAR, M. S. AND AHMAD SHAMSUDDIN A.

P-56

WILLISON K. Y. S., KAMARUZZAMAN, Y., AZHAR, N. A. M AND ROSNAN, Y. UMI KALTHOM AHMAD AND AHMAD ZAMANI AB HALIM

P57

P-58

P-59

NORHAYATI MOHD TAHIR, POH SENG CHEE, SUHAIMI HAMZAH, KHALIK HJ WOOD, SHAMSIAH ABD. RAHMAN, WEE BOON SIONG, SUHAIMI ELIAS AND NAZARATUL ASHIFA ABDULLAH SALIM ZAINOHA ZAKARIA, NURZAHWANI MOHD NOR,

OPTIMISATION OF SOLID-PHASE MICROEXTRACTION (SPME) FOR THE DETERMINATION OF TRIAZINES IN WATER SAMPLES USING EXPERIMENTAL DESIGN DETERMINATION OF SEDIMENT CONTAMINATION LEVELS BY USING ENRICHMENT FACTOR (EF) METHOD IN THE TERENGGANU RIVER, TERENGGANU, MALAYSIA. THE SPATIAL DISTRIBUTION OF ORGANIC CARBON IN PAHANG SOUTH CHINA SEA DURING THE PRE MONSOON AND POST MONSOON SEASON DEVELOPMENT OF A SUBCRITICAL WATER EXTRACTION FOR THE QUANTITATIVE DETERMINATION OF POLYCHLORINATED BIPHENYLS IN SEWAGE SLUDGE. ANALYSIS OF PM10 IN KUALA TERENGGANU BY INSTRUMENTAL NEUTRON ACTIVATION ANALYSIS

PENGHASILAN KITOSAN DARIPADA KULIT UDANG HARIMAU DAN

v

Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

NO

P-60

PENULIS/PEMBENTANG RUBIATULNISHA MOHD BAKERI DAN RAHANIZA BT ABDUL RAHIM FARIDAH MOHD MARSIN, NOR AZIAH BUANG, WAN AZELEE WAN ABU BAKAR FAUJAN HJ AHMAD, YAMIN YASIN AND MAHIRAN BASRI HONG HENG SEE, M. MARSIN SANAGI, WAN AINI WAN IBRAHIM, AHMEDY ABU NAIM UMI K. AHMAD, ABDUL RAHIM YACOB, GEETHA SELVARAJU SYAHIDAH AKMAL MUHAMMAD AND MD. SANI IBRAHIM ZURINAH ABDUL LATIFF AND MD. SANI IBRAHIM

TAJUK KERTAS KERJA KEBERKESANANNYA SEBAGAI BAHAN PEMBALUT MAKANAN CO2 / H2 METHANATION ON NICKEL OXIDE BASED CATALYST DOPED WITH VARIOUS ELEMENTS FOR THE PURIFICATION OF NATURAL GAS ESTERIFICATION OF BETULINIC ACID USING LIPASE AS CATALYST RETENTION THERMODYNAMICS IN HIGH TEMPERATURE REVERSED-PHASE LIQUID CHROMATOGRAPHY USING HYDROORGANIC AND SUPERHEATED WATER ELUENT A HOME-MADE SPME FIBER COATING FOR ARSON ANALYSIS HEALTH RISK ASSESSMENT OF PERSISTENT ORGANIC POLLUTANTS THROUGH DIETARY INTAKE (I): POPS IN FISH AND MARINE ORGANISMS HEALTH RISK ASSESSMENT OF PERSISTENT ORGANIC POLLUTANTS THROUGH DIETARY INTAKE (II): POPS IN CEREALS, MILKS, AND CHICKENS Synthesis, isolation and single crystal X-ray analysis of novel complexes CpMo(CO)2(S2P(SPhMe)2) and Cp2Mo2(µ-S)(µ-S2)(SPhMe) (Cp = (η5-C5H5)) ANALYSIS OF NANOSIZED METAL PARTICLES USING AN RF PLASMA TREATMENT TECHNIQUE ANALYSIS CHEMICAL VAPOR DEPOSITED OF NOVEL CARBON MATERIALS THERMODYNAMIC OF THE BINDING OF THE THREE – WATER SOLUBLE PROPHYRINS WITH DNA NATURAL FLUORIDE LEVELS IN NONFLUORIDATED DRINKING WATER: A CROSS-SECTIONAL STUDY IN KOTA KINABALU, SABAH SORPTION OF METAL IONS ONTO BENTONITE AND PRETREATED BENTONITE: A COMPETITIVE SORPTION KINETICS STUDY BIOPENDERIA ELEKTROKIMIA UNTUK PENGESANAN KETOKSIKAN 2,4DIKLOROFENOKSIASETIK ASID BERASASKAN ENZIM ALKALINE FOSFATASE TERPEGUN DALAM HIBRID

P-61 P-62

P-63 P-64

P-65

P-66

MEI LEE OOI, RICHARD C.S. WONG AND SEIK WENG NG ROSLAN MD NOR

P-67

P-68

P-69

TAMIL MANY THANDAVAN, LEE CHENG CHOO AND ROSLAN MD NOR MORTEZA KESHAVARZ, ABDOLKHALEGH BORDBAR SHAHARUDDIN MS, SUMARLAN S, MOHD YUNUS A, DASRILSYAH S A. M. YUSOF, C.H. CHIA

P-70

P-71

P-72

LOH KEE SHYUAN, LEE YOOK HENG, MUSA AHMAD, SALMAH ABDUL AZIZ DAN ZAMRI ISHAK

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

NO P-73

PENULIS/PEMBENTANG WONG HON LOONG, SUGENG TRIWAHYONO, MUSTAFFA SHAMSUDDIN NOR FAIROLZUKRY AHMAD RASDY, WAN AINI WAN IBRAHIM, M. MARSIN SANAGI, AHMEDY ABU NAIM

TAJUK KERTAS KERJA SOL-GEL/KITOSAN SURFACE AND ACIDITY ANALYSIS OF SOLID SUPER ACID BASED ON HZSM-5 DETERMINATION OF POLYCYCLIC AROMATIC HYDROCARBONS IN PALM OIL WASTE WATER BY SOXHLET EXTRACTION AND GAS CROMATOGRAPHY-FLAME IONIZATION DETECTION SYNTHESIS AND CHARACTERIZATION OF NANO SIZE POLY(STYRENEDIVINYLBENZENE) USING DISPERSION COPOLYMERIZATION PRELIMINARY INVESTIGATION ON THE SEPARATION OF 2,3,7,8-TCDD AND 2,3,7,8TCDF USING MICELLAR ELECTROKINETIC CHROMATOGRAPHY SEPARATION OF PROPICONAZOLE ENANTIOMES BY MICELLAR ELECTROKINETIC CHROMATOGRAPHY WITH DIFFERENT BUFFER PH HPLC METHOD FOR DETERMINATION OF ACRYLAMIDE IN HEATED FOODS EFFICIENT DECHLORINATION OF CHLOROAROMATIC COMPOUNDS BY USING ELECTROCHEMICAL METHOD KAJIAN KUALITI AIR DAN HUBUNGANNYA DENGAN AKTIVITI GUNA TANAH DI BEBERAPA BAHAGIAN SUNGAI LANGAT PRELIMINARY WORK ON THE APPLICATION OF FTIR AND CHEMOMETRICS IN HONEY ANALYSIS ASSESSMENT OF PHYTOCHEMICAL CONTITUENTS OF VARIOUS SOURCES OF ZINGIBER ZERUMBET AND CURCUMA XANTHORRHIZA

P-74

P-75

NORSYARIZA ABD AZIZ, AHMEDY ABU NAIM M. MARSIN SANAGI, WAN AINI WAN IBRAHIM, ASIAH HUSSAIN WAN AINI WAN IBRAHIM MOHD. MARSIN SANAGI, SHARAIN LIEW YEN LING AND JULIANA HUSSSAIN WAN AINI WAN IBRAHIM, DADAN HERMAWAN AND M. MARSIN SANAGI A.K. MOHAMAD KAMAL AND H. ABIDIN MURNI SUNDANG, SUGENG TRIWAHYONO, NOOR FATIMAH AWANG PANJANG, AND AISHAH ABDUL JALIL MAZLIN BIN MOKHTAR DAN LEW LOO MING

P-76

P-77

P-78 P-79

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P-81

P-82

P-83

MOHAMED NOOR HASAN AND MOHAMED NURUDDIN MOHAMED NASIR MAZITA MOHD. DIAH, THAVAMANITHEVI SUBRAMANIAM, ZABIDI RAZALI, ZANARIAH UJANG, AHMAD HAZRI ABD. RASHID AND HASNAH SIRAT C.K. GOH, Y.H. TAUFIQ-YAP, I. RAMLI AND M.Z. HUSSEIN ZAWAHIL AHMAD NADZIR1, NASHRIYAH MAT DAN ISMAIL SAHID ABDUL RAHIM YACOB, RATNA SARI DEWI DASRIL AND

P-84

P-85

THE EFFECT OF MECHANOCHEMICAL TREATMENT ON THE STRUCTURE AND CATALYTIC PERFORMANCE OF VANADIUM PHOSPHATE CATALYSTS PERBANDINGAN KAJIAN KUALITI AIR DIKAWASAN PENGAIRAN MUDA, KEDAH DAN KADA, KELANTAN COMPARISON AND PHYSICAL CHARACTERIZATION OF VARIOUS

vii

Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

NO

PENULIS/PEMBENTANG VICINISVARRI A/P INDERAN UMI K. AHMAD, SUMATHY RAJENDRAN AND SYAHIDAH ABU HASSAN D. SUHARDY, N. MUSTAFFA, H. WAN SU, K. FARIZUL HAFIZ AND S. SAIFUL AZHAR

P-86

TAJUK KERTAS KERJA SOURCE OF HIGH SURFACE AREA CARBON PREPARED BY DIFFERENT TYPES OF ACTIVATION FORENSIC ANALYSIS OF EXPLOSIVE RESIDUES FROM HAND SWABS CHARACTERIZATION OF CELLULOSE AND PULP OBTAINED FROM RICE STRAW PLANTS OF DIFFERENT PULPING PROCESS USING SCANNING ELECTRON MICROSCOPY (SEM). HARNESSING ELECTRO DRIVEN SEPARATION TECHNIQUE FOR THE SEPARATION OF SELECTED AGROCHEMICALS KANDUNGAN LOGAM BERAT DI DALAM BEBERAPA SIRI TANAH OKSISOL DI SEKITAR TASIK CHINI, PAHANG

P-87

P-88

WAN AINI WAN IBRAHIM, S. M. MONJURUL ALAM AND AZLI SULAIMAN SAHIBIN ABD. RAHIM, MUHD. BARZANI GASIM, MOHD. NIZAM MOHD SAID, WAN MOHD RAZI IDRIS, AZMAN HASHIM, SHARILNIZAM YUSOF DAN MASNIYANA JAMIL WONG KA LUN, SALASIAH ENDUD NOZIEANA KHAIRUDDIN, IDA IDAYU MUHAMAD

P-89

P-90

TIN-CONTAINING MESOPOROUS MCM-48 IN OXIDATION OF BENZYL ALCOHOL TO BENZALDEHYDE PRELIMINARY STUDY OF ANTIMICROBIAL (AM) EFFECTS OF STARCH-BASED FILM INCORPORATED WITH NISIN, LYSOZYMES AND LAURIC ACID THE APPLICATION OF SOL GEL IMMOBILIZED FLUORESCEINMANGANESE COMPLEX AS FLUORESCENT CARBON DIOXIDE SENSING MATERIAL ANALISIS MINYAK PETROL KOMERSIAL MENGGUNAKAN KAEDAH PENDARFLUOR ANALYTICAL APPLICATION OF FUNCTIONALIZED ZnS AS FLUORESCENCE LABEL FOR THE DETERMINATION OF PROTEINS MINERALIZATION STUDIES ON PARAQUAT AND MALATHION USING TiO2/ZnO BASED PHOTOCATALYST STUDIES OF POTATO PEELS EXTRACT AS NATURAL ANTIOXIDANT IN EDIBLE OIL

P-91

P-92

MUSTAFFA NAWAWI, TEE SHIAU FOON, SHEMALAH D/O RAMASUNDRAM MUSTAFFA NAWAWI, TUAN MUHAMMAD NAJIB TUAN OMAR DAN TEE SHIAU FOON MUSTAFFA NAWAWI, SHEMALAH A/P RAMASUNDRAM AND TEE SHIAU FOON SITI HABSAH HASSAN AND RUSMIDAH ALI WAN SANI WAN NIK, CHEK YIN SEZ, KU HALIM KU BULAT, MD. MUKHLESUR RAHMAN, FARID NASIR ANI

P-93

P-94

P-95

P-96

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

MINERAL AND HEAVY METAL CONTENTS IN BOTTLE DRINKING WATER Aminah Abdullah*, Roshaida Muhammad, Norrakiah Abdullah Sani* Food Science Programme, School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, 43600, Selangor, Malaysia. E-mail: kama@pkrisc.cc.ukm.my Abstract. Mineral and heavy metal contents were determined in different types of bottle drinking water: carbonated natural mineral water (A-D), un-carbonated natural mineral water (E-H), reverse osmosis water (I-L) and distilled water (M-P). Inductively Coupled Plasma- Optical Emission Spectroscopy (Perkin Elmer) was used for the analyses of cations (potassium, calcium, magnesium and sodium) and heavy metals (cadmium, lead and zinc). Ion Chromatography was used for the analysis of anions (fluoride, chloride, nitrate and sulphate). Carbonated natural mineral water (A) had high concentration of potassium (86.40 mg/L), while calcium was found higher in un-carbonated natural mineral water of E (87.20 mg/L). Brand C had high concentration of magnesium (34.67 mg/L) while sodium content was highest in D (33.40 mg/L). Both carbonated (B) and un-carbonated (E) natural mineral water had high concentration of fluoride (0.12 mg/L), while E had high concentration of chloride (21.24 mg/L). Nitrate was found highest in F (14.18 mg/L) while C (0.73 mg/L) and E (0.72 mg/L) contained the most sulphate. Cadmium was only detected in carbonated natural mineral water (B and C) at 0.01 mg/L. Lead was high in un-carbonated natural mineral water (H) with 0.03 mg/L and the highest zinc concentration was 0.54 mg/L also in un-carbonated natural mineral water (G). Based on the mineral concentrations, there were differences between the analyzed values with the labeled values. The minerals and heavy metals concentrations were mostly higher in natural mineral water but the heavy metal contents did not exceed the standard concentrations in the Food Act 1983 and Food Regulations 1985 [3]. Abstrak. Kandungan mineral dan logam berat telah ditentukan di dalam air minuman botol di pasaran Malaysia, seperti air mineral semulajadi berkarbonat (A-D), air mineral semulajadi tanpa karbonat (E-H), air osmosis songsang (I-L) dan air suling (M-P). Penentuan mineral kation (kalium, kalsium, magnesium dan natrium) dan logam berat (kadmium, plumbum dan zink) dilakukan dengan menggunakan Spektroskopi Pancaran Optik dengan Sumber Plasma Berganding Secara Aruhan (ICPOES). Penentuan anion (fluorida, klorida, nitrat dan sulfat) pula dilakukan dengan menggunakan Kromatografi Ion. Air mineral semulajadi berkarbonat (A) mengandungi kepekatan kalium paling tinggi (86.40 mg/L). Kandungan kalsium paling tinggi adalah di dalam air mineral semulajadi tanpa karbonat E (87.20 mg/L). Air mineral semulajadi berkarbonat (C) pula mempunyai kepekatan magnesium (34.67 mg/L) yang tinggi, manakala natrium (33.40 mg/L) yang paling tinggi adalah di dalam D. Kedua-dua B dan E mengandungi kepekatan fluorida yang tinggi (0.12 mg/L), manakala E mengandungi tinggi klorida (21.24 mg/L). Nitrat didapati paling tinggi di dalam F (14.18 mg/L), manakala C (0.73 mg/L) dan E (0.72 mg/L) mengandungi paling tinggi sulfat. Kepekatan kadmium hanya terdapat di dalam air mineral semulajadi berkarbonat (B dan C) pada 0.01 mg/L, manakala plumbum (0.03 mg/L) di dalam H dan zink di dalam G (0.54 mg/L) adalah yang paling tinggi. Berdasarkan kepekatan mineral, terdapat perbezaan nilai kandungan mineral yang dianalisis dengan nilai pelabelan. Kepekatan mineral dan logam berat kebanyakannya adalah paling tinggi di dalam air mineral semulajadi. Namun, kandungan logam beratnya tidak melebihi piawaian kepekatan yang ditetapkan oleh Akta Makanan 1983 dan Peraturan-peraturan Makanan 1985 [3]. Key words: Minerals, heavy metals, carbonated, un-carbonated, reverse osmosis, distilled water

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Introduction There are over 200 brands of bottle drinking water in the local market. The bottled water is of different qualities. Bottled drinking water is classified into three categories known as filtered water, mineral water and distilled water. Filtered water is being produced by filtering and bottling pipe water. Distilling pipe water and collecting evaporated water before ozonation and bottling in order to produce fresh and clean drinking water free from contamination is known as distilled water. Mineral water is produced from water sources which are at least 100 meter deep in the underground and these sources have to be far away from factories and houses in order to avoid from water contamination [5]. The industry of packaging mineral water becomes more important due to the high demand of the products and the changing of Malaysian healthy lifestyle. More than three dozens minerals have been discovered and only 19 minerals are needed to ensure of good health. The human body needs only seven minerals in higher amount known as macro minerals while 12 other minerals are needed in small amount known as micro minerals [10]. Some examples of macro minerals are calcium, magnesium, sodium, potassium and sulphur, while fluoride and nitrate are examples of micro minerals. Contamination of heavy metals in drinking water is one of the factors that concerns consumers and it should be given more attention due to the side effects it may have caused to the human health [1]. Heavy metals are toxic materials and may contaminate the environment causing bioaccumulation in the environmental composition [6]. Heavy metals like lead, cadmium and mercury are cumulative toxic in nature that can cause environmental pollution and reported of high toxicity [7]. Therefore, this research was conducted to determine the minerals and heavy metals in some bottled drinking water available in the local market. Secondly, to determine whether they meet the standards set by the Food Act 1983 and Food Regulations 1985 [3]. Experimental Four types each of bottled carbonated natural mineral water (Brand A-D), un-carbonated natural mineral water (Brand E-H), reverse osmosis water (Brand I-L) and distilled water (Brand M-P) were analyzed. Each brand was sampled three times with different expiry dates. Determination of minerals and heavy metals in bottled drinking water was divided into three stages which were samples preconcentration, elements separation and samples analyses. For samples pre-concentration and elements separation, four main processing steps were used which were conditioning, trapping, washing and elution. Pre-concentration process and separation of disturbing matrix elements were done first because of the lower concentration of heavy metals in the water samples. Ion exchange method compacted with Chelex 100 as trapping agent was used. Conditioning process were washing Chelex 100 with HNO3 followed by de-ionized water to discharge dirt that may be trapped in Chelex 100, besides to increase trapping function. A pH buffer solution was added to modify the pH of Chelex 100 and also to make it trapped better. After the treatment process was completed, modified samples were inserted into burettes for analyzed trapping. This process was known as elements trapping of water samples. Burettes were washed with de-ionized water to eliminate dirt. Finally, elution process of heavy metals that were trapped in Chelex 100 was done using HNO3. The elution products were analyzed using Inductively Coupled Plasma-Optical Emissions Spectroscopy (ICPOES, Perkin Elmer Model OPTIMA 4300) for cations (potassium, calcium, magnesium and sodium) and heavy metals (cadmium, lead and zinc), and Ion Chromatography was used for anions (fluoride, chloride, nitrate and sulphate) in all of the drinking water samples (Brand A-P). All used apparatus were immersed in nitric acid incessant for 24 hrs to eliminate any metals or dirt that may stick to the apparatus before being rinsed with de-ionized water and dried in oven at 800C for 24 hrs.

Results And Discussion Table 1 showed the mineral (cations) concentrations of bottle drinking water analyzed according to brands. Overall, distilled water and reverse osmosis water contained the lowest mineral concentrations compared to the natural mineral water. The natural mineral water industries bottled

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mineral water directly from its underground water sources from springs, wells, bores or other outlets [3]. As it flows underground, a stream picks up minerals from rocks. Carbonated natural mineral water (A) had high concentration of potassium (86.40 mg/L), while the lowest was in Brand N and P (distilled water). However potassium was not detected in Brand J and L (reverse osmosis water) and Brand O (distilled water). Potassium values according to the labels of the bottle water (A-H) were very much lower, ranging from 2.00 to 6.20 mg/L compared to the analyzed samples (6.65 to 86.40 mg/L). Calcium was found higher in un-carbonated natural mineral water of Brand E (87.20 mg/L), while the lowest was in Brand N (0.34 mg/L). Calcium values according to the labels of the bottle water (A-H) were very much lower, ranging from 11.50 to 42.00 mg/L compared to the analyzed samples (29.50 to 87.20 mg/L). Table 1. Mineral (Cation) concentrations (mg/L) in bottle drinking water Potasium Calcium Magnesium Sodium Potasium Calcium Magnesium Sodium Analysed Labelled Carbonated Natural Mineral Water A 86.40a 47.00de 4.31d nd 2.00 29.00 1.00 4.00 57.50cd 8.92c 22.57b 5.30 42.00 58.50c B 4.10 ns 40.10de 34.67a 15.10c 6.20 11.50 86.23a C 8.00 11.00 29.50e 34.03a 33.40d 3.50 12.00 71.73b D 1.60 6.00 Un-Carbonated Natural Mineral Water 87.20a 3.35de 70.67b E nd 2.80 22.40 2.90 20.50 75.80ab 5.03d 60.33c F 16.00c 5.30 24.00 4.50 ns c d a c 54.67 35.17 62.13 G 15.17 2.60 12.50 6.60 10.50 45.00de 18.67b 72.07b H 32.00a 4.30 41.00 4.40 9.90 Reverse Osmosis Water 36.13d 1.45f 0.48g I 16.67c ns ns ns ns f g 0.34 1.24 J nd 14.80cde ns ns ns ns d f fg c 1.65 1.07 35.90 K 15.16 ns ns ns ns 3.23edf 3.94f L nd 12.67e ns ns ns ns Distilled Water 0.62f 0.90g M 8.30e nd ns ns ns ns f e 0.34 2.43efg N 6.77 nd ns ns ns ns f fg 1.03 1.68 O nd nd ns ns ns ns 0.53f 0.23g P 6.65e 13.67de ns ns ns ns a-g Different alphabets in the same column for each mineral showed significant differences (p<0.05). nd = not detected. ns = not stated. Brand Brand C had high concentration of magnesium (34.67 mg/L), while the lowest was in Brand P (0.23 mg/L). Magnesium values according to the labels of the bottle water (A-H) were very much lower, ranging from 1.00 to 8.00 mg/L compared to the analyzed samples (3.35 to 35.17 mg/L). In some geographical area, magnesium content in drinking water would contribute to 20% to 40% of the daily need (Whitney 1999). However, Malaysian consumers should not depend entirely on bottle mineral water for its magnesium intake as the concentration was low ranging only from 0.02% to 4%. The sodium content was highest in Brand D (33.40 mg/L), while Brand L had the lowest (12.67 mg/L). Sodium values according to the labels of the bottle water (A-H) were very much lower, ranging from 4.00 to 20.50 mg/L compared to the analyzed samples (15.10 to 33.40 mg/L). Table 2 showed the mineral (anion) concentrations of bottle drinking water analyzed according to brands. Both carbonated (B) and un-carbonated (E) natural mineral water had high concentration of fluoride (0.12 mg/L), while Brand J (0.01 mg/L), K (0.01 mg/L) and M (0.002 mg/L) had the lowest. Fluoride values according to the labels of the bottle water (A, C and H) were very much higher, ranging from 0.20 to 1.00 mg/L compared to the analyzed samples (0.03 to 0.12 mg/L). Both the

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labeled and the analyzed values of fluoride were lower than the values given by the Food Act 1983 and Food Regulations 1985 [3] (1.50-2.00 mg/L). Table 2. Mineral (Anion) concentrations (mg/L) in bottle drinking water Flouride Chloride Nitrate Sulphate Flouride Chloride Nitrate Sulphate Analysed Labelled Carbonated Natural Mineral Water A 0.12ab 1.43d nd 0.39cd 1.00 10.00 ns 1.00 1.47d ns ns 0.12a ns 4.50 B 1.01d 0.44bc 10.24c 0.23 13.5 0.03b 6.30 8.10 C 4.70c 0.73a 0.65d ns 1.00 0.02b ns 3.00 D 0.32d 0.23cd Un-Carbonated Natural Mineral Water E 0.12a 21.24a 11.78b 0.72a ns 41.10 3.50 6.30 F 0.07ab 19.49ab 14.19a 0.67ab ns 15.00 5.30 5.40 G 0.07ab 18.22b 11.40b 0.68ab ns 13.50 3.90 3.30 b d d d H 0.03 0.41 0.86 0.36 0.20 1.00 ns ns Reverse Osmosis Water I nd 0.21d 0.23d 0.02e ns ns ns ns J 0.01b 0.33d 0.39d nd Ns ns ns ns b d d cd K 0.01 0.53 0.36 0.18 Ns ns ns ns L 0.03ab 1.17d 1.06d 0.15d Ns ns ns ns Distilled Water M 0.002c 0.38d 0.04d nd Ns ns ns ns d N nd 0.15 0.16d nd Ns ns ns ns O nd nd nd nd Ns ns ns ns P nd nd nd 0.33cd Ns ns ns ns a-g Different alphabets in the same column for each mineral showed significant differences (p<0.05). nd = not detected. ns = not stated. Brand Brand E had high concentration of chloride (21.24 mg/L), while Brand N had the lowest concentration (0.15 mg/L). Chloride values according to the labels of both the carbonated and uncarbonated natural mineral water were higher than the analyzed samples except for Brands F and G. Nitrate was found highest in F (14.19 mg/L), while the lowest was in Brand M (0.04 mg/L). Most of the labeled values for nitrate (E, F and G) were lower than the analyzed values. Both the labeled and the analyzed values of nitrate were lower than the values given by the Food Act 1983 and Food Regulation 1985 [3] (45-250 mg/L). Consumption of drinking water containing high nitrate content may contribute towards the daily intake of total nitrate as the bioavailability of nitrate in drinking water is much better than in food [2]. For e.g. the consumption of un-carbonated natural mineral water of Brand F may fulfill RDA’s requirement for nitrate as the concentration was higher than the FAO/ WHO daily needs i.e. 3.7 mg/day [9]. Brands C (0.73 mg/L) and E (0.72 mg/L) contained the most sulphate, while the lowest was in Brand I (0.02 mg/L). Sulphate values according to the labels of the bottle water (A-G) were very much higher, ranging from 1.00 to 8.10 mg/L compared to the analysed samples (0.23 to 0.73 mg/L). Both the labeled and the analyzed values of sulphate were lower than the values given by the Food Act 1983 and Food Regulations 1985 for natural mineral water [3] (200 mg/L). The lowest minerals concentrations were in distilled and reverse osmosis water due to the way they were being processed. Distillation process means purifying water through evaporation and recondensation and this process filters all inorganic minerals, while distilled water is like a vacuum without any beneficial minerals. Reverse osmosis process is actually cleaning water with a partially through membrane and a higher pressure than the osmosis pressure of water were given. Water will undergo reverse osmosis process which will force water to flow from a higher concentration section to

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a lower concentration section. Water will be able to go through the membrane but other bigger molecules will be trapped on the membrane and this purify water will be free from heavy metals, dirt and microbes. Table 3 showed the heavy metal concentrations of bottle drinking water analyzed according to brands. Cadmium concentration was only detected in carbonated natural mineral water (B and C) at 0.01 mg/L which complied with the Food Act 1983 and Food Regulations 1985 [3] (0.01 mg/L). Lead content was high in un-carbonated natural mineral water (H) with 0.03 mg/L. Similar mean concentration values of cadmium (0.01 mg/L) were obtained in carbonated (A-D) and un-carbonated (E-G) natural mineral water and reverse osmosis water (J and L). These lead values were lower than the recommended concentration (0.05 mg/L) in the Food Act 1983 and Food Regulations 1985 [3]. Zinc concentration was higher in un-carbonated natural mineral water of Brand G (0.54 mg/L) and the lowest in Brands A, D, I and L (0.06 mg/L). These values were lower than the standard zinc concentration set by Food Act 1983 and Food Regulations 1985 [3]. Heavy metals concentrations were mostly found in the natural mineral water compared to distilled and reverse osmosis water except for zinc which was also found in all of the analyzed reverse osmosis water and one distilled water (P). Lead was also found in two brands of reverse osmosis water (J and L). Lead would be able to transfer into drinking water via piping and because of water storage tank containing lead [4]. Table 3. Heavy metal concentrations (mg/L) in bottle drinking water Brand Cadmium Lead Zinc Brand Cadmium Lead Zinc Carbonated Natural Mineral Water Reverse Osmosis Water A nd 0.01b 0.06de I nd nd 0.06de B 0.01a 0.01b 0.34ab J nd 0.01b 0.07de a b cde C 0.01 0.01 0.19 K nd nd 0.29cde b cde b D nd 0.01 0.06 L nd 0.01 0.06de Un-Carbonated Natural Mineral Water Distilled Water b cde E nd 0.01 0.19 M nd nd nd F nd 0.01b 0.46ab N nd nd nd G nd 0.01b 0.54a O nd nd nd a cde H nd 0.03 0.20 P nd nd 0.25cde a-e Different alphabets in the same column for each heavy metals showed significant differences (p<0.05). nd = not detected. Conclusions Some mineral concentrations in several bottled drinking water may contribute to the daily needs of minerals in the body. Most of the cations labeled on the bottle drinking water were much lower than the analyzed samples. On the other hand, anions labeled in most of the bottle mineral water were much higher than the analyzed samples. Although heavy metals were found in almost all of the bottle drinking water, but the values did not exceed the standard concentrations in the Food Act 1983 and Food Regulations 1985 [3]. References 1. Anthony, R. K., Takano, N., Ayame, A. (1999) "Solvent Extraction of Pb (II) From Acid Medium With Zinc Hexametylenedithiocarbamate Followed by Back-Extraction and Subsequent Determination by FAAS" Analy. Chimica Acta 386. 281-286. 2. Cabarello, J. M., Rubio, A. C., de la Torre, H. (2002) "Nitrate Intake From Drinking Water on Tenerife Island (Spain) ", Toxicology Department, Faculty of Medicine, La Laguna, Tenerife, Spain 85-91. 3. Food Act 1983 and Food Regulations 1985. (2003) Percetakan Nasional Malaysia Berhad. Kuala Lumpur.

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4. Pusat Racun Negara (1995) "Logam Berat" (on-line) http://www.prn2.usm.my/mainsite/bulletin/1995/penawar1.html (22 August 2003), 1-6. 5. Noreta A. (2001) "Perlukah Anda Membeli Air Botol? Buletin Perlindungan Perngguna" (on-line) http://www.kpdnhq.gov.my/hep/BPD/11_2001/11_2001_7.htm (22 August 2003), 7. 6. Tam, N. F. Y., Wong, Y. S. (2000) "Spatial Variation of Heavy Metals in Surface Sediment of Hong Kong Mangrove Swamps". Environ. Pollution 110. 195-205. 7. Toppi, S.L., Gabbrielli, R. (1999) "Response to Cadmium in Higher Plants". Environ. Exp. Botany 41. 105-130. 8. Whitney, E. N., Roffles, S. R. (1999) "Understanding Nutrition", 8th eds. An International Thomson Publishing Company, 366-432. 9. WHO (1992) "Revision of the WHO Guidelines for Drinking Water Quality. Report of the Final Tasks Group Meeting" Geneva, Switzerland. 10. Williamson, J., Wyandt, S., Christy, M. (2000) "Minerals For Good Health" J. Drug Topic. 44. 83.

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DETERMINATION OF TOTAL, SOLUBLE AND INSOLUBLE DIETARY FIBERS IN BAKERY PRODUCTS Suriah Abdul Rahman*, Tengku Farizan Izzi Che Ku Jusoh, Norrakiah Abdullah Sani* and Noraini Burok** *Food Science Program, School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, 43600 Selangor, Malaysia. E-mail: sar@pkrisc.cc.ukm.my **Hiestand Malaysia Sdn. Bhd., Lot 2, Jalan P10/14, Bandar Baru Bangi, 43600, Selangor, Malaysia. Abstract. This study was carried out to determine the total, soluble and insoluble dietary fibers in bakery products. Ten types of frozen bakery products have been selected which were categorized as pastry and bread. Pastry samples were Butter Gipfel, Strudel Apple, Pineapple Danish and Florida Cranberry Danish. Bread samples were French Baguette, Rustico Baguette, Multigrain Roll, Wholemeal Roll, Muesli Bread and Multi Seed Bread. Pastry samples were in the form of pre-proven and bread samples as pre-baked. All of the samples were obtained in frozen condition from a bakery factory situated at Bandar Baru Bangi. The total, soluble and insoluble dietary fibers were analyzed using Enzymatic Gravimetric Method (AOAC 991.43). Results showed that soluble dietary fibers were in the range of 0.00-0.73 g/100g, insoluble dietary fibers 4.50-14.07 g/100g and total dietary fibers 4.54-14.49 g/100g. Multigrain Roll contained the highest soluble dietary fiber (0.73 ± 0.30 g/100g). Rustico Baguette contained total dietary fiber and insoluble dietary fiber in the highest amount which were 14.49 ± 1.12 g/100g and 14.07 ± 1.03 g/100g respectively. Abstrak. Kandungan gentian diet jumlah, gentian diet larut dan gentian diet tidak larut dalam beberapa produk bakeri telah ditentukan di dalam kajian ini. Sebanyak sepuluh jenis produk bakeri telah dipilih dan dikategorikan kepada dua kumpulan iaitu pastri dan roti. Sampel pastri terdiri daripada Butter Gipfel, Strudel Apple, Pineapple Danish dan Florida Cranberry Danish. Sampel roti ialah French Baguette, Rustico Baguette, Multigrain Roll, Wholemeal Roll, Roti Muesli dan Roti Multi Seed. Sampel pastri diterima dalam bentuk pra-kembang dan sampel roti dalam bentuk prabakar. Kesemua sampel diperolehi dalam keadaan sejuk beku daripada kilang roti yang terletak di Bandar Baru Bangi. Penentuan kandungan gentian diet jumlah, gentian diet larut dan gentian diet tidak larut dijalankan menggunakan Kaedah Gravimetri Enzim Penimbal Fosfat (AOAC 991.43). Hasil analisis menunjukkan julat kandungan gentian larut dalam produk bakeri ialah 0.00-0.73 g/100g, gentian diet tidak larut 4.50-14.07 g/100g dan gentian diet jumlah 4.54-14.49 g/100g. Kandungan gentian diet larut tertinggi adalah Multigrain Roll (0.73 ± 0.30 g/100g). Rustico Baguette mengandungi gentian diet jumlah dan gentian diet tidak larut yang paling tinggi iaitu sebanyak 14.49 ± 1.12 g/100g dan 14.07 ± 1.03 g/100g masing-masing. Key words: Total, soluble, insoluble, dietary fiber, bakery products

Introduction Dietary fiber is the edible parts of plants or analogous carbohydrates that are resistant to digestion and absorption in the human small intestine or partial fermentation in the large intestine [1]. These

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include cellulose, hemicellulose, lignin, gums and pectin [6]. Fibers are classified according to several characteristics, including their chemical, structure, digestibility by bacterial enzymes and solubility in water. Some fibers are insoluble, meaning that they do not dissolve readily in water and others are soluble, they do dissolve in water [11]. Food sources of soluble fibers are dried beans, oats, barley and some fruits and vegetables [5]. Wheat bran and whole grains contain the most insoluble fibers, but vegetables and beans also are good sources of insoluble dietary fiber [3]. The dietary fiber in wheat comes principally from two sources, the outer branny layers containing cellulose and non cellulosic polysaccharides and the endosperm contains cellulose and ß-glucans [10]. For baked products, the soluble dietary fiber ranged from 0.56 to 1.62 g/100g, insoluble dietary ranged from 0.85 to 8.64 g/100g and total dietary fiber varied between 1.54 g/100 for soft white bread and 9.67 g/100g for reduced calorie white bread (firm) [8]. Several disorders of the digestive system, including diverticulitis, cancer of large bowel, appendicitis, gall stones, varicose vein, haemorrhoids, ischaemic heart disease, tooth decay, obesity and diabetes are all caused by lack of fiber in the diet [4]. World Health Organization [12] recommends that healthy adults consume between 27 to 40 grams of dietary fiber per day, meanwhile [9] recommendations range between 20 to 30 grams per day. The objective of the study was to determine the total, soluble and insoluble dietary fibers in some bakery products. Experimental Collection of samples Ten different types of bakery products have been selected and were categorized in two groups, pastry and bread. Pastry samples were Butter Gipfel, Strudel Apple, Pineapple Danish and Florida Cranberry Danish. Bread samples were French Baguette, Rustico Baguette, Multigrain Roll, Wholemeal Roll, Muesli Bread and Multi Seed Bread. Pastry samples were in the form of pre-proven (PP) and bread samples were pre-baked (PB). Pre-proven products were proved in proofing room for 60 min (temp 28-31ºC, humidity 75-82% rh). Pre-baked products were baked in an oven for 14 to 25 min after proofing for 60 min (temp 29-31ºC, humidity 55-65% rh). All of the samples were obtained in frozen condition (-220C) from a bakery factory, labeled and put into an icebox before being transferred to the food laboratory in Universiti Kebangsaan Malaysia. Samples preparation The frozen samples were cut and homogenized thoroughly in a blender. The samples were further dried overnight in an oven at 105ºC. The dried samples were cooled in a desiccator and then powdered using blender to a homogenous mixture. The samples were then stored in a freezer (-220C). Determination of total, soluble and insoluble dietary fiber Samples were analyzed for soluble and insoluble dietary fiber fractions according to AOAC Method 991.43 [2], an enzymatic-gravimetric procedure. Residues were suspended in phosphate buffer and digested sequentially with heat-stable α-amylase at 95-100ºC, protease at 60ºC and amyloglucosidase at 60ºC. Enzyme digestates were filtered through tared fritted glass crucibles. Crucibles containing insoluble dietary fiber were rinsed with diluted alcohol (78% and 95%), followed by acetone and dried overnight in an oven at 105ºC. Filtrates plus washing were mixed with four volumes of 95% ethanol to precipitate materials that were soluble in the digestates. After one hour, precipitates were filtered through tared fritted glass crucibles. One of each set of duplicate insoluble fiber residues and soluble fiber residues were ashed in a muffle furnace at 550ºC for 5 h. Another set of residues were used to determine protein as Kjeldahl nitrogen x 5.70. Soluble or insoluble dietary fiber residues (% original samples weight) minus % ash and % crude protein found in the residues were taken to be the value for respective dietary fiber fraction (both the ash and crude protein were also determined in this study but not shown in this article). Total dietary fiber was calculated as the sum of soluble and insoluble dietary fiber.

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Statistical analysis The data generated on total, soluble and insoluble dietary fiber contents were analyzed using SPSS (Statistical Package for Social Sciences) version 12.0 and reported as a mean ± S.D. Results And Discussion Soluble dietary fiber (SDF), insoluble dietary fiber (IDF) and total dietary fiber (TDF) in bakery products are presented in Table 1. The SDF content ranged from 0.00 g/100g in Multi Seed Bread to 0.73 g/100g in Multigrain Roll. The IDF and TDF of bakery products ranged at 4.50 g/100g and 4.54 g/100g in Butter Gipfel and 14.07 g/100g and 14.49 g/100g in Rustico Baguette respectively. Multigrain Roll (0.73 ± 0.30 g/100g) contained the highest SDF obtaining it from wheat flour, rye flour, sesame seed, linseed, sunflower seed, millet and oats. Rustico Baguette which contained IDF and TDF in the highest amount was made from wheat flour, rye flour, ruchmehl, wheat bran, sunflower seed, sesame, linseed, oats and millet. Bran is the best source of dietary fiber [13]. Soluble dietary fiber in Florida Cranberry Danish (0.54 ± 0.36 g/100g), Strudel Apple (0.32 ± 0.31 g/100g) and Pineapple Danish (0.24 ± 0.33 g/100g) were contributed by fruits in the fillings. The filling of Florida Cranberry Danish was made from roselle, Strudel Apple contained mixture of apple and raisin and Pineapple Danish was made from pineapple. The IDF contents in Multi Seed Bread and Multigrain Roll were 13.22 ± 0.05 g/100g and 13.63 ± 1.60 g/100g respectively. These two products were rich with grains and seeds such as wheat flour, rye flour, sesame seed, sunflower seed, linseed, oats and millet. The TDF value in Muesli Bread was 10.09 ± 0.15 g/100g. The ingredients such as wheat flour, ruchmehl, rye flour, raisins, walnut, apple diced, mixed peel and oats contributed to the dietary fiber content in Muesli Bread. However, the TDF contents in French Baguette and Butter Gipfel were 9.13 ± 2.28 g/100g and 4.54 ± 0.60 g/100g respectively. Both of these products were made from wheat flour. Sample Table 1. Dietary fiber contents of bakery products (mean ± S.D.) SDF IDF (g/100g) (g/100g) TDF (g/100g) 4.54 ± 0.60 7.55 ± 0.48 11.62 ± 2.95 7.89 ± 1.17 9.13 ± 2.28 14.49 ± 1.12 14.37 ± 1.70 11.62 ± 0.46 10.09 ± 0.15 13.22 ± 0.05

Pastry 4.50 ± 0.58 Butter Gipfel 0.04 ± 0.07 7.23 ± 0.37 Strudel Apple 0.32 ± 0.31 11.38 ± 2.65 Pineapple Danish 0.24 ± 0.33 7.36 ± 0.91 Florida Cranberry Danish 0.54 ± 0.36 Bread 9.08 ± 2.28 0.23 ± 0.02 French Baguette 14.07 ± 1.03 0.42 ± 0.06 Rustico Baguette 13.63 ± 1.60 0.73 ± 0.30 Multigrain Roll 12.06 ± 0.38 0.56 ± 0.16 Wholemeal Roll 10.11 ± 0.24 0.08 ± 0.15 Muesli Bread 13.22 ± 0.05 0.00 ± 0.00 Multi Seed Bread SDF: soluble dietary fiber; IDF: insoluble dietary fiber; TDF: total dietary fiber ±: Standard deviation of duplicate analyses

Table 2 shows the serving size and quantity of dietary fiber per serving for each of the bakery products. According to FDA [7], products containing 5 g or more dietary fiber per serving can be claimed as high fiber products, while products containing 2.5 to 4.9 g per serving as good source of fiber. However, food making high fiber or good source of fiber claims must meet the definition for low fat (3 g fat per serving) or the level of total fat must appear next to the high fiber claim. From this study, only French Baguette and Multigrain Roll can be categorized as high fiber products, while Wholemeal Roll is a good source of fiber.

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Conclusions Soluble dietary fiber (SDF), insoluble dietary fiber (IDF) and total dietary fiber (TDF) in the bakery products were in the range of 0.00-0.73 g/100g, 4.50-14.07 g/100g and 4.54-14.49 g/100g respectively. Multigrain Roll contained the highest SDF (0.73 ± 0.30 g/100g) while Rustico Baguette contained IDF and TDF in the highest amount which were 14.07 ± 1.03 g/100g and 14.49 ± 1.12 g/100g respectively. French Baguette and Multigrain Roll are high fiber products, while Wholemeal Roll is a good source of fiber. Table 2. Serving size and quantity of dietary fiber per serving for each bakery products Serving size Quantity of Quantity of dietary Quantity of fat Sample (g) dietary fiber fiber per serving per serving (g) per 100g (g) Butter Gipfel 35 4.54 1.59 8.74 Strudel Apple 55 7.55 4.15 10.10 Pineapple Danish 45 11.62 5.23 8.63 Florida Cranberry Danish 55 7.89 4.34 9.93 French Baguette 195 9.13 17.80 0.59 Rustico Baguette 200 14.49 28.98 5.89 Multigrain Roll 35 14.36 5.03 1.87 Wholemeal Roll 35 12.62 4.42 0.04 Muesli Bread 350 10.09 35.32 6.51 Multi Seed Bread 350 13.22 46.27 6.51 Acknowledgement The authors wish to thank Hiestand Malaysia Sdn. Bhd. for their interest and support in this study. References 1. AACC. (2001) "The Definition of Dietary Fiber", (online) http://course.che.umn.edu/01fscn43461s/4346R (21 July 2004). 2. AOAC. (1997) "Official Methods of Analysis", 16 th Ed. Gaitherburg: AOAC International. 3. Anderson, J., Young, L. (2004) "Dietary Fiber", (online) http://www.ext.colostate.edu (21 July 2004). 4. Bingham, S. (1978) "Dictionary of Nutrition, a Consumer’s Guide to the Facts of Food", Britain: Communica-Europa. 5. Boeckner, L. (1995) "Extension Nutrition Specialist", (online) http://www.pubs@unl.edu (21 July 2004). 6. Calloway, D. H., Carpenter, K. O. (1981) "Nutrition and Health", Philadelphia: Saunders College Publishing. 7. FDA (1995) "Labelling Claim", (online) http://www.fda.gov/fdac/graphics/foodlabel.pdf (31 August 2004). 8. Li, B. W., Andrews, K. W., Pehrsson, P. R. (2002) "Individual Sugars, Soluble and Insoluble Dietary Fiber Contents of 70 High Consumption Foods" J. Food Comp.Analy. 15. 715-723. 9. Malaysia Dietary Guidelines (1998) "A Report by An Expert Panel". Kuala Lumpur: Inc Press. 10. Southgate, D. A. T. (2003) "Dietary Importance. In. Carbalerro, B., Truco, L. C., Finglas, P. M. Encyclopedia of Food Sciences and Nutrition", 2nd Ed. Vol. 3. Amsterdam: Academic Press. 11. Whitney, E. N., Cataldo, C.B., DeBruyne, L. K., Rolfes, S. R. (2001). "Nutrition for Health and Health Care", 2nd Ed. Australia: Wardsworth Thomson Learning. 12. WHO (1990) "Diet, Nutrition and the Prevention of Chronic Disease. Reports of a WHO Study Group. WHO Technical Reports Series 797", Geneva: World Health Organization. 13. Wilson, E.D., Fisher K.H., Garcia, P.A. (1979) "Principles of Nutrition", 4th Ed. New York: John Wiley & Sons.

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KINETICS AND THERMODYNAMIC FOR SORPTION OF ARSENATE BY LANTHANUM-EXCHANGED ZEOLITE Md Jelas Haron, Saiful Adli Masdan, Mohd Zobir Hussein and Zulkarnain Zainal Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia mdjelas@fsas.upm.edu.my Abstract: Zeolites are crystalline, hydrated aluminosilicate containing exchangeable alkaline and alkaline earth cations in their structural frameworks. Since zeolites have permanent negative charges on their surfaces, they have no affinity for anions. However recent studies have shown that modification of zeolites with certain surfactants or metal cations yield sorbents with a strong affinity for many anions. In this paper, modification of zeolites (zeolite A, X and ZSM5) were performed by exchange of naturally occurring cations with lanthanum ion that forms low solubility arsenate salt. The exchanged zeolites were used to sorb arsenate from aqueous solution. Among parameters investigated were effect of pH, arsenate initial concentrations, contact time and temperature. The maximum exchanged capacity of La(III) ion was obtained when using solution with initial pH of 4. Zeolite X gives the highest La(III) exchanged capacity compared to other zeolites. The results show that As(V) sorption by La-zeolites occurred at about pH 6.5 and increased as pH increases and reaching maximum at equilibrium pH about 7.8. On the other hand, almost no arsenate sorption occurred on unexchanged zeolites. This indicates that La(III) ion on the exchanged zeolites is taking part on the As(V) sorption via surface precipitation. The results also show that the sorption capacities increased with increasing initial As(V) concentrations. The sorption follows Langmuir model with maximum sorption capacities of 0.41, 0.21 and 0.19 mmol/g at 25°C for La exchanged zeolite X (La-ZX), La exchanged zeolite ZSM5 (La-ZSM) and La exchanged zeolite A (La-ZA), respectively. The amounts of sorption of As(V) by La exchanged zeolite increased as temperature increases from 25 to 70°C shows that the process is endothermic. The free energy change (∆G°) for the sorption at 25°C were -10.25, -9.65 and -8.49 kJ/mol for La-ZX, La-ZSM and La-ZA, respectively. The negative values of ∆G° means that the sorption of As(V) ions on La-exchanged zeolite is spontaneous, perhaps because the La(III) have high affinity towards the arsenic ion as indicated by a low Ksp value of of lanthanum arsenate. A slightly positive entropy change for sorption of As(V) ion on La-exchanged zeolite could be due to fixation of the ions on the La(III) exchange sites that randomly distributed on the sorbents. The kinetics study shows that the As(V) sorption follows first order kinetic model. The first-order kinetic constants for the sorption are 2.77x10-3, 2.25x10-3 and 1.60x10-3 min-1 for La-ZX, La-ZSM and La-ZA, respectively. Abstrak: Zeolit adalah kristal aluminosilikat terhidrat mengandungi kation logam alkali dan alkali bumi yang boleh ditukarganti dalam struktur bingkainya. Oleh kerana strukturnya mempunyai cas negative kekal, ia tidak mempunyai afiniti terhadap anion. Walau bagaimana pun kajian terkini mendapati pengubahsuaian dengan surfaktan atau ion logam tertentu boleh menjadikan zeolit sebagai pengerap dan mempunyai afiniti yang kuat tehadap anion. Kertas ini melaporkan penukaran kation asal zeolit (A, X and ZSM5) dengan ion logam lanthanum yang berupaya membentuk garam arsenat yang mempunyai nilai keterlarutan rendah. Zeolit yang mengandugi kation lanthanum telah digunakan untuk mengerap arsenat dari larutan akueus. Diantara parameter yang dikaji termasuklah kesan pH, kepekatan awal arsenat, masa sentuh dan suhu. Muatan pertukaran maksima La(III) kedalam zeolit berlaku pada pH 4. Zeolit X mempunyai muatan pertukaran La(III) paling tinggi berbanding zeolite lain. Keputusan kajian menunjukkan

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erapan As(V) oleh La-zeolit berlaku pada pH 6.5 dan meningkat dengan penigkatan pH, dan mencapai maksimum pada pH 7.8. Sebaliknya zeolit asal tanpa pertukaran lanthanum hampir tidak menunjukkan sebarang erapan terhadap arsenat. Ini menunjukkan La(III) pada zeolit mengambil bahagian didalam erapan arsenat berkemungkinan melalui pemendakan permukaan. Keputusan juga menunjukkan muatan erapan arsenat menigkat dengan penigkatan kepekatan awal ion arsenat. Erapan As(V) mengikuti model Langmuir dengan muatan erapan meksimum 0.41, 0.21 dan 0.19 mmol/g pada 25°C masing-masing bagi zeolit X tertukar La (La-ZX), zeolite ZSM5 tertukar La (La-ZSM) dan zeolite A tertukar La (La-ZA). Amaun erapan As(V) meningkat dengan peningkatan suhu daripada 25 ke 70°C menunjukkan proses ini adalah eksotermik. Perubahan tenaga bebas (∆G°) pada 25°C adalah -10.25, -9.65 dan -8.49 kJ/mol masing-masing untuk La-ZX, La-ZSM dan La-ZA. Nilai ∆G° negative menunjukkan proses erapan adalah spontan, mungkin disebabkan oleh afiniti yang tinggi ion lanthanum terhadap ion arsenik seperti yang ditunjukkan oleh nilai Ksp lanthanum arsenate yang rendah. Nilai perubahan entropi yang positif pula menguatkan lagi anggapan berlaku tindakbalas antara ion As(V) dengan ion lanthanum yang bertaburan secara rawak pada zeolit. Kajian kinetic menunjukkan erapan As(V) mematuhi model kinetic tertib pertama. Pemalar kinetic tertib pertama adalah 2.77x10-3, 2.25x10-3 dan 1.60x10-3 min-1 masing-masing untuk La-ZX, La-ZSM and La-ZA. Keywords:La-exchanged zeolite, arsenate sorption, thermodynamic and kinetics Introduction Although arsenic is known as a highly toxic element, the compounds are still widely used in many applications. Among others they are used for the productions of ceramics, semiconductors, pesticides and fertilizers. The arsenic also enters the environment through other anthropogenic activities such as petroleum refineries, fossil fuel power plants and non-ferrous smelting. Recently most international drinking water standards were lowered to 0.01 mgl-1. The international standard for effluent was set to 1.0 mgl-1. Consequently there is growing interest in the study on removal of arsenic ion from aqueous systems. Conventional precipitation methods for arsenic removals have not been successful to meet drinking and effluent standards for As due to limitation of solubility of the resultant product. On the other hand, adsorption appears to be one of the promising methods for removal of arsenic from water. Among others, the removal of As(V) by adsorption methods have been studied using spodic [1], aquifer material [2], oxides of antimony and manganese [3], iron hydroxide [4] iron coated catalyst [5] and ion exchanger [6]. Zeolites are crystalline, hydrated aluminosilicate minerals containing exchangeable alkaline and alkaline earth metal cations normally of group I and group II elements, in particular, sodium, potassium, magnesium, calcium, strontium, and barium, as well as water molecules in their structural frameworks. Structurally, they are complex, porous, crystalline inorganic polymers, enclosing interconnected cavities in which the metal ions and water molecules are contained. They are based on an infinitely extending three dimensional network of AlO4 and SiO4 tetrahedra linked to each other by sharing all the oxygen ions [7]. Zeolites may be represented by an empirical formula of M2/nAl2O3xSiO2.yH2O. In this oxide formula, x is generally equal to 2 or greater since AlO4 tetrahedra are joined only to SiO4 tetrahedra, n is the cation valence which neutralize the negative charge on the aluminosilicate framework and y represents the water contained in the voids of the zeolite [8]. Zeolites have excellent catalytic as well as separation properties. Several properties of zeolite minerals have been studied, including adsorption and ion exchange. These were important applications of zeolites for removal of heavy metals by both processes. Among others, the advantages of ion exchange over the chemical precipitation method for heavy metal removal are high selectivity, can be recovered and produce less sludge. The availability of natural zeolites provides a low cost ion exchanger [9]. Table 1 shows a few examples of zeolite composition [10].

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The Si/Al ratio of a zeolite indicates its cation content; the fewer aluminium atoms there are, the fewer the exchangeable cations will be present. The highly siliceous zeolites such as zeolite ZSM5 can have a Si/Al ratio that lies between 20 and 1.5 where else zeolite A and X have Si/Al ratio around 1. The zeolites with high Si/Al ratios are stable in the presence of concentrated acids [11]. Table 1: Zeolite Compositions [10] Zeolite Natural Mordenite Faujasite Clinoptilolite Synthetic Zeolite A Zeolite X Zeolite ZSM-5 Typical formula Na8[AlO2)8(SiO2)40].24H2O (Ca,Mg,Na2,K2)4.5[AlO2)59(SiO2)27].27H2O Na6[(AlO2)6(SiO2)30].24H2O Na12[(AlO2)12(SiO2)12].27H2O Na86[(AlO2)86(SiO2)106].264H2O (Na,TPA)3[(AlO2)3(SiO2)93].16H2O

Since zeolites have a permanent negative charge on their surface, they have no affinity for anions. However recent studies have shown that modification of zeolites with certain surfactants or metal cations yield sorbents with a strong affinity for many anions [12]. In order to sorb anions, the modified surface must either possess positively charged exchange sites, or there should be replacement of weakly held counter ions of the surfactant by more strongly held counter ions. Cationic-surfactant-modified zeolites were shown to remove chromate from aqueous solutions [12, 13]. Some cationic exchanged forms of zeolites showed high uptake of iodide and molybdate from solution [14] . Since As(V) presence in water as arsenate anion in wide pH range, the removal of As(V) by zeolites can be enhanced after loading it with various metal ions such as Al(III) [15] and Pb and Cu [16]. Previous reports have shown that rare earth compounds including lanthanum [17] were effective for removal of As(V) from aqueous solutions. In this paper, modification of zeolites A, zeolite X and zeolite ZSM5 were performed by exchange of their naturally occurring cations with lanthanum ion. The exchanged zeolites were used sorb arsenate from aqueous solution. Parameters investigated include effect of pH, arsenate initial concentration, contact time and temperature. The lanthanum exchanged zeolite has been used as catalysts [18]. Eksperimental Aluminium sulphate hexahydrate, sodium hydroxide, sulphuric acid, hydrochloric acid and sodium dodecyl sulphate were purchased from BDH. Propylamine, lanthanum chloride heptahydrate, sodium arsenate were purchased from Fluka while silicic acid and tetrapropylammonium bromide were purchased from Aldrich. These chemicals are 99.9% pure. Zeolites used in this study were zeolite X (Fluka) and zeolite A (BDH) and used without further treatment. Zeolite ZSM5 was synthesised as follows [19]: 2 grams of silicic acid, 1 g of tetrapropylammonium bromide (TPABr) as templating agent and 0.5 g of sodium hydroxide were mixed in 5 mL of distilled water followed by addition of 1 mL of n-propylamine and mixed well. Another solution containing 0.25 g of aluminium sulphate and a few drops of concentrated sulphuric acid was also prepared. The former solution was added to the latter and the resulting solution was thoroughly mixed, and aged in a hydrothermal bomb at 160oC for 5 days. The resulting zeolite mixture was calcined at 500oC for 2 hours. Preparation of La(III)-exchanged on Zeolites About 5.0 g zeolite was added to a 100 mL 0.10 M lanthanum chloride heptahydrate (Fluka) solutions at pH 5 (HCl) and stirred for 12 hours at ambient temperature. The zeolite was separated from the solution by centrifugation. The pH of the final solution was measured and the amount of lanthanum

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ion in the supernatant was determined by Inductively Couple Plasma-Atomic Emission Spectrometer (ICP-AES) (Perkin Elmer P1000) using recommended procedure by the manufacturer. Sorption of Arsenic Ion on Zeolites and La-exchanged Zeolites A series of NaH2AsO4 (Aldrich) solutions (25 ml) with concentration ranges from 25-125 mM were prepared. The pH of the solution was adjusted by 0.1M hydrochloric acid and about 0.1 g of zeolite sorbent was added. The mixture was then stirred at ambient temperature. At a specific contact time, the adsorbent was separated from the solution by centrifugation. The pH of the final solution was measured and the arsenate ion was quantified by ICP-AES. Parameters that can affect the sorption capacities such as pH, concentration of arsenate ions, adsorbent dosage and temperature were studied. Zeolite A, zeolite X, zeolite ZSM5 and La-exchanged zeolite X (La-ZX), La exchanged-zeolite ZSM5 (La-ZSM) and La-exchanged zeolite A (La-ZA) were used as sorbents. RESULTS AND DISCUSSION La(III) Metal Ions Uptake by Zeolite Previous researcher showed that La exchange capacity in zeolite increased by increasing the initial pH value [20]. In the present study, the highest exchange capacity of La(III) was obtained when using solutions with initial pH of 4, which changed to give a final pH value of 5-6. The results are similar with previous studies of metal sorption by natural zeolite, which revealed that the sorption capacity is low at pH below 4.0 [21, 22] Moreover, low pH values are undesirable for zeolite sorption because this would affect its physical and chemical structure [23]. Zeolite X gives the highest La exchanged capacity compared to other zeolite as shown in Table 2. This was expected since zeolite X has the biggest pore diameter [19]. The La exchange of the zeolite is carried out in acidic solution in order to avoid the hydrolysis and precipitation of lanthanum hydroxides. However, under this condition, the sodium ions in the zeolite will be exchanged not only for La(III), but also for hydronium ions from the solution [24]. Table 2 Exchanged capacities of zeolite for La Zeolite Pore diameter[19] La exchanged capacity nm mmol/g Zeolite X 1.20 1.10 Zeolite ZSM5 0.55 0.80 Zeolite A 0.42 0.58 Sorption of Arsenate Ion by Zeolites The experiments conducted by Bonnin [25] showed that natural zeolites were unable to adsorbed significant amount of As(V) from aqueous solution. Modification of the zeolite by exchanged with ferrous ion increased the As(V) sorption by 92% in the order of 25 mg/g. In the present study, sorption of arsenate ion was carried out using original and La exchanged zeolite A, X and ZSM5. Figure 1 shows the capacities at equilibrium pH for the sorption of arsenate ion on La-ZA, La-ZX, LaZSM. The results show that the As(V) sorption occurred at about pH 6.5. The sorption increased as pH increases and reaching maximum at about pH 7.8. At higher pH, La will be precipitated as La(OH)3. On the other hand, almost no arsenate sorption occurred on unexchanged zeolites as shown in Figure 2. This confirmed that La(III) ion on the zeolites was taking part on the As(V) sorption. Since the Ksp of lanthanum arsenate is reasonably low being 3.55x10-22 M2, suggesting that the As(V) removal by the La-exchanged zeolites most likely via surface precipitation. The As(V) retention is highest on the La-ZX. This could be due to highest La exchanged capacity of zeolite X compared to the other zeolites as shown in Table 2. Effect of As(V) Concentrations on Sorption The sorption of arsenate ion on La-exchanged zeolites was conducted with various initial concentrations (1 mM to 5 mM) at pH 5. The results show that the sorption capacities increased with increasing initial concentration (Figure 3). In order to establish the maximum

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0.30 As(V) sorption capacity, mmol/g 0.25 0.20 0.15 0.10 0.05 0.00 6.00

6.50

7.00

7.50

8.00

Equilibrium pH Figure 1. Sorption capacity of As(V) by La-Zeolite at equilibrium pH
La-ZX La-ZMS La-ZA

0.30 Sorption capacity, mmol/g 0.25 0.20 0.15 0.10 0.05 0.00 6.00 6.50 7.00 equilibrium pH 7.50 8.00

Figure 2. Sorption of As(V) by unexchanged zeolite at various equilibrium pHs Zeolite X Zeolite ZSM Zeolite A

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As(V) sorption capacity, the Langmuir equation of the following form was applied to the sorption equilibria at different concentrations:

Ce 1 C = + e qe bqm qm

(3)

where Ce is the concentration of metal solution at equilibrium (mmol/L), qe the amount of As(V) sorbed at equilibrium (mmol/g), qm the maximum sorption capacity and b is constant related to binding energy of sorption system. The linearity of the plots (Fig. 4) shows that the sorption follows the Langmuir model with maximum capacities of 0.41, 0.21 and 0.19 mmol/g for La-ZX, La-ZSM and La-ZA, respectively (Table 3). It appears that there is a strong correlation (R2=0.88) between As(V) sorption capacity to exchange capacity of La in zeolite. However there is no correlation found between binding constants and capacities. Table 3 Maximum sorption capacities of La-zeolites for As(V) Langmuir binding energy Zeolite La exchanged capacity As(V) maximum constant, (b) mmol/g sorption (qm) mmol/g L/mmol La-ZX 1.10 0.41 6.36 La-ZSM 0.80 0.21 14.82 La-ZA 0.58 0.19 0.49 Thermodynamic Parameters The amount of sorption of As(V) by La-exchanged zeolites increased as temperature increases from 25 to 70 °C. The following relationships has been used to evaluate the thermodynamic parameters the standard Gibbs free energy ∆G°, enthalpy ∆H° and entrophy ∆S°: ∆G°= -RT ln Kd (3)

and

∆S ° ⎛ ∆H ° ⎞ 1 log K d = −⎜ ⎟ + ⎝ 2.303 R ⎠ T 2.303R

(4)

where Kd is the equilibrium partition constant calculated as the ratio between sorption capacitiy and equilibrium concentration. The change in free energy (∆G°) for As(V) sorption were calculated to be -10.25, -9.65 and -8.49 kJ/mol for La-ZX, La-ZSM and La-ZA, respectively at 25°C. The negative values of ∆G° means that the sorption of As(V) ions on La-exchanged zeolite is spontaneous, perharps because the La have high affinity towards the arsenic ion due to low Ksp value of 3.55x10-22 [26] (for comparison, Ksp AgCl is 1.77x10-10 M2). From equation (4) a plot of log Kd versus 1/T (Fig. 5) would give ∆H° and ∆S° which are given in Table 4. The positive values of ∆H° indicate the endothermic nature of sorption process which is compared favorably with that reported using lanthanum oxide [27] . A slightly positive entropy change for sorption of As(V) ion on La-exchanged zeolite is due to fixation of the ions on the exchange sites of the randomly distributed La ions on the sorbents.

Table 4. Thermodynamic parameters for the adsorption of As(V) by La-zeolite Adsorbent ∆G° (kJ/mol) ∆H° (kJ/mol) ∆S° J/molK LaZX -10.25 14.78 0.085 LaZMS -9.65 10.21 0.067 LaZA -8.49 18.51 0.091

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0.45 Sorption Capacity, mmol/g 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 1.0 2.0 3.0 4.0 Equilibrium concentration, mmol/L Figure 3. Effect of concentration on sorption capacity of As(V) by La-Zeolite La-ZX La-ZSM La-ZA 0.0 5.0

40.0 35.0 30.0 Ce/qe, g/L 25.0 20.0 15.0 10.0 5.0 0.0 0.00 1.00 2.00 3.00 4.00 5.00 Ce mmol/L Figure 4. Langmuir isotherm of As(V) sorption by La-Zeolite
La-ZX La-ZSM La-ZA

y = 5.1871x + 10.619 R2 = 0.9913

y = 4.7703x + 0.3216 R2 = 0.9997

y = 2.4177x + 0.3801 R2 = 0.9965

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2.2 2.1 2 1.9 log Kd 1.8 1.7 1.6 1.5 1.4 0.0028 0.0029 0.003 0.0031 0.0032 0.0033 0.0034 1/T (per degree Kelvin) Figure 5. Determination of thermodynamic parameters for sorption of As(V) using various La-zeolite La-ZA La-ZX La-ZSM
y = -966.91x + 4.7324 2 R = 0.9885 y = -772.02x + 4.4006 2 R = 0.9307

y = -533.72x + 3.4836 2 R = 0.9958

0
0 2 4 6 8 10

-0.2
y = -0.0721x - 0.2493

-0.4 log(qe-q) -0.6 -0.8 -1 -1.2 Time, hr
y = -0.0587x - 0.5183 R = 0.9849
2

R = 0.9907

2

y = -0.0418x - 0.587 R = 0.9782
2

Figure 6. Sorption of As(V) by La-zeolite according to first order kinetic equation
La-ZX La-ZSM La-ZA

Sorption Kinetics of As(V) by La-zeolite The kinetics of sorption of As(V) by modified zeolite was studied in batch experiments. The pseudofirst-order rate constants for sorption of arsenic ions are determined using the following equation log(qe-qt) = logqe – k1t/2.303 (5)

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where qt refers to the amount of As(V) sorption at any time t. A straight line plot of log(qe-qt) versus t (Fig. 6) indicates the applicability of the first order kinetics for As(V) sorption. The sorption is rapid at the beginning with t1/2 of about 3, 4 and 5 hours for La-ZX, La-ZSM and La-ZA, respectively. The pseudo-first-order kinetic constants k1 for As(V) sorption for the respective zeolites were 2.77x10-3, 2.25x10-3 and 1.60x10-3 min-1. Conclusion The maximum exchanged capacity of La(III) ion was obtained when using solutions with initial pH of 4 with zeolite X gives the highest La(III) exchanged capacity compared to zeolite ZSM5 and zeolite A. The results show that the As(V) sorption by La-zeolites occurred at about pH 6.5 and increased as pH increases and reaching maximum at about pH 7.8. Almost no arsenate sorption occurred on unexchanged zeolites which indicates that La(III) ion on the exchanged zeolites is taking part on the As(V) sorption probably via surface precipitation. The results also show that the sorption capacities increased with increasing initial As(V) concentrations. The sorption follows Langmuir model with maximum sorption capacities of 0.41, 0.21 and 0.19 mmol/g at 25°C for La-ZX, La-ZSM and La-ZA, respectively. The amounts of sorption of As(V) by La-zeolite increased as temperature increases from 25 to 70°C shows that the process is endothermic. The free energy change (∆G°) for the sorption at 25°C were -10.25, -9.65 and -8.49 kJ/mol for La-ZX, La-ZSM and La-ZA, respectively. The negative values of ∆G° shows that the sorption of As(V) ions on La-exchanged zeolite is spontaneous, perhaps because the La(III) have high affinity towards the arsenic ion as indicated by a low Ksp value of the lanthanum arsenate. A slightly positive entropy change for sorption of As(V) ion on La-exchanged zeolite could be due to fixation of the ions on the La(III) exchange sites that randomly distributed on the sorbents. The kinetics study shows that the As(V) sorption follows first order kinetic model. The first-order rate constants for the sorption are 2.77x10-3, 2.25x10-3 and 1.60x10-3 min-1 for La-ZX, LaZSM and La-ZA, respectively. Acknowledgements The authors thank to Ministry of Science, Technology and Innovation for Fundamental Research Grant No: 55204. References 1. 2. 3. 4. 5. 6. 7. 8. Lindberg, J., Sterneland.J., Johansson, P.O., and Gustafsson, J.P., (1997). Spodic material for in situ treatment of arsenic in ground water. Ground Water Monitoring and Remediation 17, 125-130. Carrillo, A., and Drever, J.L. (1998). Adsorption of arsenic by natural aquifer material in the San Antonio-El Triunfo mining area, Baja California, Mexico. Environmental Geology 35, 251-257. Galer, J.M., Delmas, R. and Loos-Neskovic, C., (1997). Progress in Ion Exchange: Advances and Applications. In Progress in Ion Exchange: Advances and Applications, Dyer A., Hudson, M.J. and Williams, P.A. (eds.), The Royal Society of Chemistry, p. 187. Raven, K.P. Jain, A. and Loeppert, R.H. (1998). Arsenite and Arsenate Adsorption on Ferrihydrite: Kinetics, Equilibrium, and Adsorption Envelopes. Environmental Science and Technology 32, 344-249. Huang J.G., L.J.C. (1997). Enhanced removal of As(V) from water with iron-coated spent catalyst. Separation Science and Technology 32, 1557-1569. Vagliasindi, F.G.A., Benjamin, M.M. (1998). Arsenic removal in fresh and nom-preloaded ion exchange packed bed adsorption reactors. Water Science and Technology 38, 337-343. Bhatia, S. (1990) "Zeolite catalysis: Principles and applications" Boca Raton, Florida: CRC Press, Inc. Breck, D.W. (1974). Zeolite molecular sieves: structure, chemistry and use. New York: John Wiley and Sons Inc.

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9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

Kersaoui-Ouki, S., Cheeseman, C.R., and Perry, R (1994). Natural zeolite utilisation in pollution control: a review of applications to metal effluents. J. Chem. Tech. Biotechnol. 59, 121-126. Mark, H.F., Othmer, D. F., Overberger, C. G. and Seaborg, G. T. (1981). Molecular sieve. In Kirk Othmer Encyclopedia of Chemical Technology, 3 Edition, New York: John Wiley and Sons Publication. Smart, L., and Moore, E. (1992). Solid state chemistry an introduction. London: Chapman and Hall, pp. 183-198. Li, Z., Anghel, I., and Bowman, R.S. (1998). Sorption of oxyanions by surfactant-modified zeolite. Journal of Dispersion Science and Technology 19, 843-857. Li, Z., and Bowman, R.S. (1997). Counterion effects on the sorption of cationic surfactant and chromate on natural clinoptilolite. Environmental Science and Technology 31, 2407-2412. Chmielewska-Horvathova, E. and Lesny, J. (1996). Pollutant immobilization in column of Ag-clinoptilolite. Journal of Radioanalytical and Nuclear Chemistry 214, 209-221. Xu Y-H., O.A. and Maeda, S., (2000). Removal of arsenate, phosphate, and fluoride ions by aluminium-loaded shirasu-zeolite. Toxicological and Environmental Chemistry 76, 111-124. Elizalde-González M.P., Mattusch, J., Einicke, W.D., and Wennrich, R. (2001). Sorption on natural solids for arsenic removal. Chemical Engineering Journal 81, 187-195. Tokunaga, S., Wasay, S.A., and Park, S.W. (1997). Removal of arsenic(v) ion from aqueous solutions by lanthanum compounds. Water Science and Technology 35, 71-78. Jia, C., Beaunier, P. and Massiani P. (1998). Comparison of conventional and solid-state ion exchange procedures for the incorporation of lanthanum in H-beta zeolite. Microporous and Mesoporous Materials 24, 69-82. Masdan (2000). Thesis MS Universiti Putra Malaysia. Jia, C., Massiani, P., Beaunier P. and Barthomeuf, D., (1993). Solid-state exchange of lanthanum in beta zeolite. Applied Catalysis A: General 106, L185-L191. Blanchard, G., Maunaye, M. and Martin, G. (1982). Removal of heavy metals from water by means of natural zeolites. Wat. Res. 18, 1501-1507. Ali, A.A.-H., and El-Bishtawi, R. (1997). Removal of lead and nickel ions using zeolite tuff. J. Chem. Tech. Biotechnol. 69, 27-34. Vaughan, D.E.W. (1988). The synthesis and manufacture of zeolites. Chem. Eng. Prog. 82, 25-31. Scherzer, J. (1989). Rare Earths, Extractions, Preparation and Application. In: Rare earths in cracking catalysts, R.G. Bautista and Wong, M. M. ( eds). (California: TMS Publication), pp. 317-331. Bonnin, D. (1997). Arsenic Removal from Water Utilizing Natural Zeolites. In Proceedings of the Annual Conference of the American Water Works Association. pp. 421-441. Firsching, F.H. (1992). Solubility Products of the Trivalent Rare-Earth Arsenates. Journal of Chemical Engineering Data 37, 497-499. Rawat A., M.M. (1998). Adsorption of the Oxyanions of Arsenic onto Lanthanum Oxide. TMS Annual Meeting 1998, 13-23.

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CARPAINE FROM Carica Papaya L. var. EKSOTIKA I M.F. Mohamad Bukhori1, N. Abdul Rahman 2, N. Khalid1 & V. Pillai3
1

. Institute of Biological Sciences, 2. Department of Chemistry, Faculty of Sciences, University of Malaya, Lembah Pantai, 50603 Kuala Lumpur, Malaysia. 3 . Strategic, Environment and Natural Resources Research Centre, MARDI Headquaters, P.O. Box 12301, 50774 Kuala Lumpur, Malaysia. khallila@yahoo.com

Abstract. In nature, plant synthesizes an extensive array of secondary metabolites, often with highly complex chemical structures. In the Caricaceaea family, the most abundant secondary metabolite reported to be the alkaloid, carpaine. Carpaine has been isolated from leaves of various carica papaya namely, the Nigerian variety and Solo and its absolute structure has been determined. Our study is concentrated on the extraction of carpaine from the leaves of a local variety, Eksotika I. The isolation procedures used is an adaptation from the methods5, 17; with major modifications to the solvent system and separation methodology for greater purification. The chemical structure of carpaine was determine using nuclear magnetic resonance (NMR) and gas chromatography mass spectrometry (GCMS) spectroscopy Abstrak. Dalam keadaan semulajadi, tumbuhan menghasilkan banyak metabolit sekunder dan biasanya dengan struktur kimia yang komplek. Dalam famili Caricaceaea kebanyakan metabolit sekunder yang dihasilkan adalah alkaloid karpain seperti terdapat dalam kebanyakan laporan. Karpain telah pun diekstrak daripada daun betik dari varieti Nigerian dan Solo dan struktur sebenar karpain telah pun ditentukan. Dalam kajian ini, penyelidikan adalah lebih menjurus dan fokus kepada pengekstrakan karpain daripada daun betik varieti tempatan iaitu Eksotika I. Kaedah pengasingan yang digunakan adalah adaptasi daripada kaedah5, 17. Walaubagaimanapun, pengubahsuaian major terhadap kaedah pengasingan kompaun ini telah pun dilakukan kerana kaedah pengasingan yang lama banyak menghasilkan kompaun sampingan atau artifak dalam ekstrak. Struktur kimia karpain telah ditentukan dengan penggunaan spektrometri resonan magnet nukleus (NMR) dan spektrometri mass kromatografi gas (GCMS).

Keywords index : Carica papaya, alkaloid, carpaine, nuclear magnetic resonance (NMR), gas chromatography mass spectrometry (GCMS).

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1. INTRODUCTION Carica papaya L. is an indigenous species widely used, both in the Malaysian diet and traditional herbal medical practice. Among of the secondary metabolites present in papaya is the carpaine, the most commonly found alkaloid in the leaves of the plant. The structure of carpaine consists of two identical substituted piperidine rings bridged by two ester groups(2), as shown in Figure 1.

Figure 1: Structure of carpaine It has been reported that the extraction of carpaine from papaya leaves will inadvertently include some precursors to carpaine such as piperideine and pseudocarpaine(17, 2, 5 and 1). Hence, only a small amount of carpaine will be extracted each time. As part of our study to develop carpaine for biological and analytical use, we have developed a methodology to extract carpaine in a more pure form by modifying the existing methods. In addition to developing a reliable method for harnessing carpaine from papaya leaves naturally, this work aims to enhance the natural production of carpaine in papaya through suspension cultures. In order to further understand how carpaine could be produced and enhanced in vitro, a consistent and efficient developmental system of cell suspension has to be developed. Here, the preliminary finding of developmental system of cell suspension is also reported.

2. EXPERIMENTAL 2.1 General Remarks for Experimental Procedure - Isolation of carpaine was achieved by using normal extraction technique involving acid and base properties. The esterification of carpaine was monitored using thin layer chromatography (TLC). The purity of the compound extracted was characterized by its melting point, 1H NMR and mass spectrum.

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2.2 Plant Material - Carpaine was isolated from ground matured leaves of Carica papaya L. var. Eksotika I obtained from the Malaysian Agricultural of Research and Development Institute (MARDI), Selangor, Malaysia. 2.3 Isolation of Carpaine - Batches of 500 g of papaya leaves were soaked for 2 weeks in a mixture of absolute ethanol, distilled water and glacial acetic acid in a ratio by volume of 95.5:5:0.5 respectively. The mixture was filtered through a Whatman filter paper (Qualitative Circles 150 mm dia.) and the filtrate was subjected to centrifugation at 5000 rpm for 15 minutes. The resulting pellet was discarded and the supernatant was collected. The combined supernatant was evaporated in vacuo at 60°C to obtain a thick dark brown slurry product. The dark brown syrup was then added into a mixture of distilled H2O/acetic acid (49:1 v/v). The mixture was then washed with diethyl ether (50 mL) to remove the non-polar compounds and the aqueous layer was retained. The aqueous layer was adjusted to pH 11 with K2CO3 solution and then re-extracted with Et2O (50 mL). The ether extract was then washed with distilled H2O (50 mL) and 5 % HCl (50 mL) was added. The acidic aqueous layer was separated from the ether layer and the pH was again adjusted to 11. The aqueous layer was then further extracted with Et2O (50 mL). Ether extract was dried over Na2SO4 anhydrous; the solvent was evaporated in vacuo at 30°C. The brown slurry obtained yielded a precipitate of dull yellow needle-like carpaine crystals upon chilling at -20°C. 2.4 Characterisation of Carpaine – Carpaine was obtained as dull yellow needle-like crystals, Rf (BuOH-HOAc-H2O, 4:1:5 v/v/v) 0.6. Its melting point (m.p.) was determined to be 119-121 oC on a WRS-1 melting apparatus (literature m.p. 119-120 oC)16. The 1H NMR spectrum recorded on a JEOL NMR spectrometer revealed the following signals for carpaine: δ 1Η(400 MHz, CHCl3) : 1.01 (6 H, d, J = 7 Hz, 2 CHCH3), 1.3-1.7 (28 H, m, 2 (CH2)7), 1.9- 2.6 (10 H, m, cyclic-H), 2.85 (2H, q, J = 7 Hz, 2 CHCH3) and 4.7 (1H, bs, 2H, 2 NH). This spectrum is identical to that reported by Sato and co-workers.13 Mass spectrometry found the M+ at 478.377 representing the empirical formula of C14H25NO2, which is half that of the carpaine.17

2.5 Regeneration of cell suspension system for papaya -The excised zygotic embryo were placed on solidified CIM medium in Petri dishes, and placed in the dark at ± 25 ºC (18 and 6) until the embryogenic callus developed.
The embryogenic callogenenesis medium, CIM, consisted of Murashige half strength MS salts Murashige and Skoog, 1962 enriched with myo-inositol (50 mg/L), full strength MS vitamins, adenine sulphate (45 mg/L), glutamine (100 mg/L), 2,4-D (10 mg/L), sucrose (6 % w/v) and solidified with phytagel (1.95 g/L). The friable embryogenic callus was removed, and transferred to liquid medium, LIM. Embryogenic cell suspension were maintained in liquid induction medium, LIM with similar composition as CIM supplemented with 2,4-D ranging 2-5 mg/L and the concentration of 2,4D will be reduced periodically in order to maintain the mature culture. Single cells of embryogenic

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suspension (5 mL) were transferred into the GM medium where the cells will grow into plantlets. The plantlets were sub-cultured every 2 weeks for a period of 3-4 months until they mature. Germination and regeneration of embryo were carried out on modified MS, GM, medium supplemented with 0.2 mg/L BAP and NAA, respectively. After 4-5 months, the plantlet were transferred to maturation medium consisting of a full strength MS with selected phytohormone, 1mg/L Giberallic acid, GA3, 2.5 µM/L IBA, and 10 µM/L Riboflavin. The pH in the all media was adjusted to 5.8 with 1.0 M NaOH and 1.0 M HCl prior to autoclaving at 121°C for 20 minutes. Ideally, Acetyl Co-enzyme A and L-Lysine(1) should be incorporate into the culture medium to enhance and catalyst the formation of carpaine naturally. However, this step has not been carried out at this stage.

RESULTS AND DISCUSSION
Normal solid/liquid and acid-base extraction is particularly useful for the separation of alkaloids from plant samples. In this study, 18.5 g crystalline carpaine was extracted from 500 g of papaya leaves. The purity of carpaine extracted from the matured leaves of Eksotika I was comparable to those reported earlier(2,
5 and 17)

since only two pseudo-carpaine compounds were isolated, namely However, in this study one of the pseudo-carpaine, i.e.

dehydrocarpaine I and dehydrocarpaine II.

the dehydrocarpaine I, was isolated only in very minor quantity. Both the pseudo-carpaines are not easily separated from carpaine through the conventional purification techniques such as recrystallization. Coke and Rice5 and Tang17 had used ethanol/water/acetic acid in a ratio of 89:10:1 to soak the leave samples for the carpaine extract. In our method, the same solvent mixture was used but in a ratio of 94.5:5:0.5. Presumably, the more non-polar medium encourages better extraction of the non-polar carpaine. In addition, we had subjected the extracts to centrifugation to remove all solid particles, which help to provide cleaner sample for crystallization. Work is in progress to further purify the carpaine, thus eliminating the minor artifact. The proton NMR and mass spectrum of the carpaine isolated is identical to those reported by Tang17 and by Sato and co-workers.13

The embryogenic callus obtained from immature embryo after 3 weeks of culture on CIM appeared crystals-like11 between the embryo petals. It was shown in this experiment that the enhancement of somatic embryo could be produced by the existence of adventive embryogenesis on single cells as in a globular embryo clumps. Physiological status of the cells such as nutrition, pre-treatment, environment and age of cell determine the competence of cell culture. In this work, the plant growth regulators such 2,4-D play a major role in affecting the competency of the single cells in liquid culture. Teenage embryo of explant used to obtain embryogenic callus would also assist in the success of obtaining mass proliferation of embryogenic suspension cells. High frequency of somatic embryogenesis from immature embryos of Eksotika I in suspension culture resulting in mass propagation of single cells in 364

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150 ml flask liquid culture was also obtained. Matured single embryos transferred to the GM medium resulted in the formation of distinct structural and developmental features with organizational of embryo cells to form specific organs (shoot/root) before reaching the stage of mature plantlet. The complete process of plant regeneration encompasses a series of defined developmental stages from cell aggregate to heart shaped to cotyledonary between 3 to 8 weeks. Several sub-culture on GM medium, supplemented with BAP and NAA (0.2 mg/L) is crucial for normal plant development since the synergistic concentration of BAP and NAA will affect the normal growth of plants.
CONCLUSIONS Carpaine was extracted with 2 artifacts, one being very minor which could be eliminated with further purification. The ratio of EtOH/H2O/HOAc at 94.5:5:0.5 is probably a better solvent system for carpaine extraction since one of the artifacts is very minor. The additional centrifugation might also have contributed to the greater purity.

Somatic embryo was induced from cultured somatic cells to form embroids somatic embryos which progressed through characteristic developmental stages and became independent from the parental callus. The best growth of single cell cultures were obtained when early stage of somatic embryos or friable embryogenic callus were used as initial inoculum. The germination of single cells from embryogenic suspensions culture showed somatic embryos germinated in a highly synchronous manner where the hypocotyls part first became swollen followed by rapid shoot multiplication. Concurrently, chlorophyll development became obvious in the cotyledons and subsequently these shoots formed normal plants.
ACKNOWLEDGEMENTS The authors wish to thank the Yayasan Felda for their financial support through the Yayasan Felda Grant # 8123222 and the University of Malaya for the moral support throughout the project. REFERENCES 1. Bevan, C. W. L. and Ogan, A. U. Studies On West African Medicinal Plants-I. Biogenesis Of Carpaine In Carica Papaya Linn. Phytochemistry, 1964, 3, 591-594. 2. Burdick, E. M. Carpaine : An Akaloid of Carica Papaya-Its Chemistry and Pharmacology. Econ. Bot., 1971, 25(4), 363-365. 3. Caldentey, K. M. O. and Inze, D. Plant cell factories in the post-genomic era: new ways to produce designer secondary metabolites. TRENDS in Plant Science, 2004, 1360-1385.

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4. Cheng, Y. C. and Tsai, S. W. Enantioselestive esterification of (RS)-2-(4-chlorophenoxy)propionic acid via Carica papaya lipase in organic solvents. Tetrahedron : Asymmetry 15, 2004, 2917-2920. 5. Coke, J. L. and Rice, W. Y. The Absolute Configuration of Carpaine. J. Org. Chem., 1965, 30, 3420-3423. 6. Fitch Maureen, M. M., & Manshardt, R. M. Somatic embryogenesis and plant regeneration from immature zygotic embryos of papaya (Carica papaya L.). Plant Cell Reports, 9(1990): 320-324. 7. Govindachari, T. R. Five decades in the study of natural products. Proc. Indian Acad. Sci. (Chem. Sci.), 2002, 114, 175-195. 8. Govindachari, T. R. and Narasimhan, N. S. Constitution of Carpaine. Chemical Laboratories, 1953, 537, 2635-2637. 9. Hornick, C. A., Sanders, L. I. and Lin, Y. C. Effect of carpaine, a papaya alkaloid, on the circulatory function in the rat. Research Communications in Chemical Pathology and Pharmacology (22), 1978, 2, 277-299. 10. Kabaleeswaran, V., Rajan, S. S., Krishnakumari, G. N. and Govindachari, T. R. Conformational flexibility of carpaine and its hydrobromide derivative. Acta Crystallographica, Section C : Crstal Structure Communications, 1999, 55 (11), 1935-1937. 11. Litz, R. E., & Conover, R. A. High-frequency Somatic Embryogenesis from Carica Suspension Cultures. Annals of Botany, 51(1983): 683-686. 12. Mahmood, K. and Abd. Rahman, N. 1997. Kaedah Spektroskopi dalam Pengenalpastian Sebatian Organik. Kuala Lumpur, Penerbit Universiti Malaya, 13. Newman, D. J., Cragg, G. M. and Snader, K. M. Natural products as Sources of New Drugs over the Period 1981-2002. J. Nat. Prod., 2003, 66, 1022-1037. 14. Sato, T., Aoyagi, S. and Kibayashi, C. Enantioselestive Total Synthesis of (+)-Azimine and (+)-Carpaine. Organic Letters (5), 2003, 21. 3839-3842. 15. Sengbusch, v. P. Botany online: The Secondary Metabolism of Plants. 2003, 1-5. 16. Spiteller-Friedman, M and Spitteler, G. Application Of Mass Spectrometry To The Elucidation Of Alkaloids, V. The Structure Of Carpaine. Phytochemistry, 1964, 98, 12341241 17. Tang, C. S. New Macrocyclic ∆1_Piperidine Alkaloids From Papaya Leaves: Dehydrocarpaine I And Ii. Phytochemistry, 1979, 18, 651-651 18. Vilasini, P., Latipah, Z., & Salasiah, A. Induction of somatic embryogenesis and plant regeneration from immature embryos of Eksotika papaya (Carica papaya). Journal of Tropical and Food Sciences. 28(2)(2000): 121-126. 19. Wilson, R. K., Kwan, T. K., Kwan, C. Y. and Sorger, G. J. Effects of papaya seed extract and benzyl isothiocyanate on vascular contraction. Life Sciences, 2002, 71 (5), 497-507.

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COMPARATIVE STUDY ON CLEANUP PROCEDURES FOR THE DETERMINATION OF ORGANOPHOSPHORUS PESTICDES IN VEGETABLES (Alvin Chai Lian Kuet1 and Lau Seng2)
2

Agriculture Research Centre, Semongok, Kuching, Sarawak Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak

1

Abstract. A study was carried out to compare the cleanup procedures for the determination of organophosphorus pesticides in vegetables. Eleven organophosphorus pesticides were extracted with acetone and methylene chloride. Extracts were cleanup up by solid-phase extraction (SPE) mixedmode column using quaternary amine and aminopropyl (SAX/NH2) or octadecyl (C18) sorbents. The pesticides were determined by gas chromatography with flame photometric detector. The recovery results obtained from the SPE SAX/NH2 and C18 cleanups in carrot, cucumber and green mustard samples were in the range of 71.0 % to 115 %. Lower recoveries were obtained for polar pesticides, methamidophos and dimethoate. These results were compared to the method currently used in the laboratory which does not include any cleanup.

Abstrak. Satu kajian telah dijalankan untuk membandingkan kaedah-kaedah pembersihan untuk menentukan racun perosak organopfosforus di dalam sayur-sayuran. 11 racun perosak organofosforus diekstrak dengan aseton dan dwiklorometana. Ekstrak dibersihkan dengan ekstraksi fasa pepejal (SPE) menggunakan amina quaterina dan aminopropil (SAX/NH2) atau turus oktadesil, C18. Racun perosak ditentukan dengan kromatografi gas yang dilengkapkan dengan pengesan fotometrik nyala. Pengembalian racun perosak dalam tiga jenis sayur-sayuran iaitu lobak merah, timun dan sawi hijau adalah di antara 71.0 % dan 115 %. Pengembalian yang rendah diperolehi untuk racun perosak yang polar iaitu methamidophos dan dimethoat. Keputusan ini dibandingkan dengan kaedah tanpa pembersihan yang digunakan di makmal pada masa ini.

Key words: Solid-phase extraction, organophosphorus pesticides, gas chromatography.

Introduction

Organophosphorus (OP) pesticides have replaced the organochlorine pesticides as concern about the persistence and polluting effect of these compounds to the environment. However, due to its low persistency, a greater number of applications to a crop may be necessary during the course of the growing season. Numerous methods have been developed for the analysis of OP pesticides in fruits and vegetables. Some of these methods advocate the use of solid-phase extraction (SPE) cartridges. A method using acetonitrile for extraction of pesticide residues in fruits and vegetables was reported [1]. The pesticides were detected on gas chromatograph (GC) equipped with flame photometric detector (FPD). A simple and efficient cleanup method for GC determination of 23 OP pesticides in crops was reported [2]. The sample was extracted with acetone and benzene. Cleanup was performed on silica catridges. It was reported that water-soluble pesticides such as dichlorvos and dimethoate gave poor recoveries in all crops. A method for the determination of 28 OP pesticides in fatty and non- fatty foods was reported [3]. Extraction was carried out using acetone and mixture of acetone-water. Carbon-celite was used as cleanup. A multi-residue method for determination of 43 OP insecticides in plant and animal tissues was reported [4]. The OP insecticides were extracted with methanol-

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dichloromethane (1 : 9) and cleaned up using gel permeation chromatograph and silica gel mini columns. Determination of OP pesticides in fruits and vegetables using octadecyl, carbon and aminopropyl cartridges was reported [5]. The pesticides were determined by GC equipped with mass selective detector. Gravity-fed C18 SPE as cleanup for detection of pesticides in spinach, oranges, tomatoes and peaches was also reported [6]. The method was used to analyze 48 OP pesticides. A method using SPE cleanup of chlorpyrifos, methidathion and methyl parathion in oranges was reported [7]. Samples were extracted with anhydrous sodium acetate with ethyl acetate. The ethyl acetate extract was concentrated and cleaned up by passing through tandem SAX and PSA SPE columns. In this study, we study the cleanup procedures for the determination of OP pesticides in vegetables using the SPE mixed-mode column; quaternary amine and aminopropyl (SAX/NH2) and octadecyl (C18) sorbents.

Experimental Eleven OP pesticide; namely methamidophos, dimethoate, diazinon, tolcofos-methyl, fenitrothion, chlorpyrifos, phenthoate, prothiofos, triazofos, cyanofenfos and azinphos-ethyl were selected for this study. These pesticides were fortified in carrot, cucumber, and green mustard at 0.5 and 0.1 ppm levels. Three replicate fortifications for each matrix type were prepared. Extraction was carried out based on procedures described by Steinwandter [8]. 10 g of sample was homogenised in a blender containing 100 ml acetone, 75 ml dichloromethane and 15 g sodium chloride for three minutes. The organic phase was transferred to a beaker and 3 g of sodium sulphate was added to remove the remaining water. For SPE SAX/NH2 cleanup, the tube was conditioned with 10 ml of acetone : petroleum ether (1 : 2). 2 ml of extract was transferred to the SPE tube, followed by eluting with 10 ml of conditioning solvent at the flow rate of 1 ml/min. For SPE C18, the tube was conditioned with 10 ml hexane : petroleum ether (1 : 1 v/v). 2 ml of extract was transferred to the tube, followed by eluting with 10 ml conditioning solvent. The eluates collected were analysed by GC-FPD on a HP 5, 15 m x 0.53 mm x 1.5 µm column. A Hewlett-Packard GC 5890 Series II equipped with FPD was used for the determination of the OP pesticides. GC conditions were: injector temperature, 260 °C; detector temperature 250 °C; carrier flow (nitrogen) 4 ml/min; oven temperature, 120 °C (1.0 min), rate 30 °C/min to 150 °C, rate 5 °C/min to 270 °C (10 min); air flow : 80 ml/min; hydrogen flow : 67 ml/min.

Results and Discussion Recoveries of the OP pesticides at 0.5 ppm and 0.1 ppm levels from carrot samples with SAX/NH2, C18 cleanup and without cleanup are shown in Table 1. At 0.5 ppm fortification level, the recoveries obtained for eleven OP pesticides using the method without cleanup were within the acceptable range of 70 -120 % [9] The recoveries for these pesticides ranged from 76 % to 108.3 % with CV of 5.0 11.0 %. Comparable results were obtained for the SPE SAX/NH2 cleanup for nine OP pesticides. Their recoveries were in the range of 92.0 % to 115.0 % with CV of 4.0 - 7.2 %. A low recovery of 30.3% was obtained for dimethoate, while methamidophos was totally absorded in the SPE during the cleanup. Both dimethoate and methamidophos were more polar as compared to the other OP pesticides. The solubility of methamidophos and dimethoate in water are 200,000 mg/L and 25,000 mg/L respectively as compared to 0.05 mg/L for prothiofos [10]. The low recoveries of these polar pesticides were because of their strong retention by polar anion exchange sorbents, SAX/NH2. For the C18 cleanup, the recoveries obtained were within the acceptable range except for methamidophos. Their recoveries were in the range of 80.0 % to 107.7 % with CV of 1.5% to 5.9 %. The recovery obtained for methamidophos was 6.7 %.

Table 1. Recovery of OP pesticides from carrot samples using different cleanup methods

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Pesticide

SAX/NH2 % Rec.(a) ± CV 0* 30.3*±17 92.0±6.1 113.3±5.8 108.7±5.7 112.3±5.8 109.7±5.7 115.0±7.2 105.3±4.0

0.5 ppm C18 % Rec.(a) ± CV 6.7*±2.5 107.7±2.3 92.7±3.2 92.0±1.7 91.7±1.5 84.7±5.9 92.3±2.1 80.0±1.0 102.0±2.0

No cleanup % Rec.(a) ± CV 76.0±5.0 108.3±6 89.3±9.3 98.7±10.7 102.0±6.1 99.3±11 97.0±8.9 103.7±10 95.7±8.1

SAX/NH % Rec.(a) ± CV 0* 71.0±6.6 95.7±3.5 94.7±4.0 92.3±4.7 92.7±1.2 99.3±2.5 93.0±4.4 98.0±6.1
2

0.1ppm C18 % Rec.(a) ± CV 40.7*±5. 9 99.7±8.7 93.7±3.1 93.3±6.7 97.0±4.6 97.0±2.0 91.0±2.7 85.3±2.5 92.7±5.7

Methamidop hos Dimethoate Diazinon Tolcofosmethyl Fenitrothion Chlorpyrifos Phenthoate Prothiofos Triazofos

No cleanup % Rec.(a) ± CV 108.3±10 .5 118.0±3. 5 97.3±4.2 97.0±2.7 110.1±1. 2 96.0±2.0 94.0±1.7 94.0±1.0 124.0±1. 0 99.7±0.6 112.0±1. 0 104.2 10.9 (a) n = 3

Cyanofenfos 112.7±4.0 88.0±1.7 99.7±7.2 90.3±5.5 96.0±4.4 Azinphos102.3±6.8 102.3±5.0 97.3±9.2 97.0±4.4 92.0±6.6 ethyl AV 107.9 91.7 98.1 92.4 93.8 SD 7.2 7.2 4.1 8.0 4.0 AV = average mean CV = coefficient of variation SD = standard deviation

At 0.1 ppm fortification level, all the OP pesticides except methamidofos showed good recoveries of 71 % to 99.3 % using the SAX/NH2 cleanup with CV of 1.2 % to 6.6 %. None of the methamidofos was recovered in this cleanup. The higher recovery obtained for dimethoate as compared to 0.5 ppm level showed that the amount of semi polar pesticide being retained by the SAX/NH2 varied depending on the concentration of the pesticides. Better recoveries were obtained for method without cleanup. The recoveries for the OP pesticides without cleanup ranged from 94.0 % to 124.0 % with CV of 0.6 % to 10.5 %. For C18 cleanup, the recoveries obtained for 10 OP pesticides were from 85.3 % to 99.7 % with CV of 2.0 % to 8.7 %. Methamidophos had lower recovery of 40.7 %. Recoveries of the OP pesticides at 0.5 ppm and 0.1 ppm levels from cucumber samples with SAX/NH2, C18 cleanup and without cleanup are shown in Table 2. At 0.5 ppm fortification level, the recoveries for 9 OP pesticides using the SAX/NH2 cleanup were within the acceptable range of 80.3 % to 95.3 % with CV of 2.5 % to 7.1 %. Low recovery of 51.7 % was obtained for dimethoate. Methamidophos was totally absorbed during the cleanup. Good recoveries were obtained for method without cleanup. Their recoveries ranged from 69.3 % to 111.0 % with CV of 1.2 % to 6.2 %. For the C18 cleanup, the recoveries were from 76.3 % to 104.0 % with CV of 1.5 % to 7.9 %. Low recovery of 8 % was obtained for methamidophos.

Table 2. Recovery of OP pesticides from cucumber samples using different cleanup methods

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Pesticide

SAX/NH2 % Rec.(a) ± CV 0* 51.7*±6.0 86.7±4.6 87.0±5.3 89.0±6.1 82.0±7.0 95.3±5.9 80.3±7.1 94.0±3.5

0.5 ppm C18 % Rec.(a) ± CV 8.0*±4.4 104.0±6.2 82.7±3.2 76.3±1.5 79.7±2.1 87.0±7.2 92.3±7.0 92.3±3.2 98.3±7.9

No cleanup % Rec.(a) ± CV 69.3±1.5 111.0±6.2 96.3±4.0 94.3±3.1 98.3±1.5 91.7±4.2 97.3±1.2 92.3±4.0 97.0±2.0

SAX/NH % Rec.(a) ± CV 0* 43.3*± 8.5 80.7±1.5 71.0±1.0 77.3±3.5 92.3±3.1 85.3±3.8 95.3±2.5 79.7±1.5
2

0.1ppm C18 % Rec.(a) ± CV 30.7*±1. 5 111.0±9. 5 86.7±7.1 94.3±8.1 95.7±8.7 88.7±7.0 103.0±6. 0 82.7±1.2 104.3±1. 2 95.7±7.5 89.0±7.9

Methamidop hos Dimethoate Diazinon Tolcofosmethyl Fenitrothion Chlorpyrifos Phenthoate Prothiofos Triazofos

No cleanup % Rec.(a) ± CV 92.0±2.7 100.3±10 .7 98.7±1.5 94.0±2.7 101.0±2. 7 94.3±1.5 87.0±7.9 93.7±7.1 99.3±7.1 91.0±1.7 111.7±2. 1 98.6 11.0 (a) n = 3

Cyanofenfos 91.3±6.1 86.7±7.4 89.3±1.5 80.3±4.0 Azinphos91.3±2.5 96.3±6.7 101.0±4.0 88.3±6.4 ethyl AV 88.5 88.0 95.3 79.3 93.3 SD 5.1 7.5 3.7 14.6 7.3 AV = average mean CV = coefficient of variation SD = standard deviation

At 0.1 ppm fortification level, the recoveries obtained for the nine OP pesticides using SAX/NH2 cleanup ranged from 71 % to 95.3 % with CV of 1.0 % to 6.4 %. A low recovery of 43.3 % was obtained for dimethoate, while methamidophos was totally absorbed in the sorbent. Good recoveries were obtained for method without cleanup. Their recoveries ranged from 87.0 % to 111.7 %, with CV of 1.5 % to 10.7 %. For C18 cleanup, the recoveries obtained for 10 OP pesticides ranged from 86.7 % to 111 % with CV of 1.2 % to 9.5 %. Low recovery of 30.7 % was obtained for methamidophos. The recovery results obtained from green mustard samples with SAX/NH2, C18 cleanup and without cleanup are shown in Table 3. At 0.5 ppm fortification level, the recoveries obtained for 10 OP pesticides using SAX/NH2 cleanup were within the acceptable range. They were in the range of 82.0 % to 102.0 % with CV of 2.7 % to 9.9 %. The only pesticide with low recovery was methamidophos. As compared to carrot and cucumber, higher recoveries were obtained for dimethoate. For semi polar pesticides such as dimethoate, the amount of absorption into the SAX/NH2 also varies depending on the samples types. Therefore, there is a wide variation of recoveries obtained among the three types of vegetables tested. Good recoveries were obtained from the method without cleanup. Their recoveries were in the range of 74.3 % to 118.3 % with CV of 0.6 % to 6.1 %. For C18 cleanup, the recoveries obtained were in the range of 71.0 % to 97.3 % with CV of 2.0 to 8.3 %. Lower recovery of 38.0 % was obtained for methamidophos.

Table 3 Recovery of OP pesticides from green mustard samples using different cleanup methods

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Pesticide

SAX/NH2 % Rec.(a) ± CV 15.3*±4.5 102.0±9.9 88.3±6.5 92.0±4.6 91.3±4.7 94.0±5.2 89.7±4.5 90.7±7.4 86.0±2.7

0.5 ppm C18 % Rec.(a) ± CV 38.0*±27.8 97.3±8.3 84.0±2.0 80.0±2.0 80.0±7.6 71.0±7.9 85.7±7.6 71.0±3.6 77.5±7.9

No cleanup % Rec.(a) ± CV 74.3±1.5 118.3±6.1 92.3±1.5 89.7±1.5 93.3±5.9 96.7±2.1 93.3±3.2 97.3±0.6 89.7±4.0

SAX/NH % Rec.(a) ± CV 0* 34.5*±9. 2 84.0±5.7 80.5±0.7 77.0±5.7 77.5±2.1 88.0±1.4 78.0±2.8 80.5±7.8
2

0.1ppm C18 % Rec.(a) ± CV 37.0*±10 .2 96.7±8.7 94.7±3.8 104.3±2. 3 96.3±3.8

Methamidop hos Dimethoate Diazinon Tolcofosmethyl Fenitrothion Chlorpyrifos Phenthoate Prothiofos Triazofos

No cleanup % Rec.(a) ± CV 93.3±10. 4 99.0±7.6 91.3±2.5 89.0±4.0 100.3±6. 8 90.3±3.2 86.3±4.9 91.3±6.7 96.3±6.7 88.3±4.0 114.7±8. 1 94.7 8.4 (a) n = 3

83.7±3.1 99.3±6.0 84.0±5.0 105.7±4. 0 Cyanofenfos 88.0±5.3 80.7±5.5 91.3±3.2 84.5±2.1 88.0±1.7 Azinphos82.0±4.6 83.0±3.6 92.3±6.1 87.5±3.5 106.3±6. ethyl 7 AV 90.4 81.0 95.5 77.2 95.8 SD 5.3 7.6 8.4 15.5 8.9 AV = average mean CV = coefficient of variation SD = standard deviation

At 0.1 ppm fortification level, the recoveries obtained for nine OP pesticides using SAX/NH2 cleanup ranged from 77.0 % to 88.0 % with CV of 0.7 % to 5.7 %. As encountered in the cucumber and carrot samples, the recovery for dimethoate was low; 34.5 %, while, methamidophos was totally absorded by the SPE sorbent. Good recoveries were obtained for method without cleanup. Their recoveries ranged from 86.3 % to 114.7 % with CV of 2.5 % to 10.4 %. For the C18 cleanup, the recoveries obtained were in the range of 83.7 % to 106.3 % with CV of 1.7% to 8.7 %. The recovery obtained for methamidophos was 37.0 %. The chromatograms for SPE cleanups and without cleanup are shown in Figure 1. All the chromatograms showed no interference peaks which co-eluted with the 11 OP pesticides. The FPD in phosphorus mode is selective and specific responding only to phosphorus compounds. It was noted that a large peak at 21 min has substantially reduced after the SAX/NH2 and C18 cleanups.

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(A)

TIME (min)

(C)

(C)

Figure 1. Typical GC chromatogram of vegetable sample without cleanup (A), after SAX/NH2 cleanup (B) and C18 cleanup (C). Peaks : 1, methamidophos; 2, dimethoate; 3, diazinon; 4, tolcofosmethyl; 5, fenitrothion; 6, chlorpyrifos; 7, phenthoate; 8, prothiofos; 9, triazofos; 10, cyanofenfos; 11, azinphos-ethyl

Conclusion The studies on the SPE cleanup methods showed that SAX/NH2 and C18 have the potentials to be used as cleanup method for OP pesticides in the laboratory. The advantage of the SPE method over the current method without cleanup is that the former removed a substantial amount of coloured compounds and matrix interferences from the samples. This can reduce the cost of GC maintenance especially for the GC inlet and column. Except for more polar pesticides such as methamidofos and dimethoate, comparable results were obtained for the other OP pesticides using SPE cleanup methods. No interfering peaks were encountered in the chromatograms. In spite of the limited range of this study, it can be predicted that the SPE cleanup can be successfully extrapolated to other pesticides and vegetables. The benefits of the SPE method compared to the other cleanup methods were the use of organic solvents is reduced, possibility of concentration the samples, less sorbent is used, no crosscontamination and shorter analysis time.

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Acknowledgement The authors wish to thank the Universiti Malaysia Sarawak and Director of Agriculture Sarawak and Senior Assistant Director (Research) for their support of this project and permission to publish this paper. The technical assistance rendered by staff of the Pesticide Residue Laboratory especially Mr. Phillip Gudom is greatly acknowledged.

References 1. Lee, S.M., Papathakis, M.L., Feng, H.M., Hunter, G.F. & Carr. J.E. (1991) Multipesticide residue method for fruits and vegetables. Fresenius J. Anal. Chem., 339, 376 - 383 2. Sasaki, K., Takashhi, S. & Saito, Y. (1987) Simplified cleanup method and gas chromatographic determination of organophosphorus pesticides in crops. J. Assoc. Off. Anal. Chem., 70, 460 - 464 3. Leoni, V., Caricchia, A.M. & Chiavarini, S. (1992). Multiresidue method for quantitation of organophosphorus pesticides in vegetable and animal foods. J. AOAC Int. 75, 511 - 518. 4. Holstege, D.M., Scharberg, D.L., Richardson, E.R. & Moller, G. (1991). Multiresidue screen for organophosphorus insecticides using gel permeation chromatography-silica gel cleanup. J. Assoc. Off. Anal. Chem. 72, 394 - 399 5. Fillion, J., Saure, F. & Selwyn, J. (2000) Multiresidue methods for the determination of residues of 251 pesticides in fruits and vegetables by gas chromatography with florescence detection. J. AOAC Int., 83, 698 - 713 6. Cook, J., Beckett, M.P., Reliford, B., Hammock, W. & Engel, M. (1999) Multiresidue analysis of pesticide s in fresh fruits and vegetables using procedures developed by the Florida Department of Agriculture and Consumer Services J. AOAC Int., 82, 1419 - 1435. 7. Yamazaki, Y. & Ninomiya, T. (1999) Determination of benomyl, diphenyl, o-phenylphenol, thiabendazole, chlopyrifos, methidation and methyl parathion in oranges by solid-phase extraction, liquid chromatography and gas chromatography. J. AOAC Int, 82, 1474 - 1478 8. Steinwandter, H. (1985) Universal 5-min on-line method for extracting and isolating pesticide residues and industrial chemicals. Fresenius Z. Anal. Chem., 322, 752 – 754 9. Parker, G.A. (1991). Validation of methods used in the Florida Department of Agriculture and Consumer Services Chemical Residue Laboratory. J. Assoc. Off. Anal.Chem. 74, 868 - 871. 10. Worthing, C.R., & Hance, R.J. (1991). The Pesticide Manual, 9th Ed. Surrey : Unwin Brothers Limited.

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MENENTUKAN KANDUNGAN PROTEIN LARUT DALAM SARONG TANGAN LATEK GETAH ASLI Salihan Bin Siais,* Abdul Aziz Awang Jabatan Kimia, Fakulti Sains, Universiti Putra Malaysia, *Lembaga Getah Malaysia, Sungai Buloh, Selangor Terdapat ramai pengguna di dunia ini yang menghadapi masalah alahan disebabkan ia menggunakan produk-produk barangan tertentu. Masalah alahan protein ( protein allergy) yang dihadapi oleh para penguna terhadap pengunaan sarong getah, penyelidikan untuk dengan menggunakan kaedah ASTM D5712-1999 Kaedah ini dilakukan dengan membuat pengukuran ketumpatan optik pada jarak gelombang 750 mn. Ia merupakan satu kaedah yang mudah yang hanya melibatkan langkah-langkah penganalisaan ringkas. Walau bagaimana pun ia merupakan satu kaedah yang standard dan telah digunakan secara rutin untuk mengawasi kandungan protein sarong tangan yang diperbuat di kilang-kilang di Malaysia untuk di eksport. Kajian ini, melibatkan penentuan kandungan protein yang terdapat dalam dua jenis sample sarong tangan getah, iaitu sample saorang tangan getah yang dibuat daripada bahan getah jenis bertepung dan sarong tangan getah yang diperbuat dari bahan getah yang telah diklorinkan. Semua sample sarong tangan ini diambil dari kilang yang sama di Selangor pada dalam bulan Mac, Jun dan September 2004. Ia terdiri daripada saiz kecil (s), serdahana (M) dan beasr (L) Hasil kajian analisa ini, mendapati bahawa, julat kepekatan proteinnya masing-masing ialah (143-250) µg/g, (136- 24) µg/g dan (122-233) µg/g masing-masing bagi saiz kecil, serdahana dan besar untuk sample bahan getah bertepung,. Kepekatan protein bagi bahan sample bahan getah berklorin pula ialah (26-32.) µg,/g, (24–33) µg/g dan 23-26) µg/g masing-masing bagi saiz kecil, serdahana dan besar bagi sample bulan Mac. Ini menunjukkan bahawa kandungan protein di dalam sarong tangan bahan getah bertepung adalah lebih tinggi berbanding sarong tangan bahan getah yang di klorinkan. telah mendorong kita melakukan satu menentukan kadar kandungan protein di dalam produk barangan tersebut

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INTRODUCTION Natural rubber latex ( NRL) gloves have been acknowledge as the best protective devices available for protecting health care personnel and their patients against viral transmission and infectious fluids (Yip and Marlene, 2003 ). According to Moneret-Vautrin, (1993) in Europe, latex protein llergy affects about 0.1% of the general population and America who frequently use latex examination gloves and surgical gloves are more susceptible. Populations in South East Asia, where rubber is an important crop, appear to be less susceptible to the problem of latex allergy, (Hasma, 1998). Latex protein allergy is of type hypersensitivity in nature. The allergic reactions are generally immediate, and progressed rapidly within minutes after severe but extremely rare case, anaphylaxis, which is a potentially fatal reaction that affects various parts of the body simultaneously (Figure 1).

Figure 1 :

An allergic urticarial reaction to NR latex proteins, with pruritic eruption characterized by transient wheals and hives.

skin

2. OBJECTIVE The objective of this study is to determine the total extractable protein content in natural rubber powdered gloves and chlorinated gloves (Figure 2) which cause the allergy to the users a) Powdered Glove b) Chlorinated Glove

Figure 2: The differences between powdered glove and chlorinated glove

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METHODOLOGY There are three diffrents step were carried out in this project, namely, sample collection, ASTM D5712-1999 method procedures and determination of protein content Sample Collection The samplings were carried out for three months, started on March 2004, June 2004 and September 2004 respectively, from company A gloves manufacturer. The company was chosen because the company is one of the biggest glove manufacturers and exporters in Malaysia, well established and complete with the new technologies of gloves manufacturing and producing the powdered gloves and chlorinated gloves as well. The Standard Malaysian Gloves (SMG) Survey sampling method was used to collect the samples. Two types of gloves were collected, which are powdered gloves and chlorinated gloves. They were different in sizes which are S, M and L respectively. The SMG sampling method was used as stated below. From 1 carton of 1000 pieces (10 dispensers x 100 pieces) 1 dispenser was taken and 20 pieces removed, S, 13 cartons, 33% (260 pieces), M 19 cartons, 48% (380 pieces) and L , 8 cartons 19% (160 pieces) Each of glove size, which are 13 pieces were chosen for determination of extractable protein content.

Standard for ASTM D5712-1999 Method Procedures Ovalbumin Serum Albumin ( 0.01g) was dissolved with in 10 mL Phosphate Buffered Saline solution (extraction buffer). The absorbance was determined at 280 nm using a UV-1601 PC (shimadzu) spectrophotometer. The absorbance, was divided by 0.64, to calculate the actual concentration of ovalbumin stock solution, approximately 1000 µg/mL To establish a working standard, a series of standard solution were prepared ( 2 µg/mL, to 200 µg/mL). See figure 3.

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Extraction and assay procedure The procedure involved were the extraction of the natural rubber latex gloves, acid precipitation and concentration of protein, color development and analysis of data obtained. The steps were as stated below:

Glove Extraction Prior to the analysis, the weight of the glove must be taken and cut pieces ) of whole glove were placed in a 250 mL polypropylene container. Phosphate Buffered Saline (PBS) solution (in the proportion of 10 mL PBS to 1 g of glove) was added, the concentration was at least 0.025 M (to maintain the extraction at pH 7.4 ± 0.2). The glove sample was agitated in PBS solution at room temperature for 120 ± 5 min. Agitation to expose all surface to extraction solution were done at the start, in middle and after 120 min. The test specimen was removed from the extraction solution and the extract was centrifuged at 3000xg for 15 minutes to remove insoluble matter Acid precipitation and concentration of protein 1.0 mL each of the reagent blank (extraction buffer), standard protein solutions (Ovalbumin standards) and natural rubber latex glove extraction solution. 0.1 mL of Sodium Deoxycholate (0.15%, was added, mixed and allowed to stand for 10 minutes.. 0.2 mL of a freshly prepared solution of 50:50 Trichloroacetic Acid (72%) and Phosphotungstic Acid (72%) were then added to precipitate the protein. The content was mixed allowed to stand for 30 minutes. The precipitate was centrifuged at 6000 x g for 15 minutes and the supernatant liquid decant off and drained by inverting each centrifuge tube on an absorbent paper towel Color Development The precipitate was redissolved in 0.35 mL Sodium Hydroxide (0.2 M). Should some protein precipitates remain, a measured quantity of the sodium hyroxide solution was further added up to a total of 1 mL. 0.35 mL each of the redissolved specimen extract, standard protein, and standard solution was pipetted into a separate micro centrifuge tubes and 0.73 mL Reagent C was added to each. The content was mixed and allowed to stand for 15 minutes.0.087 mL of reagent D was further

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added to each tube. Each was mix well immediately upon adding the reagent using a vortex mixer. The color was allowed to develop for 30 minutes. Each of the solution was transferred to the cuvette and the absorbance of sample against blank was measured at 750 nm using spectrophotometer UV1601 PC ( ShimDZU). Then the calibration curve was automatically plotted by the instrument Analysis of data The absorbances of the test extracts were converted to µg/mL using a calibration curve. The concentration of the protein analyte in the test extract was read from the calibration curve. The extractable protein (EP) content in the natural rubber latex gloves is presented in µg/g unit which are determined.( ASTM D5712-99 Standard Test Method ,1999 ) Determination Of Protein Content The UV-Visible Spectrophotometer, model name UV-1601PC (Shimadzu), Dual-Beam UV-VIS Spectrophotometer design are used to determination of protein content in this study. RESULTS AND DISCUSSION Standard Calibration Curve A calibration curve is constructed by every set of protein assays the typical curve obtained is shown in figure 3.. The ideal calibration standard would be a pure preparation of the same protein being quantitated.
1.6 1.4 1.2 Absorbance 1 0.8 0.6 0.4 0.2 0 0 50 100 150 200 250 Protein concentration ug/mL

Figure 3: ASTM D5712 – 1999 method standard curve

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Extractable Protein ( EP ) Contents in Powdered and Chlorinated Gloves Thirteen replicates (SMG Procedure, 1998) of gloves S, M and L sizes were sampled on March, June and September 2004 from company A were extracted and tested using ASTM D5712 – 1999. The types of gloves were tested are NR powdered and chlorinated gloves. The EP content were obtained from analysis is given in table 1 and figure 4 for powder gloves.

Table 1 :

Extractable Protein Content in Powdered Gloves Extractable Protein Content ( µg/g ) M Sept. March June Sept. March 162 142 135 152 138 135 135 136 157 130 143 149 139 143 10 247 230 249 251 230 238 236 264 248 264 240 239 260 246 12 211 216 241 262 222 247 217 225 228 274 234 240 231 234 18 122 159 147 136 22 158 120 145 142 120 136 120 147 136 15 230 222 232 221 241 225 233 222 225 229 215 224 238 227 7

March Glove 1 Glove 2 Glove 3 Glove 4 Glove 5 Glove 6 Glove 7 Glove 8 Glove 9 Glove 10 Glove 11 Glove 12 Glove 13 Mean Std. Dev. 226 227 229 256 234 223 215 216 230 230 220 234 226 228 10

S June 263 249 251 238 241 250 255 224 230 231 269 264 288 250 18

L June 225 226 231 227 225 248 218 225 235 232 227 275 241 233 15

Sept 122 118 119 130 121 120 118 119 128 130 125 119 120 122 4

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300 Protein C ontent ug/g 250 200 150 100 50 0 March June MONTH September
S
M
L

Figure 4 :

Variation of Extractable protein content in Powdered Glove

From the data in table 1, shown, that the EP content for S, M and L sizes on March and June 2004 is not much difference with the ranges between (227 – 250) µg/g. The EP content have not much changes between these two months, we can assumed that, the company did nottake action to improved it. The EP content for glove samples for all sizes were sampling on September 2004 are reduced to ranges between (122 -143) µg/g that is about 50% reducing the EP content. Research by Amir Hashim, 1993 ) shows they have several factors were effected the level of protein content in the gloves those are, pre–vulcanised latex and post vulcanization process, wet gel and dry leaching, leaching times and temperatures and effect of thickness

Pre –vulcanised latex and post vulcanization process Pre-vulcanised refer to compounded latex heated at 70°C for 2h. Compounded latex that are matured at room temperature for five days. The film from the latter heated at 110 °C for 15 minutes is the post-vulcanised. Heating increases the EP content in dipped films. Thus, the EP content in the unleached pre-vulcanised latex films is high. If the pre-vulcanised (PV) latex film is leached in the wet stage, most of this EP is removed. Is the films are then dried at room temperature, the EP content remains low.

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Wet gel and dry leaching Since heating increases the EP content, latex films leached after drying (dry leaching) have lower EP content than wet gel leached films for short leaching times. However, a combination of wet leaching and dry leaching is the more preferable method.

Leaching times and temperatures During leaching, the extraction of the soluble proteins is initially rapid but the rate of extraction decreases quickly and levels off after 5- 10 minutes. The reduction of the EP content also, can be improved slightly by leaching at an elevated temperature.

Effect of thickness Leaching is a diffusion controlled process. Thus, thinner products would require a shorter leaching time to reduce the EP content compared to thicker ones. Chlorinated gloves were produce by dipping the gloves into a chlorinated solution (for example, in sodium hypochlorite acidified with hydrochloric acid solution). After chlorination, the solution should be drained off and the gloves washed with water continuously until all remaining acid washed away. The gloves are then dipped in a solution of ammonia to neutralize any residual acid, the pH of the washing is checked to ensure adequate neutralization (pH of the water after washing should be in the range of 6.8 – 7.0). The gloves are then ready to be dried and tumble dried preferably at a low temperature with range (50 – 60)°C followed by cooling and airing. The EP content values were obtained is given in table 2 and figure 5 for chlorinated gloves. It was found out that the protein was in the ranged between (23 to 32) µg/g for all glove sizes collected in March, June and September 2004. The comparison of the EP content between powdered gloves and chlorinated gloves is given in figure 6.. Nor Aishah, 1993, reported the value of EP content of the gloves exposed to chlorine water for 15 minutes decreases significantly from the control sample where the EP content is rerduced from (85923) µg/g ( 0.01% Cl ). According Eng, (2001) in the rubber glove industries, chlorination is being

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widely used to produce powder free glove and research by Yip et al. (1994) ; Turjanmaa and Reunala (1990) found that, the EP level of the chlorinated vulcanized NR gloves were reduced drastically. In this study, it reveals that the EP content in powdered gloves was higher compared to the EP content in chlorinated gloves. The variations between the two types of gloves are shown in Figures 19. Researchers have shown in several papers (Beezhold and Beck, 1992 ; Beezhold et. al., 1994) that cornstarch powder binds the latex protein in the NR gloves. Comparison studies by Beezhold et. al.,(1997) using the Lowry test methods between NRL powdered gloves and NRL powder free gloves the results range are (60 -1039) µg/g ( median of 204 ) and (20- 108) µg/g (median of 33) . Table 2: Extractable Protein Content in Chlorinated Gloves
Extractable Protein Content S March Glove 1 Glove 2 Glove 3 Glove 4 Glove 5 Glove 6 Glove 7 Glove 8 Glove 9 Glove10 Glove11 Glove12 Glove13 Mean Std.Dev 30 29 39 29 24 35 26 37 35 32 34 31 29 32 4 June 27 24 26 28 26 25 27 29 34 22 25 27 22 26 2 Sept. 29 20 26 29 20 25 28 20 19 30 28 22 23 25 4 March 39 40 32 37 26 28 32 33 35 25 29 37 33 33 5 M June 30 32 35 30 29 29 26 31 25 34 32 33 29 30 3 Sept. 21 20 24 20 25 23 21 27 29 23 24 24 26 24 3 March 21 25 36 19 26 19 33 15 27 30 35 22 24 26 7 ( µg/g ) L June 23 24 21 24 26 23 21 27 20 23 20 20 21 23 2 Sept 20 24 22 25 27 19 24 22 22 28 26 23 20 23 3

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35
protein content ug/g

30 25 20 15 10 5 0 March June MONTH September

S

M

L

Figure 5 :

Variation of Extractable protein content in Chlorinated Glove

Protein content ug/g

300 250 200 150 100 50 0
C hl or in at ed (M C ar hl or ch in C ) at hl or ed in (J at un ed e (S ) ep te m be r) (M ar ch (J un e em be r) ) )

S

M

L

P ow de

P ow de

Figure 6: Comparison of the EP content between Powdered Gloves and Chlorinated Gloves.

P ow de

re d

(S ep t

re d

re d

5. CONCLUSION In this study, aqueous soluble proteins in natural rubber latex were extracted in a buffer solution. The extractable protein determined by using ASTM D5712-1999 where the proteins were precipitated to concentrate them and than separated from water-soluble substances. The precipitated proteins are re-dissolved and quantified colorimetrically by using ovalbumin (OVBM) as protein standard.

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The conclusions from this studies that the calibration curves of ASTM D5712-99 was incurved and the protein assay showed that the EP content for powdered glove in March and June 2004 the results range are (223 to 250)µg/g. The samples were sampling on September 2004 shows the EP content was reduced which the results range are (23-32) µg/g almost 50% decreased. This range is under detection limit (50 µg/g) of ASTM D5712-1999 assay method (ASTM standard test method, 1999). Reported by Hasma et. al. 2004, the EP content using ASTM D5712-1999 method gave a better correlation (r2 = 0.76) with the allergen content. They were observed, that the EP limit of <60 µg/dm2 ( ≈ 80µg/g) had corresponded to low to moderate allergen while EP of >60 µg/dm2 corresponded to high allergen. This impiled that NR gloves with values between 60 µg/dm2 to the recommended limit of 200 µg/dm2 ( ≈ 260µg/g) could contain high allergen.

REFERENCES Amir H. (1993). Effect of Leaching on Extractable Protein Content Latex Proteins and Glove Industry. Rubber Research Institute of Malaysia 5: 51 ASTM D5712-99 (1999) Standard Test Method for the Analysis of Aqueous Extractable Protein in Natural Rubber and its Products Using the Modified Lowry Method. American Society for Testing and Materials, 1-7 Beezhold, D. and Beck, W.C. (1992) Surgical glove powders bind latex antigen. Archives of Surgery 127:1354-1357 Beezhold, D., Zehr, B and Kostyal, D. 1997) Prevention of latex allergen transfer by glove liners. Guthrie J. 66:15-19. Beezhold, D., Kostyal, D. and Wiseman, J. (1994) The transfer of protein allergens from latex Eng, A.H. ( 2001) Effects of Chlorination on Nitrosamines and Nitrosatable Substances in Natural Rubber Gloves. J. Rubb. Res., 4(1), 11-16 Hasma, H, Nurul Hayati, Y and Lau, C.H. Ruhida A.Rand Amir Hashim ( 2004) Correlation between Total EP and Antigen Contents of NR Gloves and Chemical Iterference On Protein Assay. J Rubb, Res 7(1) 56-70)

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Hasma, H., Shahnaz, M., Yip, E., Azizah, M., Mok, K.L. and Nasaruddin, B.B. (1998) Binding Patterns of IgE Antibodies in Sera of Rubber tappers to Fresh Hevea Latex Serum Proteins. J. Rubb. Res., 1(3), 146-153. Moneret-Vautrin, D.A., Beaudouin, E., Widmer, S., Maouton, C., Kanny, G., Prestat, F., Kohler, C., Feldmann, L., (1993) Prospective Study og Risk Factors in Natural Rubber Latex Hypersensitivity. J Allergy Clin. Immunol., 92, 668-677 Nor Aisah AA ( 1993) Latex Protein and Gloves Industry: Chlorination of Gloves, Rubber Research Institute of Malaysia, 53-60 Shimadzu Corporation (1997) Instruction Manual UV-1601PC User’s System Guide. Standard Malaysian Glove (SMG) (1998) Technical Requirements for Standard Malaysian Glove, Malaysian Rubber Board. Yip, E., Turjanmaa, K., Ng, K.P. and Mok, K.L. (1994) Allergic responses and levels of extractable proteins in NR l and dry rubber products J. nut Rubb. Res. 9(2), 79. Yip, E. and Marlene, R. (2003) Latex Protein Allergy and Your Choice of Gloves: A Balanced Consideration Vol. 12/No.1.

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VALIDATION OF Ra-226 AND K-40 MEASUREMENT IN ENVIRONMENTAL SAMPLES USING GAMMA SPECTROMETRY SYSTEM Yii Mei Wo and Zaharudin Ahmad Industrial Technology Division Malaysian Institute For Nuclear Technology Research (MINT) Bangi, 43000 KAJANG, MALAYSIA Tel: 03-8925 0510 Fax: 03-89282997 E-mail: yii@mint.gov.my ABSTRACT: Mineral and natural resources usually contains long half-life natural radionuclides (such as U-238, Th-232 and K-40) and had been greatly exploited for different utilizations. When these materials are processed, their concentration become higher in the wastes. Since all these radionuclides have a very long half-life, when concerning about the safety of members of public, it became a requirement by regulation under the Atomic Energy Licensing Act (Act 304) to control the limits of discharge. Both U-232 and Th-228 are less soluble and less mobile compared to their decay daughter products (such as Ra-226 for U-238 and Ra-228 for Th-232). Therefore the determination of these soluble radionuclides, i.e. Ra-226, Ra-228 and K-40 in the environmental samples becomes more important due to their high mobility and solubility. Gamma Spectrometry System was used in the measurement of radium isotopes and K-40, because it is one of the easiest methods to be performed. This measuring method must be validated for several parameters include specificity, precision (repeatability), bias (accuracy), linearity, range, detection limit, robustness and ruggedness in order to ensure it fits for the purpose. This work summarizes how these parameters were fulfilled for this analytical method using several types of certified reference materials. The same validation method would be considered workable on Ra-228 as well, since both Ra-226 and Ra-228 are isotopic, thus have similar physical and chemical properties.

Keywords: Ra-226, K-40, Method Validation, Gamma Spectrometry System

INTRODUCTION For the past few decades, there has been an increasing interest in radionuclides present in the environment and their possible effect, either acute or chronic, on human health [1]. These radionuclides can be transported across long distances from their source of emission, removed from the atmosphere, then move into the biosphere and hydrosphere where finally will affect the human population by several pathways. Among these, one of them is from the radionuclides that present naturally in the environment [2]. Mineral and natural resources had been greatly exploited for different uses. Usually, these materials contain natural radionuclides such as from the uranium and thorium series, and K-40, which were known as the Natural Occurring Radioactive Material (NORM). When the NORM is processed, the concentration for these radionuclides became higher in the wastes [3]. Since all these radionuclides have a very long half-life, when concerning about the public safety, it became an urge by regulation under the Atomic Energy Licensing Act (Act 304) to control the limits of discharge.

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Naturally, in the earth cluster, majority of uranium presents as U-238 and thorium presents as Th-232. Both uranium and thorium are less soluble and less mobile compared to their decay daughter products (such as Ra-226 for U-238 and Ra-228 for Th-232). Therefore the determination of these soluble radionuclides, i.e. Ra-226, Ra-228 and K-40 in the environmental samples become more important because of their high mobile ability and solubility. Gamma Spectrometry is one of the most widely used nuclear instrumentations in determining the activity concentration of Ra-226, Ra-228 and K-40 inside a sample. This is because no chemical treatments and no complicated sample preparation techniques were required during the measurement and the samples usually were not being destructed after the analysis. Basically, the instrument needs to be calibrated for the energy and efficiency prior to a measurement. After calibration, Ra-226, Ra228 and K-40 inside a sample can be measured either directly or let it be in secular equilibrium with its daughter products. K-40 can be detected easily at its own energy line at 1460.83 keV. While, Ra226 and Ra-228 are detected using the equilibrium daughters’ energy lines, i.e. Pb-214 (295.22 keV and 351.93 keV) and Bi-214 (609.31 keV) for Ra-226 and Ac-228 (911.20 keV) for Ra-228 [4]. Usually, all solid samples were directly packed homogenously into the container as its original form. When necessary, the sample was crushed or cut into small pieces to have more samples to be put inside the container. On the other hand, for the water samples, large volume of water (1-10 litres) was pre-concentrated in order to have a higher activity inside the container. In order to ensure that this method is able to give a representative result for the Ra-226 and K-40, the method was first validated using some traceable and certified material. Certified reference material was used in most of the test in order to reduce the systematic errors that could arise from the complicated sample preparations. Method validation is a prerequisite for a laboratory going for accreditation to international standards. The parameters required for method validation of an analytical method include specificity, precision (repeatability), bias (accuracy), linearity, range, detection limit, robustness and ruggedness [5-7].

EXPERIMENTAL Preparation and counting of standards, samples and certified reference material All standards, samples and certified reference material were prepared in a same size container to obtain the similar counting geometry. The high-purity germanium (HPGe) detector was calibrated by using a prepared commercial gamma multinuclides standard that is traceable to NIST. The energy and efficiency calibration were then be validated by using the Certified Reference Material, CRM, (IAEA, Soil-6) present in the same counting geometry. All the samples (including standard) were counted directly in the system with a suitable counting time. Data was collected through counting different types of certified reference material and also from some prepared samples, by using standard source solution for Ra-226 that is traceable to NIST. K-40 solution was prepared from the 99.5% purity Potassium Dichromate that supplied by the May & Baker Ltd. All the data was used to calculate the specificity, precision (repeatability), bias (accuracy), linearity, range, detection limit, robustness and ruggedness.

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Specificity Specificity is the ability of an analytical method to distinguish the analyte to be measured (hence Ra226 and K-40) from other substances present in the sample. Specificity is necessary to assure the accuracy of the measurement and the quality of the analysis results. The specificity of the instrument can be determined by counting the SRM IAEA-326, which contains both Ra-226 and K-40 radionuclides. The interested radionuclides shall appear at the expected energy line to prove the specificity of the technique. Precision/Repeatability Precision is a measure of the degree of repeatability for a test or measurement method under normal conditions. It is usually expressed as the standard deviation(s) or the percent relative standard deviation (RSD) for a statistically significant number of samples. Precision depends only on the distribution of random errors but is not associated with the true value. To check the precision of this method, it was determined by calculating the percent of RSD for at least seven measurement results from this method [7]. Bias/Accuracy Accuracy (mostly caused by systematic errors) is the closeness of the samples true content of a specific analyte with the average result from several measurements. The accuracy of a measurement method shall be determined by comparing the arithmetic mean (average) of at least seven measurement results [7] obtained from this specific method with the known value (activity or concentration) of the standard reference source containing the specific analyte. In this work, a certified reference material (Soil-6) will be used to serve this purpose. To achieve this target, the U score value shall be calculated by using the equation below.

U score =

x lab − u CRM UncCRM + Unclab
2 2

(1)

where xlab is the mean value from measurement; uCRM is the certified value, UncCRM and Unclab is the standard deviation for the certified value and analytical value respectively. The calculated U score value shall not be greater than 1.5 to prove that the method is not bias [8]. The accuracy of the analytical measurement can also be estimated via a statistical equation known as z-score defined as below:

z=

xi − x ref

σ i 2 − σ ref 2

(2)

where xi is the measured value of analytes in control sample σI is the standard deviation of the sample xref is reference value or certified value of the control sample σref is the standard deviation of the control sample Hence the classification of accuracy can be based on the basis of z-scores:

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if z < 2, the quality of measurement is satisfactory if 2 < z < 3, the quality of measurement is questionable, and if z > 3 , the quality of measurement is poor thus require further analysis The certified value of the Ra-226 in the certified reference material, Soil-6, is 79.92 Bq/kg with the 95% confidence level within 69.56 to 93.42 Bq/kg. Whilst, the certified value of the K-40 in the certified reference material, IAEA-326, is 580 Bq/kg with the 95% confidence level within 571 to 589 Bq/kg. Linearity and Working Range Linearity is the ability of the method to elicit test results that are directly proportional to radionuclide activity within a given range. Linearity is usually reported as the variance of the slope of the linear regression of a standard curve. Measurements shall be made with the method for the range of standard or reference concentrations available to the laboratory (from background concentrations to the highest concentration available in standard). The measurement points shall be graphed and the variance of the linear fit shall be calculated. The range shall be expressed in the same unit (such as Bq/kg) as the test results obtained with the method. The gamma-ray spectrometry used with the test methods at the laboratory have been shown by the instrument vendors to be linear over the entire range of measurements possible at the laboratory. The linearity of the analyte measurement was carry out by preparing a series of different radioactivity from NIST traceable Ra-226 and pure K2Cr2O7 for K-40. Limit of Detection The detection limit (DL) is an estimation of the minimum activity or concentration that can be measured with a specific level of confidence. The type of detection limits used at the laboratory was calculated based on the Currie Limit equation. The minimum detectable activity (MDA) is generated and reported for sample with calculated activity lower than MDA.

MDA = 1.645 ×

Background counts Live time(s )
Robustness

(3)

Robustness is a measure of the sensitivity of an analytical method in the presence of minor deviations in the experimental conditions of the method. This can be done by having different operators to analyse the same reference material [7]. Ruggedness/Reproducibility Ruggedness is a more measure of the method performance under variations of condition more severe than determine for robustness. Ruggedness is a measure of how effectively the method performs under less-than-ideal laboratory conditions. The ruggedness of the method was determined from the analysing data of the same reference material using several different equipments [7].

RESULTS AND DISCUSSION Specificity Since the SRM IAEA-326 contains both Ra-226 and K-40, the measured results from this sample are used to study the specificity for the method. Ra-226 is determined through its daughter energy line, i.e. Pb-214 (295.22 keV and 351.93 keV) and Bi-214 (609.31 keV). Meanwhile, the K-40 is identified through its energy line at 1460.83 keV. All the energy lines used were suggested values

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[4]. From the experiment, both Ra-226 and K-40 could still be identified easily at the suggested energy lines. This shows that the method was able to specify both radionuclides at it particular energy line with a little variance in energy. Table 1 below summarised the energy line measured by the counting system to identify both the Ra-226 and K-40 for several measurements. Table 1. Ra-226 and K-40 energy lines for several measurements Ra-226 No of Measurement Peak 1 Peak 2 Peak 3 Measurement 1 295.11 351.86 609.25 Measurement 2 295.11 351.86 609.12 Measurement 3 295.11 351.74 609.12 Measurement 4 295.24 351.86 609.00 Measurement 5 295.11 351.86 609.12 Measurement 6 295.24 351.86 609.00 Measurement 7 295.11 351.86 609.12 Measurement 8 295.24 351.86 609.25

K-40 1460.40 1460.52 1460.15 1460.27 1460.40 1460.02 1460.40 1460.52

The slight different in the energy line could be due to the stability of the electronic components for the system [9]. However, this slight different in the peak energy (less than 1 keV) is meaningless when compared to the whole working range of the system beginning from 40 to 2000 keV. Therefore, from this method, the radionuclides of Ra-226 and K-40 can be identified easily using the gamma spectrometry. Precision/Repeatability The CRM Soil-6 had been used to check Ra-226 while CRM IAEA-326 was used to check K-40 in order to determine the precision of the method. The CRM Soil-6 was used to check the Ra-226 because the activity is higher when compared to the CRM IAEA-326 and the activity is also close to the background level of Malaysia [10]. Therefore this value is more suitable to be used when dealing with environmental samples. The average value reported was 84.83 ± 5.25 Bq/kg and 559.7 ± 47.4 Bq/kg, for Ra-226 and K-40 respectively. The counting results on both CRM were shown in the following control charts (Figure 1 and 2) with horizontal lines indicating the acceptance limit (mean ± 1σ), warning limit (mean ± 2σ), and action limit (mean ± 3σ) respectively. From the control charts, the changes of counting results over time would be monitored and proper corrective action will be taken if the counting results depart from the acceptable range. From the accumulated results, 72.7 % % for Ra-226 and 87.5% for K-40 of the results falls within the acceptable limit (mean ± 1σ) while about 96.4 % of the Ra-226 in Soil-6 and all K-40 for IAEA-326 falls within the warning limit (mean ± 2σ). The precision expressed in the relative standard deviation (RSD) of this analysis was 6.2% and 8.5 % for Ra-226 and K-40 respectively. Generally, the precision for this method is considered to be fairly good.

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Ra-226 in Soil 6 105 100 95 90 85 80 75 70 65
29 03 J 09-Fa 24 n01 -Feb-04 -Feb 0 02-Me 08-Mab-04 09-Mar-04 09-Mar-04 12-Mar-04 29-Mar-04 06-Mar-04 1109-A ar-04 17--M pr-04 A pr 24-Ma r-04 31-May-04 4 - -04 07May-04 21-J ay-04 y 07-Jun-04 0 12-Jun-04 19-Jul-04 2 - u- 4 27 020-Jull-04 09-AJul-04 u 16-AJu l-04 30-Aug-04 0 01-Aug-04 06-Sug-04 13-Seg-04 11-Sep-04 4 18-Oep-04 -0 0235-Ocpt-04 08--N ct-04 O 24-Noct-04 07-Nov-04 13-Dov-04 20-Dev-04 27-Dec-04 4 03 Dec-04 11-J ec-04 c 18-Jan-04 24 0 08 -Jan-04 15 --Fan-05 Ja 24-Men-05 24-Mab-05 24-Mar-05 24-Mar-05 24-Mar-05 24-Mar-05 04-Mar-05 07-A ar-05 -Apr-05 pr-05 r-05 05 5
+3σ +2σ +1σ Mean -1σ -2σ -3σ

Activity (Bq/kg)

Date
Figure 1. Control Chart of Soil-6 analysis for Ra-226
K-40 in IAEA-326

700 Activity (Bq/kg) 650 600 550 500 450 400 1 2 3 4 5 6 7 8 Me asure me nt

+3σ +2σ +1σ Mean -1σ -2σ -3σ

Figure 2. Control Chart of IAEA-326 analysis for K-40 Bias/Accuracy Bias is calculated based on the U score between the experimental values (the same data obtained from Figure 1 and Figure 2 above) and the CRM’s certified value. From the measurements data, the calculated U score is found to be 0.62 and 0.43 for Ra-226 and K-40 respectively, which means that there is no significant bias in this method [8]. However, if there is a possibility of occurring bias, correction from the bias can be made by applying a correction factor in the equation when calculating the activity for the Ra-226 or K-40 in a sample. The calculated Z score is found to be 0.60 and 0.01 for Ra-226 and K-40 respectively, showing that the accuracy of the measurement is satisfactory. Linearity and Working Range The results of the linearity tests were shown in Figure 3 below, which indicating a strong linear correlation between the measured activities for Ra-226 and K-40 and the amount of standard added. The correlation coefficient found to be 0.9970 and 0.9796 for Ra-226 and K-40 respectively. Since the figure indicating a good linearity, the figure can be extrapolates towards minimum and maximum to extend the working range. Slight deviation for the linearity of K-40 could be probably due to disturbance from the natural potassium present in the distill water used. The minimum working range

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for this method will be at it’s detection limit. Meanwhile the upper working range was considered happen when the activity inside the sample causes detector of the system have a high dead time, such as 20 % and above. At such situation, the distance between the sample and the detector will be increased to reduce the dead time. New calibration will be established for the system prior to a measurement of sample.
Activity vs Amount used
Ra-226
700 Activity (Bq/kg) 600 500 400 30 0 20 0 10 0 0 0 1 2 3 4 Amount (g) 5 6 7 8
y = 24.297x - 1.6 8 R2 = 0.9796 y = 143.43x - 0.173 4 R2 = 0.997

K-40 Linear (Ra-226) Linear (K-40)

Figure 3. Linearity test of Ra-226 and K-40 showing relation between added standard and measured activity. Limit of Detection Detection limit is not the minimum amount ever to be detected inside the sample. It is proportional to the square root of background and thus also proportional to the square root of the measurement time. For the activity measurement, the limit of detection usually been expressed as the minimum detectable activity (MDA). However, this value is very much depending on background counts, counting time, detector efficiency, emission probability and the gamma-ray energy [11-12]. If a lower limit of detection value is needed, usually a very long counting time (2-3 days) will be required. This is only necessary when dealing with samples for research purpose. Basically a brand new counting system will be able to achieve a lower MDA value. When the detector getting older, the MDA value will start to increase. In such case, the MDA of the measurement was determined by counting a distil water sample for more than 60 hours. The values are found to be 0.09 Bq/kg and 0.19 Bq/kg for Cs137 and Ra-226 when using a new counting system (shelf life < 1 year) compared to 0.84 Bq/kg and 2.43 Bq/kg for Cs-137 and Ra-226 for an old counting system (shelf life > 15 years). The Soil-6 was also be counted at different counting time range of 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours and 16 hours to find the minimum counting time that can still produce quantitative results. Experimental results (range from 76.97 to 89.12 Bq/kg) show that the minimum counting time of 30 minutes is sufficient enough to enable the spectrometry system to detect Ra-226 quantitatively and the measured value was also closed to the certified value. The same behaviour is found when counting Cs-137 in food samples. Therefore, as a whole, all samples shall be counted for at least 30 minutes to obtain representable results. Robustness Robustness of the method was determined by replicate measuring of the CRM Soil-6 by several different operators using the same instrument. The results of the measurement were summarised as in the table 2. From this table, we found that the mean value for the measurement is very close to each

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other, showing the high precision among the operators. The RSD from the three operators are below 7%. The robustness of the method was checked, by performing the student t-test [13] using the above data. The calculated t value is found to be less than the t critical value, at 95% confidence level when comparing the sets of data. This means that the slight different among the measurement results arose can still be tolerance. No similar experiments had been conducted for the K-40. However, since the Ra-226 can produce robust results, the K-40 will considers being robust if both radionuclides are present in the environmental samples. The calculated Z score value is 0.36, 1.85 and 1.98 for operator 1, 2 and 3, respectively. This score indicates that the precision of the measurement carried out by the respective operator is still within the satisfactory level.

Operator Reading 1 Reading 2 Reading 3 Reading 4 Reading 5 Reading 6 Reading 7 Reading 8 Reading 9 Reading 10 Reading 11 Reading 12

Table 2. 1 90.78 93.24 86.74 83.40 84.27 88.06 77.56 86.21 87.95 85.62 85.46 86.83

Ra-226 measurement data for three different operators 2 3 Operator 1 2 90.08 90.37 Reading 13 84.97 69.95 86.08 86.11 Reading 14 88.40 88.04 92.75 87.74 Reading 15 95.00 85.46 74.81 Reading 16 89.99 84.11 82.82 Reading 17 88.95 86.15 83.16 Reading 18 85.99 81.85 Reading 19 85.35 84.58 Reading 20 84.45 79.05 Reading 21 85.79 73.72 Reading 22 81.03 75.15 Reading 23 76.84 76.18 Reading 24 Mean 86.39 85.65 Std. Dev. 3.62 5.82 Rel. Std Dev. 4.19 6.80 Z-score 0.36 1.85 Ruggedness/Reproducibility

3 75.67 91.24 81.99 88.23 85.83 89.19 80.36 83.97 89.36 86.85 81.78 89.87 83.33 5.41 6.49 1.98

The certified value for the Ra-226 in the Soil-6 and K-40 in the SRM IAEA-326 was the average results obtained from the inter laboratories comparison organized by the IAEA which involves the usage of various method and equipments. In other words, the certified value is an acceptable value internationally. Therefore, if the laboratory is able to measure the activity for Ra-226 and K-40 that close to the certified value, it shows that the reproducibility ability of the laboratory. Since the activities measured for both Ra-226 and K-40 were close to the certified value, the reproducibility for this method is therefore no doubtful. Moreover, the results from the ANOVA statistical test performed using the sets of data in table 2 above found that the probability of this result, assuming the null hypothesis, is 0.165. This shows that the results were not significant (P > 0.05) and therefore the probability of the result occurring by chance is high [13]. Since both Ra-226 and Ra-228 are isotopes that carry the similar chemical behaviour, the use of Ra226 to validate the measurement method above shall also be representative for Ra-228 as well.

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CONCLUSIONS The method used in the measurement was found to be fairly good to measure the activity of radium isotopes and K-40 in the environmental samples. There is no doubt for the system (this method) to sense the present of Ra-226 and K-40 inside a sample. The precision of the method is found to be fairly good, with the RSD value less than 10% and the calculated z-score values are less than 2, indicate that the precision of the measurement is still within the satisfactory level. The average value obtained for the CRM is close to the certified value with the U score value less than 1 showing no significant bias of this method. Also, a wide linearity range was observed showing that the ability of the system to work from the minimum detectable activity till it maximum range. Minimum counting time of 30 minutes is found to be sufficient enough to enable the spectrometry system to detect Ra226 quantitatively. Meanwhile, the MDA values are found ranging from 0.19 to 2.43 Bq/kg for several different counting systems. The method validation has also demonstrated that, this measuring technique is robust and rugged. Overall, this analytical technique is able to produce quality and reliable analytical result for measuring of radium isotopes and K-40 in the samples.

RUJUKAN 1. 2. 3. 4. Povinec P. P., 1994. Sources of Radioactivity in the Marine Environment and their Relative Contributions to Overall Dose Assessment from Marine Radioactivity. IAEA-MEL-R2/94. Instituto de Pesquisas Energeticas e Nucleares, 1998-1999. Progress Report. Brazil. 87. Malaysian Institute for Nuclear Technology Research, 2002. Radiation Safety, Second Edition. Malaysia. 341. Lawrence Berkeley National Laboratory, 1999. Source from “The Lund/LBNL Nuclear Data Search Version 2.0, February 1999” website http://nucleardata.nuclear.lu.se/nucleardata /toi/.. Accessed on 21 March 2005. Department of Standards Malaysia, 1999. ISO/IEC 17025: General Requirements for The Competence of Testing and Calibration Laboratories. Malaysia. 16. Ellison, S.L.R., Rosslein, M. and Williams, A., 2000. EURACHEM, Quantifying Uncertainty in Analytical Measurement, Second Edition. UK. National Association of Testing Authorities, 1997. Format and Content of Test Methods and Procedures for Validation and Verification of Chemical Test Methods., Technical note. ISBN 0947289151. Australia. Peter, V., 2003. Method Validation, 4th Regional Training Course on QA/QC of Nuclear Analytical Techniques. 20-24 October 2003. KAERI, Taejon, Korea. Yii, M. W., Zaharudin, A., and Kamarozaman, I., 2003. The Effect Of Surrounding Conditions On The Radioactivities Measurement Using Gamma Spectrometry System. In MTC Proceeding. 22-24 July 2003. MINT, Malaysia.

5. 6. 7.

8. 9.

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10.

11. 12.

Omar, M., Ibrahim, M. Y., Hassan, A., Lau, H.M., and Ahmad, Z., 1990. Enhanced Radium Level in Tin Mining Areas in Malaysia. In: Proceedings of an International Conference on High Levels of Natural Radiation. M. Sohrabi, J.U. Ahmad and S. A. Durrani (editors). 3-7 Nov 1990. Ramsar, Islamic Republic of Iran. 191-195. Debertin, K. and Helmer, R. G., 1988. Gamma and X-Ray Spectrometry with Semiconductor Detectors. Elservier Science. The Netherlands. 399. Dovlete, C. and Povinec, P.P., 2002. Quantification of Uncertainty in Gamma Spectrometric Analysis of Environmental samples. 2nd Regional Training Course on QA/QC of Nuclear Analytical Techniques. 12-16 August 2002. Kuala Lumpur. LGC Limited, 2001. Method Validation Training Course. 3-5 December 2001. SCK•CEN, Mol, Belgium.

13.

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INVESTIGATION ON NEW TRANSITION METAL COMPLEXES OF THE SCHIFF BASES DERIVED FROM SALICYLALDEHYDE AND 2-HYDROXYACETOPHENONE Karimah Kassim∗, Yong Soon Kong, Hadariah Bahron, Nor Hadiani Ismail, Syarifah Rohaiza Syed Omar, Ernie Eileen Rizlan Ross Department of Chemistry, Faculty of Applied Sciences, Universiti Teknologi MARA, 40450 Shah Alam, Selangor. New metal complexes of the Schiff bases N,N’-ethylenebis(salicylideneimine), N,N’-phenylenbis (salicylideneimine), Bis(2-hydroxyacetophenone)ethylenediimine and bis(2-hydroxyacetophenone) phenylendiimine with Co(II), Ni(II), Cu(II), Mn(II), Fe(III), Sb(III) and Bi(III) are synthesized and 1 elucidated by elemental analyses, infrared spectroscopy and H and 13C NMR spectroscopies. The various Schiff base ligands are prepared by condensation reaction between appropriate diamine compounds with salicyaldehyde or 2-hydroxyacetophenone in the ratio of 1:2. The metal complexes are then prepared by the insertion method in the ratio of 1:1 (Schiff base ligand : metal cation). The results suggest that the Schiff bases are bivalent anion with bidentate donors derived from phenolic oxygens and azomethine nitrogens. The new complexes are also screened for their antibacterial activities. Keywords: tetradentate Schiff base, metal complexes, antibacterial activities Introduction Schiff bases are typically formed by the condensation of a primary amine and an aldehyde or ketone. The resultant functional group, R1HC=N-R2, is called an imine and is particularly effective for binding metal ions via the N atom lone pair, especially when used in combination with one or more donor atoms to form polydentate chelating ligands or macrocycles. Examples of a few compounds that have been commonly investigated are given in Figure 1 below [1].
R
H H N NH N NH
H2N N NH CH3

N H

NH

Figure 1. Example of Schiff Base Schiff base ligands assure a constant coordination geometry and an electronically flexible environment to metals. The relative arrangement of the two vacant or functionalizable coordination sites is of a crucial importance for reactivity, but is almost exclusively dependent on the ionic radius of the metal ion [2]. The structures of the Schiff base ligands investigated in the present study are shown in Figure 2 (a), (b), (c) and (d).

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OH

HO

OH

HO

N

N

N

N

SalenH2 SalOPDH2

OH

HO

OH

HO

N H3C

N CH3 H3C

N

N CH3

HapenH2

HapenOPDH2

Figure 2. General structure of Schiff bases investigated Experimental Method Synthesis of SalenH2 Schiff base ligand N,N-ethylenebis(salicylideneimine) (SalenH2) was synthesized at room temperature. 1 mmol of ethylenediamine and 2 mmol of salicylaldehyde was dissolved in ethanol. 1 mol of salicylaldehyde was each added drop wise into 1 mmol of ethylenediamine. Colourless solution of ethylenediamine changed to yellow solution indicating condensation reaction has occurred. Ligand containing solution was then left to crystallized at room temperature, resulting in formation of yellow crystals. Schiff base ligand was then filtered, washed with cold ethanol and dried in a silica filled dessicator. Synthesis of SalOPDH2 Schiff base ligand N,N-phenylenebis(salicylideneimine) (SalOPDH2) was synthesized at room temperature. I mmol of o-phenylenediamine and 2 mmol of salicylaldehyde was dissolved in ethanol. Colourless solution of o-phenylenediamine changed to orange solution. Ligand containing solution was then was then left to crystallized at room temperature resulting an orange crystals. Schiff base ligand was then filtered, washed with cold ethanol and dried in a silica filled dessicator.

Synthesis of HapenH2 A solution of 2 mmol 2-hydroxyacetophenone in absolute ethanol and 1 mmol of ethylenediamine in absolute ethanol were mixed and stirred for half an hour. The color of the mixture change to yellow upon stirring. The mixture was then refluxed for 1 hour and cool to the room temperature to produce

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yellow semicrystalline solids The yellow semicrystals were then filtered and washed with ethanol and air dried. Synthesis of HapOPDH2 2 mmol of 2-hydroxyacetophenone and I mmol of o-phenylenediamine were dissolved separately in absolute ethanol. On the mixing, a yellow solution was produced. The the mixture was heated under reflux for 1 hour and the colour changed to golden brown upon refluxing. No solid appeared upon cooling. After a day, some yellow long needle shaped crystals were obtained. The crystals were filtered off and washed with cold ethanol. Synthesis of SalenH2 and SalOPDH2 complexes The method used in complexation were direct insertion. Complexation of metal compounds were done by using acetate salts of manganese, cobalt, nickel, copper and ferric chloride, each were dissolved in 50 mL of methanol. Addition of 1 mmol of Schiff base ligand (SalenH2 or SalOPDH2) in 20 mL of dichloromethane into methanolic solution of iron and copper salts gave dark brown and green precipitate respectively. For manganese(II), nickel(II) and copper(II), dark brown, red and green solutions were observed respectively. Precipitate was then filtered, washed with a few drops of dichloromethane and dried with silica gel in a desiccator. Complex solutions of manganese and nickel were at room temperature to allow evaporation of solvents. Dark brown and red crystals were collected from manganese and nickel complexes of N,N-ethylenebis(salicylideneimine). N,NEthylenebis(salicylideneimine)nickel(II) complex and N,N-ethylenebis(salicylideneimine)copper(II) gave red and green needles. Synthesis of HapenH2 and HapOPDH2 complexes The 1 mmol ligand (HapenH2 or HapOPDH2) was dissolved in 20 ml freshly distilled THF in a 100 ml round bottom flask under a stream of nitrogen. The ligand was stirred for half an hour. The acetate metal salts of nickel(II), copper(II), cobalt(II), tin(II), antimony(III) and bismuth(III) I mmol in 20 ml freshly distilled THF under a stream of nitrogen was added to the ligand. The mixture was stirred overnight. For nickel, copper, cobalt, tin, antimony and bismuth the green, dark red, dark brown, white and yellow percipitate were obtained respectively. The percipitate were filtered off with cold ethanol and air dried.
HO H

HO H 3C

OH

HO

2
O

2

OH
O

HO

H 2N
EtOH,

NH 2

N H

N H

EtOH reflux 1 hr
H 3C

N

N CH 3

room tempreture

SalenH2

HapenH2

Complexation

Complexation

+ MX 2 + MX 2
MeOH
O O

+

+

MX 3

MX 3

MeOH, KOH, N 2 , reflux 1 hr

THF, N 2

MeOH
X
O O

O

O

O

X M

O

M
N H N H

M
N H N H

M
N H 3C N CH 3

N H 3C

N CH 3

+

2HX

+

2HX

+ 2HX

+ 2HX

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Figure 3. Overall synthesis path of tetradentate Schiff base ligands and complexes containing ethylenediimine

HO H
HO H3C

OH

HO 2
O

OH

HO

2

H2N
N H N H EtOH, room tempreture

NH2

O

N EtOH reflux 1 hrs H3 C

N CH3

SalOPDH2 Complexation

HapOPDH2 Complexation

+ MX2 +
O

MX2

+

+

MX3

MX 3

MeOH, KOH, N2 , reflux 1 hr

MeOH, KOH, N2 , reflux 1 hr X M

MeOH

MeOH X
O O O O

O

O

O

M
N H N H H N

M
N H H3 C N

M
N CH3 H3C N

N CH3

+ 2HX

+

2HX

+ 2HX

+ 2HX

Figure 4. Overall synthesis path of teradentate Schiff base ligands and complexes containing o-phenylendiimine

Results and Discussion (a) Elemental Analysis Elemental analyses of Schiff base ligands and complexes show good concordance to the predicted values calculated by using ACD Labs Chemsketch 5.0. Schiff base complexes of nickel and copper were divalent, whilst trivalent for manganese, cobalt and iron, bismuth and antimony.

Table 1. Elemental analysis of SalenH2 and SalOPDH2 ligands and complexes Compound SalenH2 Ni(Salen) Cu(Salen) Yield (%) 98 86 88 Found (Calc.) (%) C H 71.66(71.62) 6.11(6.01) 58.54(59.13) 58.68(58.26) 4.69(4.34) 4.55(4.28)

N 11.07(10.44) 8.87(8.62) 8.39(8.49)

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Co(Salen) Mn(Salen) Fe(Salen) SalOPDH2 Ni(SalOPD) Cu(SalOPD) Co(SalOPD) Mn(SalOPD) Fe(SalOPD)

72 90 81 96 94 79 68 92 81

59.54(59.09) 57.07(56.85) 45.00(53.74) 75.99(75.93) 62.89(64.40) 62.77(61.53) 54.57(61.12) 61.73(61.69) 58.35(59.22)

4.85(4.34) 4.75(4.51) 5.19(3.95) 5.16(5.10) 3.99(3.78) 4.43(4.43) 4.08(3.96) 4.09(4.00) 3.94(3.48)

8.46(8.61) 7.21(7.37) 11.23(7.83) 9.07(8.86) 7.24(7.51) 7.01(6.83) 5.56(6.48) 6.34(6.54) 6.34(6.91)

Table 2. Elemental analysis of HapenH2 and HapOPDH2 ligands and complexes Compound HapenH2 Ni(Hapen) Cu(Hapen) Co(Hapen) Sb(Hapen) Bi(Hapen) Sn(Hapen) HapOPDH2 Ni(HapOPD) Mn(HapOPD) Co(HapOPD) (b) Infrared Spectroscopy Table 3. Infrared spectroscopy analysis of SalenH2 and SalOPDH2 ligands and complexes Compound SalenH2 Ni(Salen) Cu(Salen) Mn(Salen) SalOPDH2 Ni(SalOPD) Cu(SalOPD) Co(SalOPD) Mn(SalOPD) Fe(SalOPD) Infrared bands (cm-1) v(C=N) 1613 1619 1625 1635 1609 1601 1605 1596 1605 1604 v(C−O) 1041 1086 1026 1050 1044 1123 1126 1039 1127 1029 Yield (%) 47.32 32.48 49.25 39.54 46.25 55.89 36.87 46.58 46.25 21.59 37.15 Found (Calc.) (%) C H 73.29(72.95) 7.24(6.80) 61.75(61.24) 5.84(5.14) 60.41(59.25) 5.04(5.17) 61.20(60.38) 5.14(4.96) 30.29(32.00) 3.35(4.03) 38.16(33.42) 3.93(4.21) 30.26(34.99) 3.11(4.40) 76.72(74.59) 5.85(6.19) 50.87(54.89) 4.60(5.09) 48.61(55.96) 5.02(4.98) 48.01(48.86) 4.96(4.43) N 10.23(9.45) 8.02(7.93) 7.83(7.69) 7.93(6.97) 4.77(6.22) 5.37(6.50) 4.45(6.80) 12.86(14.09) 2.56(1.68) 4.36 (4.25) 4.31(2.07)

Infrared spectra of Schiff base ligands and complexes (Table 3) were recorded and compared to identify shifting of bands upon complexation. The important bands in infrared spectra are stretching vibrations mode of C=N and C−O at 1500 cm-1, stretching vibrations mode of aromatic ring C-H -1 -1 -1 (3031-3057 cm ) and C=C stretching in the region of 1456-1632 cm . The peaks at 3100-3128 cm in the ligand spectra, which were absent in the metal complex spectra were assigned to N-H bending -1 -1 mode vibration. Bands at 600 cm and 400 cm region which indicate M-O and M-N coordination

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bonds were difficult to be observed due to noise. Coordination of ligand to the central metal ion can be deduced by the shifting of C=N and C−O stretching vibration bands and absence of broad band at 3300 - 3500 cm-1 which is associated with N-H bending mode vibration of imine group and O-H stretching mode of hydroxyl group of Schiff Base ligand [3]. The C=N band of SalOPDH2 shifts to lower value compared to their metal complexes, which corresponds with the loss of hydrogen bond and the formation of a new chelate ring between imine nitrogens and central metal ion [4]. The C-O band of ligand however shifts to a higher value compared to complexes, this is due to participation of phenolic O atoms in coordination to metal ions [5]. Table 4. Infrared spectroscopy analysis of HapenH2 and HapOPDH2 ligands and complexes Compound HapenH2 Ni(Hapen) Cu(Hapen) Co(Hapen) Sb(Hapen) Bi(Hapen) Sn(Hapen) HapOPDH2 Ni(HapOPD) Mn(HapOPD) Co(HapOPD) Infrared bands (cm-1) ν(OH) ν(C=N) 3350 1703 1601 1607 1613 1611 1609 1621 3224 1607 1619 1614 1622 ν(M-O) 667 645 639 675 646 649 653 681 663

Some important infrared absorption frequencies of the ligands HapenH2 and HapOPDH2 and their metal complexes are given in the Table 4 with their probable assignments. The free Schiff bases show their characteristic OH frequencies at around 3350 cm-1 (HapenH2) and 3224 cm-1 (HapOPDH2). The OH stretching vibration disappears in the spectra of the complexes indicating strong participation of the OH group in chelate formation. The ν(OH) bands disappear due to the deprotonation of the OH group upon complexation [6]. A strong band at 1703-1601 cm-1 in the spectra of the free Schiff bases is characteristic of the azomethine (C=N) group. It is expected that coordination of the nitrogen to the metal atom would reduce the electron density in the azomethine link and thus lower the C=N absorption. In the Schiff base complexes the C=N absorption band has been shifted to the region around 1621-1601 cm-1 indicating the coordination of the Schiff bases through the nitrogen atom. The appearance of new bands in the IR spectra of the metal complexes in the region 681-639 cm-1 is due to the M-O bonds, developed through complexation [7]. (c) Nuclear Magnetic Resonance Spectroscopy The absence of peak at 4.30 – 5.40 ppm in all 1H NMR spectra shows amine group of diamines has 1 been converted to imine group by Schiff Base condensation. The H NMR spectra of the ligands present sharp multiplets in the region of 8.30-6.90 ppm due to the presence of aromatic ring. The singlets observed in the region δ 13.0 – 13.3 ppm were due to extensive hydrogen bonding from by the hydroxyl group of the ligand and the absence of this peak in the spectra of their respective metal complexes suggest coordination to the metal has taken place [3]. The 13C NMR of the complexes presented many noise peaks and could not be well interpreted. The presence of the noise peaks was probably due to suspended particles in the sample which could not be removed even after several filtration.

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Table 5. 1H Nuclear Magnetic Resonance spectroscopy analysis of SalenH2 and SalOPDH2 ligands and complexes Compound SalenH2 SalOPDH2 Ni(Salen) Ni(SalOPD) Residue Phenyl rings CH2 OH Phenyl ring OH Phenyl rings CH2 Phenyl rings CH3 No of H 8 4 6 12 6 8 4 8 4 δH (ppm) 6.95 – 8.30 3.85 13.3 7.33-8.60 13.0 6.50– 8.20 3.44 6.67– 8.27 7.32

Table 6. 13C Nuclear Magnetic Resonance Spectroscopy Analysis of SalenH2 and SalOPDH2 ligands and complexes Compound H2SE H2SOP C1 165.7 165.1 C2 161.3 162.9 C3 133.7 133.6 Chemical Shift, δ (ppm) C4 C5 C6 C7 130.8 118.8 120.1 116.2 131.6 118.8 121.1 116.7
HO

C8 59.8 142.7

C9 129.1

C10 126.8

C7 C3 C6 C4 H C2 C5

OH

C1 N C8

N H C 10 C9

Table 7. 1H Nuclear Magnetic Resonance spectroscopy analysis of HapenH2 and HapOPDH2 ligands and complexes Compound HapenH2 HapOPDH2 Bi(Hapen) Ni(HapOPD) Residue Phenyl rings CH2 CH3 Phenyl ring CH3 Phenyl rings CH2 CH3 Phenyl rings CH3 No of H 8 4 6 12 6 8 4 6 8 4 δH (ppm) 6.76 – 7.45 3.91 2.16 6.71-7.67 2.17 6.76 – 7.54 3.98 2.41 6.87 – 7.74 2.16

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Table 8. 13C Nuclear Magnetic Resonance spectroscopy analysis of HapenH2 and HapOPDH2 ligands and complexes Compound HapenH2 HapOPDH2 C1 C2 163.45 163.35 164.60 157.56 Chemical Shift, δ (ppm) C3 C4 C5 C6 C7 C8 133.86 131.48 129.48 127.43 119.82 50.40 132.16 130.42 118.45 121.24 115.86 146.71
C7 C3 C6 C4 H3C C2 C5 C1 N C8 C 10 C9 N CH3 OH HO

C9 C10 128.31 123.37

(d) Antibacterial Activity Table 9. Antibacterial activity of of HapenH2 and HapOPDH2 ligands and complexes HapenH2 24h E. coli Pseudomona s sp. Streptococcu s sp. Staphylococc us aureus Salmonella typhi 24h Sn(Hapen) 48 24h h Sb(Hapen) 24h 15 mm 24 mm 15 mm 48h 16 mm 25 mm 15 mm Bi(Hapen) 24 48h h HapOPDH2 24h 48h Co(HapOPD) 24h 7 mm 48h -

Antibacterial activities of the ligands and the complexes of HapenH2 and HapOPDH2 were measured by the disc diffusion method against the bacteria E. coli, Pseudomonas sp., Streptococcus sp., Staphylococcus aureus. The test solution were prepared in methanol. The results of the antibacterial activities are summarized in Table 9. In general, it was observed that the Co(Hapen) and Sb(HapOPD) have the highest antibacterial activity than their respective free ligands against Streptococcus sp.and Staphylococcus aureus. Such an increased activity for the metal chelats as compared to the free ligands can be explained on the basis of the chelation theory. Chelation considerably reduces the polarity of the metal ion because of the partial sharing its positive charge with the donor groups and possible π electron delocalization over the chelate ring. Such chelating could increase the lipophilic character of the central metal atom, which subsequently favour the permeation through the lipid layer of cell membrane. The mode of action of the complexes may involve the formation of the hydrogen bond through azomethine group (>C=N) with the active metal centers of the cell constituents resulting in the interference with normal cell process [9][10]. Sb(HapOPD) has higher activity against the Streptococcus sp.and Staphylococcus aureus compared to Co(Happen).

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Conclusions The new metal complexes of the Schiff bases salicylideneimine, salicylic-o-phenylendiimine, (2hydroxyacetophenone)ethylenediimine and 2-hydroxyacetophenone-o-phenylendiimine with Co(II), Ni(II), Cu(II), Mn(II), Sb(III) and Bi(III) have been synthesized and characterized. The results showed that the Schiff bases are bivalent anion with bidentate donors derived from phenolic oxygens and azomethine nitrogens. The complexes are more active than their parent ligands in the antibacterial activity. Acknowledgement The author is grateful to acknowledge for the schlorship given by Universiti Teknologi MARA. This research was funded by grant from Institute of Research, Development and Commercialization (IRDC)-10519.

References 1. A.L.El-Ansary, A.A. Soliman, O.E. Sherif and J.A. Ezzat, Synth. React. Inorg. Met. Org. Chem., 2002, Vol. 32(7), 1301. 2. M.T.H. Tarafder, K.H. Jin, K.A. Crouse, A.M. Ali, B.M.Yamin, H.K. Fun, Polyhedron, 2002, Vol. 21, 2547. 3. Singh, M. S. and Singh, P. K. 2003, Synth .and React. Inorg. Met.Org. Chem. 33, 2, 271 – 280. 4. Amirnasr, M., Schenk, K. J. & Vafazadeh, R. 2001, Polyhedron. 20, 695 – 702. 5. 6. Teoh, S. G., Yeap, G. Y., Loh, C. C., Foong, L. W., Teo, S. B. & Fun, H. K. 1997, Polyhedron. 16, 13, 2213 – 2221. 7. A.A. Solimon and W.Linert, Thermochimica Act., 1999, 67 8. D.M. Boghaei and S. Mohebi, Tetraheron 58, 2002, 5357 9. N. Kathale, N.S. Roa, N.N. Roa and K.N. Munshi, Synth. React. Inorg. Met. Org. Chem., 2001, Vol. 31(5), 859. 10. R. Prabhakaran, A. Geetha, M. Thilagavathi, R. Karvembu. V. Krishnan, H. Bertagnolli, K. Natarajan, J. Inorg. Biochem., 2004, Vol. 98, 2131. 11. T.Daniel Thangadurai, D.Anitha and K. Natarajan, Synth. React. Inorg. Met. Org. Chem., 2002, Vol. 32(7), 1329.

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HUBUNGAN PERMINTAAN KLORIN DENGAN KUALITI AIR MENTAH Lim Fang Yee & Md. Pauzi Abdullah Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains & Teknologi, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor. Tel: 03-89215447; Fax: 03-89215410; E-mail: mpauzi@pkrisc.cc.ukm.my Abstrak. Klorin sebagai agen disinfeksi dalam air minuman telah digunakan secara meluas sejak ia berjaya dipraktikkan dalam bekalan air minuman di Jersey City pada 1908. Kebanyakan loji rawatan air di Malaysia menggunakan klorin sebagai agen disinfeksi untuk membunuh patogen dan bahan pencemar yang membahayakan para pengguna. Oleh kerana klorin merupakan agen disinfeksi yang kuat, ia boleh bertindak balas dengan komponen-komponen kimia seperti mangan, ferum, hidrogen sulfida, ammonia, dan fenol dalam air. Tindak balas ini berlaku dengan pantas, dan klorin tidak akan bertindak sebagai agen disinfeksi sehingga semua sebatian organik dan inorganik yang hadir dalam air telah bertindak balas dengan klorin. Komponen-komponen kimia yang boleh bertindak balas dengan klorin akan menyebabkan permintaan klorin dalam air. Permintaan klorin dalam air perlu dipenuhi sebelum klorin bebas terhasil. Klorin bebas ini seterusnya akan mengurai kepada asid hipoklorus dan ion hipoklorit yang penting dalam proses disinfeksi untuk membunuh patogen dan bahan pencemar dalam air. Kebanyakan loji rawatan air mengekalkan klorin bebas sebanyak 0.2 mg/l dalam sistem pengagihan sebelum sampai kepada pengguna. Kajian ini melibatkan penentuan parameter-parameter yang dipercayai boleh bertindak balas dengan klorin di sembilan stesen pensampelan di sepanjang Sungai Semenyih dan empat stesen di loji rawatan air. Parameterparameter yang ditentukan terdiri daripada ammonia, sianida, sulfida, fenol, fosforus, nitrit, mangan, ferum, dan karbon organik jumlah. Secara keseluruhannya, kajian ini mendapati ammonia dan karbon organik jumlah merupakan sebatian yang paling banyak bertindak balas dengan klorin untuk menghasilkan trihalometana dan kloramina. Selain itu, kepekatan sebatian-sebatian sianida, sulfida, fenol, fosforus, nitrit, mangan, dan ferum juga menurun selepas proses pengklorinan. Hasil kajian ini boleh digunakan menilai tahap permintaan klorin di Sungai Semenyih. Kata Kunci: Permintaan klorin, ammonia, jumlah karbon organik, lembangan Sungai Semenyih

Pengenalan Disinfeksi merupakan proses yang paling penting dalam proses rawatan air mentah kerana proses ini berupaya membasmi penyakit yang disebabkan oleh mikroorganik patogen [1]. Sebarang bentuk proses disinfeksi adalah perlu untuk menyingkirkan mikroorganisma yang boleh membahayakan. Klorin digunakan secara meluas sebagai disinfektan kerana ianya murah dan berkesan. Hampir semua loji rawatan air di Malaysia menggunakan klorin dalam proses disinfeksinya [2,3,4,5]. Di sebalik keberkesanan proses pengklorinan bagi membunuh patogen dalam air, pengklorinan didapati dapat menjanakan hasil sampingan seperti trihalometana (THM) yang merupakan sebatian halo-organik yang terbukti bersifat karsinogenik dan mutagenik [6,7]. Klorin yang digunakan sebagai agen disinfeksi dalam proses rawatan air telah menyebabkan isipadu air terawat mengandungi sebatian berklorin yang tinggi [8]. Klorin merupakan agen pengoksidaan yang kuat. Gas klorin sangat larut dalam air dan dapat membentuk asid hipoklorus (HOCl) dan ion hipoklorit (OCl¯) dengan cepat [9]. Klorin bebas ialah jumlah HOCl dan OCl¯ yang hadir. HOCl adalah agen disinfeksi yang lebih cekap daripada OCl¯.

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

Keberkesanan pembasmian kuman HOCl adalah 40 kali lebih cekap daripada OCl¯. Klorin adalah kurang berkesan sebagai disinfektan pada pH di bawah 6 dan pH yang melebihi 8 [10]. Klorin boleh mengoksidakan banyak komponen yang hadir dalam air. Apabila klorin dilarutkan dalam air, sebatian organik dan agen penurun yang lain akan bertindak balas dengan klorin bebas ini dan menyebabkan permintaan klorin dalam air. Klorin yang selebihnya akan memusnah dan merosakkan protein dan asid nukleik mikroorganisma dalam air [11,12]. Kepekatan sebatian organik dan pepejal terampai yang tinggi dalam air mentah sering menyebabkan permintaan klorin yang tinggi dan mengurangkan keberkesanan klorin sebagai disinfektan [13,14]. Akibatnya, klorin yang lebih perlu didoskan untuk membunuh patogen dalam air. Disinfeksi air minum dengan menggunakan klorin akan menghasilkan bahan sampingan yang boleh memudaratkan kesihatan orang awam [15,16]. Kebimbangan yang dinyatakan akhir-akhir ini tentang adanya kepekatan surihan sebatian organoklorin dalam air adalah penyebab barah apabila diuji pada dos yang tinggi terhadap haiwan. Kewujudan trihalometana dalam air minum di Malaysia boleh membimbangkan masyarakat kerana ianya berhubungkait dengan kesihatan manusia [17]. Penemuan saintifik ini telah menyebabkan isu THM menjadi satu perkara yang dibimbangkan dan perlu diberi penekanan dan perhatian pada peringkat kebangsaan dan juga antarabangsa [18]. Selain itu, agen penurun yang hadir dalam air boleh bertindak balas secara reaktif dengan klorin. Permintaan klorin ini mesti dipenuhi sebelum klorin dapat bertindak sebagai disinfektan. Adalah perlu supaya kajian dan penyelidikan dibuat dengan memberikan kawalan proses yang berhati-hati dan penggunaan dos klorin yang sesuai dalam proses disinfeksi. Penggunaan dos klorin yang optimum supaya tidak terlalu kurang dan tidak menentu luar daripada yang diperlukan adalah penting. Kajian ini bertujuan untuk mengenalpasti komponen-komponen yang boleh bertindak balas dengan klorin serta puncapuncanya, supaya pengurusan dan tindakan dapat diambil untuk menyingkirkan pelopor yang boleh menyebabkan permintaan klorin. Experimen Kawasan Kajian Loji pembersihan air Sungai Semenyih terletak di Presint 19, Putrajaya. Loji ini mendapatkan sumber air mentahnya dari Sungai Semenyih. Air mentah dipamkan melalui saluran paip sepanjang 8 km dari Jenderam Hilir ke loji pembersihan air untuk proses perawatan. Pensampelan air dilakukan di empat stesen di dalam loji pembersihan air. Keempat-empat stesen ini adalah mengikut susunan proses rawatan air, iaitu air mentah, air mendap, air tapisan, dan air bersih. Pendosan klorin dilakukan selepas air dituraskan dengan pasir deras untuk menyingkirkan jisim pepejal dalam air, iaitu klorin ditambahkan ke dalam air tapisan. Selepas proses pengklorinan, air disalurkan ke tangki air bersih dan seterusnya dipam ke kolam imbang. Dari kolam imbang, air bersih disalurkan secara graviti ke kolam agihan sebelum disalurkan kepada pengguna. Sumber air mentah iaitu Sungai Semenyih mengalir melalui tiga mukim, iaitu Hulu Semenyih, Semenyih, dan Beranang. Dalam kajian untuk menentukan punca-punca yang menyebabkan permintaan klorin, sebanyak sembilan stesen telah dipilih bagi mewakili kawasan kajian. Terdapat beberapa aktiviti yang dijalankan di lembangan Sungai Semenyih yang berpotensi mencemarkan Sungai Semenyih, iaitu perindustrian, pembinaan, pertanian, dan penternakan. Kawasan kajian mempunyai topografi yang seimbang dengan adanya kawasan landai, beralun, dan tanah tinggi. Lebih kurang 65 % lembangan Sungai Semenyih adalah bergunung-ganang dan kawasan hilir sungai adalah berpaya. Semua stesen pensampelan yang tersebut di atas ditunjukkan dalam rajah 1. Pemilihan sembilan stesen pensampelan ini adalah berasaskan kepada beberapa kriteria dan kesesuaian pensampelan dilakukan. Selain itu, kehomogenan air, bentuk muka bumi, sumber pencemaran, serta pertemuan anak-anak sungai dengan Sungai Semenyih merupakan faktor yang dipertimbangkan dalam pemilihan stesen-stesen seperti yang ditunjukkan dalam Jadual 1. Jadual 1 Lokasi Stesen Pensampelan di Sungai Semenyih

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

Stesen SS1 SS2 SS3 SS4 SS5 SS6 SS7 SS8 SS9

Latitud N 03° 04.572’ N 03° 00.520’ N 02° 56.866’ N 02° 56.615’ N 02° 54.308’ N 02° 54.213’ N 02° 53.160’ N 02° 53.021’ N 02° 53.021’

Longitud E 101° 53.034’ E 101° 52.125’ E 101° 50.891’ E 101° 50.808’ E 101° 48.690’ E 101° 48.533’ E 101° 48.373’ E 101° 47.560’ E 101° 49.163’

Lokasi Empangan Sungai Semenyih Sungai Saringgit Sungai Semenyih Sungai Rinching Sungai Semenyih Sungai Beranang Sungai Semenyih Stesen Pam Air Mentah

SS1

SS2

SS4

SS3

SS6 SS8 SS9 SS7 SS5

Rajah 1 Peta kawasan kajian dan stesen-stesen pensampelan di lembangan Sungai Semenyih Bahan dan Kaedah

408

Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

Dalam kajian ini, sebanyak 14 kali pensampelan telah dijalankan antara bulan Disember 2004 hingga bulan Julai 2005. Sampel diambil dengan menggunakan kaedah “grab sampling”, iaitu dengan menggunakan bekas untuk menimba air di permukaan sungai atau lebih kurang 1 m dari permukaan sungai. Empat botol sampel yang berisipadu 1 liter digunakan, iaitu dua botol kaca dan dua botol plastik. Semasa pensampelan air, beberapa parameter kualiti air diukur secara in situ. Pengukuran parameter in situ dilakukan dengan menggunakan YSI 556 Multiprobe System (MPS) dan Ultrameter keluaran syarikat Myron L. Company. Parameter-parameter yang diukur secara in situ ialah suhu, konduktiviti, kemasinan, jumlah pepejal terlarut, oksigen terlarut, pH, klorofil A, dan keupayaan pengoksidaan dan penurunan. Parameter-parameter kimia seperti ammonia, sianida, sulfida, fenol, fosforus, nitrit, karbon organik jumlah, ferum, dan mangan dianalisis di makmal kimia Universiti Kebangsaan Malaysia. Semua sampel dianalisis secepat mungkin setelah pensampelan dilakukan. Dalam kes dimana sampel air tidak dapat dianalisis dengan serta merta, sampel air diawet dengan menggunakan asid sulfurik atau asid nitrik kepada pH 2 dan disimpan pada suhu 4 °C. Kesemua parameter ini dianalisis mengikut kaedah piawai yang telah ditetapkan [19]. Keputusan dan Perbincangan Permintaan klorin terhasil daripada pelbagai tindak balas dimana klorin digunakan untuk mengoksidakan komponen-komponen yang hadir dalam air. Data yang terhad selama lapan bulan ini menunjukkan permintaan klorin di loji rawatan air Sungai Semenyih adalah disebabkan oleh ammonia, sianida, sulfida, fenol, fosforus, nitrit, mangan, ferum, dan karbon organik jumlah. Ammonia Dalam proses pengklorinan, tindak balas di antara ammonia dan klorin tidak dapat dielakkan. Tindak balas klorin dengan ammonia akan menghasilkan satu siri sebatian kloro-ammonia yang dipanggil kloramina. Rajah 2(a) di bawah menunjukkan pengurangan kepekatan ammonia di sepanjang proses rawatan air. Paras ammonia didapati berkurang sebanyak 88 % daripada air tapisan (L3) kepada air bersih (L4) selepas klorin didoskan. Rajah 2(b) menunjukkan tahap pencemaran ammonia di sepanjang Sungai Semenyih sebelum air dipamkan ke loji rawatan air. Punca pencemaran ammonia berlaku di stesen 5 (SS5) dan stesen 7 (SS7). Kedua-dua SS5 dan SS7 terletak berhampiran dengan Pusat Bandar Rinching dan Bandar Tasik Kesuma. Pencemaran ammonia boleh berpunca daripada kumbahan domestik dan sistem rawatan kumbahan yang belum siap. Permintaan klorin adalah berkadar dengan banyaknya ammonia yang hadir dalam air mentah. Tindak balas klorin dengan ammonia bergantung kepada suhu, pH, kepekatan ammonia, dos klorin, dan masa sentuhan [20].
Am m onia m g/l 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 L1 L2 L3 L4
2.5 2 mg/l 1.5 1 0.5 0 1 2 3 4 5 Stesen 6 7 8 9

Ammonia

2(a) Rajah 2

2(b)

(a) Tahap pengurangan ammonia di loji rawatan air (b) Punca pencemaran ammonia di 9 stesen pensampelan di Sungai Semenyih

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

Sianida Dalam loji rawatan air Sungai Semenyih, sianida hadir dalam kepekatan yang rendah. Rajah 3(a) menunjukkan kepekatan sianida adalah kurang daripada 0.5 ppb dalam air bersih. Kajian yang dijalankan mendapati kepekatan sianida berkurang sebanyak 73.68 % selepas klorin didoskan. Punca pencemaran sianida berlaku di SS3, SS5, dan SS7 sepeti yang ditunjukkan dalam rajah 3(b). Pencemaran sianida ini adalah disebabkan oleh pembuangan industri dan aktiviti pelupusan sampah yang terletak berhampiran dengan sungai. Sianida dihasilkan terutamanya oleh industri yang berasaskan elektropenyaduran dan racun insektisid [10].
Sianida m g/l 0.0035 0.003 0.0025 0.002 0.0015 0.001 0.0005 0 L1 L2 L3 L4

Sianida
0.007 0.006 0.005 0.004 0.003 0.002 0.001 0 1 2 3 4 5 Stesen 6 7 8 9

3(a) Rajah 3

mg/l

3(b)

(a) Tahap pengurangan sianida di loji rawatan air (b) Punca pencemaran sianida di 9 stesen pensampelan di Sungai Semenyih

Sulfida Ion sulfida biasanya dilepaskan daripada sisa industri yang berasaskan kimia, kertas, tektil, dan aktiviti menyamak kulit binatang. SS5 dan SS7 mencatatkan bacaan sulfida yang tertinggi seperti yang ditunjukkan dalam rajah 4(b). Rajah 4(a) menunjukkan pengurangan kepekatan sulfida sepanjang proses rawatan air. Ion sulfida mengalami pengurangan sebanyak 61.46 % daripada air tapisan (L3) kepada air bersih (L4). Tindak balas sulfida dengan klorin berlaku dengan pantas dan menghasilkan sulfat dan hidrogen klorida.
Sulfida m g/l 0.1 0.08 0.06 0.04 0.02 0 L1 L2 L3 L4 mg/l 0.05 0.04 0.03 0.02 0.01 0 1 2 3 4 5 Stesen 6 7 8 9

Sulfida

4(a) Rajah 4

4(b)

(a) Tahap pengurangan sulfida di loji rawatan air (b) Punca pencemaran sulfida di 9 stesen pensampelan di Sungai Semenyih

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

Fenol Alley [10] mendapati klorin dapat bertindak balas dengan fenol dengan mudah, dan sekiranya klorin hadir, kepekatan fenol dapat dikurangkan kepada tahap yang paling rendah. Rajah 5(a) menunjukkan pengurangan fenol dalam loji rawatan air sebelum dan selepas proses pengklorinan. Fenol didapati berkurang sebanyak 42 % selepas klorin ditambahkan. Rajah 5 (b) menunjukkan pencemaran fenol yang berlaku di SS5 dan SS7 yang mencatatkan bacaan sebanyak 0.26 mg/l dan 0.28 mg/l masingmasing. Pencemaran fenol di kedua-dua stesen ini adalah berpunca daripada aktiviti perindustrian yang menyalurkan sisanya ke dalam Sungai Semenyih.
Fenol m g/l 0.1 0.08 0.06 0.04 0.02 0 L1 L2 L3 L4

Fenol
0.03 0.025 mg/l 0.02 0.015 0.01 0.005 0 1 2 3 4 5 Stesen 6 7 8 9

5(a) Rajah 5 Fosforus

5(b)

(a) Tahap pengurangan fenol di loji rawatan air (b) Punca pencemaran fenol di 9 stesen pensampelan di Sungai Semenyih

Fosforus yang hadir secara semulajadi biasanya wujud dalam bentuk fosfat. Kehadiran fosfat juga boleh disebabkan oleh detergen sintetik dan bahan pencuci. Rajah 6(a) menunjukkan tahap pengurangan fosforus sepanjang proses rawatan air. Fosforus berkurang sebanyak 78.69 % selepas klorin ditambahkan. Rajah 6(b) menunjukkan trend pencemaran fosforus di sembilan stesen di lembangan Sungai Semenyih. Punca pencemaran fosforus berlaku pada SS1, SS2, dan SS5. Selain detergen, kehadiran fosforus juga boleh dikaitkan dengan penggunaan baja dalam aktiviti pertanian.

Fosforus m g/l 0.5 0.4 0.3 0.2 0.1 0 L1 L2 L3 L4 mg/l 3 2.5 2 1.5 1 0.5 0 1 2 3

Fosforus

4

5 Stesen

6

7

8

9

6(a)

6(b)

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

Rajah 6

(a) Tahap pengurangan fosforus di loji rawatan air (b) Punca pencemaran fosforus di 9 stesen pensampelan di Sungai Semenyih

Nitrit Nitrit merupakan sebahagian daripada bentuk nitrogen yang hadir dalam kitaran nitrogen. Nitrit terbentuk sebagai proses perantaraan dalam proses nitrifikasi [19]. White [1] mendapati nitrit boleh menghasilkan permintaan klorin yang tinggi apabila bertindak balas dengan klorin. Rajah 7(a) menunjukkan pengurangan nitrit selepas klorin ditambahkan pada L4. Secara amnya, nitrit yang hadir tidak memberikan permintaan klorin yang tinggi di loji rawatan air Sungai Semenyih. Pengurangan nitrit selepas proses pengklorinan ialah sebanyak 42.86 %. Kajian ini mendapati pencemaran nitrit adalah rendah di lembangan Sungai Semenyih kecuali SS5. Rajah 7(b) menunjukkan trend pencemaran nitrit di sembilan stesen di lembangan Sungai Semenyih. Kehadiran nitrit di SS5 boleh dikaitkan dengan sisa buangan industri dan aktiviti penternakan yang berhampiran.
Nitrit m g/l 0.014 0.012 0.01 0.008 0.006 0.004 0.002 0 L1 L2 L3 L4 0.2 0.15 mg/l 0.1 0.05 0 1 2 3 4 5 Stesen 6 7 8 9

Nitrit

7(a) Rajah 7 Mangan

7(b)

(a) Tahap pengurangan nitrit di loji rawatan air (b) Punca pencemaran nitrit di 9 stesen pensampelan di Sungai Semenyih

Mangan merupakan logam berat yang boleh disingkirkan melalui proses pengoksidaan dengan klorin. Ini menunjukkan tindak balas boleh berlaku antara mangan dan klorin dan mangan boleh menyebabkan permintaan klorin dalam air. Pengurangan mangan dalam air bersih ialah 49.37 % selepas klorin didoskan seperti yang ditunjukkan dalam rajah 8(a).

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

Mangan m g/l 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0 L1 L2 L3 L4
0.15 0.1 0.05 0 1 2

Mangan

mg/l

3

4

5 Stesen

6

7

8

9

8(a) Rajah 8

8(b)

(a) Tahap pengurangan mangan di loji rawatan air (b) Punca pencemaran mangan di 9 stesen pensampelan di Sungai Semenyih Pencemaran mangan di lembangan Sungai Semenyih adalah paling tinggi di SS7 seperti yang ditunjukkan dalam rajah 8(b). Pencemaran di SS7 adalah disebabkan oleh tapak pelupusan sisa pepejal yang berhampiran dengan Sungai Beranang. Selain itu, mangan juga hadir secara semulajadi dalam kepekatan yang rendah. Ferum Ferum biasanya wujud sebagai ferus bikarbonat yang larut separa dalam air. Klorin akan bertindak balas dengan ferum untuk menghasilkan ion ferik. Rajah 9(a) menunjukkan pengurangan ferum sepanjang proses rawatan air. Kebanyakan ferum boleh disingkirkan setelah air dimendapkan. Walaubagaimanapun, kepekatan ferum telah berkurang sebanyak 46 % selepas proses pengklorinan. Tindak balas ferum dengan klorin boleh berlaku dalam julat pH yang luas (pH 4-10). Kehadiran ferum dalam loji rawatan air adalah berpunca daripada SS5 dan SS7 seperti yang ditunjukkan dalam rajah 9(b). Sumber pencemaran ferum di kedua-dua SS5 dan SS7 berpunca daripada larut lesap dari tapak pelupusan sampah, kakisan daripada jambatan yang berhampiran dengan sungai, dan sisa buangan industri.
Ferum m g/l 1.4 1.2 1 0.8 0.6 0.4 0.2 0 L1 L2 L3 L4 mg/l 1 0.8 0.6 0.4 0.2 0 1 2 3 4 5 Stesen 6 7 8 9

Ferum

9(a) Rajah 9

9(b)

(a) Tahap pengurangan ferum di loji rawatan air (b) Punca pencemaran ferum di 9 stesen pensampelan di Sungai Semenyih

Karbon Organik Jumlah

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

Karbon yang ditentukan dalam kajian ini ialah karbon organik terlarut (DOC). DOC meliputi semua bentuk karbon seperti karbon organik, karbonat tak organik, karbon dioksida, dan spesis yang lain seperti HCN yang larut dalam air. Rajah 10(a) menunjukkan kepekatan DOC dalam loji rawatan air, dimana DOC mengalami pengurangan sebanyak 33.91 % selepas proses pengklorinan. DOC merupakan pelopor yang boleh bertindak balas dengan klorin untuk menghasilkan THM, asid haloasetik, dan haloasetonitril yang boleh membawa kesan kepada kesihatan manusia. Oleh itu, punca pencemaran DOC perlu dikenalpasti. Rajah 10(b) menunjukkan SS5 dan SS7 merupakan dua stesen yang paling tercemar dengan karbon organik. Secara amnya, kepekatan DOC adalah paling tinggi berbanding dengan parameter-parameter yang lain yang boleh menyebabkan permintaan klorin. Jadi pengawalan pencemaran DOC dari buangan domestik, industri, dan pertanian adalah penting dalam menangani masalah permintaan klorin.
DOC m g/l 6 5 mg/l 4 3 2 1 0 L1 L2 L3 L4 10 8 6 4 2 0 1 2 3 4 5 Stesen 6 7 8 9

DOC

10(a) Rajah 10

10(b)

(a) Tahap pengurangan DOC di loji rawatan air (b) Punca pencemaran DOC di 9 stesen pensampelan di Sungai Semenyih

Kesimpulan Kajian ini bertujuan untuk menentukan faktor-faktor dan punca-punca yang menyebabkan permintaan klorin di loji rawatan air Sungai Semenyih, yang merupakan salah satu sumber bekalan air utama di daerah Hulu Langat. Loji rawatan air Sungai Semenyih mendapatkan sumber air mentahnya dari Sungai Semenyih. Data yang terkumpul selama lapan bulan ini mendapati komponen-komponen yang menyebabkan permintaan klorin ialah ammonia, sianida, sulfida, fenol, fosforus, nitrit, mangan, ferum, dan karbon organik terlarut. Jadual 2 menunjukkan kepekatan komponen-komponen ini dalam air tapisan (sebelum pengklorinan). Keputusan kajian menunjukkan karbon dan ammonia boleh menyebabkan permintaan klorin yang tinggi berbanding dengan komponen-komponen yang lain. Jadual 2 Kepekatan komponen-komponen yang menyebabkan permintaan klorin dalam air tapisan Komponen Ammonia Sianida Sulfida Fenol Forforus Nitrit Mangan Min (mg/l) 0.1255 0.0019 0.0056 0.0095 0.1525 0.0035 0.0797 Julat (mg/l) 0-0.36 0.001-0.003 0.001-0.008 0.004-0.006 0.11-0.25 0.001-0.005 0.018-0.116

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

Ferum DOC

0.0141 4.0070

0-0.020 2.454-5.125

Secara puratanya, kandungan karbon dan ammonia berkurang sebanyak 1.36 mg/l dan 0.111 mg/l masing-masing selepas klorin ditambahkan. Selain itu, kepekatan parameter-parameter yang lain seperti sianida, sulfida, fenol, nitrit, fosforus, mangan, dan ferum juga berkurang selepas proses pengklorinan. Parameter-parameter ini tidak mempengaruhi permintaan klorin dengan banyak berbanding dengan DOC dan ammonia kerana hadir dalam kepekatan yang rendah. Kajian ini turut mendapati terdapat dua stesen di lembangan Sungai Semenyih yang menjadi punca yang menyebabkan permintaan klorin, iaitu SS5 dan SS7. Punca pencemaran yang dikenalpasti di SS5 dan SS7 ialah aktiviti perindustrian, kumbahan domestik, pertanian, penternakan, dan larut lesap dari tapak pelupsan sisa pepejal. Langkah-langkah yang bersesuaian perlu diambil untuk melaksanakan pendekatan pengurusan yang bersistematik supaya pencemaran sungai dapat diminimumkan, dan seterusnya permintaan klorin dapat dikurangkan untuk menjimatkan kos rawatan air. Penghargaan Setinggi-tinggi penghargaan ditujukan kepada Konsortium Abass Sdn. Bhd. yang membiayai projek ini atas sumbangan Geran Penyelidikan STGL-011-2005 dan kemudahan yang disediakan oleh Pusat Pengajian Sains Kimia dan Teknologi Makanan, Universiti Kebangsaan Malaysia serta kepada semua pihak yang terlibat dalam menjayakan projek ini. Rujukan 1. White, G.C. 1992. Handbook of Chlorination and Alternative Disinfections. Ed. ke-3. New York: Van Nostrand Reinhold Publisher, Inc. 2. Sukiman, S. & Pauzi, A. 1993. Chemical Quality of Malaysian Drinking Water Sources. Drinking Water Quality: Microbiological and Public Health Aspects, UKM: 63-69. 3. Johari, M. A. 1994. Perkembangan Teknologi Dalam Sistem Bekalan Air. Paper Presented at SKAM-7, Kuala Lumpur. 27-29 Sept. 4. Wan, K. J., Ki, D. K., Jong, I. D. & Yong. C. 2005. Multi-route Trihalomethane Exposure in Households Using Municipal Tap Water Treated with Chlorine or Ozone-Chlorine. Science of the Total Environment. 339: 143-152. 5. Sukiman, S. & Pauzi, A. 1993. Chemical Quality of Malaysian Drinking Water Sources. Drinking Water Quality: Microbiological and Public Health Aspects. UKM: 63-69. 6. Rook, J. J. 1974. Formation of Haloforms During Chlorination of Natural Water. Water Treat. Exam. 23: 234-243. 7. Bellar, T. A., Lichtenberg J. J. & Kroner, R. C. 1974. The Occurrence of Organohalogens on Chlorinated Drinking Waters. J. AWWA. 66: 703-706. 8. Tachkikawa, M., Aburada, T., Tezuka, M. & Sawamura, R. 2005. Occurrence and Production of Chloramines in The Chlorination of Creatinine in Aqueous Solution. Water Research 39: 371-379. 9. Macolo, G., Lopez, A., James, H. & Fielding, M. 2001. By-Products Formation During Degradation of Isoproturan In Aqueous Solution. II: Chlorination. Water Research 35(7): 1705-1713. 10. Alley, E. R. 2000. Water Quality Control Handbook. New York: McGraw-Hill Inc. 11. Chanratchakool, P. 1995. White Patch Disease of Black Tiger Shrimp (Penaeus Monodon). AAHRI Newsletter. 4(1): 3. 12. Aacher, A., Fischer, E., Turnheim, R., Manor, Y. 1997. Ecologically Friendly Wastewater Disinfection Techniques. Water Research. 31(6): 1398-1404. 13. Hopkins, J.S., Hamilton, R.D. II, Sandifier, P.A., Browdy, C.L., Stokes, A.P. 1993. Effects of Water Exchange Rate On Production, Water Quality, Effluent Characteristics and Nitrogen Budgets of Intensive Shrimp Ponds. J. World Aquacut. Soc. 24(3): 304-320.

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14. 15. 16. 17. 18. 19. 20.

Dierberg, F. F., Kiattisimkul, W. 1996. Issue, Impacts, and Implications of Shrimp Aquaculture in Thailand. Environ. Man. 20(5): 649-666. Rook, J. J. 1976. Haloforms in Drinking Water. J. AWWA. 68: 168-172. Cotruvo, J. A. & Wu, C. 1981. Controlling Organics. Why Now?. J. AWWA. 70: 590-594. Morrow, C. M. & Minear, R. A. 1987. Use of Regression Models to Link Raw Water Characteristics to Trihalomethanes Concentrations in Drinking Water. J. Wat. Res. 21: 41-48. El-Dib, M. & Ali, R. 1995. THMs Formation During Chlorination of Raw Nile River Water. Egypt. J. Wat. Res. 29: 375-378. APHA. 1998. Standard Method For the Examination of Water and Wastewater. Ed. ke-20. Washington: American Public Health Association. Faust, S. D. & Aly, O. M. 1998. Chemistry of Water Treatment. Ed ke-2. New York: Lewis Publishers.

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REMOVAL OF DYE BY IMMOBILISED PHOTOCATALYST LOADED ACTIVATED CARBON Zulkarnain Zainal*, Chang Sook Keng and Abdul Halim Abdullah Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, Serdang 43400 UPM, Selangor Darul Ehsan, Malaysia * Corresponding author E-mail: zulkar@fsas.upm.edu.my

Abstract The abilities of activated carbon to adsorb and titanium dioxide to photodegrade organic impurities from water bodies are well accepted. Combination of the two is expected to enhance the removal efficiency due to the synergistic effect. This has enabled activated carbon to absorb more and at the same time the lifespan of activated carbon will be prolonged as the workload of removing organic pollutants is shared between activated carbon and titanium dioxide. Immobilisation is selected to avoid unnecessary filtering of adsorbent and photocatalyst. In this study, mixture of activated carbon and titanium dioxide was immobilised on glass slides. Photodegradation and adsorption studies of Methylene Blue solution were conducted in the absence and presence of UV light. The removal efficiency of immobilised TiO2/AC was found to be two times better than the removal by immobilised AC or immobilised TiO2 alone. In 4 hours and with the concentration of 10 ppm, 1.5 g of immobilised TiO2 loaded activated carbon gave an almost total removal (99.50%). Keywords: Photodegradation; Adsorption; Methylene Blue, Titanium Dioxide; Activated Carbon __________________________________________________________________________________ ___ 1. Introduction The idea of conserving the environment does not come in one or two days time. It has become a necessity to enlighten each an everyone of us of the problem faced by the Mother Nature. Pollution in water bodies is escalating and the fear of living in dirty environment has encouraged researchers to come out with or design techniques to overcome the problem. Developing techniques ranging from distillation, ion exchange, filtration, ultrafiltration, reverse osmosis, ultraviolet (UV) radiation to carbon adsorption has become a necessity. Combined adsorption-photodegradation process is another alternative in the development of wastewater treatment method and is expected to give better results in removing organic pollutants [13]. Adsorption can be done by using a variety of materials such as sawdust, coconut shell, rice husk, banana pith, chitosan, chitin, orange peel, activated carbon and so on. On the other hand, photodegradation is applied by using semiconducting compound such as TiO2, SnO2, SiO2, ZrO2 and many more. It is proven that titanium dioxide photocatalyst, anchored or embedded onto co-adsorbent with large surface area such as activated carbon produced promising results due to the high capacity of activated carbon to adsorb most of the pollutants and the synergistic effect between these two materials [4-8]. In order to ensure the water treatment system works effectively, immobilisation is introduced. A variety of supporting substrates such as glass, aluminium plates, beads and glass rings may be used in the system. The purposes of immobilising the photocatalyst-adsorbent are to avoid the filtration step, reduce losses of the materials and increase the efficiency of the whole system [9-12].

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2. Experimental 2.1 Preparation of immobilised TiO2/AC TiO2/AC prepared from TiO2 and activated carbon powders were studied using Malvern Mastersizer. Size and characteristics of the particles may affect the stability, chemical reactivity, opacity, flowability and material strength of many materials. With the ratio of 20:80, 0.30 g titanium dioxide powder (BET surface area = 50 m2/g, pH 4 suspension, Aeroxide Degussa P25) was mixed with 1.20 g activated carbon (iodine number = 1150-1200, carbon total content = 70%-80%, bulk density = 0.40-0.44, ash = maximum 5%) before adding 15.0 mL of distilled water. The mixture was stirred for 1 hour to ensure homogeneity. The binder was prepared by adding 5.0 g polyvinylalcohol into 80.0 mL formaldehyde at 70 °C under continuous stirring. A transparent, sticky polymer glue was formed and was kept in a sealed bottle to prevent it from rapid hardening. Later, pieces of glass slides with dimension of 2.54 cm x 7.62 cm were applied with a thin layer binder. Then, TiO2/AC suspension was brushed onto the layer of binder and let to dry. The samples were kept in boxes to prevent preactivation. 2.2 Preparation of Methylene Blue Methylene Blue stock solution with the concentration of 200 ppm was prepared by dissolving Methylene Blue powder in distilled water. Dye solutions used in the removal process were prepared from the stock solution. 2.3 Photocatalytic Degradation with Ultraviolet Lamp The photodegradation experiments were run by illuminating the dye solution (200 ml) containing immobilised TiO2/AC using an ultraviolet lamp. Air was continuously pumped into the solution to ensure a constant supply of oxygen. The experimental set-up was covered to avoid unnecessary exposure to light and the temperature was maintained at 28 ºC using a water bath. UVVisible analysis was performed on the sample solutions using Perkin Elmer UV/VIS Lambda 20 Spectrophotometer.

3. Results and Discussion 3.1 Particle Size of TiO2 and Activated Carbon Figure 1 illustrates the size distribution of titanium dioxide and activated carbon. From the figure, it was found that activated carbon has a wide range of sizes whereas titanium dioxide is more uniform whereby it has about the same cumulative percentages in the range of 10 µm to 100 µm. The particle size of titanium dioxide is smaller than activated carbon as it shows higher cumulative percentage at smaller particle size. On the other hand, the cumulative percentage of activated carbon soars higher than titanium dioxide after 350 µm. This shows the compatibility between these two materials when they are mixed together. TiO2 with smaller particle size will go in between or into the pores of activated carbon that have bigger particle size. This enables TiO2 and activated carbon to perform effectively in the adsorption-photodegradation process.

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100

80

Cumulative (%)

60

Titanium dioxide Degussa P25

40 Activated carbon

20

0 -1 0 1 2 3

Log Size

Figure 1: Graph of particle size of TiO2 and activated carbon powders. 3.2 Comparison between Titanium Dioxide, Activated Carbon and Titanium Dioxide/Activated Carbon The removal of Methylene Blue by using the three different samples is depicted in Figure 2. From the graph, it shows that a mixture of TiO2/AC gives a better efficiency by achieving 99.50% of the dye removed after 4 hours. With the usage of activated carbon immobilised samples, 74.33% of the dye was removed and the percentage of removal goes down to 46.60% by using TiO2 immobilised samples. These results proved that the combination of titanium dioxide and activated carbon is the best option in removing Methylene Blue. The amount removed by TiO2/AC is 6.70 x 10-6 mol followed by AC (4.65 x 10-6 mol) and TiO2 (2.76 x 10-6 mol). Photodecomposition of adsorbed Methylene Blue enhances the adsorption rate of this dye by keeping the adsorptive capacity of activated carbon unsaturated [8]. Dye molecules adsorption by activated carbon followed by a transfer of the molecules to TiO2 where photocatalysis occurred has created a mutual combination for the enhancement of the dye removal system. This has enabled the adsorption capacity of activated carbon to be maintained and to perform effectively for a longer period of time.

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1 0.8 0.6 C/Co 0.4 0.2 0 0 50 100 150 Time (min) AC TiO2 TiO2/AC 200 250 300
Figure 2: Graph C/Co versus time for the removal of Methylene Blue by using different samples: AC, TiO2 and TiO2/AC. [Conditions: 5 pieces of glass slides, 200 ml of 10 ppm Methylene Blue solution and temperature (28 ºC)]. 3.3 Effect of Amount of TiO2/AC Figure 3 shows the effect of amount of TiO2/AC on the removal of Methylene Blue. The figure illustrated that the uptake of dye molecules increased with time. Increasing amount of TiO2/AC resulted the equilibrium time to decrease as the surface area provided is greater for more adsorption to occur. The removal effect of TiO2/AC was studied by using three kinetic models: first-order, pseudosecond-order and intraparticle diffusion rate model. The removal effect of TiO2/AC by using the firstorder-model is depicted in Figure 4 whereas pseudo-second-order model can be seen in Figure 5. A plot of qt against the square root of t for the intraparticle diffusion rate model for Methylene Blue onto TiO2/AC can be found in Figure 6. The results in Table 1 shows the correlation coefficient, r12 and first-order apparent rate constant, k1 compared with correlation coefficient, r22, sorption capacity, q2, sorption rate constant of pseudo-second-order rate constant, k2 and initial sorption rate, h and correlation coefficient, ri2 and intraparticle diffusion rate constant, ki at various amount of TiO2/AC, m. From this table, the results can be better represented by pseudo-second-order based on the correlation coefficients (> 0.990). As the amount of TiO2/AC increases, sorption capacity, q2 decreases. This trend is in agreement with Ho and McKay [13] where by these researchers studied the sorption of Basic Red 22 and Acid Red 114 by biosorbent waste product pith. Correlation coefficients for the intraparticle diffusion rate parameter are the lowest and this suggests that the diffusion controlled mechanism is not predominant. Figure 7 illustrates the variation of apparent rate constant, k1 and amount of removal against the amount of TiO2/AC. 1.5 g gives the highest apparent rate constant and greatest amount of removal with the value of 4.91 x 10-2 min-1 and 6.70 x 10-6 mol respectively. The results revealed that Methylene Blue removal efficiency increases up to the optimum amount (1.5 g), beyond which the amount of removal decreases. It is due to the difficulty of light penetration into the immobilised samples for photocatalysis to occur.

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35 Sorption Capacity, qt (mg/g) 30 25 20 15 10 5 0 0 20 40 0.5 g 60 80 Time (min) 1.0 g 1.5 g 100 2.0 g 120 140

Figure 3: Graph of sorption capacity versus time for the effect of amount of TiO2/AC on the sorption of Methylene Blue onto TiO2/AC. [Conditions: 5 pieces of glass slides, 200 ml of 10 ppm Methylene Blue solution, light source (1 ultraviolet lamp) and temperature (28 ºC)].

0 0 -1 ln C/Co -2 -3 -4 -5 Time (min) 0.5 g 1.0 g 1.5 g 2.0 g 10 20 30 40 50 60 70 80 90

Figure 4: Graph ln C/Co versus amount of TiO2/AC used in the removal of Methylene Blue. [Conditions: 5 pieces of glass slides, 200 ml of 10 ppm Methylene Blue solution, light source (1 ultraviolet lamp) and temperature (28 ºC)].

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140 120 t/qt (min g/ mg) 100 80 60 40 20 0 0 50 100 150 Time (min) 0.5 g 1.0 g 1.5 g 2.0 g 200 250 300

Figure 5: Graph of pseudo-second-order sorption kinetics of Methylene Blue onto TiO2/AC at various amount of TiO2/AC. [Conditions: 5 pieces of glass slides, 200 ml of 10 ppm Methylene Blue solution, light source (1 ultraviolet lamp) and temperature (28 ºC)].

40 Sorption Capacity, qt (mg/g) 35 30 25 20 15 10 5 0 0 2 4 Time 0.5 g 1.0 g 6
0.5

8
0.5

10 2.0 g

12

(min ) 1.5 g

Figure 6: Graph of intraparticle diffussion of Methylene Blue onto TiO2/AC at various amount of TiO2/AC. [Conditions: 5 pieces of glass slides, 200 ml of 10 ppm Methylene Blue solution, light source (1 ultraviolet lamp) and temperature (28 ºC)].

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5 Appa rent rate constant, k1 4.8 (10 min )
-1

7 6.8 6.6 6.4 6.2 6 0 0.5 1 1.5 2 2.5 Amount of TiO2/AC (g) Apparent rate constant, k1 Amount of removal Amount of Removal -6 (10 mol)
1.9369 0.4613 0.2888 0.1083

4.6 4.4 4.2 4

Figure 7: Graph of apparent rate constant and amount of removal versus amount of TiO2/AC in the removal of Methylene Blue. Table 1: Parameters for the effect of amount of TiO2/AC in the removal of Methylene Blue. m r22 q2 k1 ki k2 h r12 ri2 -2 -2 (g) (mg/g) (x10 (mg/g (x10 ((mg/g per g/mg per per min0.5) mmin-1) min) min) 0.5 1.0 1.5 2.0 0.9979 0.9917 0.9987 0.9990 34.2466 7.7220 5.2219 2.0247 0.43 1.49 4.00 9.06 5.01 0.89 1.09 0.37 0.9767 0.9856 0.9797 0.9873 4.02 4.14 4.91 4.87 0.8536 0.8438 0.8958 0.8079

4. Conclusion Combination of activated carbon and titanium dioxide has proven to be efficient in removing dye molecules compared with neat TiO2 and neat AC. Moreover, shorter time frame is required in achieving equilibrium when TiO2/AC is applied to the dye removal system. TiO2/AC able to remove 95.50% of Methylene Blue whereas activated carbon gives 74.33% efficiency and the dye removal by TiO2 only shows 46.60%. The dye removal system is initialized by the adsorption of Methylene Blue on activated carbon followed by a mass transfer to titanium dioxide to photodegrade the dye molecules. The removal rate of Methylene Blue was affected by the amount used and the sorption of this dye followed the pseudo-second-order chemical reaction kinetics model in which best correlation coefficients were shown. Acknowledgement The authors wish to acknowledge the Department of Chemistry, Universiti Putra Malaysia for the provision of laboratory facilities.

-2

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References 1. Qourzal, S., Assabbane, A., Ait-Ichou, Y. (2004) “Synthesis of TiO2 via Hydrolysis of Titanium Tetraisopropoxide and its Photocatalytic Activity on a Suspended Mixture with Activated Carbon in the Degradation of 2-naphthol” Journal of Photochemistry and Photobiology A: Chemistry. 163. 317321. 2. Takeda, N., Iwata, N., Torimoto, T., Yoneyama, H. (1998) “Influence of Carbon Black as an Adsorbent Used in TiO2 Photocatalyst Films on Photodegradation Behaviors of Propyzamide” Journal of Catalysis. 177. 240-246. 3. Yuan, R., Guan, R., Zheng, J. (2005) “Photocatalytic Degradation of Methylene Blue by a Combination of TiO2 and Activated Carbon Fibers” Journal of Colloid and Interface Science. 282. 87-91. 4. Carpio, E., Zúñiga, P., Ponce, S., Solis, J., Rodriguez, J., Estrada, W. (2005) “Photocatalytic Degradation of Phenol Using TiO2 Nanocrystals Supported on Activated Carbon” Journal of Molecular Catalysis A: Chemical. 228. 293-298. 5. Tryba, B., Morawski, A.W., Inagaki, M. (2003) “Application of TiO2-mounted Activated Carbon to the Removal of Phenol from Water” Applied Catalysis B: Environmental. 41. 427-433. 6. Herrmann, J.-M., Matos, J., Disdier, J., Guillard, C., Laine, J., Malato, S., Blanco, J. (1999) “Solar Photocatalytic Degradation of 4-chlorophenol Using the Synergistic Effect Between Titania and Activated Carbon in Aqueous Suspension” Catalysis Today. 54. 255-265. 7. Matos, J., Laine, J., Herrmann, J.-M. (1998) “Synergy Effect in the Photocatalytic Degradation of Phenol on a Suspended Mixture of Titania and Activated Carbon” Applied Catalysis B: Environmental. 18. 281-291. 8. Fu, P., Luan, Y., Dai, X. (2004) “Preparation of Activated Carbon Fibers Supported TiO2 Photocatalyst and Evaluation of its Photocatalytic Reactivity” Journal of Molecular Catalysis A: Chemical. 221. 81-88. 9. Gelover, S., Mondragón, P., Jiménez, A.(2004) “Titanium Dioxide Sol-gel Deposited Over Glass and its Application as a Photocatlayst for Water Decontamination” Journal of Photochemistry and Photobiology A: Chemistry. 165. 241-246. 10. Ventaka Subba Rao, K., Rachel, A., Subrahmanyam, M., Boule, P. (2003) “Immobilization of TiO2 on pumice stone for the photocatalytic degradation of dyes and dye industry pollutants” Applied Catalysis B: Environmental. 46. 77-85. 11. Venkata Subba Rao, K., Subrahmanyam, M., Boule, P. (2004) “Immobilized TiO2 Photocatalyst During Long-term Use: Decrease of its Activity” Applied Catalysis B: Environmental. 49. 239249. 12. Serpone, N., Borgarello, E., Harris, R., Cahill, P., Borgarello, Pelizzetti, E. (1986) “Photocatalysis Over TiO2 Supported on a Glass Substrate” Solar Energy Materials. 14. 121-127. 13. Ho, Y.S., McKay, G. (1999) “A Kinetic Study of Dye Sorption by Biosorbent Waste Product Pith” Resources, Conservation and Recycling. 25. 171-193.

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THE PRELIMINARY STUDY ON PHYTOCHEMICAL CONSTITUENT FROM QUASSIA INDICA Mohd Hussin Zain, 1Zalilawati Mat Rashid, 1Juriffah Ariffin, 1 Habsah Mohamad, and 2Mohd Razali Salam. 1 Department of Chemical Sciences, 2Department of Biological Sciences, Faculty of Science and Technology, University College of Science and Technology Malaysia (KUSTEM), 21030 Kuala Terengganu, Terengganu, Malaysia Corresponding fax / e-mail: 09-6694660 / mhuzain@kustem.edu.my ABSTRACT: The purpose of this study is to isolate and characterise the chemical constituent from Quassia indica. This plant is from Simaroubaceae family. Eurycoma longifolia jack is one of genera in this family which is well known among Malaysian people as for refining a male aphrodisiac and energy. In Malaysia, this plant was traditionally identified as ‘kayu pahit’. Traditional folks throughout the world have used plants from Simaroubaceae species for medicinal purpose such as a tonic, purgative, for treatment of colic, diarrhea, dysentery and skin diseases. The phytochemical screening showed the presence of alkaloid and cardiac glycoside compounds. Thin layer chromatography and column chromatography were used for isolation and purification of the compound. Then, gas chromatography was used to further confirm the purity of two isolated compounds. Various modern spectroscopy and spectrometry techniques such as Ultra Violet (UV) spectroscopy, Infra-red (IR) spectroscopy and Gas Chromatography-Mass (GC-MS) spectrometry were used to identify the compound structures. According to UV, IR spectrum and GC-MS fragmentation pattern, the compounds were preliminary group as benzoic acid and phenolic derivatives. Keywords: Quassia indica, Simaroubaceae, alkaloid, cardiac glycoside.
1

INTRODUCTION Malaysian Forest with wet and dry season has various species of tree. It is give many commercial advantages such as potential medicines, supplement vitamins and cosmetic uses. Since decades ago, traditional folks have used plants from Simaroubaceae family for medical purposes and also used to against a wide variety of illnesses [1]. In Malaysia, a tropical dry forest is dominant in lowland areas that have prolonged dry season or rainfall at any time. The Simaroubaceae species are mostly found in this lowland forest [2]. It has been reported that the Simaroubaceae family comprising of 30 to 31 genera and 200 species throughout the world which are 8 genera and 10 species are found in Malaysia. The genera are Ailanthus, Allantospermum, Brucea[3,4], Eurycoma[5,6,7], Harrisonia[8], Irvingia , pricrasma and Quassia [9,10]. The Simaroubaceae species from genera of Ailanthus, Brucea, Eurycoma, Harisonia are known rich of quassinoids[11], alkaloids, terpenes, chromes and limonoids. Only a few studies on Quassia indica have been reported. Currently, in Malaysia the study for potential usages of chemical constituent from Quassia indica have not found although this species abundantly growth in specific swamps area. Therefore this early report was prepared to give some information to other people and this plant may well prove to be the source of new medicinal usages.

METHODOLOGY Extraction method was used to extract a chemical constituent in stem and leaves of Quassia indica by soaking the sample in different polarity of solvent. The expected crude extract were then subjected to isolate and purify by using chromatography techniques. The isolated compounds were characterised

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by using spectroscopy methods such as Uv-Visible spectroscopy, Infrared spectroscopy and Gas chromatography-Mass spectrometry. A phytochemical screening Plant of Quassia indica was collected from Chalok river basin. A small amount of stem and leaf was chemically screening for reference. Leaf and stem were tested for alkaloid and cardiac glycoside compounds. This species give a positive result of alkaloid with white precipitate using Reagen Mayer. Meanwhile, Legal Test was showed a positive result of existence aglycone moiety with dark-red color in test solution. The existence of sugar moiety of cardiac glycoside was test by using Keller-Killiani Test. The positive result was showed with occurrence layer of red-brown color in test solution. Isolation and purification of chemical constituent 1kg of leave and stem dried powder was extracted three times with petroleum ether. After the solution was decanted off, the residue was kept extracted with chloroform. Then the extraction procedure were repeated by using chloroform and followed with methanol. The crude chloroform extract was evaporated to remove a solvent and selected for further procedure. This concentrated extract was subjected to isolate and purified. The best and suitable solvent system that monitored by thin layer chromatography for chemical compound isolation in chloroform extract was hexane:chloroform with 30:70 in ratio. The concentrated extract was mixed with small amount of silica gel and then the solvent was removed to yield a dried powder sample. The chemical constituents in dried powder sample were isolated and purified by using column chromatography technique. The sample was eluted with above solvent system by increasing polarity until the ratio of 30:70. For every 50 ml elute was collected and then analyzed by Gas chromatography to elucidate the purity of isolated compound. The elute (fraction) with similar chromatogram was combined together. The column chromatography technique was repeated for purified the combination of elute by using the solvent system of hexane: ethyl acetate. After the isolated elute was analyzed by Gas chromatography, only two compounds were successfully isolated and purified as predicted as benzoic acid derivative and phenolic derivative respectively.

RESULT AND DISCUSSION Characterization of benzoic acid derivative According to Ultra-violet spectrum of sample, its showed a maximum absorption (λmax MeOH) at 250nm which was assigned band to a chromophore conjugated with aromatic ring(Silverstei et el). Meanwhile, in IR spectrum, the sharp absorption in region 2856cm-1 and 2926cm-1 was indicated the C-H stretching of methyl group. The next most intense absorption band at 1731cm-1 was assigned to C=O stretching vibration of ester group. The presence of C-O stretching band of ester group was appeared at 1277cm-1 and 1161cm-1 while the presence of C=C stretching band of aromatic ring was assigned at 1664cm-1. The positive ion mass at m/z 149 was corresponded to the molecular ion mass and base peak of [C9H9O2]+ of benzoic asic derivative. The other positive ion mass contains ions derived by loss of the redical groups from this molecule ion which lead to [C2HO2]+ and [C3H3O2]+ fragmentation ions at m/z71 and m/z 57 respectively. The predicted structure of benzoic derivative is shown below.

Characterization of phenolic derivative

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According to Ultra-violet spectrum, the maximum absorption (λmax MeOH) at 288nm was predicted band to existence of cyclic and aromatic compound. In IR spectrum, the sharp broad absorption band at 3423cm-1 was due to the presence of OH stretching of phenolic compound. Meanwhile the strong band at 1775cm-1 was clearly indicated the presence of C=O of lactone group as a backbone. The presence of C-O stretching vibration of lactone group was appeared at 1249cm-1 and 1172cm-1 while the presence of C=C stretching band of aromatic ring and alkene group were was presence at 1772cm-1 and 1665cm-1 respectively. The mass spectrum of phenolic derivative compound showed the positive ion mass at m/z 203 was corresponded to the molecular ion mass. The other positive ion mass contains ions derived by loss of the radical groups from this molecule ion which lead to [C10H7O3]+ ,[C8H5O3]+ , [C8H3O]+ and [C2HO2]+ fragmentation ions at m/z 175, 149, 115 and 91 respectively. Further fragment ion at m/z149 gives an ionic species of [C6H5]+ at m/z 77. The predicted structure of benzoic derivative is shown below.

CONCLUSIONS The preliminary study on Quasssia indica from Simaroubaceae species indicated that plant has alkaloid and cardiac glycoside compounds. Although these compounds were not successfully isolated, two other compounds were purified. Based on the characteristic data of UV, IR and GS-MS spectra, it showed that those compounds could be predicted as benzoic acid and phenolic derivatives compound.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. Whitemore, T. C. (1983). Tree flora of Malaya: A manual for foresters. Forest Department, Ministry of Primary Industries Malaysia. Longman Malaysia. Vol.1-4, 2nd ed. Kuala Lumpur, pp. 345-352. Campbell, N. A., Rece, J. B., Mitchell, L. G. (1999). Biology. 5th ed. Addition Wesley Longman, Inc. U.S.A, pp. 1044-1051. Ik, H. K., Ryoto, s., Yukia, H., Kooichi, T. (2003). Three Novel Quassinoids, Javanicolides A and B From Seed of Brucea javanica. Tetrahedron 59: 9985-9986. Lumanadio, L., Nanjoo, S., Harry, H.S.F., John, M. P., Douglas, A.K. (1996). A lignan and Four Terpenoids from Brucea javanica That Induce Differentiation with Cultured HL60 Promyelocytic Leukemia Cells. Phytochemistry. 43 52:127-133. Ang, H. H., Hitotsuyanagi, Y., Fukaya, H., Takeya, K. (2000). Quassinoids from Eurycoma longifolia. Phytochemistry 59(8):833-837. Ang, H. H., Hitotsuyanagi, Y., Takeya, K. (2000). Eurycolactonas A-C, Novel Quassinoids from Eurycoma longifolia.Tetrahedron Letters 41:6849-6853. Ang, H. H., Kit, L. C., Joon, W. M.(2002). Effects of 7 Day Daily Replacement of Culture Medium Containing Eurycoma longifolia Jack constituents on The Malaysia Palsmodium falciparum isolates. Journal of Ethopharmacology. 49:171-175. Mohamed, S. R., Frank, R. F., Dulcie, A. M., Joseph, K. R.(1999). 11β,12 βdiacetoxyharrisonin, A Tetranortriterpenoid from Harrisonia abyssinica. Phytochemistry 52:127-133. Koike, K., Ohmoto, T.(1994). Quassinoids from Quassia indica. Phytochemistry 35(2):459463.

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10. 11.

Saxena, S., Pant, N., Jain, D.C., Bhakuni, R.S. (2003). Antimalaria agent from Plant Sources. Med. Rev. Art. 85(9). Yishan, O., Katsuyoshi, M., Kazuo, K., Taichi, O. (1995). Alkaloids and Quassinoids of Brucea Mollis Var. Tonkinensis. Phytochemistry 39(94):911-913.

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ARIMA AND INTEGRATED ARFIMA MODELS FOR FORECASTING AIR POLLUTION INDEX IN SHAH ALAM, SELANGOR Lim Ying Siew, Lim Ying Chin and Pauline Mah Jin Wee International Education Centre (INTEC), Universiti Teknologi MARA Section 17 Campus 40200 Shah Alam, Selangor. e-mail: yslim590@salam.uitm.edu.my Abstract. Air pollution is one of the major issues that has been affecting human health, agricultural crops, forest species and ecosystems. Since 1980, Malaysia has had a series of haze episodes and the worst ever was reported in 1997. As a result, the government has established the Malaysia Air Quality Guidelines, the Air Pollution Index (API) and Haze Action Plan, to improve the air quality. The API was introduced as an index system for classifying and reporting the ambient air quality in Malaysia. The API for a given period is calculated based on the sub-index value (sub-API) for all the five air pollutants, namely sulphur dioxide (SO2), nitrogen dioxide (NO2), ozone (O3), carbon monoxide (CO) and particulate matter below 10 micron size (PM10). The forecast of air pollution can be used for air pollution assessment and management. It can serve as information and warning to the public in cases of high air pollution levels and for policy management of many different chemical compounds. Hence, the objective of this project is to fit and illustrate the use of time series models in forecasting the API in Shah Alam, Selangor. The data used in this study consists of 70 monthly observations of API (from March 1998 to December 2003) published in the Annual Reports of the Department of Environment, Selangor. The time series models that were being considered were the Integrated Autoregressive Moving Average (ARIMA) and the Integrated Long Memory Model (ARFIMA) models. The lowest MAE, RMSE and MAPE values were used as the model selection criteria. Between these two models considered, the integrated ARFIMA model appears to be the better model as it has the lowest MAPE value. However, the actual value of May 2003 falls outside the 95% forecast interval, probably due to emissions from mobile sources (i.e., motor vehicles), industrial emissions, burning of solid wastes and forest fires. Keywords: Air Pollution Index (API), Integrated Autoregressive Moving Average (ARIMA), Fractionally Integrated Autoregressive Moving Average (ARFIMA) Introduction Air pollution is one of the major issues that has been affecting human health, agricultural crops, forest species and ecosystems. Air quality monitoring is part of the initial strategy in the pollution prevention program in Malaysia. Since 1980, six major haze episodes were officially reported in Malaysia that is in April 1983, August 1990, June 1991, October 1991, August to October 1994 and July to October 1997. The 1997 haze episode was the worst ever experience in the country [3]. As a result, the government has established the Malaysian Air Quality Guidelines, the Air Pollution Index (API) and the Haze Action Plan in an attempt to improve the air quality. There are possible health effects of exposure to air pollution. Recent studies have examined possible health effects of the 1997 forest fires. For example, respiratory disease outpatient who visited the Kuala Lumpur General Hospital increased from 250 to 800 per day and the data assembled indicated an increase in cases of asthma, acute respiratory infection, and conjunctivitis [1]. A study conducted by Nasir et al. [8] suggested that in the 1997 haze episode the total health effects were estimated to include 285,227 asthma attacks, 118,804 cases of bronchitis in children, 3889 cases of chronic bronchitis in adults, 2003 respiratory hospital admission, 26,864 emergency room visits and 5,000,760 restricted activity days. In addition, among the five pollutants, ozone was demonstrated to cause stress to the skin. It possesses a strong oxidizing potential and is therefore very reactive to the affected part [7]. Blockage of sunlight may also promote the spread of harmful bacteria and viruses that would otherwise be killed by ultraviolet B. Components of smoke haze, including polycyclic aromatic hydrocarbons known as carcinogens are also potentially dangerous and their effects may not

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be apparent for years. The consequences may be more severe to children, for whom the particulates inhaled are high relative to their body size [4]. Since 1996, the National Environmental Research Institute [9], Denmark, has developed a comprehensive and unique integrated air pollution forecasting model system, called the THOR system. The system has been used to forecast the air pollution from accidental releases such as power plants, industrial sites and natural or human made fires. Dominici et al. [6] studied on the improved semi-parametric time series models of air pollution and mortality. Clearly then from the above discussions, the modelling and ability to forecast API in Malaysia can be useful to many organisations like Environmental Agencies, Medical Research Institutes, Hospitals, etc. and the public at large. Therefore, the objective of this research is to model the API time series data in Shah Alam, Selangor so that such a model may be able to provide somewhat an estimate of the future API values. Methodology The Data Set The Air Pollution Index (API) was introduced as an index system for classifying and reporting the ambient air quality in Malaysia. The API for a given period is calculated based on the sub-index value (sub-API) for all the five air pollutants, namely sulphur dioxide (SO2), nitrogen dioxide (NO2), ozone (O3), carbon monoxide (CO) and particulate matter below 10 micron size (PM10) which are included in the Malaysia API system. The API reference value has been based on the Malaysia Ambient Air Quality Guidelines (MAQG) of 1989 as shown in Table1 [2]. Table 1. API and Health Effect API readings 0-50 51-100 101-150 151-200 210-300 301-500 >500 Alert level No alert No alert Early alert Early alert On alert Warming Emergency Health Effect Descriptor Good Moderate Unhealthy Unhealthy Very unhealthy Hazardous Hazardous

Alam Sekitar Malaysia Berhad (ASMA) is a company that monitors the ground level ambient air continuously 24 hours a day. ASMA is responsible to install, operate, and maintain a network of 50 continuous air quality monitoring stations throughout Malaysia for the Department of Environment, DOE [7].

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Series

1 40.

1 20.

1 00.

80.

60.

40.

20. 0 1 0 20 30 40 50 60 70

Figure 1: The time series plot of the monthly API data observed at the Shah Alam monitoring station from March 1998 to December 2003 The data used in this study consists of 70 monthly observations of API (from March 1998 to December 2003) published in the Annual Reports of the Department of Environment, Selangor [10]. The time series plot of API is shown in Figure 1. Time Series Modelling Procedure For the purpose of time series modelling in this study, the first 58 observations (March 1998 to December 2002) were used to fit the ARIMA and integrated ARFIMA models while the subsequent 12 observations (from January 2003 to December 2003) were kept for the post sample forecast accuracy check. A brief description of the time series models and definitions used in this study are as follows. A stationary ARMA (p, q) model is defined as a sequence of random variables { X t } , given by

{ Z t } is a sequence of uncorrelated random variables with zero mean and constant variance, denoted as { Z t } ~ WN ( 0, σ 2 ), [5]. A process { X t } is called an ARIMA (p, d, q) process [5] if d is a nonnegative integer such d that (1 − B ) X t is a causal ARMA (p, q) process. The ARIMA (p, d, q) processes satisfies the d difference equation of the form φ ∗ (B ) ≡ X t φ (B )(1 − B ) X t = θ (B )Z t , { Z t } ~ WN ( 0, σ 2 ), where φ ( z ) and θ (z ) are polynomials of degrees p and q respectively, and φ ( z ) ≠ 0 for z ≤ 1 . The φ ∗ ( z ) has a zero of order d at z = 1 . The process { X t } is stationary if and only if d = 0 , in which case it
X t − φ1 X t −1 − L − φ p X t − p = Z t + θ 1 Z t −1 + L + θ q Z t − q where

reduces to an ARMA (p, q) process. A long memory process [5] or a fractionally integrated ARMA, ARFIMA (p, d, q) processes with 0 < d < 0.5 is a stationary process with much more slowly decreasing autocorrelation function satisfy the difference equation of

(1 − B )d φ (B )X t = θ (B )Zt , where { Z t } ~ WN (0, σ 2 ), φ ( z ) = 1 − φ1 z − ... − φ p z p satisfying φ ( z ) ≠ 0 and θ ( z ) = 1 + θ1 z + ... + θ q z q , satisfying θ ( z ) ≠ 0 d for all z such that z ≤ 1 , and B is the backward shift operator. The operator (1 − B ) is defined by
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ρ (k ) at lag k as k → ∞ which satisfies the property of ρ (k ) ~ Ck 2 d −1 . The ARFIMA processes

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the binomial expansion of (1 − B ) =
d

∑π
j =0

j

B j with n 0 = 1 and π j =

k −1− d for j = 0 , 1, k 0< k ≤ j

2, ….

be the observed time series. Since there is a gradual decrease in level of { y t }, we differenced the series at lag 1 to obtain a new series that is more or less constant in its level and this we denote it by { X t } . To this { X t } , we fitted an ARMA (p, q) process. The entire process of model fitting was done using the computer software “ITSM 2000, version 7.0”, [5]. The criteria chosen to measure the accuracy of the forecast in this study are the mean absolute error (MAE), the root mean squared error (RMSE) and the mean absolute percentage error (MAPE) are given below.

For the purpose of this study, we let { Yt } be the time series that represents the API and

{ yt }

× 100% n n n ˆ where xi and xi are the actual observed values and the predicted values respectively while n is the

MAE =


i =1

n

ˆ xi − xi
,

RMSE =

ˆ ∑ (x i − x i )
i =1

n

2


,

n

MAPE =

i =1

ˆ xi − xi xi

number of predicted values. Results In this section, we present the results of the study. Let { Yt } be the API and X t = ∇Yt where { X t } is an ARMA (p, q) process. The best model fitted based on the AICC criterion that is given as follows: X t = 1.047 X t −1 − 0.6656 X t − 2 − 0.1976 X t −3 + Z t − 1.771Z t −1 + 1.772 Z t − 2 − 0.7764Z t −3 where

{Z t } ~ WN (0, 0.027020) .

The monthly forecast results of the API values using the ARIMA (3, 1, 3) model for the year 2003 are shown in Table 2. Table 2. Forecasts of the API values from January 2003 to December 2003 using the ARIMA (3, 1, 3) model Month Actual Forecast 95% Confidence Interval January 62 61.23 (44.36, 84.50) February 62 63.20 (45.25, 88.27) March 56 65.61 (44.43, 96.88) April 57 66.76 (43.81, 101.73) May 143 65.70 (42.73, 101.04) June 83 63.20 (41.08, 97.22) July 70 60.93 (39.60, 93.73) August 70 60.18 (39.03, 92.79) September 36 61.15 (39.08, 95.68) October 43 62.96 (39.12, 95.68) November 49 64.17 (38.88, 105.92) December 59 63.81 (38.23, 106.52) In Figure 2, the graph of the predicted values given by the ARIMA (3, 1, 3) model and the actual values of the API, together with their 95% forecast intervals are shown. We note that the actual API values fall within the 95% confidence interval.

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160

140

120

Air Pollution Index

100

80

60

40

20

0 1 2 3 4 5 6 Months 7 8 9 10 11 12

Actual values

Predicted values

95% lower bound

95% upper bound

Figure 2: Graph of the API values with 12 predicted values of the ARIMA (3, 1, 3) model and the actual values from January 2003 to December 2003 For ARFIMA modelling, again, we let { Yt } be the API and X t = ∇Yt where an ARFIMA (p, d, q) process given by,

{ X t } is now

(1 − B )−0.5 X t

= Z t − 0.001520 Z t −1 + 0.3578Z t − 2 , where {Z t } ~ WN (0,0.036980 ) .

The monthly forecast results of the API values using the integrated ARFIMA (0, −0.5, 2) model for the year 2003 are shown in Table 3. Table 3. Forecasts of the API values from January 2003 to December 2003 using the integrated ARFIMA (0, −0.5, 2) model Month January February March April May June July August September October November December Actual 62 62 56 57 143 83 70 70 36 43 49 59 Forecast 59.18 58.56 58.53 58.13 57.70 57.26 56.84 56.44 56.05 55.68 55.32 54.97 95% Confidence Interval (33.60, 85.98) (30.31, 87.25) (25.18, 92.55) (22.56, 94.44) (20.60, 95.52) (19.00, 96.22) (17.63, 96.71) (16.41, 97.05) (15.31, 97.29) (14.31, 97.46) (13.38, 97.58) (12.51, 97.65)

Figure 3 shows the predicted values given by the integrated ARFIMA (0, −0.5, 2) model and the actual values of the API, together with their 95% forecast intervals. The actual API values all fall within the 95% forecast intervals.

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160

140

120

Air Pollution Index

100

80

60

40

20

0 1 2 3 4 5 6 Months 7 8 9 10 11 12

Actual values

Predicted values

95% lower bound

95% upper bound

Figure 3: Graph of the API values with 12 predicted values of the integrated ARFIMA (0, −0.5, 2) model and the actual values from January 2003 to December 2003 In Table 4, the MAE, MAPE and RMSE values of the ARMA (3, 1, 3) and the integrated ARFIMA (0, −0.5, 2) models are shown. Table 4. The MAE, MAPE and RMSE values of the ARMA (3, 1, 3) and the integrated ARFIMA (0, −0.5, 2) models Model MAE RMSE MAPE ARIMA (3, 1, 3) 16.786 25.821 24.70% 15.896 27.299 20.86% ARFIMA (0, −0.5, 2) The MAE and MAPE values of the integrated ARFIMA (0, −0.5, 2) model are smaller when compared to those of the ARIMA (3, 1, 3) model. The RMSE value for ARIMA (3, 1, 3) is found to be smaller than the ARFIMA (0, −0.5, 2) model. The actual API value for May 2003 falls outside the 95% forecast intervals when forecasting using both the ARIMA (3, 1, 3) and ARFIMA (0, −0.5, 2) model. The ARIMA (3, 1, 3) model seem to be unable to forecast well as the actual API value for September 2003 is also found to be below the 95% lower bound of the forecast interval. Conclusion Based on this data set, the integrated ARFIMA model appears to have a slightly better forecasting performance compared to that of the ARIMA although both models are unable to forecast all values within the 95% forecast interval. The actual API value of May 2003 which falls outside the 95% upper bound of the forecast interval may be due to the emissions from mobile sources like motor vehicles, industrial emissions, burning of solid wastes and forest fires. As such, factors which could affect the API should be taken into consideration in modelling of API for a better forecast ability as the modelling processes in this project were done based on the data of API only. References 1. Afroz R., Hassan M.N. and Ibrahim N.A. (2003), Review Of Air Pollution And Health Impacts In Malaysia, Environmental Research, Volume 92, Issue 2, pp 71-77. 2. Awang M.B., Hassan M.N., Noor Alshurdin M.S., Abdullah A.M. (1997), Air pollution in Malaysia, in IMR Quaterly Bulletin, No. 43, pp 26-42.

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3. 4. 5. 6. 7. 8.

9. 10.

Awang M. B., Jaafar A. B., Abdullah A. M., Ismail M.B., Hassan M.N., Abdullah R., Johan S. and Noor H. (2000), Air Quality In Malaysia: Impacts, Management Issues And Future Challenges, Respirology, Vol. 5, pp 183-196. Beardsley R., Bromberg P.A., Costa D.A., Devlin R., Dockery D. W., Frampton M. W., Lambert W., Samet J. M., Speizer F. E., Utell M.(1997), Smoke Alarm: Haze From Fires Might Promote Bacterial Growth. Science America, pp 24-25. Brockwell, P.J. and Davis, R.A. (2002), Introduction To Time Series And Forecasting, 2nd Edition, Springer-Verlag, New York. Dominici, F., McDermott, A., Hastie, T. J.(2004), Improved Semi-Parametric Time Series Models Of Air Pollution And Mortality. http://wwwstat.stanford.edu/~hastie/Papers/dominiciR2.pdf. Accessed on 27 April 2004. Faridah Mohamad (2002), Impacts Of Eexposure To Ambient PM10 On Hospital Outpatient Visits For Haze-Related Diseases And School Children Lung Function, Masters Thesis. Universiti Putra Malaysia, Malaysia. Nasir M.H., Choo W.Y., Rafia A., Theng L.C., Noor M. M. H (2000), Estimation Of Health Damage Cost For 1997-Haze Episode In Malaysia Using the Ostro model, Proceeding Malaysian Science And Technology Congress, Confederation Of Scientific And Technological Association In Malaysia,COSTAM, Kuala Lumpur, in Press. National Environment Research Institute, NERI (2003), THOR. http://www.dmu.dk/1_viden/2_Miljoetilstand/3_luft/4_Spredningsmodeller/5_thor/default_en.asp. Accessed on 27 April 2004. ----- (1998-2003), Annual Report, Department of Environment, Selangor.

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FLUCTUATION ACTIVITIES OF 210PO IN WATER THE WATER COLUMN AT BAGAN LALANG, SELANGOR PHANG FEONG KUAN1, ZAHARRUDIN AHMAD2 and CHE ABD. RAHIM MOHAMED1* 1 Marine Science Program, School of Environmental Science and Natural Resources, Faculty Science and Technology, National University of Malaysia (UKM), 43600 Bangi, Selangor, Malaysia. 2 Malaysian Institute of Nuclear Technology Research (MINT), 43000 Bangi, Selangor, Malaysia. ∗ Corresponding author;e-mail:carmohd@pkrisc.cc.ukm.my Tel.: +603-89213209

ABSTRACT The concentration activity of 210Po was measured in water samples collected from six stations at Bagan Lalang, Selangor. Results showed that the dissolved and particulate activities of 210Po were varied from 3.03 x 10-4 Bq l-1 to 16.75 x 10-4 Bq l-1 and 2.41 x 10-2 Bq g-1 to 56.69 x 10-2 Bq g-1 (dry wt), respectively. Overall, the activities of 210Po in dissolved phase are relative low due to the insoluble of 210Po in water. More than 99% of 210Po in the water column occurred in the particulate phase due to the high affinity of 210Po to particulate phase. In addition, variation of 210Po activities was influence by the tidal current and the chemical behavior of 210Po in water column for both sampling. Keywords: 210Po, water column, dissolved phase, suspended particulate matter Introduction Polonium-210 (210Po) is the final alpha-emitting daughter nuclide in the natural 238U decay series entering the marine environment via the natural radioactive decay of 222Rn gas, 226Ra in solution, and through wet and dry atmospheric deposition of 210Bi, 210Pb and 210Po [1, 2]. A small amount of 210Po will be formed in-situ in the water column of marine environment as a result of uranium decay [3]. In the water column, 210Po is particle-reactive radionuclides and tend to associate with the particulate phases [4]. Plankton also absorbs polonium and forms as complexes with organic matter, while lead reveals as a stronger tendency to be sorbed onto mineral suspended matter [3, 5, 6, 7]. 210Po is strongly accumulated by marine organisms and it is approximately three times enriched in phytoplankton and 12 times in zooplankton with respect to 210Pb [8]. Bagan Lalang, Selangor is coastal area that located at the edge of Sepang around 15km from the centre town (Figure 1). The major river system in this area is Kuala Sepang Kechil which is drains into the Malacca Straits. The objective of this study is to determine the distribution of 210Po in the water column along the Kuala Sepang Kechil, Selangor.

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Experimental Sample collection was made at six stations throughout the Bagan Lalang estuary on 20 Mac and . About 15 l of water samples were collected using Van Dorn water sampler and in situ paremeters, i.e., salinity, pH and water depth also collected using calibrated portable meter (Model: YSI-SCT6810). In the labaratory, water samples were filtered through the pre-weighted of 0.45 µm pore size membrane filter paper. Both dissoleved and suspended particulate matter samples (TSM) were processed using the suggested method by [9]. The activities of 210Po was measured using alpha spectrometer.

Figure 1. Sampling stations at Bagan Lalang, Sepang.

Results and Discussions The dissolved and particulate of 210Po activities from six stations obtained at Bagan Lalang, Sepang were measured and listed in Table 1. Table 1. Aktiviti 210Po dalam sampel air dan TSM. 20 Mac 2004 Station Dissolved 210Po Particulate 210Po Location (Layer) x 10-4 (Bq l-1 ± x 10-2 (Bq g-1± 1σ) 1σ) 1 (S) 02° 36' 55'' 3.11 ± 1.76 23.00 ± 2.65 N 101° 41' 07'' E 2 (S) 02° 36' 35'' N 4.28 ± 1.12 17.79 ± 2.39 8 Sept 2004 Dissolved 210Po Particulate 210Po x 10-4 (Bq l-1 ± x 10-2 (Bq g-1± 1σ) 1σ) 7.77 ± 4.32 20.64 ± 3.88

3.03 ± 7.54

19.36 ± 6.02

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101° 40' 22'' E 3 (S) 02° 36' 30'' N 101° 39' 19'' E 02° 36' 05'' N 101° 38' 36'' E 02° 36' 11'' N 101° 36' 56'' E 02° 36' 06'' N 101° 35' 38'' E 4.17 ± 1.28 19.95 ± 2.94 10.49 ± 2.82 40.41 ± 13.45

4 (S) (M) (B) 5 (S) (M) (B) 6 (S) (M)

6.89 ± 2.43 4.82 ± 1.17 4.78 ± 1.28 4.53 ± 1.90 10.25 ± 2.95 6.60 ± 1.73 49.43 ± 0.61

44.95 ± 10.08 12.89 ± 2.56 7.71 ± 15.64 26.67 ± 8.40 35.99 ± 10.52 34.12 ± 7.13 222.66 ± 2.74

6.76 ± 2.55 4.07 ± 2.92 16.75 ± 10.39 9.97 ± 3.01 10.88 ± 4.28 3.24 ± 3.12 72.96 ± 1.73

56.69 ± 22.88 43.48 ± 11.59 2.41 ± 24.55 33.31 ± 22.22 50.61 ± 23.24 49.17 ± 16.95 316.08 ± 5.89

(B) Total Activity (-) – No data

The activity of 210Po was varied from 3.11 x 10-4 Bq l-1 to 10.25 x 10-4 Bq l-1 and 3.03 x 10-4 Bq l to 16.75 x 10-4 Bq l-1 for dissolved 210Po obtained on 20 March 2004 and 8 September 2004, respectively. Overall, the activities of 210Po in dissolved phase are relative low due to the insoluble of 210 Po in water [10], where 210Po will be adsorbing into particulate phase once it enters into the water column. Besides that, the activities of 210Po in surface layer were higher than in middle and bottom layer. This was probably due to the supply of deposition of atmospheric 210Pb originated from the decay of 222Rn [4]. 210 Po concentrations in suspended particulate matter are varied from 7.71 x 10-2 Bq g-1 to -2 44.95 x 10 Bq g-1 (dry wt) and 2.41 x 10-2 Bq g-1 to 56.69 x 10-2 Bq g-1 (dry wt) for both sampling on 20 March 2004 and 8 September 2004, respectively. The standard deviation value for few stations was higher than the concentrations value in suspended matter samples caused by the high background value and low concentration of these nuclides in the samples. Overall, the activities of 210Po is higher in particulate phase compare to dissolved phase (p<0.01). The 210Po activities principally occurred in the particulate phase and more than 99 % of total activity (dissolved + particulate) of all samples was found associated with the particulate fraction (Table 1). This is due to the very insoluble feature of this radionuclide in the seawater [1, 10] and tends to associate with particles. In addition, particulate 210Po are higher in surface water is related to the deposition of atmospheric 210Pb. Once 210Pb is deposited, it is rapidly absorbed by particles [11]. Besides, the high activities of 210Po in surface layer was probably due to dissolution of sinking particle and biological remobilization of 210Po [5, 12, 13, 14, 15]. In addition, the activities of 210Po at the bottom layer is slightly higher than the middle layer. This is propably due to the resuspension of 210Po from the surface sediment [10] and decay of 226Ra in sediment.
-1

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Conclusion Measurement of dissolved and particulate activities of 210Po at water column of Bagan Lalang, Selangor were obtained. Results showed that more than 99% of 210Po were occured in particulate phase due to the high affinity of 210Po to particulate phase. The variation of 210Po activities was influence by tidal current and the chemical behavior of 210Po in water column. Acknowledgement This research was supported by the grant of IRPA 09-02-02-0045-EA141. The author would like to thanks all staffs from Malaysian Institutes of Nuclear Technology Research (MINT) for their useful opinions in completing this research. The author is also like to thank the laboratory assistants from National University of Malaysia (UKM) in sample collections and technical support. References 1. 2. 3. 4. 5. 6. Wildgust, M. A., McDonald, P. & White, K. N. 1998. Temporal changes of 210Po in temperate coastal waters. The Science of Total Environment 214: 1-10. Turekian, K. K., Nozaki, Y. & Benninger, L. K. 1977. Geochemistry of atmospheric radon and radon products. Annu. Rev. Earth Planet. Sci. 5: 227-255. Skwarzec, B. & Bojanowski, R. 1988. 210Po content in sea water and its acculumution in southern Baltic plankton. Marine Biology 97: 301-307. Yang, C. H. & Lin, H. C. 1992. Lead-210 and Polonium-210 across the frontal region between Kuroshio and East China Sea, Northeast of Taiwan. TAO 3 (3): 379-394. Bacon, M.P., Spencer, D.W. & Brewer, P.G. 1976. 210Pb/226Ra and 210Po/210Pb disequilibria in seawater and suspended particulate matter. Earth and Planetary Science Letters 32: 277-296. Bacon, M. P., Brewer, P. G., Spencer, D. W., Murray, I. W. & Goddard, I. 1980. Lead210, polonium-210, manganese and iron in the Cariaco Trench. Deep sea Research 27A: 119-135. Fellows, D.A., Karl, D.M. & Knauer, G.A. 1981. Large particle fluxes and the vertical transport of living carbon in the upper 1500 m of northeast Pacific Ocean. Deep Sea Research 28: 921-936. Shannon, L. V., Cherry, R. D., Orren, M. J. 1970. Polonium-210 and lead-210 in the marine environment. Geochim. Cosmochi,. Acta. 34: 701-711. Theng, L. T. & Mohamed, C. A. R. 2005. Aktivities of 210Po and 210Pb in the water column at Kuala Selangor, Malaysia. Journal of Environmental Radioactivity 80: 273-286. Tanaka, N., Takeda, Y. & Tsunogai, S.1983. Biological effect on removal of Th-234, Po-210 and Pb-210 from surface water in Funka Bay, Japan. Geochimica et Cosmochimica Acta 47: 1783-1790. Helz, G. R., Setlock, G. H., Cantillo, A. Y., Moore, W. S. 1985/86. Processes controlling the regional distribution of 210Pb, 226Ra and anthropogenic zinc in estuarine sediments. Earth and Planetary Science Letters 76: 23-24. Nozaki, Y. & Tsunogai, S. 1976. 226Ra, 210Pb. and 210Po disequilibria in the western North Pacific. Earth and Planetary Science Letters 32: 313-321. Thomson, J.K. & Turekian, K.K. 1976. 210Po and 210Pb distributions in ocean water profiles from the eastern South Pacific. Earth and Planetary Science Letters 32: 297-303. Cochran, J.K., Bacon, M.P., Krishnaswami, S. & Turekian, K.K. 1983. 210Po and 210Pb distributions in the central and eastern Indian Ocean. Earth and Planetary Science Letters 65: 433-452. Chung, Y. & Finkel, R. 1988. 210Po in the western Indian Ocean: distribution, disequilibria and partitioning between the dissolved and particulate phases. Earth and Planetary Science Letters 88: 232-240.

7. 8. 9. 10. 11. 12. 13. 14. 15.

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HEAVY METAL CONCENTRATIONS IN THE RAZOR CLAMS (SOLEN SPP) FROM MUARA TEBAS, SARAWAK Devagi Kanakaraju*, Connie anak Jios., and Shabdin Mohd Long Faculty of Resource Sciences and Technology, University of Malaysia Sarawak, 94300 Kota Samarahan, Sarawak E-mail: kdevagi@frst.unimas.my Abstract The razor clams (Solen spp) or locally known as ‘ambal’ in Sarawak collected from Muara Tebas were studied for their heavy metals contents in tissues and shells. Sediment samples were also tested for their metal contents. Concentrations of Pb, Fe, Zn, Cu, Cd and Mn were determined by using Flame Atomic Absorption Spectrophotometer (FAAS). Tissues of razor clams showed highest concentrations of Fe and Zn, while shells accumulated highest concentrations of Pb and Mn. The lowest metal concentrations found were Cu and Cd. In general, the levels of metals in ‘ambal’ were within the permissible limit recommended by international standard, the Food and Agricultural Organization (FAO). However, the study revealed that the sediments at Muara Tebas fall under the category of slightly polluted (for Pb) when compared to the guidelines suggested by United States Environment Protection Agency (USEPA). Keywords: razor clams, ‘ambal’ heavy metals, sediment Abstrak Razor clam (Solen spp) atau lebih dikenali sebagai ambal di Sarawak telah dipungut dari Muara Tebas untuk kajian kandungan logam berat dalam bahagian tisu dan cengkerangnya. Sampel sedimen juga telah dikaji kandungan logam beratnya. Kepekatan logam Pb, Fe, Zn, Cu, Cd dan Mn telah ditentukan dengan menggunakan Spektroskopi Serapan Atom Nyala (FAAS). Bahagian tisu ambal mengandungi kepekatan Fe dan Zn yang tertinggi manakala cengkerang pula menunjukkan kepekatan Pb dan Mn yang paling tinggi. Kepekatan logam yang paling rendah ialah Cu dan Cd. Secara keseluruhannya, tahap kepekatan logam dalam ambal berada dalam had yang disyorkan oleh piawai Food and Agricultural Organization (FAO). Walau bagaimanpun, kajian ini menunjukkan bahawa sedimen di Muara Tebas berada dalam kategori sedikit tercemar (untuk Pb) apabila perbandingan dibuat dengan piawai yang disyorkan oleh United States Environment Protection Agency (USEPA). Kata Kunci: razor clam, ambal, logam berat, sedimen

Introduction Various species of edible bivalve mollusks such as clams, oyster and cockles are found on the mangrove mudflats and intertidal sandy beaches in Peninsula Malaysia as well as Sarawak. Razor clam (Solen spp) or locally known as “ambal” is found abundantly in the intertidal sandy beaches in Kuching and Samarahan Division of Sarawak. There are three different species of Ambal in the genus Solen that commonly found in Sarawak [1]. The three species are Solen corneus, Solen species and Solen vagina and they are locally known as “Ambal Biasa’, ‘Ambal Jernang’, and ‘Ambal Riong’ respectively. Heavy metals pollution has been a hot issue in environmental studies for many years. Even though, metals occurs naturally in the environment but due to the anthropogenic inputs which originate from various human activities the concentrations have been rising. Heavy metals tend to accumulate in the food chain and eventually will be consumed by organisms. Bivalve mollusks are

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well-known to accumulate heavy metals and have been widely used as bioindicator for monitoring heavy metal pollution in aquatic environment [2-4]. There is very little documented information available about the metal contents in ‘ambal’ despite the popularity as a source of seafood item in Sarawak. Owing to limited studies on ‘ambal’, various aspects on ‘ambal’ are still unexplored such as the feeding behavior, biology and population dynamics. A study was conducted on the stock assessment and some biology perspective of ‘ambal’ [5]. Some work was also done on the bacterial density and quality of water in ‘ambal’ from Asajaya Laut and Kampung Buntal, Sarawak [6]. Thus, this study was undertaken to determine the amounts of heavy metals (Pb, Fe, Zn, Cu, Cd and Mn) in tissues and shells of razor clam and sediments at Muara Tebas. The suitability of ‘ambal’ as a seafood item was evaluated by comparing with an international standard (FAO). Experimental Sample Collection This study was carried out along the beach at Muara Tebas during low tide in the month of September and November 2004, the harvesting periods of ‘ambal’ (Figure 1). The sampling stations was divided into 3 positions namely low tide station (S1), middle tide station (S2 and S3) and high tide station (S4, S5).

The razor clams were collected using long, elongated, slender stick of four feet in length a mixture of limestone powder, ashes and salt. Razor clam samples ranging between 3.0 and 10.0 cm in length were collected. About 10-15 individuals were collected at each station to prepare a pooled sample. Immediately after collection, the samples were washed with seawater to remove sediment before being kept in the labeled glass jar. The samples were kept in a cool box. Sediment samples were collected from each sampling stations around the area inhabited by the razor clam using the PVC tubes. Three to four random surface sediment samples were collected from each station were divided into two sub-ranges, 0 - 5 cm and 5 - 10 cm.

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Chemical analysis Razor clam samples were thawed and carefully washed with tap water and deionized water to remove any extraneous material. Tissues and shell samples were freeze-dried. Subsequent to the drying process, the samples were ground to fine powder using pestle and mortar and stored in polyethylene bottles. The composite homogenates were divided into three sub-samples for replicate analyses. Acid wet digestion method modified from [2] was employed to the razor clam samples. One gram of tissue sample was weighed and digested with 6 ml of concentrated nitric acid (63 %) and 1 ml of 30 % hydrogen peroxide using hot plate digester. While for the shell samples, one gram of shell samples was digested with the mixture of 2 ml of concentrated nitric acid (63 %), 5 ml of concentrated hydrochloric acid (37 %) and 1 ml of 30 % hydrogen peroxide. The samples were dissolved for one hour and then digested for another one hour. The digested samples were filtered and diluted to 50 ml with deionized water. Oven-dried sediment samples were ground to the size of 50 mesh. One gram of sediment samples was digested for three hours with freshly prepared mixture 1:3 of 20 ml of nitric acid and hydrochloric acid using hot plate digester. The levels of heavy metals in the filtrate were determined with Flame Atomic Absorption Spectrophotometer (Perkin – Elmer Model 3110). The quantitative measurement was made using calibration curves obtained with five standard solutions of each elements analyzed in this study. Fresh working standard solutions were prepared using AAS stock solution (1000 ppm). Acid blank for every batch was analyzed to evaluate any contamination. All results reported in this study are expressed in dry weight. Statistical Analysis One-way analysis of Variance (ANOVA) is used to study differences in metal concentrations at different sampling period and at two different depths for sediments. Correlation coefficient (r) was used to test the relations between the concentrations of metals in sediments with tissues and shells of razor clam.

Results and Discussions Metals concentrations in sediments The pH of the surrounding sediments of Muara Tebas was found to be alkaline (8.13 – 8.36) (Table 1). Temperature and dissolved oxygen (DO) was in the range of 28.5 – 33.7 oC and 5.2 -7.4 mg/L. Metals contents in sediments at two different depths (0 – 5 cm and 5 – 10 cm) Muara Tebas are shown in Table 2.

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Table 1. Mean pH, temperature and DO of sediments at Muara Tebas Station Low tide Mid tide High tide pH 8.13 8.36 8.30 Temperature (oC) 28.5 30.3 33.7 DO (mg/L) 7.4 5.2 6.6

Table 2. Metal contents (µg/g dry wt) in sediments from Muara Tebas
Sampling Station S1* S2 S3 S4 S5 S1** S2 S3 S4 S5 Pb 0-5 36.67 ± 0.03 35.50 ± 0.04 51.83 ± 0.04 41.83 ± 0.06 38.17 ± 0.05 49.17 ± 0.07 53.83 ± 0.07 51.83 ± 0.04 54.50 ± 0.03 52.50 ± 0.04 5-10 35.83 ± 0.03 36.50 ± 0.06 53.17 ± 0.06 44.50 ± 0.08 45.17 ± 0.08 47.83 ± 0.03 54.17 ± 0.06 53.17 ± 0.06 56.33 ± 0.05 53.67 ± 0.04 0-5 5.17 ± 0.01 4.33 ± 0.01 5.17 ± 0.01 5.17 ± 0.01 5.17 ± 0.01 5.17 ± 0.01 4.83 ± 0.01 5.17 ± 0.01 5.67 ± 0.01 4.33 ± 0.01 Cu 5-10 4.67 ± 0.01 4.67 ± 0.01 5.67 ± 0.01 5.67 ± 0.01 4.67 ± 0.01 4.33 ± 0.01 5.83 ± 0.01 5.67 ± 0.01 5.67 ± 0.01 4.33 ± 0.01 0-5 529.17 ± 0.05 520.33 ± 0.02 557.00 ± 0.04 546.33 ± 0.03 497.83 ± 0.02 534.17 ± 0.04 571.50 ± 0.02 546.33 ± 0.04 566.50 ± 0.03 539.33 ± 0.05 Fe 5-10 477.67 ± 0.05 499.00 ± 0.04 523.67 ± 0.01 484.00 ± 0.03 463.67 ± 0.03 514.83 ± 0.05 571.50 ± 0.05 523.67 ± 0.01 573.33 ± 0.06 527.83 ± 0.05 0-5 32.50 ± 0.01 32.50 ± 0.01 37.67 ± 0.01 32.67 ± 0.01 29.33 ± 0.01 30.83 ± 0.02 31.50 ± 0.01 37.67 ± 0.01 34.83 ± 0.01 33.67 ± 0.01 Zn 5-10 34.33 ± 0.01 33.33 ± 0.01 32.83 ± 0.01 35.33 ± 0.01 29.17 ± 0.01 30.67 ± 0.01 35.17 ± 0.01 32.83 ± 0.01 33.50 ± 0.01 30.33 ± 0.01 0-5 3.33 ± 0.01 2.83 ± 0.01 1.67 ± 0.01 2.67 ± 0.29 1.67 ± 0.01 1.50 ± 0.01 1.83 ± 0.01 1.67 ± 0.01 1.83 ± 0.01 1.33 ± 0.01 Cd 5-10 3.33 ± 0.01 1.83 ± 0.01 1.67 ± 0.01 2.67 ± 0.01 1.83 ± 0.01 2.00 ± 0.01 2.50 ± 0.01 1.67 ± 0.01 2.33 ± 0.01 0.83 ± 0.01

S1* : Data obtained during first sampling S1**: Data obtained during second sampling

The amounts of Fe, Pb, Zn, Cu and Cd was found in the range from 463.67 µg/g – 573.33 µg/g, 35.50 µg/g – 56.33 µg/g, 29.17 µg/g – 37.67 µg/g, 4.33 µg/g – 5.83 µg/g and 0.83 µg/g – 3.33 µg/g respectively. Fe occurs in the highest level whereas Cd was found in the least amount. Fe is naturally abundant in the earth’s crust. There was no much difference observed for the metals concentrations at two different depths. The amounts were quite close for both depths. Even, statistical analysis also showed that there was no significant difference between the depths (p < 0.05). The

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sediments were assessed by doing comparison with United States Environment Protection Agency (USEPA) guidelines [7]. The sediments at Muara Tebas fall under the category slightly polluted with Pb (40-60 µg/g). This could be due to the anthropogenic sources which contribute to Pb contamination. The major source of Pb in the environment is related to the burning of fossil fuels and via atmosphere [8]. Hence, Pb contamination in Muara Tebas may be resulted from burning of fossil fuels from boats used for fishing and also leisure activities at Kuching Bay. Table 3. USEPA guidelines classification for sediments (µg/g) Elements Pb Cu Zn Cd Unpolluted <40 <25 <90 Slightly polluted 40-60 25-50 90-200 Heavily polluted >60 >50 >200 -

Metal concentrations in razor clam Table 4 represent the metal concentrations and standard deviation of six elements analyzed in ‘ambal’ collected from Muara Tebas. The metals contents in tissues and shells of ‘ambal’ at Muara Tebas revealed major variations. It was further proved by statistical analysis (p < 0.05) which showed that there were significant differences of Pb, Fe, Zn, Cu, Cd and Mn in tissues and shells. The most abundant elements in tissues of razor clam were Fe and Zn which ranged from 276.17 µg/g – 1161.00 µg/g and 72.17 – 97.00 µg/g respectively. The shells indicated the highest levels of Pb (52.00 µg/g– 58.17 µg/g) followed by Mn (30.83 µg/g – 67.00 µg/g), Cu (12.67 µg/g – 23.83 µg/g) and Cd (7.33 µg/g – 9.00 µg/g). The wide variations of metals in two different parts of ‘ambal’ could be expressed by the complex relationship between environmental concentrations and bioaccumulation [9]. There are various factors which influence the metals accumulation in bivalves. Among the factors known include metal bioavailability, season of sampling, size, hydrodynamics of the environment and reproductive cycle [3]. Cd was also found in a very trace amount in tissues and shells likewise in sediments. There was moderate significant negative correlation for Cd in shells with sediments at Muara Tebas (r = - 0.51., P = 0.00). This may be attributed to the low bioavailability of Cd in sediments and water environment. Metals concentrations obtained in this study were compared with the international standards for metals in mollusks/shellfish compiled by Food and Agricultural Organization (FAO) of the United Nations [9] (Table 4). All metals were within the regulated limits except for Pb contents in shells. FAO permissible limit for Pb was 5 – 30 µg/g and the results obtained ranged 52.00 µg/g – 58.17 µg/g. The Pb concentration possibly originated from the surrounding sediments as Pb was found in slightly polluted range. However, poor correlation (r = 0.17) was attained between Pb concentrations in shells and sediments at Muara Tebas. The results need to be further verified with other techniques as contamination could have occurred during the sample preparation and analysis. ‘Ambal’ collected from Muara Tebas can be classified as safe for human consumption even though the level of Pb in the shells exceeded the permissible limit as ‘ambal’ are famous for

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their tissues. The shells are thrown away during the cooking process and only the tissues are consumed. Table 4. Metal concentrations in tissues and shells (in µg/g dry weight) at Muara Tebas
Station Tissue S1* S2 S3 S4 S5 S1** S2 S3 S4 S5 15.00 0.02 15.33 ± 0.08 16.17 ± 0.07 15.33 ± 0.03 15.50 ± 0.07 9.67 ± 0.02 9.50 ± 0.03 14.83 ± 0.04 9.50 ± 0.04 13.83 ± 0.03 Pb Shell 56.83 ± 0.04 52.17 ± 0.01 56.00 ± 0.02 58.17 ± 0.01 55.33 ± 0.06 52.17 ± 0.02 56.33 ± 0.03 54.83 ± 0.02 55.17 ± 0.05 52.00 ± 0.01 Tissue 2.67 ± 0.01 2.17 ± 0.01 2.17 ± 0.01 2.33 ± 0.01 10.67 ± 0.01 4.33 ± 0.01 4.67 ± 0.01 4.33 ± 0.01 3.83 ± 0.01 4.17 ± 0.01 Cu Shell 19.50 ± 0.01 22.17 ± 0.01 22.00 ± 0.01 21.17 ± 0.01 20.67 ± 0.01 12.83 ± 0.01 12.67 ± 0.01 16.83 ± 0.01 23.83 ± 0.01 19.33 ± 0.01 Tissue 289.33 ± 0.02 298.00 ± 0.01 276.17 ± 0.01 312.50 ± 0.03 377.83 ± 0.04 782.50 ± 0.06 940.33 ± 0.08 1161.00 ± 0.03 459.17 ± 0.01 323.83 ± 0.06 Fe Shell 165.17 ± 0.02 171.83 ± 0.02 105.83 ± 0.01 97.67 ± 0.02 157.67 ± 0.01 95.17 ± 0.01 143.00 ± 0.02 134.17 ± 0.03 82.67 ± 0.01 125.83 ± 0.01 Tissue 77.83 ± 0.01 85.50 ± 0.01 86.17 ± 0.01 82.17 ± 0.01 75.67 ± 0.01 78.83 ± 0.01 83.17 ± 0.01 72.17 ± 0.01 97.00 ± 0.04 89.67 ± 0.01 Zn Shell 11.67 0.01 7.83 ± 0.01 7.33 ± 0.01 6.17 ± 0.01 4.33 ± 0.01 6.33 ± 0.01 7.83 ± 0.01 17.67 ± 0.01 19.17 ± 0.01 20.17 ± 0.01 Tissue 1.17 ± 0.01 1.17 ± 0.01 1.17 ± 0.01 0.67 ± 0.01 1.83 ± 0.01 2.17 ± 0.01 1.17 ± 0.01 2.00 ± 0.01 2.83 ± 0.01 2.17 ± 0.01 Cd Shell 7.33 ± 0.01 7.33 ± 0.01 7.33 ± 0.01 7.83 ± 0.01 8.67 ± 0.01 8.33 ± 0.01 7.83 ± 0.01 8.17 ± 0.01 8.67 ± 0.01 9.00 ± 0.01 Tissue 16.17 ± 0.01 19.17 ± 0.01 15.17 ± 0.01 18.67 ± 0.01 16.17 ± 0.01 24.17 ± 0.01 24.67 ± 0.01 41.83 ± 0.01 15.33 ± 0.01 11.33 ± 0.01 Mn Shell 47.17 ± 0.01 64.83 ± 0.01 38.67 ± 0.01 37.67 ± 0.01 67.00 ± 0.01 30.83 ± 0.01 35.67 ± 0.01 31.33 ± 0.01 40.33 ± 0.01 43.33 ± 0.01

FAO limits

5-30 µg/g

50-150 µg/g

-

200-500 µg/g

10 µg/g

-

S1* : Data obtained during first sampling

S1**: Data obtained during second sampling

Conclusions Accumulation of the selected metals Pb, Fe, Zn, Cu, Cd and Mn in the tissues and shells of razor clam was evaluated. Tissues showed highest accumulation of essential elements like Fe and Zn while shells accumulated highest concentrations of Pb and Mn. The level of Pb in shells exceeded the limit designated by FAO.

Acknowledgment The authors are grateful to University of Malaysia Sarawak for their financial support.

References 1. Pang, S.C., 1992. Razor clams (Solen sp) Fishery in Sarawak. Jabatan Perikanan Kementerian Pertanian Malaysia, Kuching Sarawak, 1 - 6.

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2. 3.

Lau, S., Mohamed, M., Tan Chi Yen, A. and Su’ut, S., 1998. Accumulation of Heavy Metals in Freshwater Mollusks. The Science of the Total Environmental 214, 113 - 121. Otchere, F.A., 2003. Heavy metals concentrations and burden in the bivalves (Anadara (Senilia) senilis, Crassostrea tulipa and Perna perna) from Lagoons in Ghana:Model to describe mechanism of accumulation/excretion. African Journal of Biotechnology 2(9), 280 - 287. Liang, L.N., He, B., Jiang, G.B., Chen, D.Y. and Yao, Z.W., 2004. Evaluation of Mollusks as Biomonitors to investigate Heavy Metal Contaminations along the Chinese Bohai Sea. Science of the Total Environmental 324, 105 - 113. Pang, S. C., 1993. A Study of Some Aspects of the Biology and Stock Assessment of Razor clam (Solen brevis) in Sarawak, Malaysia. Fishery Research Institute Report, Kuching, Sarawak. Apun, K., Ridan, T. M. M. and Bujang, B., 2001. Microbiological quality of Razor clam (ambal) and Water of their Growing Area. Proceedings of the Regional Conference on Natural Resources and Environmental Management, 18-20 October 2001, Kuching, Sarawak. Cheggour, M., Chafik, A., Langston, W.J., Burt, G.R., Benbrahim, S. and Texier, H., 2001. Metals in Sediments and the Edible Cockle Cerastoderma edule from two Moroccon Atlantic lagoons: Moulay Bou Selham and Sidi Moussa. Environmental Pollution 115, 149-160. Avelar, W. E. P., Mantelatto, F. L. M., Tomazelli, A. C., Silva, D. M. L., Shuhama, T. and Lopes, J. L. C., 2000. The Marine Mussel Perna Perna (Molluscsa, Bivalvia, Mytilidae) as an Indicator of Contamination of Heavy Metals in the Ubatuba Bay, Sao Paulo, Brazil. Water, Air and Soil Pollution 118, 65-72. Wagner, A. and Boman, J., 2004. Biomonitoring of Trace Elements in Vietnamese Freshwater Mussels. Spectrochimica Acta Part B: Atomic Spectroscopy 59, 1125-1132.

4.

5.

6.

7.

8.

9.

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PERTUKARAN FASA ZEOLIT ASLI KE FASA ZEOLIT SINTETIK YANG DICIRIKAN OLEH XRD BAGI MENGHASILKAN BAHAN PENUKAR ION Zainab Ramli*, Dewi Jamaliah Kamsiar dan Hasidah Mohd Arsat Jabatan Kimia, Fakulti Sains,Universiti Teknologi Malaysia, 81310 UTM Skudai, Johor, Malaysia. *e-mail: zainab@kimia.fs.utm.my Abstrak- Dalam kajian ini, modernit asli diubahsuai kepada zeolit yang rendah nisbah Si/Al untuk mendapatkan sifat penukar ion yang lebih baik. Pengubahsuaian dilakukan secara pemanasan hidroterma pada suhu 100˚C dan pada julat masa 0 hingga 24 jam. Hasil yang diperolehi dicirikan menggunakan kaedah XRD dan spekstroslopi Inframerah. Keputusan pencirian mendapati fasa hablur yang terhasil ialah campuran zeolit X dan P. Zeolit P lebih dominan untuk pemanasan melebihi 6 jam manakala zeolit X yang lebih tulin terbentuk pada pemanasan 6 jam. Keupayaan penukar ion sampel zeolit asli dan sampel pada pemanasan 6 jam dan 24 jam menggunakan ion Ca2+ sebagai kation contoh memberikan hasil penukaran ion sampel 24 jam (83.57%) diikuti dengan sampel pad 6jam (72.50%) dan yang paling rendah zeolit asli (69.45%). Ini menunjukkan sampel yang mengandungi zeolit P mempunyai kapasiti penukar ion Ca2+ paling baik iaitu sebanyak 21 mg Ca2+/g zeolit, peningkatan sebanyak 23% berbanding zeolit asli. Katakunci: Zeolit Asli, Penukar Ion, Zeolit X, Zeolit P Abstract In this study, natural mordenite was modified to other zeolites phases having low Si/Al in order to increase the cation exchange capacity of the material. Modification was carried out hydrothermally at 100oC in time range between 0 to 24 hours. The samples obtained were characterized by XRD and infrared spectroscopy. Results showed that a mixture of zeolite X and P were formed zeolite X was the dominant zeolite at 6 hrs heating time while zeolite P were dominant after 6hrs. Ion Exchange capacity of natural mordenite, samples at 6hr and 24 hrs heating, performed using Ca2+ cation gave cation exchange in the decreasing order of 83.57% , 72.50%, 69.45% for sample 24 hrs, 6hrs and natural mordenite respectively. It indicates that sample having zeolite P phase is the best cation exchange capacity with 21 mg Ca2+/g zeolite with an increased of 23% capacity compared to natural zeolite. Keywords: Natural zeolite, ion exchange, zeolite X, zeolite Y

Pengenalan Zeolit asli jenis mordenit merupakan ahli kumpulan zeolit mineral dengan formula kimianya (Ca,Na2,K2)Al2Si10O24 [1] Kegunaan umum mordenit asli adalah dalam rawatan air berammonia dan sebagai baja dimana zeolit berfungsi sebagai penukar ion [2-4]. Mordenit asli jarang digunakan bagi penghasilan zeolit lain tetapi penggunaannya masih boleh dikembangkan. Berasaskan kandungan utama sebatian yang terdapat dalam zeolit asli iaitu silika dan alumina, ia berkemungkinan diubah kepada fasa zeolit lain, dengan mengubah nisbah oksida bahan mula bagi mensintesis zeolit yang dikehandaki. De las Pozas et al [5] telah melaporkan pertukaran klinoptilolit asli kepada fasa zeolit Y dan P manakala Shan Wan et al [6] telah berjaya menyediakan MCM-41 mesoporous daripada mordenit bagi tujuan permangkinan. Abu terbang yang juga mengandungi campuran silika dan alumina sebagai bahan utama telah berjaya menghasilkan fasa zeolit P [7-9] telah berjaya menghasilkan berbagai zeolit yang mempunyai kandungan Si/Al yang rendah. Mordenit asli yang didapatkan daripada lapangan biasanya tidak tulen dan mengandungi oksida logam lain sehingga merendahkan keupayaan penukarionannya. Oleh itu dalam kajian ini mordenit asli akan cuba diubahsuai kepada fasa zeolit lain terutama zeolit yang mempunyai nisbah

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Si/Al yang lebih rendah seperti zeolit A, X, Y dan P untuk meningkatkan kapasiti penukarionan zeolit asli tersebut. Eksperimen Pengubahsuaian zeolit asli secara hidroterma Dalam sintesis ini komposisi oksida yang digunakan adalah 6 NaO2 : Al2O3 : 8 SiO2 : 112 H2O. Mordenit (5.0 g) dan RHA (3.5 g) dimasukkan ke dalam botol teflon. NaAlO2 (1.6 g) dan NaOH (5.4 g) dilarutkan dalam 40 mL air suling dan larutan aluminat ini dimasukkan ke dalam botol teflon yang mengandungi mordenit asli dan RHA tadi.. Campuran diaduk semalaman untuk menghomogenkan dan seterusnya dipanaskan dalam ketuhar pada suhu 96-100 ºC. Pemanasan dilakukan pada julat masa tertentu antara 0-24 jam. Pepejal yang terhasil pada setiap waktu pemanasan dituras dan dicuci dengan air suling sehingga pH air basuhan antara 7 - 10. Hasil sintesis dikeringkan semalaman di dalam ketuhar pada suhu 100°C. Setelah kering hasil sintesis dicirikan dengan menggunakan XRD dan IR untuk mengesahkan jenis fasa zeolit yang terbentuk. Kapisiti Penukar Ion Kation Ca2+ digunakan sebagai model kation contoh untuk mengkaji kapisiti penukar ion hasil sintesis berbanding mordenit asli. Bagi penyediaan penukargantian ion ini, berat sampel, kepekatan dan isipadu larutan kalsium silikat yang digunakan adalah ditetapkan. Di dalam proses penukarionan ini sebanyak 0.2 g sampel dan 50 mL 100 ppm kalsium silikat digunakan. Analisis kepekatan ion Ca2+ dilakukan menggunakan kaedah fotometeri nyala Keputusan dan Perbincangan Perubahan Fasa Zeolit Asli Difraktogram semua sampel selepas sintesis pada masa pamanasan yang berlainan ditunjukkan dalam Rajah 1. Daripada difraktogram jelas menunjukkan terdapat perubahan fasa zeolit asli terhadap masa pemanasan. Sampel yang dipanaskan selama 1 jam menunjukkan kebanyakan puncak bagi fasa mordenit telah berubah kefasa baru. Kehadiran puncak pada sudut rendah 2θ 6.49°, adalah salah satu petunjuk atau ciri bagi pembentukan fasa zeolit X yang menunjukkan pembentukan keliangan besar pada sistem bingkaian fasa hablur baru. Sampel selepas pemanasan 3 jam telah mula bertukar menunjukkan pembentukan fasa zeolit X sebagai fasa utama dengan memberikan 5 puncak pembelauan yang mewakili puncak zeolit X dengan beberapa puncak fasa yang tidak boleh dikenalipasti. Sampel pada pemanasan selama 6 jam, memberikan pola pembelauan bagi fasa zeolit X yang paling utama. Puncak-puncak pembelauan tersebut adalah pada 2θ 10.07°, 15.50°, 17.57°, 20.17°, 24.40°, 26.74°, 28.61°, 31.14° dan 31.79°. Manakala puncak pembelauan 2θ 12.44° dikenal pasti adalah fasa zeolit P. Difraktogram pemanasan pada 9 jam, beberapa puncak bagi fasa zeolit P mula terbentuk dengan jelas yang masih bercampur dengan fasa zeolit X. Pemerhatian ini mencadangkan fasa zeolit X telah mula berubah bentuk kepada fasa zeolit P yang lebih stabil secara termodinamik. Keputusan ini juga menunjukkan yang fasa zeolit X adalah metastabil yang mengalami penyusunan semula bingkaian melalui pemelarutan semula bingkaian zeolit X dan pembentukan nuklius zeolit P. Puncak pembelauan utama zeolit P ditunjukkan pada 12.50°, 17.74° dan 21.70° dan 28.06. Pertukaran fasa zeolit X berlaku sepenuhnya kepada fasa zeolit P seperti yang ditunjukkan oleh difraktogram bagi sampel pada pemanasan 12 jam. Puncak pembelauan fasa zeolit P adalah 2θ 12.49°, 17.67°, 28.08° dan 33.42° dengan satu puncak yang tidak dapat dikenalpasti terdapat pada 21.66°. Kestabilan zeolit P terhadap masa pemanasan dibuktikan oleh sampel pada pemanasan 24 jam sudut pembelauan 12.49°, 17.64°, 21.65°, 28.06° dan 33.35° bagi zeolit P yang tulen. Rajah 2 memperlihatkan graf ringkasan kepada perubahan fasa zeolit asli terhadap masa yang menunjukkan zeolit X adalah fasa yang lebih dominan pada pemanasan selama 6 jam dan zeolit P pada pemanasan lmelebihi 6 jam

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

Dalam spektrum inframerah pada Rajah 3 menunjukkan pola regangan yang biasa bagi zeolit modernit, zeolit X (sampel 6 jam) dan zeolit P. Jika diperhatikan dengan lebih teliti, sampel pada masa 6 jam ada menunjukkan sedikit kehadiran zeolit P (ditanda * dalam spektrum). Zeolit asli memberikan tiga getaran utama pada 1011.6 cm-1, 607.5 cm-1 dan 437 cm-1 yang berkaitan dengan regangan asimetri TO4, regangan simetri TO4 dan bengkokan T-O. Sampel hasil sintesis pada 6 jam dan 24 jam menunjukkan regangan asimetri TO4 telah beranjak ke frekuensi lebih rendah, 1002.0 cm1 . Anjakan ke frekunsi rendah menunjukkan kebanyakan ikatan Si-O-Si dalam sampel zeolit asli telah bertukar membentuk ikatan Si-O-Al [10]. Ini disebabkan sampel ini mempunyai ikatan Al-O yang lebih panjang berbanding ikatan Si-O. Ini membuktikan zeolit yang lebih rendah nisbah Si/Al telah terbentuk. Penyusutan nombor gelombang daripada zeolit X dan seterusnya zeolit P menunjukkan
400
300
200
100

Zeolite asli

0

5

10

20

30

40

50

300

x
200

1 jam
m

100

0

2

10

20

30

40

50

400
300
200
100
0 2

X X

P

X * * X X
20

P

3 jam
* X
30 40 50

*

10

500

x

Kiraan/saat

400
)

300
200
100
0 5
600

X

P

X

X

X
20

X

X

X

X

6 jam
X

10

30

40

50

500
L in (

X * P X P P X
20

400

P X
30

9 jam
P

300

200

100

X
10

*

02
400
300
200
100
0 5

40

50

P P

P

P P

12 jam
20

10

30

40

50

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

700
600
500
400
300
200
100
0

P

P
P
5

P

P

24 jam
40

10

20

30

50

2-theta (degree) Rajah 1 : Difraktogram bagi sample zeolit asli yang disintesis pada masa yang berlainan

Zeolite asli 100%
Kehabluran

Zeolite X

Gismondine Zeolite NaP

100%

0

3

6

9

12

15

18

21

24

Masa(jam)
Rajah 2. Ringkasan perubahan fasa zeolit asli terhadap masa pemanasan

Zeolit asli
% Transmittance

6 jam * 24 jam

* regangan zeolit P
1600 1400 1200 1000 800 600 400

Wave number (1/cm)

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

Rajah 3 : Spektrum Inframerah mordenit asli dan hasil sintisis pada 6 jam dan 24 jam

fasa zeolit X adalah dalam keadaan metastabil dalam medium sel beralkali dan mudah bertukar kepada fasa yang lebih stabil iaitu zeolit P.

Keupayaan Penukar Ion Zeolit asli dan zeolit yang disintesis secara amnya adalah dalam bentuk natrium, Na-zeolit. Proses penukarion yang berlaku adalah ion kation Na+ dalam zeolit dengan ion yang ingin ditukar ganti. Persamaan penukar ion diberikan oleh persaman dibawah. 2 Na-zeolit (p) + Ca2+(ak) Ca-zeolit (p) + 2Na+(ak)

Sampel hasil sintesis pada masa 6 jam (fasa utama zeolit X) dan 24 jam(fasa zeolit P) bersama zeolit asli dilakukan pertukaran ion dengan ion Ca2+. Keupayaan penukar ion setiap sampel ditunjukkan dalam Rajah 4. Secara am proses penukaran ion berlaku dengan pantas dengan semua sampel dapat melakukan penukaran ion sebanyak 50% dalam masa 15 minit. Keputusan menunjukkan yang kapasiti penukar ion adalah berkadar langsung dengan masa. Semua sampel selepas pengubahsuaian memberikan keupayaan penukarion sebanyak > 55% dalam masa 15 minit manakala zeolit asli memberikan 53%. Sampel pada masa 24 jam yang merupakan sampel zeolite P menunjukkan keupayaan penukar ion paling baik dengan peratus penukar ion sebanyak 84 % pada masa pengadukan 60 minit. Seterusnya kapasiti penukar ion bagi setiap sampel adalah zeolit asli, sampel pada masa 6 jam dan sampel pada masa 24 jam ialah 17, 18 dan 21 mg Ca2+/g zeolit. Dalam hal ini peningkatan sebanyak 23 % kapasiti penukar ion zeolit yang disentisis berbanding sampel zeolite asli.

90 80 70 60 50 40 30 20 10 0 0

% penukar ion

zeolit asli

sampel 6 jam

sampel 24 jam

20

40

60

Masa (minit) Rajah 4 : Graf kapasiti penukar ion Ca2+ bagi sampel zeolit asli, sampel pada 6 jam dan 24 jam

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Kesimpulan Dalam kajian ini, mordenit asli telah berjaya ditukarkan kepada fasa zeolit lain iaitu zeolit X dan zeolit P. Zeolit X adalah merupakan zeolit yang metastabil dan berubah kepada zeolit P yang lebih stabil pada pemanasan yang melebihi 6 jam. Zeolit P didapati merupakan zeolit dominan untuk pemanasan melebihi 6 jam. Keupayaan penukar ion sampel dengan fasa zeolit P menunjukkan sifat penambahan sifat penukar ion dengan peningkatan % penukar ion sebanyak 23% berbanding zeolit mordenit asli. Penghargaan Penghargaan ditujukan kepada MOSTI di atas pembiayaan projek dibawah IRPA. No Projek 09-0206-0057 SR0005/09-03. Rujukan 1. 2 L. B. Sand and F. A. Mumpton (1978), “Natural Zeolites, Occurrence, Properties, Use”, Tucson : Pergamon R. Moreno-Tost, J. Santamaría-González, E. Rodríguez-Castellón, A. Jiménez-López, M. A. Autié, E. González, M C. Glacial and C. De las Pozas (2004 )Selective catalytic reduction of nitric oxide by ammonia over Cu-exchanged Cuban natural zeolites, Applied Catalysis B: Environmental 50, 279-288. L.R. Weatherley and N.D. Miladinovic (2004) Comparison of the ion exchange uptake of ammonium ion onto New Zealand clinoptilolite and mordenite Water Research, 20, 43054312 Marlene Seijó Echevarría, Ruben Del Tóro Déniz, Eugenio Martinez Castellanos, Gerard A. Sherbakov, Juan Jose Rodriguez Moya (1997) “Uses of natural zeolite in the removal of Pb2+ from contaminated water” Eclética Química, 22. Carlos de las Pozas, David Diaz Quintanilla,Joaquin Perez-Pariente, Rolando RoqueMalherbe and M. Magi (1989) Hydrothermal transformation of natural clinoptilolite to zeolites Y and P1: Influence of the Na, K content’ Zeolites, 9, 33-39 Shan Wang, Tao Dou, Yuping Li, Ying Zhang, Xiaofeng Li and Zichun Yan (2004) “Synthesis, characterization, and catalytic properties of stable mesoporous molecular sieve MCM-41 prepared from zeolite mordenite” J. Solid State Chem. 177, 4800-4805. Miki Inada, Yukari Eguchi, Naoya Enomoto and Junichi Hojo, (2005) Synthesis of zeolite from coal fly ashes with different silica–alumina composition Fuel, 84, 299-304. Hidekazu Tanaka, Yasuhiko Sakai and Ryozi Hino (2002) Formation of Na-A and -X zeolites from waste solutions in conversion of coal fly ash to zeolites’ Material Research Bulletin, 37, 1873-1884 Nor Idah Taib (1999) Sintesis Zeolit Daripada Abu Terbang, Tesis Projek Sarjana Muda. UTM E.M. Flanigen, H. A Szymanski dan H.Khatami(1971), Infrared Structural Studies of Zeolite Framework., Adv. Chem.Ser., 201-229

3. 4. 5. 6 7. 8. 9. 10

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DILUTE SOLUTION PROPERTIES OF BRANCHED POLYSTYRENE Maimunah Sokro*, Arba’at Hassan, Hanita Othman
*

Pusat Pengajian Sains Kimia dan Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia. email: mysukro@pkrisc.cc.ukm.my

Abstract: The dilute solution properties of polystyrenes are reported for osmotic second virial coefficients and for intrinsic viscosities in three common organic solvents. As observe for other branched polymers, branching decreases the second virial coefficient in good solvents and lowers the theta temperature for a polymer–solvent system. For linear polystyrene in methyl-cyclohexane, the theta temperature is 36 ± 2°C. A correspondence between intrinsic viscosity and second virial coefficient, valid for hard-spheres solutions, holds in good solvents; this correspondence improves with decreasing branch molecular weight. The osmotic-pressure data are interpreted with a colloidlike thermodynamic framework using a van der Waals-type equation of state. The reference state is the hard sphere and the perturbation is given by an attraction decaying with the sixth power of the center-to-center distance between polymers. The hard-sphere diameter is obtained from intrinsicviscosity data. Predicted and observed osmotic pressures are in good agreement. Abstrak: Virial koeffisen kedua tekanan osmosis dan kelikatan intrinsik larutan polistirena dalam tiga jenis pelarut organik dibincangkan. Percabangan menurunkan virial koeffisen kedua tekanan osmosis polimer dalam pelarut baik, dan menendahkan suhu theta system polimer-pelarut yang dikaji. Suhu theta polistirena dalam metal-sikloheksana adalah pada 36 ± 2°C. Hubungan antara kelikatan intrinsik dan virial koeffisen kedua bagi molekul berbentuk sfera dalam pelarut baik masih kekal, dan semakin bertambah baik dengan penurunan berat molekul cabangan polimer. Data tekanan osmosis dianalisis dengan menggunakan persamaan keadaan van der Waals. Rujukan keadaan polimer adalah sfera padu dan isipadu tak terusiknya dikira berasaskan jarak pusat-ke-pusat diantara molekul polimer. Garis pusat sfera padu dikira daripada data kelikatan intrinsic, dan didapati data eksperimen selari dengan data yang dikira dari teori persamaan keadaannya. Keywords: polystyrene; Osmotic second virial coefficient; Intrinsic viscosity; theta temperature

Introduction Several experimental studies suggest that branched polymers behave like hard spheres in dilute solution. Static and dynamic light-scattering experiments in toluene give the molecular-weight dependence of the translational diffusion coefficient, radius of gyration, and second virial coefficient; this dependence follow the behavior typical of hard spheres [1]. The molecular-weight independence of intrinsic-viscosity at fixed temperature in toluene and cyclohexane is also a typical feature of solutions of hard spheres [2]. In the same solvents, the hydrodynamic radius varies with the molecular weight raised to the 1/3 power, a typical characteristic of hard-sphere behavior [2]. Measurements in the semi dilute regime, however, showed a progressive structural stiffening effect as the branching density increases [3]. In concentrated polymer solutions, branching increases the sorption of poor solvents but the effect upon sorption of good solvents is negligible [4]. In dilute solutions, branched polymers present peculiar characteristics. The theta temperature (the temperature where the osmotic second virial coefficient is zero [1] for branched polystyrenes in cyclohexane is lower than that for the linear homolog [5]. There is evidence that the difference between the theta temperatures for linear and for branched polymers increases with decreasing branch molecular weight [2] in agreement with experimental evidence for other kinds of branched polymers [6].

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The purpose of this work is to investigate further the influence of polymer architecture on intermolecular interactions in dilute solutions. With a membrane osmometer, osmotic second virial coefficients, B22 were measured for linear and for branched polystyrenes in toluene (good solvent) and in cyclohexane (theta solvent), and for branched polystyrene in methyl-cyclohexane (poor solvent). Intrinsic viscosity measurements, reported here, in cyclohexane and methyl-cyclohexane provide information on the ‘coil-to-globule’ transition [7], and [8]. These measurements also provide the hydrodynamic radii for the polymers studied here. As suggested by previous studies, a perturbedhard-sphere model is suitable for calculating osmotic pressures [9], [3].. In the McMillan–Mayer [10] framework, the osmotic-pressure data were correlated with a colloid-like potential of mean force, using a hard sphere as reference [11] and a perturbation described by an attractive potential decaying with the sixth power of the center-to-center distance between polymer molecules. It is well established experimentally that the osmotic pressure of a solution containing a macromolecular solute is higher when the temperature differs higher from a critical solution temperature. Most osmotic pressure measurements have been carried out in dilute solutions with the aims to determine the relative molecular weight and the osmotic second virial coefficient, B22. A number of theories for the thermodynamic behaviour of dilute macromolecular solutions have been developed [12], [1], [13], [14] and [15]. The concentration, c2 dependence of the reduced osmotic pressure, π is expressed by Morawetz [13] and Rudin [14] as, (π/c2) = [(RT/M) + {(RTV1O)/(2M 2)}c2 + {(RT(V1O)2)/(2M 3 )}c22 + {RT(V1O)3/(2M 4)]c23 + …] .. (1) Where B22 = (V1O)/(2M2) is the osmotic second virial coefficient, C23 = (V1O)2/(2M3) is the osmotic third virial coefficient , and D24 = (V1O)3/(2M4) is the osmotic fourth virial coefficient. The osmotic pressures, π of the polymer solution are then expressed as; {(π/ RT)} = (1/M)c2 + B22 c22 + C23 c23 + D24 c24 + ……. …… (2) Where the solute concentration, c2 is expressed in grams per millilitre; V1o is the molar volume of pure solvent; M is number average molecular weight; R is the gas constant; and T is the absolute temperature. Table 1. Polymer characterization data Polymer PSL PSB-A PSB-B PSB-C PSB-D Whole sample M (g/mol) 53100 66700 727000 5030000 22300000 Number of branches MW/Mn 1.06 1.18 1.18 1.11 1.15 12 108 826 2900

Experimental Reagents Four samples of branched polystyrene, PSB and one sample of linear polystyrene, PS-L from Pressure Chemical Company, Pittsburgh, Pansyal was used in all measurements. Polymers characteristics are

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shown in Table 1. Solvents used were toluene (from Fisher Science), a good solvent for polystyrene, cyclohexane (from Fisher Science), a theta solvent, and methyl-cyclohexane (from Aldrich), a poor solvent. Solvent purity was at least 99%. Solvents were used as received, while polymers were kept under vacuum for a few days before the osmotic-pressure measurements to ensure the absence of lowmolecular-weight impurities. The cellulose-acetate membrane with molecular-weight of 20,000 g/mol was found suitable. Table 2. Experimental results from osmotic pressure, π measurements on polystyrene c2 (g/l) in Toluene 38.6oC 47.7oC 0.2619 0.2843 0.3409 0.3693 0.4073 0.4405 0.5162 0.5570 0.6666 0.7174 0.7510 0.8071 0.8925 0.9571 1.0125 1.0840 1.0951 1.1712 1.2709 1.3564 1.3824 1.4737 π PSB-A in (KPa) in cyclohexane in methyl-cyclohexane o 32.2 C 32.6oC 41.6oC 0.2528 0.2378 0.2435 0.3232 0.2982 0.3116 0.3805 0.3467 0.3674 0.4713 0.4220 0.4567 0.5910 0.5187 0.5759 0.6556 0.5698 0.6409 0.7606 0.6510 0.7475 0.8467 0.7159 0.8357 0.9044 0.7588 0.8953 1.0240 0.8456 1.0197 1.0976 0.8979 1.0970

6.38 8.09 9.47 11.64 14.47 15.99 18.42 20.39 21.71 24.41 26.05

Procedure Osmotic pressure Osmotic pressures of polymer solutions were measured with a Jupiter Instrument Company Membrane Osmometer model 231 (Jupiter, FL, USA). Samples PSB-A and PSB-B were measured in toluene and in cyclohexane. PSB-A were also measured in methyl-cyclohexane. Osmotic pressure is a colligative property and a function of the number of molecules in solution. The osmotic pressure, π of polystyrenes, PSB and PSL solutions was measured over the temperature range of 5 to 50°C at concentrations from 2 to 30 g/dl. Measured osmotic pressures, π are given in Table 2, Figure 1, and Figure 2.

Viscometry Intrinsic-viscosity measurements were performed with a standard viscometer of the Ubbelohde type. The temperature was maintained constant within ±0.2°C using a water bath. [η] for PSB-A, PSB-B, PSB-C, and PSB-D polymers in cyclohexane, and for PSB-A in methyl-cyclohexane, were measured at different temperatures, to observe the coil-to-globule transition near the theta temperature. The molecular weight measurements for branched polystyrene, PSB-A in methyl-cyclohexane were measured at 32.6 and at 41.6°C. Molecular-weight data for branched polystyrene, PSB-A obtained by light-scattering experiments in cyclohexane, reported else where [16].

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1.5

1.0 π (KPa) 0.5 0.0 0.0 4.0 8.0 12.0 16.0 20.0 24.0 28.0 c 2 (g/liter)
Fig. 1. Osmotic pressures for polystyrene PSB-A in different solvents. in toluene at 38.6°C, in toluene at 47.7°C, in cyclohexane at 32.2°C, in methyl-cyclohexane at 32.6°C, and in methyl-cyclohexane at 41.6°C. Result From the customary virial expansion of the osmotic pressure π as a function of polymer concentration, c2 we obtain the osmotic second virial coefficient, B22 [16] (Figure 1 and 2). B22 is positive for a polymer in a good solvent, negative for a polymer in a poor solvent and zero at the theta temperature [1] and [17]. Results for the osmotic second virial coefficient, B22 are listed in Table 3. The results observed in toluene and cyclohexane generally agree with those obtained with light scattering [16]. The observed differences may be due to the higher concentrations required to measure osmotic pressures: small contributions from three-body interactions may be reflected in the data from osmometry. The experimental number-average molecular weights from membrane osmometry are in good agreement with the polymer specifications given in Table 1 for the branched polystyrene of PSB-A. In a good solvent, B22 is lower for branched polymers than that for linear homologs. Branching lowers the theta temperature for a branched polymer in a solvent, when compared to that of the linear homolog. By interpolation, osmotic-pressure data for branched polystyrene PSB-A in methylcyclohexane at two different temperatures yield a theta temperature of 36 ± 2°C. The theta temperature for linear polystyrene, PSL in methyl-cyclohexane lies between 60 and 70°C [18]. At 32.2°C, the osmotic second virial coefficient for branched polystyrene PSB-A in cyclohexane is positive, indicating that the theta temperature for branched polystyrene, PSB-A in cyclohexane is lower than that for the linear homolog (34.5°C) [19]. No additional experiments were performed to determine the theta temperature, already known for a similar polymer–solvent system [2[.

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0.16

0.12 π (KPa)

0.08

0.04

0 0 4 8 12 16 c 2 (g/liter)
Fig. 2. Osmotic pressures for polystyrene PSB-B; in toluene at 38.5°C, in toluene at 47.8°C, in cyclohexane at 32.2°C, and ▲ in cyclohexane at 43.0°C. The intrinsic viscosity, [η], for long polymer chains is a function of the radius of gyration according to [25]. [η] = {Φ(<S2>)3/2}/MV ….. (3) Where Φ, <S2>, Mv are, respectively, Flory's viscosity factor [1], the min square radius of gyration squared, and the polymer viscosity-average molecular weight [19]. The factor Φ is essentially constant for a given polymer architecture [20]. A linear polymer contracts to a compact form when the solvent quality decreases as, for example, when lowering the temperature [21]. For high-molecularweight linear chains, the hydrodynamic-radius contraction associated with a coil-to-globule transition is significant. Table 3. Number-average molecular weight Mn, second osmotic virial coefficient, B22 Radii, R, from intrinsic viscosity, and H. Polymer PSL PSB-A PSB-A PSB-B PSB-B PSB-A PSB-B PSB-B PSB-A PSB-A solvent Toluene Toluene Toluene Toluene Toluene cyclohexane cyclohexane cyclohexane Methyl-cyclohexane Methyl-cyclohexane T(oC) 49.2 38.6 47.7 38.5 47.8 32.2 32.2 43.0 32.6 41.6 Mn(103 g/mol) 59.5 ± 1.4 68.1 ± 0.2 64.5 ± 1.2 940 ± 60 800 ± 20 61.8 ± 0.8 592 ± 4 787 ± 3 65.7 ± 1.2 67.6 ± 0.8 B22 (10-4 cm3 mol g-2) 2.6 ± 0.2 2.715 ± 0.03 2.16 ± 0.17 0.148 ± 0.03 0.15 ± 0.03 0.32 ± 0.10 0.060 ± 0.003 0.20 ± 0.04 - 0.68 ± 0.10 0.49 ± 0.08 R (Ǻ) 48 48 112 112 47.4 102.6 103.3 45 46 H (kBT) 0.526 0.314 0.949 0.547 0.835 0.045 0.270 1.227 0.961

20

24

28

32

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Contraction is due to a collapse to very small dimensions within a narrow temperature region below the theta temperature, and a slight expansion at higher temperatures [22] and [23]. However, for lowmolecular-weight chains, the coil-to-globule transition does not imply a significant collapse. Because of the compact structure that characterizes branched polymers made by low-molecular-weight branches [2], their coil-to-globule transition likely resembles that of low-molecular-weight chains.

0.01 0.009
[η] (Liter/g)

0.008 0.007 0.006 0.005 0.004 0 5 10 15 20 25 30 35 40 45 Temperature, T (oC)

Fig. 3. Intrinsic viscosity, [η] as a function of temperature for branched polystyrenes. = Polystyrene PSB-A in methyl-cyclohexane, = Polystyrene PSB-A in cyclohexane, = Polystyrene PSB-B in cyclohexane, = Polystyrene PSB-C in cyclohexane, = Polystyrene PSB-D in cyclohexane. Figure 3 shows intrinsic viscosity, [η] as a function of temperature. For sample polystyrene PSB-A, in cyclohexane, their intrinsic viscosity, [η] does not depend significantly on temperature, while polystyrene PSB-A, in methyl-cyclohexane, there is a strong dependence. The data suggest that the polystyrene PSB-A sample does not display a coil-to-globule transition in cyclohexane, although [η] seems to decrease below 10 –15°C, in agreement with a theta temperature lower than that for linear polystyrene in the same solvent. For a polymer similar to PSB-A, Gauthier and coworkers found a theta temperature near 15°C [2]. Because the coil-to-globule transition occurs at temperatures close to the theta temperature, the intrinsic-viscosity data in methyl-cyclohexane agree with the theta temperature determined by membrane osmometry for this polymer – solvent system. For the polystyrene PSB-B sample in cyclohexane, [η] decreases at temperatures below 25°C, suggesting a coil-to-globule transition. For the polystyrene PSB-C sample in cyclohexane, [η] is almost constant with temperature; therefore, there is no evidence of a coil-to-globule transition, possibly because of the globular structure developed upon building the PSB-B molecules from the PSB-A homolog. This polymer may present a compact, globular, very dense morphology at every temperature in the range studied. Further shrinking becomes unfeasible due to steric effects. For the polystyrene PSB-D sample in cyclohexane, [η] decreases below 33°C, suggesting a transition in

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cyclohexane that very close to the theta temperature of linear polystyrene in the same solvent. PSB-D sample polymers should present a denser structure than PSB-C, this experimental evidence is contrary to the general observation that branching lowers the theta temperature for a given polymer–solvent system.

Discussion For a dispersion of hard spheres, the Einstein equation relates intrinsic viscosity to hydrodynamic radius RH as; [η] = (10π/3)(NA)(R3H/M) ….. (4) where NA and M are Avogadro's number and the mass of the sphere. For rigid-sphere molecules with negligible attractive interactions, it is possible to obtain an ‘effective’ radius RT from B22 [24]. Using B22 = (16π/3)(NA){(R3T)/M2} ….. (5) For monodisperse spheres of uniform density, the hydrodynamic radius is equal to the effective radius [6]. The morphology of a hyperbranched polymer, like an branched polymer, is similar to that of a hard sphere [3] and [4]. Figure 4 shows the second virial coefficient, B22 measured by membrane osmometry or by light scattering as a function of molecular weight, together with B22 calculated from intrinsic-viscosity data in toluene. The polymer weight-average molecular weight was used for M. The symbols are larger than the experimental uncertainty. Because intermolecular attractions are neglected, we cannot expect to obtain a good estimate of B22 by combining Equation (4) and (5). However, the correspondence for branched polymers of the same sample in a good solvent is reasonable and improves with decreasing branch molecular weight. Therefore the correspondence improves with increasing spherical morphology of the polymer in solution. In a poor solvent, cyclohexane, the correspondence remains generally valid, but only at temperatures higher than the theta temperature, because the relation between [η] and B22 fails at the theta temperature and Equation (5) holds only for positive second virial coefficients.

Osmotic pressure from a theoretical equation of state Static and dynamic light-scattering experiments, intrinsic-viscosity measurements at constant temperature [4], and other experimental evidence suggest that branched polymers in dilute solutions can be viewed as essentially spherical macromolecules. [25]. Therefore their thermodynamic properties in solution may be described with a colloid-like framework. The osmotic-pressure data are readily reproduced with a van der Waals-type equation of state. The reference state is the hard-sphere system [11]; the perturbation is given by an attractive potential energy that scales with the sixth power of the inverse distance between polymers r {π/ρkT} = {(1 + η + η2 - η3)/(1 - η)3} + {2πρ ∫∞{1 – exp [-(H/kT)(σ/r)6]} r2 dr } (6)

In Eq.(6), π, ρ, η, and σ, are respectively, the osmotic pressure, the polymer number density, the packing fraction (η = (π/6)ρσ3) and the hard-sphere diameter; H represents the long-range attractive energy parameter. The parameters used to fit Eq. (6) to the experimental data are listed in Table 2. Because the hydrodynamic radius is obtained from [η], the only adjustable parameter is H; our results for H are similar to Hamaker constants reported for polymer solutions [25]. The fitted osmotic pressures are compared for the PSB-A (Fig. 1) and PSB-B (Fig.2) polymers in different solvents. In all cases fits are reasonable, confirming the colloid-like nature of these dissolved polymers.

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35 B22 (x 10-5 cm3 mole/g2)

25

15

5

-5 0 1 2 3 4 5 M (x 105 g/mole)
Fig. 4. B22 for branched polystyrenes in toluene as a function of molecular weight. = are for polystyrene PSB-A for experimental data and prediction from Eq. (5) respectively. , = are for polystyrene PSB-C for experimental data and prediction from Eq. (5) respectively. , = B22 measured in this work for experimental data and prediction from Eq.(5) respectively.

,

Conclusions The osmotic second virial coefficients B22 are reported for solutions of branched polystyrenes in toluene, cyclohexane and methyl-cyclohexane. In a good solvent, due to the smaller radius of gyration, B22 for a branched polymer is always lower than that for the homologous linear polymer. In a theta solvent, branching tends to increase solubility, lowering the theta temperature. For branched polystyrene, PSB-A in methyl-cyclohexane, the theta temperature is 36±2°C. Intrinsic-viscosity data were used to obtain polymer size and to estimate osmotic second virial coefficients. In good solvents, predicted and observed second virial coefficients agree well. Agreement improves with decreasing branch molecular weight. Because these polymers are considered to be essentially spherical macromolecules, the experimental osmotic pressures have been fitted with a molecularthermodynamic equation suitable for colloids. The good fits obtained provide further evidence for the perturbed-hard-sphere behavior of branched polymers in dilute solutions. Acknowledgements The author (MS) expressed her thank you and gratitude to Universiti Kebangsaan Malaysia for the financial support to enable her attending this seminar.

References [1]. Flory, P. J. (1953). Principles of polymer chemistry, Cornell University Press, Ithaca, NY. [2]. Gauthier, M., Li, W. and Tichagwa, L. (1997) Polymer 38, p. 6363.

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[3]. Gauthier, M., Möller, M. and Burchard, G., (1994) Macromol Symp 77, p. 43. [4]. Lieu, J. C., Prausnitz, J. M. and Gauthier, M. (2000) Polymer 41, p. 219. [5]. Gauthier, M., Chung, J., Choi, L. and Nguyen, T., T. (1998) J Phys Chem B 102, p. 3138. [6]. Grest, G. S., Fetters, L. J., Huang, J. S., Richter, D. (1996) In: Prigogine, I., Rice, S. A., editors. Advances in chemical physics, vol. 94, p. 67. [7]. Sun, S. F. and Fan, F. (1997) Polymer 38, p. 563. [8]. Baysal, B. M. and Uyanik, N. (1992) Polymer 33, p. 4798. [9]. Gauthier, M. and Möller, M., (1991) Macromolecules 24, p. 4548. [10]. McMillan, W. G. and J.E. Mayer, J. E. (1945) J Chem Phys 13, p. 276. [11]. Carnahan, N. F. and K.E. Starling, K. E. (1970) J Chem Phys 53, p. 600. [12]. Allan, P., W., (1959), Techniques of Polymer Characterization, Butterworth’s Scientific Pub., London. [13]. Morawetz, H., (1975), Macromolecules in Solution, John Wiley & Sons, New York. [14]. Rudin, A., (1982), The Elements of Polymer Science and Engineering, An Introductory Text for Engineers and Chemists, Academic Press. Inc. New York. [15]. van’t Hoff, J.H., (1887) Z Phys Chem 1), pp. 481–508. [16]. Maimunah Sokro (1980), Compatibility studies on oligomeric poly(dimethylsiloxanes). Unpublished doctoral thesis, University of Salford, United Kingdom. [17]. Tombs, M. P. and Peacocke, A. R. (1974) The osmotic pressure of biological macromolecules, Clarendon Press, Oxford. [18]. Elias, H. -G. In: Brandrup, J., Immergut, E. H. and Grulke, E. A., (1999) Editors, Polymer handbook (4th ed.), Wiley, New York. [19]. Young, R. J. and Lovell, P. A. (1991). Introduction to polymers (2nd ed.),Chapman and Hall, London). [20]. Roovers, J., Hadjichristidis, N. and Fetters, L. J. Macromolecules 16, p. 214. [21]. Bauer, D. R. and Ullman, R.(1980), Macromolecules 13 p. 392. [22]. Wang, X., Qiu, X. and Wu, C.(1998) Macromolecules 31, p. 2972. [23]. Wang, X. and Wu, C.(1999) Macromolecules 32, p. 4299. [24]. Yamakawa, H. (1971) Modern theory of polymer solutions, Harper and Row, New York. [25]. Hempenius, M. A., Zoetelief, W. F.,Gauthier, M., and Möller, M.(1998) Macromolecules 31, p. 2299.

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EXTRACTION OF CONDENSED TANNINS FROM MANGROVE BARKS Maimunah Sokro*, Liew Siew Har, Liew Han Woon, Arba’at Hassan, Hanita Othman *Pusat Pengajian Sains Kimia dan Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia. email: mysukro@pkrisc.cc.ukm.my Abstract: The condensed tannins of mangrove ‘kurap’ or Rhizophora mucronata and mangrove ‘minyak’ or Rhizophora apiculata were extracted using water, methanol or acetone at different concentrations with or without acidification. Among solvents tested, 70% acetone, containing 1% concentrated HCL, extracted a maximum amount of condensed tannins from mangrove bark. Rhizophora apiculata contained (11.0 %) lower amount of condensed tannin compared to Rhizophora mucronata (11.85 %). Dried leaves of Rhizophora mucronata contained the lowest amounts of condensed tannins (2.3 %) compered to all other parts of the plant. The percentage of the condensed tannins extracted depend on the age of the mangrove tree, the type and percentage of the solvent used, as well as the type and the concentration of the acid used, and the condition of keeping the extracted tannins aliquot. Abstrak: Tannin daripada kulit bakau ‘kurap’, ‘Rhizophora mucronata’ dan bakau ‘minyak’, ‘Rhizophora apiculata’ diekstrak dengan menggunakan air, metanol, atau aseton pada kepekatan yang berbeza dengan atau tanpa asid. 70% aseton yang mengandungi 1% HCl merupakan pelarut yang paling optimum. Kandungan tannin pada Rhizophora apiculata (11 %) lebih rendah jika dibandingkan dengan kandungan dalam kulit Rhizophora mucronata (11.85 %). Peratus kandungan tannin pada daun kering Rhizophora mucronata paling rendah. Peratus kandungan tannin yang diekstrak bergantung kepada usia pokok, jenis dan peratus pelarut yang digunakan, dan juga jenis dan peratusan asid yang digunakan. Cara penyimpanan dan keadaan bahagian-bahagian pokok bakau itu sendiri akan menentukan peratus kandungan tanninnya. Keywords: tannins, mangrove tannins, Condensed tannins, extraction Introduction Mangrove trees contain phenolic substances, including tannins [1], [2]. Phenolics are secondary compounds widely distributed in the plant kingdom. Tannins are a special group of phenolics, with high molecular weight, that occur only in vascular plants such as flowers, fruits, seeds, leaves, stems, barks, and woods. The chemical diversity of tannins confers many kinds of reactions. Tannins (polyphenols) are produced via condensation of simple phenolics that are secondary metabolites and are widespread in the plant kingdom. Tannins do not constitute a unified chemical group, but have a variety of molecular structures. They are generally divided into hydrolysable (galloyl and hexahydroxydiphenoyl esters and their derivatives) and condensed proanthocyanidins or known as well as condensed tannins, CD (polymers of flavan-3-ols; [3]. Hydrolysable tannins are made up of sugars (primarily glucose) and gallic acids, while condensed tannins consists of oligomers and polymers of flavanoids. Tannins are biologically active compounds and may have beneficial or adverse nutritional effects and physiological consequences. Endogenous tannins protect unharvested seeds from attack by insects, birds and herbivores, as well as certain diseases and untimely germination [4]. Possible harmful effects of certain biological compounds, such as phenolics, trypsin inhibitors and phytates, have received considerable attention [5], [6], and [7]. These compounds occur naturally in the plant kingdom, as well as seeds of legumes and cereals and, if present in sufficient quantities, may lower nutritional value and biological availability of dietary proteins and minerals.

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Fig. 1. Molecular structures: (a) catechol (b) (-)-epicatechin Condensed tannins, CD which are also known as proanthocyanidins, PA are polymers of flavan-3-ol unit’s i.e.polyhydroxyflavan-3-ol (Fig. 1, Fig.2, Fig. 3 and Fig. 4). The properties of Condensed tannins depend on their structure in terms of monomer units (degree of hydroxylation and 2,3-cis- or 2,3-trans-stereochemistry), their degree of polymerization (DP) and the linkage-type between flavan3-ols (4–8 as shown in Fig. 3 and Fig. 4, or 4–6 branched structures as shown in Fig. 4) with a considerable range of structural variation. For the procyanidin, PC- type polymers the constituent flavan-3-ol units are either catechin (trans) or epicatechin (cis) with R = H (Fig. 2 and Fig. 3), while prodelphinidin, PD- type polymers contain either gallocatechin (trans) or epigallocatechin (cis) with R = OH (Fig. 2, Fig. 3 and Fig.4), and many polymers are found to be mixtures of the two classes.

Fig. 2. The basic repeating unit in condensed tannins. If R1 = R2 = OH, R3 = H, then the structure is that for (-)-epicatechin. The groups at R1 and R3 for other compounds are indicated below the structure. R2 = O - galloyl in the catechin gallates. The extractable condensed tannins, CT were estimated to have molecular weight, in the range 5300 – 5900 g/mole. The identity of the individual units that make up condensed tannins, CT polymer, and the length of the polymer chain can be estimated by subjecting the polymer to strong acid-catalyzed cleavage in the presence of phloroglucinol or benzyl mercaptan. These reactions result in the release of terminal units as free flavan-3-ols, whereas extender units are distinguished as benzylthioether adducts which are formed by nucleophilic capture of the carbocations generated under the acid conditions of the reaction. The average molecular weights of condensed tannins, CT determined by cleavage with phloroglucinol was in the range of 1900 – 2200 g/mole. Tannins are naturally-occurring uncrystallisable colloidal substances with pronounced astringent properties. Their main characteristic is that they bind and precipitate gelatin from solution and form

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insoluble compounds with gelatin-yielding tissue which is the property which enables them to convert raw hide and skin into leather, consolidating the dermal network of hide into firmer and drier structures of improved thermal stability, durability and water resistance. Because of their proteinbinding properties, tannins are of considerable importance in food processing. Tannins have been reported to exert other physiological effects; e.g., they can reduce blood pressure, accelerate blood clotting, decrease the serum lipid level, modulate immune-responses and produce liver necrosis. The dosage and kind of tannins are critical for these effects. Tannins in wine have potent antioxidance against low-density lipoprotein (LDL) of which the oxidized form are a precursor of coronary heart disease. Plant phenols can be considered a nuisance because they can complex proteins by hydrogen bonding.

Fig. 3. Model structure for condensed tannin. If R = H or OH then the structure represents a procyanidin or prodelphinidin. The 4→ 6 linkage (dotted line) is an alternative interflavan bond. The terminal unit is at the bottom of such a multi-unit structure. The objective of this study was to evaluate the effects of various solvent extraction systems on the recovery of mangrove tannins, and to determine the distribution of tannins and phenolic acids in mangrove bark. Experimental Mangrove bark The bark of mangrove ‘kurap’ or Rhizophora mucronata and mangrove ‘minyak’ or Rhizophora apiculata were collected from Port Dickson Beach, Negeri Sembilan, Malaysia in September of 2004. The bark and wood were separated manually. The total fresh weight and recovery of barks were recorded and samples used for moisture determination immediately after collecting. Samples were dried and then kept at room temperature for further study. The bark was ground using an IKA MF10

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WERKE grinder and subsequently sieved with a 60-mesh sieve and used immediately for subsequent analysis. Reagents The chemical standards were of the highest purity grade. Tannic acid, Gallic acid, catechin, vanillic acid, caffeic acid, p-coumaric acid, protocatechuic acid, ferulic acid, p-hydroxy-benzoic acid and phydroxy-benzaldehyde were all obtained from Sigma–Aldrich Chemie. Folin–Ciocalteau and rhodamine were also obtained from Sigma–Aldrich Chemie. Methanol, Na2HPO4, butanol, glacial acetic acid, ethyl acetate, hydrochloric acid, HCL and sodium sulphate was obtained from Syeterm.

Fig. 4. Thiolysis reaction of condensed tannin polymers with procyanidin (R = H) and/or prodelphinidin (R = OH) units. The trans-stereochemistry is associated with catechin and gallocatechin (not shown), while cis-stereochemistry is associated with epicatechin and epigallocatechin. All the terminal units in the polymer were released as flavan-3-ols, and the extender units as flavan-3-ol benzylthioethers.

Procedure Extraction of tannins Mangrove bark chips sample was extracted with different solvents as follows. A 2g mangrove bark together with 10 ml water (20%, w/v) were mixed and heated in a boiling water bath for 30 minutes, cooled then centrifuged and the supernatant collected in a clean flask. The procedure was repeated two more times and the combined extracts were evaporated using a rotary evaporator at 40 °C to almost dry and the residue was then dried in the vacuum oven at 40oC to constant weight. A 2 g mangrove bark chips together with 10 ml acidified water (1%, v/v, HCl in water) (20%, w/v) were mixed and heated in a boiling water bath for 30 minutes, cooled then and the supernatant collected in a clean flask. This procedure was repeated two more times and the combined extracts

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were evaporated using a rotary at 40oC to almost dry and residue was then dried in the vacuum oven at 40oC to constant weight. A 2 g mangrove bark chips were extracted three times with 10 ml (20%, w/v) absolute methanol, absolute acetone, 90, 80, 70, 60 and 50% methanol or 90, 80, 70, 60 and 50% acetone. In another experiment, 100, 90, 80, 70, 60 and 50% methanol, as well as similar acetone solutions, acidified with 1% concentrated HCl, respectively, were used as extraction solvents. Samples were cooled and then subsequently centrifuged and supernatants collected in a clean flask. This procedure was repeated two more times and combined extracts were evaporated using a rotary evaporator at 40 °C to almost dry and residue was then dried in vacuum oven at 40oC to constant weight. 2g of all samples of the dry residue was then dissolved in 25 ml absolute methanol for further analysis. Extraction of polyphenols Ground mangrove bark samples (2 g) were placed in a flask of 10 ml capacity on ice. Methanol– water, 1:1 v/v (10 ml), was added and the flasks were suspended on a shaker for a minute. The flasks were then centrifuged for 10 min. The supernatants were collected, filtered and stored in a deep freezer. The pigments were removed using diethyl ether in 1% acetic acid [8]. Extraction of phenolic acids The samples (2 g) of ground mangrove bark were extracted twice each with ethyl acetate (10 ml). The ethyl acetate was dried over sodium sulphate and then the sample was concentrated by rotary evaporation at 30 °C. The concentrates were transferred into small sample bottles and stored in a freezer for analysis. Determination of condensed tannins (vanillin’s assay) The condensed tannins were assayed colorimetrically by the method of Price, Van Scoyoc and Butler [9]. To 0.2–1 ml of methanolic solution of condensed tannins, 5 ml of 0.5% vanillin reagent were added; a 5 ml volume of 4% concentrated HCl in methanol was used as a blank. The absorbances of samples and blank were read at 500 nm after standing for 20 minutes at room temperature. Catechin (3.5 moles of water per mole of catechin) was used as a standard in these experiments. The content of condensed tannins in the mangrove bark was expressed as mg or g catechin equivalents per 100- g sample. Quantification of the polyphenol In methanol solution, the total phenolics were quantified using the Folin–Ciocalteau method, gallotannins by the rhodamine assay and flavanols by the vanillin assay. All the methods were adopted from Makkar [8]. Results and discussion In previous studies they have demonstrated that most of plant kingdoms served as an excellent source of condensed tannins, CT [8] and [10]. In the literature, different solvent systems have been used for extraction of condensed tannin, CT from plant materials as the extraction efficacy of condensed tannin, CT depends on their chemical nature, solvent system used and extraction conditions employed [11].. The crude extracts of condensed tannin, CT contain low-molecular-weight phenolics as well as condensed tannin, CT. The low- molecular-weight phenolics may include phenolic acids, as well as several subclasses of flavonoids [12]. In addition the extracted condensed tannin, CT consist of a series of oligomeric and polymeric compounds [1] that differ in their sensitivity toward the reagents used for their determination [11]. This makes the selection of appropriate methods for quantization of phenolics a difficult task. Recently, Merken and Beecher [12] reviewed High Pressure Liquid Chromatography, HPLC methodologies for measurement of food flavonoids. According to these

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authors the existing HPLC methods can only separate a limited number of flavonoids and other phenolics and a method for simultaneous determinations of all prominent flavonoids is still needed. The molecular composition of crude mangrove bark condensed tannin, CT extracts is still unknown and therefore it is difficult, based on available data, to select both appropriate standards and HPLC methodologies for separation and quantitation of phenolics involved. Since the aim of this study was to evaluate the effectiveness of solvent systems for extraction of condensed tannin, CT from mangrove bark, we selected the vanillin assay for quantification of condensed tannin, CT crude extract. This method is commonly used for quantification of condensed tannin, CT due to its specificity toward flavanols and dihydrochalcones [11]. Moreover, according to Oszmainski & Bourzeix [14], the vanillin method provides the most accurate estimate of the content of condensed tannin; CT. Methanol is usually used for carrying out the vanillin assay because, in methanol, the vanillin reaction is more sensitive toward polymeric condensed tannin, CT than monomeric flavanols [15]. However, it may still lead to over estimation of condensed tannin, CT content of crude extracts that are rich in monomeric components. The content of condensed tannins of mangrove bark in Rhizophora mucronata and Rhizophora apiculata was 12 % and 11.0% respectively (Table 2). The content of condensed tannins in Rhizophora mucronata and Rhizophora apiculata was almost the same. The content of condensed tannins in different plant parts of mangrove was significantly different (2 to 17%); the highest amount was present in fresh brownish bark (16.95%), followed by dark brown barks (12%), branches plus stems (9.0%), fresh green leaves (2.7%), and dried brown leaves (2.0%). The synthesis of tannins in different plant parts may depend on the metabolic rate of tannin synthesis at a particular site. Another reason may be higher polymerization of existing polyphenolic compounds in the bark to highmolecular-weight compounds during maturation. These results indicate that, as the maturity progressed, delocalization of condensed tannins from leaves to stems to branch to bark occurred and this was followed by their polymerization into high-molecular-weight compounds [16]. Several factors, such as plant type, cultivar, age of the plant or plant parts, stage of development, and environmental conditions, govern the tannin content in plants. The changes observed during development or maturation was mostly due to metabolism of polyphenolic compounds or polymerization of existing phenolic compounds. Table 1. Effect of different solvents on extraction of condensed tannins from mangrove barks solvent Water Water + HCl 100% Methanol 90% Methanol 80% Methanol 70% Methanol 100% acidified Methanol 90% acidified Methanol 80% acidified Methanol 70% acidified Methanol 100% acetone 90% acetone 80% acetone 70% acetone 100% acidified acetone 90% acidified acetone 80% acidified acetone 70% acidified acetone Rhizophora mucronata (%) 0.21 0.45 2.10 2.15 1.60 0.95 2.55 2.90 4.45 4.65 0.20 4.15 8.55 10.30 0.36 6.60 10.80 11.85 Rhizophora apiculata(%) 0.19 0.38 1.89 1.95 1.45 0.86 2.35 2.88 4.30 4.45 0.18 4.13 8.35 9.50 0.29 6.40 10.30 11.00

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The condensed tannins in different plant parts of mangrove tree were extracted into 70% acidified acetone; results are presented in Table 1. Mature bark had a higher tannin content than physiologically immature bark (light in weight, light green colour, relatively smaller size tree). Thus, as the maturity stage progressed, the concentration of condensed tannins increased. Fresh green leaves (2.7%), and dried brown leaves (2.0%) had a lower content of tannins than all other plant parts. This increase in tannin content may be due to a higher polymerization of existing polyphenolic compounds in the bark to high-molecular-weight compounds during maturation. Similar results and conditions have been reported by earlier workers for beans [6].

Table 2. Condensed tannins in different plant parts of mangrove bark Plant part Fresh green leaves Dried leaves Branches Fresh bark Dried bark Condensed tannins (g/100g barks) 2.75 2.30 9.20 16.95 11.78

Conslusion In conclusion, fresh dark brown mature bark contained higher amounts of tannins than their immature counterparts. Acidified acetone-water served as an efficient system for recovery of a maximum amount of condensed tannins from mangrove tree. Acknowledgements The author (MS) expressed her thank you and gratitude to Universiti Kebangsaan Malaysia for the financial support to enable her attending this seminar.

References [1]. Salunkhe, D. K., Chavan, J. K., & Kadam, S. S. (1990). Dietary tannins: consequences and remedies (pp. 29–76, 122–134). Boca Raton, FL: CRC Press. [2]. Salunkhe, D. K., Jadhav, S. J., Kadam, S. S., & Chavan, J. K., (1982), Chemical, biochemical and biological significance of polyphenols in cereals and legumes. CRC Critical Reviews in Food Science and Nutrition 17 (1982), pp. 277–305. [3]. Haslam, E., (1989). Plant polyphenols-vegetable tannins, Cambridge University Press, Cambridge. [4]. Hulse, J., H., (1979), Introduction and recommendations. In: J.H. Hulse, Editor, Polyphenols in cereals and legumes, Presented at the Annual Meeting of the Institute of Food Technologists, St. Louis, MO (1979), pp. 3–8.

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[5]. Deshpande, S., S., & Cheryan, M., (1985), Evaluation of vanillin assay for tannin analysis of dry beans. Journal of Food Science , 50, pp. 905–910. [6]. Deshpande, S., S., Sathe, S., K., and Salunkhe, D., K., (1984), Chemistry and saftey of plant polyphenols. In: M. Friedman, Editor, Nutritional and toxicologocal aspects of food saftey, Plenum Press, New York, NY, pp. 457–467. [7]. Reddy, N., R., Sathe, S., K., and Salunkhe, D., K., (1982), Phytates in legumes and cereals. Advances in Food Research 28, pp. 1–18. [8]. Makkar, H., P., S., Blummel, M., and Becker,. K., (1995) , In-vitro effect of and interactions between tannins and saponins and fate of tannins in the rumen. Journal of the Science of Food and Agriculture 69, pp. 481–493. [9]. Makkar, H., P., S., Blummel, M., Borowy, N., K., and Becker, K., (1993), Gravimetric determination of tannins and their correlations with chemical and protein precipitation methods. The Journal of the Science of Food and Agriculture 61, pp. 161–165. [10]. Shahidi, F., Chavan, U., Naczk, M., and Amarowicz, R., (2001), Nutrient Distribution and phenolic antioxidants in air-classified fractions of beach pea (Lathyrus maritimus, L.). Journal of Agricultural Food Chemistry 49, pp. 926–933. [11]. Shahidi, F., and Naczk, M., (1995), Food phenolics: sources, chemistry, effects and applications. Lancaster, Technomic Publishing Company. [12]. Merken, H., M., and Beecher, G., R., (2000), Measurement of food flavonoids by highperformance liquid chromatography: a review. Journal of Agricultural and Food Chemistry 48, pp. 577–599. [14]. Oszmianski, J., and Bourzeix, M., (1966), Comparison of methods for determining the content and polymerization of proanthocyanidins and catechins. Polish Journal of Food Nutrition and Science 5, pp. 43–50. [15]. Price, M., L., Van Scoyoc, S., and Butler, L., G., (1978), A critical evaluation of the vanillin reaction as an assay for tannin in sorghum grain. Journal of Agricultural and Food Chemistry 26, pp. 1214–1217. [16]. Price, M., L., Hagerman, A., E., and Butler, L., G., (1980), Tannin content of cowpeas, chickpeas, pigeonpeas and mung beans. Journal of Agricultural and Food Chemistry 28, pp. 459–461.

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THE PERCEPTIONS ON ENVIRONMENTAL ISSUES OF SELECTED RESIDENTS IN KUALA PERLIS Maimunah Sokro*, Arba’at Hassan, Hanita Othman *Pusat Pengajian Sains Kimia dan Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia. email: mysukro@pkrisc.cc.ukm.my Abstract: This is paper presents findings of a research study on the attitudes of selected residents of a fishing village in Kuala Perlis, Perlis related to environmental aspects. The environmental issues like supplies of clean water and electricity, management of sanitation and garbage disposal, level of education, status of employment, and standard of housing were investigated. Data were gathered through field observations, interviews, and written questionnaire surveys. The data were collected, then tabulated, organized, and analysed. Findings indicated that the (1) conditions of basic facilities like supplies of clean water and electricity, appropriateness management of garbage disposal, and housing standard were very poor, (2) residents’ level of sanitation and level of income were low, (3) social activities in the community were absent, and (4) the levels of education and knowledge concerning social and environmental awareness of this fisherman community were low. The study revealed implications on environmental education goals, i.e.: knowledge, awareness, skills and active participations related to the aspects in improving the quality and standard of living. Recommendations included the need to establish immediate actions on the betterment level and programs of education, health programs, improvement of basic facilities and infrastructure, re-organization of economic distribution, and recommendations for further detailed research. Abstrak: Kertaskerja ini membentangkan dapat kajian berkenaan sikap penduduk kampung nelayan Kuala Perlis tentang aspek-aspek alam sekitar. Isu berkaitan bekalan asas air bersih, elektrik, dan sampah sarap, aras pendidikan, status pekerjaan, dan kualiti perumahan telah dikaji. Data-data dikumpul melalui pemerhatian, temubual, dan soalselidik bertulis. Data tersebut kemudiannya disusun, diolah, dan dianalisis. Keputusan mendapati bahawa (1) tahap bekalan air bersih dan elektrik, pengurusan sampah sarap, dan kualiti perumahan penduduk adalah dhaif, (2) pengurusan pembuangan najis dan tahap pendapatan bulanan penduduk sangat rendah, (3) aktiviti kemasyarakatan tidak ada, dan (4) aras pendidikan dan pengetahuan penduduk kampung nelayan ini berkaitan alam sekitar tersangat rendah. Kajian mendapati implikasi berkaitan matlamat pendidikan alam sekitar seperti aras pengetahuan, kesedaran, kemahiran, dan penyertaan aktif untuk meningkatkan tahap kualiti dan aras hidup lebih tinggi adalah negatif. Cadangan-cadangan kajian termasuk keperluan mengadakan tindakan seperti meningkatkan tahap pendidikan, program berkaitan kesihatan, kemudahan bekalan asas dan lain-lain aspek yang berkaitan dengan meningkatkan kualiti taraf hidup mereka. Cadangan lain termasuk meneruskan kajian lanjutan berhubung pembentukan semula dalam membahagikan kemudahan infrastruktur dan menaiktaraf aras ekonomi penduduk. Keywords: environmental knowledge, awareness, clean water, garbage disposal, sanitation, environmental education goals

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Introduction Kuala Perlis is about 13 km distance from Kangar town (Fig. 1). It is a fishing village and port town, located at the estuarine of Sungai Perlis. There are about 10,000 people residing in this somewhat muddled but uniquely attractive town. Fishing is an industry and everything else related to it is a major item in the shops and warehouses. Kuala Perlis is noted for its local food, such as laksa or rice noodles dipped in spicy fish-soup with sliced onion and cucumbers. Kuala Perlis is also the second largest town in the state of Perlis and is the entry and exit point to Langkawi, as well as Phuket Island and other coastal towns in Southern Thailand. It is a good place to relax at dusk and watch a spectacular sunset or the fishermen returning with their catch. Majority of the Kuala Perlis residents are from low income families from fishing activities. The community is having major developmental and socio-economical problems due to unstable employment, low level of education, and absence of public health. Due to the low standard of education and environmental knowledge, the ignorance of the community has affected this residential area. The environment has become filthy and smelly, thus attention and immediate actions for development are needed. Objectives The objectives of studying Kuala Perlis village are to (i) evaluate socio-economic and environmental status, (ii) estimate the degree of problems, (iii) assist the policy-maker in identifying the abovementioned problems, and (iv) recommend possible solutions to minimize the problems.

Fig. 1: Map of Malaysia: Kuala Perlis, west of Kangar (study area)

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Fig. 2: Kuala Perlis Problems Several major problems afflicting the community are shortage supply for clean water and electricity, lack of proper sanitation, improper management of garbage disposal, less number of schools and low standard of education, employment, and housing facilities. i) Supply of Clean Water Kuala Perlis residents are very much fortunate to have proper water daily supply from the pipe line provided. However, small number of residents are still depending their daily water usage from the shallow man-made wells they constructed in the area which are not suitable for everyday health use (i.e.: for cooking and drinking). The physical properties of this well water are salty, brownish, and smelly. ii) Supply of Electricity Kampung Kuala Perlis is located about 13 km from Kangar, the state capital. The town is very fortunate to have good electricity supply provided by Tenaga Nasional Berhad (TNB). iii) Imroper Sanitation A minor problem that is faced by the community is sanitation. Proper places for sanitation or public toilets are not found publicly. This is because most of the residents built their toilets in their houses. However, couples stilt toilets outside their residential area can be seen along the beach or between bushes.

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iv) Management of Garbage Disposal Kuala Perlis residents are deprived of proper garbage disposal places. Most of the times, they dump their garbage in the sea, throw them in the surrounding ground, or anywhere they want without concern for the community’s health. This littering habit is in-fact dirty their own living area and the environment. vi) School and Education Neither school has been built in the study area of fisherman village in Kuala Perlis. Children of the village go to school at the adjacent area. Even though available schools in the vicinity are quite near the village but majority of the families cannot afford to send their children to school due to their low of income. This is resulting to the very minimal standard of education for the community and thus contributing to the low quality of their living. vii) Unemployment More than 50% of the residents in Kuala Perlis are fishermen. Majority of them only have initial or older fishing boats. Their income is minimal which can only suffice to feed their family members on day-to-day basis. viii) Condition of Housing Good and proper housing scheme for settlement is also another major factor that determines the quality of living. Residential houses in this village are built without proper planning. The house standard is low. Majority semi permanent wooden houses are constructed very close to each other, side-by-side. As a result, the residents are facing difficulty in obtaining better access road for communication and/or facilities to promote a better way of living. Methodology The study involved several stages of collecting data, starting from preliminary preparation, field observation to the community, personal oral interviews, distributing questionnaire surveys, data organization, tabulation, analysis, interpretation, and writing the reports. i) Field observations Field observations to the community of the village were conducted to collect required data. ii) Personal oral interviews About 100 residents of the village participated in personal interviews during the field observations. The objective of this interview was to collect primary data as much as possible. iii) Questionnaire surveys and the report Written questionnaire surveys were distributed to the residents through random selection. They were asked to complete the demographic data, their knowledge and/or perceptions and their need related to environment. The respondents indicated perceptions concerning their knowledge and environmental needs based on a five-point Likert scale as listed below: 4 = A complete extent 3 = A moderate extent 2 = A little extent 1 = No extent 0 = Do not know The completed questionnaire surveys were collected for analysis, interpretation and written presentation for the research.

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Findings and Analysis The data gathered were collected for analysis using Statistical Analysis System (SAS) 6.06 program (Cody, 1987; Jaffe, 1989). Computation of the percentage and mean were used. Based on the available data obtained from the study, the problems were divided into two categories: (I) demographic, and (II) residents’ perceptions on environmental knowledge and need. The results were reported in terms of percentage and mean. The data obtained from the different phases of investigation had served as the framework in the writing and the paper. Table 1: Demographic Data Gender RESPONDENTS (%) 52 48 100 Marital Status RESPONDENTS (%) 96 4 100

ITEM Male Female Total

ITEM Marred Single Total

Range of ages (years) ITEM RESPONDENTS (%) < 30 12 31 - 35 20 36 - 40 36 > 40 32

Number of children in the family ITEM RESPONDENTS (%) 1-2 8 3-4 50 5-6 25 >6 17

I). Demographic Data Of the total resident samples (n = 230) for this study, 52% were males and 48% were females. Their ages ranging from 30 years (12%) to above 40 years old (32%), with 96% of them were married and 4% were single. Most families had children between one to more than six in a family. The detail information of the data is found in Table 1. Majority of the 100 residents surveyed, 8% found out to be working with the government, 40% fishermen, 20% self-employed, and 32% did not have steady employments. Almost majority of them (60%) having incomes below RM 300.00, 36% between RM 301.00 to RM 500.00, and 4% with monthly income of above RM 501.00 (Table 1 shows demographic data, Table 2 shows the detail distribution of occupations and their monthly income). Samples from this study area provided balanced respondents. About 40% of the Kuala Perlis residents were fishermen. In term of length of stay, they have been living in the community ranging from less than five (20%) to more than 16 years (24%) (see Table 3). Table 2: Residents’ Occupations and Monthly Incomes Occupations of the residents ITEM RESPONDENTS (%) Government servants 8 Fishermen 40 Self-employed 20 Not employed 32 Range of Incomes (RM) ITEM RESPONDENTS (%) < 300.00 60 301.00 – 500.00 36 > 501.00 4 Total 100

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Table 3: Residents’ Length of Stay Length of stay (Years) ITEM RESPONDENTS (%) <5 20 6 - 10 40 11 - 15 16 > 16 24 II). Perceptions on Environmental Knowledge and Need Table 4 illustrates residents’ perceptions in terms of basic facilities and need in disposing their household garbage. The findings were reported in the form of means on the degree of agreement based on a five-point Likert scales mentioned earlier. Table 4: Needs for Basic Facility Need for Basic Facilities ITEMS MEAN RESPONSES Clean water 4.00 Proper electricity 3.96 supply Sanitary 3.96 Proper garbage 3.96 disposal

Water Resources ITEMS MEAN RESPONSES Man-made wells 3.92 Ponds 1.52 Rivers 1.28 Neighbourhood 1.72 Pipes 0.10 a. Needs of Basic Facility

Use of Water Resources ITEMS MEAN RESPONSES Cooking 3.36 Drinking 3.36 Washing/Bathing 3.52 Everything 3.40

Data analysis found that these communities were lacking from all kinds of basic facilities (like supply of clean water, proper sanitation, and garbage disposal places). Final data analysis indicated that majority of the residents got their water for daily use from pipe line, while some are from man-made wells or from their neighbour nearby. The water obtained from manmade wells, however, is not suitable for daily use, especially for drinking and cooking purposes. Detail results on the perception of basic facility needs are presented in Table 4. b. Perceptions on Environmental Issues Responses on the need of proper sanitation showed that majority of residents have their toilets built in their houses. This seems that they have proper sanitation. However, small numbers of them seem to build stilts toilet somewhere along the beach or between bushes. People with this improper sanitation need to be helped (Table 5).

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In the case of household garbage, majority of the residents are disposing their garbage in the sea or on the beach, by burying in the ground, or by throwing it anywhere surrounds their houses (Table 5). Analysis data for Kuala Perlis residents on the extent of knowledge on environment found to be that majority of them only had “a little” knowledge related to issues on environment. Majority of them (79%) have knowledge on environment ranging from “don’t know” (20%) to “a little extent” (55%). Very little ‘(1%) of them has the environmental knowledge at “a complete extent” (Table 6). Table 5: Needs for Environmental Facility Method of Sanitation ITEMS MEAN RESPONSES Own toilet Bush Beach/Sea Anywhere convenient ‘ EXTENT RESPONSES A complete extent A moderate extent A little extent No extent Don’t know 4.00 0.16 1.32 1.64 Method of Disposing Garbage ITEMS MEAN RESPONSES Burning 3.56 Burying 1.64 Beach/Sea 3.88 Anywhere 1.48 convenient

Table 6: Residents’ Knowledge on Environment RESPONDENTS (%) 1 20 55’ 4 20 Key: 4 = A complete extent 3 = A moderate extent 2 = A little extent 1 = No extent 0 = Do not know

Conclusions Based on the findings presented in this paper, the following conclusions were drawn: 1. The community of Kuala Perlis needed proper basic facilities like supplies of clean water and electricity, proper management of sanitation and garbage disposal, etc. at “A Complete Extent.” 2. The residents had low level of monthly income, below the standard of living (below RM 300.00) 3. They absent of social activities in the community. 4. Their levels of education and knowledge concerning the awareness on environment were very low. 5. All results found in this research seem to have significant impact on environment (EIA= environmental impact assessment) and social (SIA=social impact assessment).

Recommendations The conclusions resulting from the study suggest four major recommendations for improvement: 1. Immediate actions on improving the level of education, health program, infrastructure developments, and basic facilities be established at the highest level of priority.

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2. The community should be organized economically, educationally, and developmentally to keep abreast to the development. In order to do this, the government sectors, private agencies, and/or nongovernment organizations can play their roles. 3. Improve the economy of the community by building them advance fishing boats so that they would be able to increase their income and at the same time to uplift their standard of living. Special arrangement of instalments can be arranged in the plan. 4. Conduct follow up and further research study for the community with full supports.

References [1]. Cody, R.P. 1987. Applied statistics and the SAS programming language. New York: Elsevier Science Publishing Co. [2]. Department of Statistics. 1991. Banci penduduk dan perumahan malaysia. Kota Kinabalu: Department of Statistics, Sabah. [3]. Jaffe, J.A. 1996. Mastering the SAS system. New York: Van Nostrand Reinhold. [4]. Kudat, Sabah. 2003. http://www.sabah.gov.my [5]. Kuala Perlis fishing village. 2005. http://www.virtualmalaysia.com/destination/view.cfm? [6]. Kuala Perlis. June 6, 2005 http://sinisini.com/malaysia/states/perlis/perlis1.htm

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OPTIMISATION OF SOLID-PHASE MICROEXTRACTION (SPME) FOR THE DETERMINATION OF TRIAZINES IN WATER SAMPLES USING EXPERIMENTAL DESIGN Md Pauzi Abdullaha, Kamarrudddin Asrib, Mohamad Salleh Ramlib & Maimunah Sulaiman @ Wahida
a

School of Chemical Sciences & Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor b Department of Chemistry Malaysia, Jalan Sultan, 46661 Petaling Jaya Selangor.

Abstract. In this paper, a sensitive capillary SPME-GC-MS method for the analysis of fourteen triazines has been developed. Direct immersion SPME has been applied to the water sample. Carbowax-divinylbenzene (CW-DVB) and poly-dimethyl siloxane-divinylbenzene (PDMS-DVB) fibers were evaluated and CW-DVB was selected for further work. Extraction parameters namely extraction times, extraction temperature, ionic strength and stirring rate were optimised using a twolevel full factorial design expanded further to a central composite design. Sample pH was evaluated later using the statistical test. The proposed optimized analytical procedure using 10 mls sample, were 50 min extraction time, extraction temperature at 50 0 C, ionic strength at 20% sodium chloride (w/v), pH neutral (6-7) and stirring rate at 60% of stirrer maximum rate. The desorption time and temperature were 5 min and 250 0 C respectively. Abstraks. Teknik Mikroekstraksi fasa pepejal, kromatografi gas-spektrometer jisim (SPME-GC-MS) secara rendaman terus digunakan untuk menganalisis empatbelas racun rumpai triazin di dalam sampel air. Fiber yang dinilai ialah karbowaks-divinilbenzena (CW-DVB) dan poli-dimetil siloksandivinilbenzena dan fiber CW-DVB dipilih untuk kajian seterusnya. Faktor keadaan sampel yang telah dikaji ialah tempoh ekstraksi, suhu ekstraksi, kekuatan ionik dan kelajuan putaran sampel. Pendekatan rekabentuk eksperimen faktor, 24 digunakan untuk menganalisis faktor yang mempengaruhi kecekapan ekstraksi dan seterusnya menggunakan rekabentuk permukaan respon untuk proses optimisasi. Optimisasi pH sampel ditentukan kemudian dengan menggunakan ujian statistik untuk mengesahkan pH optimum. Prosedur analisis lengkap yang digunakan ialah isipadu sampel 10 ml bagi tempoh ekstraksi selama 50 minit, suhu sampel 50 0 C, kekuatan ionik pada paras 20% NaCl (w/v), pH neutral (6-7) dan kadar putaran sampel 60% daripada kadar putaran maksimum alat. Masa desorpsi dan suhu desorpsi ialah masing-masing 5 minit dan 250 0 C Keywords : Optimisation, water analysis, Experimental design, pesticides Introduction Triazines are applied as selective pre- and post-emergence herbicides for the control of weed in many agricultural crops like corn, wheat, barley, pineapple, sugarcane, bananas, and cocoa as well as in railways, roadside verges and golf course [1,2 &3]. Ametryn and atrazine are the triazines registered with Malaysian Pesticides Board [4]. A number of triazines (ametryn, atrazine, simazine, cyanazine and terbuthylazine) are regulated under draft National Standard for Drinking Water Quality, Engineering Services Division, Ministry of Health Malaysia. The maximum acceptable value is within 0.6 to 50 ug/l depend on the specific triazines [5]. A recent EU directive, Council Directive 98/83/EC states the level must not exceed 0.1 ug/L for individual compound and some of their degradation product, (0.5 ug/L for all compound) in water intended for human consumption [6]. To monitor sources of water intended for human consumption in compliance with Malaysian draft Standard and EU legislation, analytical methods with detection limits well below 0.1 ug/L are needed. SPME technology eliminates the uses of organic solvent and has the advantages of simplicity. This solvent-free extraction technique involves two steps. In the first step, a fused silica fiber coated with polymeric stationary phase is exposed to the sample matrix where the analyte partition between the matrix and the polymeric stationary phase. Then, in the second step the analyte/fiber is transferred

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to the analytical instrument for desorption, separation and quantitation[7]. Gas chromatography-mass spectrometry was selected for the analysis of triazines in water because of its high specificity. The parameters affecting the SPME method are type of fiber employed, extraction temperature and time, ionic strength, sample agitation and pH, and desorption temperature and time [7]. Procedural optimisation can be achieved in a traditional trial, studying each factor separately, or using chemometric approach based on the use of an optimum set of experiment which allows the simultaneous variation of all experimental parameter studied, and the distinguishing of interactions among them that are not detectable with classical experimental methods [8, 9 &10]. The chemometric methodology also reduces the number of experiment required. The aim of the work reported here is to select the best fiber and to optimise the conditions for the detection and quantification of triazines and their degradation products in water sample. CWDVB and PDMS-DVB fiber were evaluated because previous studies have shown that these fibers were more efficient than the polyacrilate (PA) and poly-dimethyl siloxane PDMS [11, 12, 13 &14]. After selecting the type of fiber, the effect of experimental parameters; extraction times, extraction temperature, ionic strength and stirring rate were evaluated using a two-level full factorial design expanded further to a central composite design. The pH sample was evaluated after the final optimised conditions were achieved and the exact percent of sodium chloride were confirmed by checking the significant P-value using statistical T-test.

Experimental Chemical and Reagents Mix triazines and their degradation products standard and the internal standard were supplied by Dr. Ehrenstorfer. These fourteen standards are desisopropyl atrazine, desethyl atrazine, simazine, atrazine, propazine, terbuthylazine, sebuthylazine, desmetryn, metribuzin, ametryn, prometryn, terbutryn, cyanazine and hexazinone were dissolved in acetonitrile containing 100 ug/ml of each analyte. The internal standard (ISTD); 1-bromo-2-nitrobenzene with 99.5 % purity was used to prepare a stock solution containing 100 ug/ml in methanol. The stock solution standard and internal standard were diluted to the required concentration with methanol. These entire standard were stored at 4 0 C in a refrigerator. The buffers were prepared by mixing ammonium acetate, acetic acid and/or ammonium hydroxide depending on the required value of pH. Ultrapure milli-Q water (Millipore) was used to daily prepare the working aqueous solution in the required working concentration range. Instrumentation SPME was performed with commercially available 65 um CW-DVB and 65 um PDMS-DVB coated fiber and housed in the appropriate manual holder (Supelco, Bellefonte, PA, USA). All SPME fibers were new at the beginning of the study and were conditioned according to supplier’s instruction. After direct immersion in high salt content sample matrix, the fiber have to immerse in deionised water for few second before it is transferred to the GC injector port in order to dissolve the salt from the fiber coating as well as to avoid the salt deposition at the GC injector port. Chromatographic analyses were performed with a Shimadzu QP5050A Gas chromatographymass spectrometry with quadrupole mass filter and electron impact ionization (EI) at 70 ev as ionization source. The injector and detector temperatures were set at 250 and 280 0 C, respectively. The split/splitless injector was maintained in splitless mode during 5 min of SPME fiber desorption. All compounds were resolved in a DB-5MS column (30 m x 0.25mm ID x 0.25 um film, using helium as carrier gas with a flow rate of 1.0 ml/min. The oven temperature programmed was as follows: initial temperature 60 0 C (held 2 min), increased by 15 0 C /min to 140 0 C, increased to 158 0 C (1 0 C/min), and finally increased to 250 0 C (10 0 C/min) and held at this temperature for 8 min.

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Table 1 : Selected ions for GC-MS identification and confirmation of target compound Selected Characteristic Ion1 (m/z) No Compound RT 9.294 17.890 18.360 22.486 23.054 23.544 24.526 27.364 28.190 28.445 29.319 29.504 29.976 30.835 37.292 1.924 1.975 2.420 2.481 2.533 2.639 2.944 3.033 3.061 3.155 3.175 3.226 3.318 3.852 RRT Molecular ion 201 173 187 201 215 229 229 229 213 214 227 241 241 240 252 Q 155 173 172 201 200 214 214 200 213 198 227 241 226 225 171 C1 157(100) 145(79) 174(30) 186(66) 215(53) 229(58) 173(45) 202(32) 198(68) 199(44) 212(63) 184(108) 185(91) 198(81) 128(23) C2 201 (77) 158(89) 187(26) 173(54) 202(32) 187(34) 216(32) 214(14) 171(28) 182(11) 185(27) 226(60) 170(77) 240(31) 172(16) 228(16) 199(28) 241(55) C3 203(72) 160(29) 145(19) 202(14) 173(30) 216(32) 229(25) 229(11) 214(15)

1 1-bromo-2nitrobenzene (ISTD) 2 Desisopropyl atrazine (DIA) 3 Desethyl atrazine (DEA) 4 Simazine (SIM) 5 Atrazine (ATR) 6 Propazine (PROP) 7 Terbuthylazine (TERB) 8 Sebuthylazine (SEBU) 9 Desmetryn (DESM) 10 Metribuzin (METRI) 11 Ametryn (AMET) 12 Prometryn (PROM) 13 Terbutryn (TERBN) 14 Cyanazine (CYA) 15 Hexazinone (HEXA)
1

Q : Quantitation ion C1, C2, C3 : characteristic ions Values in bracket is the percent of abundance ion to the quantitation ion.

The specific ion monitoring (SIM) mode was performed in which four ions for each compound except metribuzin, cyanazine and hexazinone (three ions) were chosen for the analysis. The selection ions as the identification and confirmation of the compound were based on the discussion and solution briefly discussed by Baldwin, R. et al. [15], Stein, S.E.[16], Andre, F. et al. [17] and Bethem, R. et al. [18]. The relative retention time (RRT) of the compound of interest to the internal standard within the reference standard and the RRT of the compound of interest to the internal standard within the sample shall be ±0.01min. Ion abundance ratios of the characteristic ion to the quantitation ion were used as second confirmation criteria. Table 1 shows the details of compound identification and confirmation. Procedures Fiber selection Extractions were performed by immersion of the fiber in 10 ml of milli-Q water, fortified with 200 ng/ml mix triazines and ISTD in screw-cap vials supplied with a PTFE-lined septum. 5mm magnetic stir bar and magnetic stirrer with heating module Pierce were used. Extraction time was 30 min, at room temperature and stirring rate was fixed at 20% of maximum stirrer rate (the knob of the stirrer

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equipment was set at number 2). pH sample was fixed at range 6.5 - 7 using the buffer system no sodium chloride was added. Desorption time and temperature were 5 min and 250 0 C respectively. The relative total ion chromatographic (TIC) area of the compound to the internal standard was monitored as extraction efficiency. Extractions were repeated for 8 times. Significant different in the extraction efficiency was evaluated using statistical T-test using SPSS Version 12 software. SPME parameter optimisation The parameters optimised under experimental design were extraction time, stirring rate, extraction temperature and ionic strength. The extractions were performed at constant pH and the conditions of the sample during the extraction were adjusted according to the two-level full factorial design and central composite design. The area of the quantitation ion for each compound was monitored as the extraction efficiency. Screening by Full Factorial Design (FFD) The FFD, 24 was chosen and was created using Design-Expert Software [20], involves 16 experiment undertaken in random order. Four replicate of centre point were incorporated in the design. The 10 ml ultra pure milli-Q water was fortified with 200 ng/ml mix triazines standard and internal standard. Table 2 shows the details of this design. Table 2 : Parameter levels in FFD, 24 Parameters Extraction time [A: min.] Stirring Rate [B:% of equipment maximum stirring rate] Knob for 100% at level 10. 20 40 60 Ionic strength [C: % NaCl w/v] Extraction temperature [ D : 0C ]

Levels

-1 0 +1

15 40 60

0 5 10

30 45 60

Optimisation by Central Composite Design, CCD In the second stage of optimisation, the CCD, α = 1.682 was created using Design-Expert software, involves 34 experiment comprise of duplicate on axial and factorial point. The 10 ml ultra pure milliQ water was fortified with 200 ng/ml mix triazines standard and internal standard. Table 3 lists the values given to each parameter. Ionic strength and pH confirmation The ionic strength confirmation was done using the comparison of the extraction mean value between 15% and 20% NaCl. The effect of the sample pH to the extraction efficiency was evaluated separately from the experiment design. Three pH point; 4, 6.5 and 8 were selected and the experiments were done in three replicate for every point.. The final decision of the optimum pH was made based on the analysis of variance (ANOVA).

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Table 3 : Parameter levels in CCD, α = 1.682 Levels /Parameters Low (-) High (+) Levels Centre Axial (-α ) Axial (+α ) Results and discussion Fiber screening The results of this screening (Table 4) show that there were significant different in extraction efficiency for the fibers tested. Only 10 out of 14 compounds were extracted at the fortified level 200 ng/ml. All the P-values were less than 0.05 means that there is significant different at 95% confident level. The extraction efficiency was better for the CW-DVB fiber. The CW-DVB fiber can extracts more polar triazines; simazine and metribuzin which are not extracted by the PDM-DVB fiber. Therefore, CW-DVB fiber was selected for method optimisation. The earlier research done by Ferrari et al.[11], Hernandez, F. et al. [12] and Hu et al. [13] agreed that the CW-DVB fiber is the best to use for the analysis of triazine herbicides in water and these finding were contradicted with the finding by Frias, S. et al. [14]. Table 4: Fiber extraction efficiency (results are expressed as average of the relative TIC area of the compound to the internal standard, n=8) Fiber/ Compound PDMS-DVB fiber CW-DVB fiber P-value SIM nd 0.150 nr ATR 0.141 0.272 0.000 PROP 0.274 0.402 0.000 TERB 0.497 0.761 0.000 SEBU 0.297 0.636 0.000 DESM 0.102 0.338 0.000 METRI AMET PROM TERBN nd 0.041 nr 0.073 0.174 0.019 0.288 0.663 0.000 0.319 1.315 0.000 Extraction time [A: min.] 30 60 45 20 70 Ionic strength [ B: % NaCl w/v] 10 20 15 5 25 Extraction temperature [ C : 0C ] 40 50 60 35 65

Note : nd ,not detected; nr, not relevant; refer Table 1 for compound full name. According to Barcelo, D. & Hennion, M. C. [20], the polarity of a molecule is strongly correlated with log K ow . Although simazine and cyanazine are having the same log K ow value, but cyanazine is not extracted by CW-DVB fiber because of their different characteristic in water solubility. The more soluble the more difficult to extract from aqueous media [20]. The other polar triazines and degradation products; cyanazine, hexazinone, desisopropyl atrazine and desethyl atrazine are not extracted by both fibers due to low fortified level. Table 5 shows the physicochemical properties of triazines herbicides. Table 5 : Physicochemical properties of triazines herbicides Compound Water solubility at 20 - 25oC (g/liter) Log Kow pKa Polarity classification

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1 2 3 4 5 6 7 8 9 10 11 12 13 14

Desisopropyl atrazine Desethyl atrazine Simazine Atrazine Propazine Terbuthylazine Sebuthylazine Desmetryn Metribuzin Ametryn Prometryn Terbutryn Cyanazine Hexazinone

3.2 0.67 0.0062 0.033 0.005 0.0085 0.035 0.58 1.05 0.2 0.033 0.022 0.171 33

1.1 1.4 2.1 2.5 2.91 - 3.02 3.04 3.31 2.4 1.58 2.63 3.1 3.65 2.1 1.05

na na 1.7 1.7 1.7 2 na 4 na 4.1 4.1 4.3 1 na

Polar Polar Semi polar Semi polar Semi polar Semi polar Semi polar Semi polar Polar Semi polar Semi polar Semi polar Semi polar Polar

Note : na, not available. Table 6 : The effect of each parameter to the extraction efficiency Percent Effect (%) Compound Quantitation ion (m/z) A: Extraction time 50.4 34.7 39.6 35.9 40.7 42.0 23.0 40.2 36.1 44.7 B: Stirring rate 0.2 1.2 0.2 3.0 1.7 0.6 1.2 3.1 2.5 4.6 C: Ionic strength 8.5 15.9 13.3 13.5 14.7 16.4 20.0 12.8 9.6 4.5 D: Extraction temperature 3.1 27.5 29.1 22.4 22.5 18.4 8.5 23.4 21.7 21.3

1 2 3 4 5 6 7 8 9 10

Simazine Atrazine Propazine Terbuthylazine Sebuthylazine Desmetrin Metribuzin Ametryn Prometryn Terbutryn

201 200 214 214 200 213 198 227 241 226

SPME optimization A two-level full factorial was preliminarily used to study the effect of the main and interaction parameters to the extraction efficiency. The data obtained from FFD and CCD were evaluated using the Design-Expert software with the guidance of Montgomery, D. C [10] discussion and guidelines. The effect value of each parameter, ANOVA and plot of main and interaction parameter are evaluated. The effects of each parameter to the extraction efficiency were showed in Table 6. Generally, all parameters shows the positive effect on extraction efficiency which means that the higher the value the better extraction efficiency. Parameter extraction time shows the highest where as stirring rate shows the lowest effect to the extraction efficiency. The ionic strength shows the second

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highest effect for the polar compound; simazine and metribuzin. Its shows that the ionic strength is the second parameter may affect the extraction efficiency of polar and low water solubility compounds. The effect evaluation was then proceed to analysis of varian, ANOVA in order to confirm the effect of each parameter studied and their interaction. Table 7 shows the details of ANOVA at 95% confident level. In this table, and for the sake of simplicity, only P-values were given. The P-value tests the statistical significance of each parameter and their interaction. As can be seen, extraction time was found to be significant for all compounds, whereas extraction temperature and ionic strength was not significant only for simazine and terbutryn respectively. Parameter stirring rate was not significant for all compounds with the exception of atrazine, sebuthylazine and ametryn. The interaction between parameter extraction time and ionic strength; extraction time and extraction temperature appears statistically significant different in extraction efficiency for most of the compound. The interaction between parameter stirring rate, B and three parameters; A, C and D shows no significant different for most of the compound. Due to the significant and positive effect of parameter extraction time, ionic strength and extraction temperature, the range value of each parameters were adjust to the higher side for the optimisation purpose in CCD as shown in Table 3. High percent of sodium chloride, % w/v was not considered in FFD because to prolong the fiber lifetime even though Pawliszyn, J.[7] have stated that higher ionic strength are required for the extraction polar compound. Hernandez F, et al. [12] have found that very fast degradation of the fiber occurred when using 30% NaCl, it being possible to use the same fiber for only about 10 - 20 extractions. In view to set the value of stirring rate, whether to take 20% or 60% of the maximum equipment rate, the interaction effect plots between this parameter and others parameter are considered. If the decision is based only on the P- value, there is no extraction efficiency different in setting 20% or 60% of equipment maximum rate. The interaction effect plots results (the results are not shown) shows thatthe increasing extraction efficiency occurs when the stirring rate parameter interact at the higher value of extraction time, ionic strength and extraction temperature. The main effect also shows the positive effect to extraction efficiency, therefore the stirring rate is set constantly at 60% of the equipment maximum rate in the optimisation by CCD. CCD is one of the various types of response surface method (RSM). RSM are represent a collection of experimental design and multiple regression based techniques that can be used to analyse problems where several factors may influence a response. The RSM goal is to optimise system performance [21]. The purpose of CCD in this study is to find the optimum condition from the combination of three parameters studied in FFD which give the significant effect to the SPME extraction efficiency. Quadratic model have been selected for this purpose. The main effects and two parameter interaction effects are not discuss in CCD finding because the results show the same significance pattern as in FFD. The main focus of the CCD is to find the best combination parameter values to produce the maximum response.

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Table 7 :

ANOVA results showing the significant of main effect and interaction Main effects Interaction effects AB AC AD BC BD CD

Compound Simazine Atrazine Propazine Terbuthylazie Desmetryn Metribuzin Ametryn Prometryn Terbutryn

A: Extraction time 0.002 0.000 0.005 0.003 0.001 0.005 0.0002 0.002 0.003

B: Stirring rate 0.684 0.050 0.619 0.144 0.029 0.447 0.330 0.025 0.195 0.097

C: D: Ionic Extraction strength temperature 0.038 0.001 0.023 0.018 0.006 0.008 0.007 0.002 0.032 0.097 0.139 0.000 0.008 0.008 0.000 0.007 0.035 0.001 0.007 0.010

0.201 0.099 0.502 0.614 0.577 0.782 0.469 0.231 0.454 0.391

0.021 0.713 0.238 0.002 0.011 0.007 0.134 0.047 0.286 0.060 0.043 0.106 0.006 0.003 0.007 0.064 0.069 0.100 0.010 0.217 0.028 0.007 0.006 0.063 0.071 0.033 0.118 0.103 0.033 0.085

0.029 0.577 0.368 0.484 0.730 0.611 0.026 0.556 0.732 0.574

0.200 0.030 0.165 0.171 0.016 0.100 0.259 0.035 0.143 0.744

Sebuthylazine <0.0001

Values are significant at P<0.05

The results were graphically showed in three dimension response surface comprise of the combination of two parameters and the area response of each compound. The target optimum combination being the top of the surface. All the diagrams were created using Design-Expert software. Figure 2a, 2b and 2c shows the response surface for ametryn (expressed as quantitation ion area) as an example for the whole process to justify the maximum condition for all compounds. The overall peak on the top of the response surface for all compounds shows the optimum ionic strength within 15 to 20% NaCl (%w/v), extraction temperature at 45 to 55 0 C and extraction time; 40 to 60 min. In view to select the exact extraction time, the gas chromatography run time was considered, which is almost 50 min therefore 50 min was selected as the optimum extraction time. The selected extraction temperature is 50 0 C after considers the intermediate value for the coverage of all compounds. The results from the comparison means of extraction efficiency between the extraction using 15% and 20% NaCl shows that there was significant different in extraction efficiency for the more polar analyte namely simazine, atrazine, and metribuzin. Therefore, ionic strength at 20% NaCl w/v was selected as an optimum condition. The ANOVA evaluations shows that there were no significant different in extraction efficiency between pH 4, 6.5 and 8 except for simazine and desmetryn. Simazine shows lower efficiency at pH 8 where as desmetryn at pH 4. After comparing the extraction efficiency mean value at pH 6.5 and 8, all compound shows no significant different in extraction efficiency, therefore optimum pH was accepted as neutral pH, 6.5 to 7.5. Our finding for the optimum conditions are comparable to earlier finding by Hu et al. [13], Frias et al. [14] and Calves et al. [22]

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even

though

they

studied

the

SPME

optimisation

parameter

by

parameter.

4033.03

3219.24

2405.46

AMET

1591.67

777.89

25.00
70.00

20.00
57.50

15.00

45.00

B: ionic strength

10.00
5.00 20.00

32.50

A: Extraction time

Figure 2a : Estimated response surface for ametryn quantitation ion area using the central composite design by plotting extraction time vs ionic strength, % NaCl (extraction temperature at 50 0 C)

3756.74

2947.87

2139

AMET

1330.13
521.259

65.00

70.00
57.50

57.50
50.00

45.00

C: extraction temperature 42.50
35.00 20.00

32.50

A: Extraction time

Figure 2b : Estimated response surface for ametryn quantitation ion ara using the central composite design by plotting extraction time vs extraction temperature (ionic strength at 15% NaCl)

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3726.24
3057.75
2389.27

AMET

1720.79
1052.31

65.00
25.00

57.50
20.00

50.00

15.00
10.00

C: extraction temperature 42.50
35.00 5.00

B: ionic strength

Figure 2c : Estimated response surface for ametryn quantitation ion ara using the central composite design by plotting ionic strength vs extraction temperature (extraction time at 50 min) 4. Conclusion.

The uses of a fiber CW-DVB for the SPME procedure in the determination of triazine compounds was shown as the best fiber for the extraction of triazine from water samples. Experimental designs used in this studies; full factorial and central composite design have demonstrated to be a very useful tools for fast and efficient selection of optimum condition when working with SPME. The optimised procedure were 50 min extraction time, ionic strength at 20% NaCl (%w/v), extraction temperature at 500 C stirring rate at 60% of the maximum equipment stirring rate and pH sample at 6.5 to 7.5 . The sample volume, adsorption time and adsorption temperature were constantly set at 10 mls, 5 min and 2500 C respectively. At this point, and having completed these studies just described, further research is required in order to determine the method performance characteristic. References 1. 2. 3. Worthing, R. C. (1987) “The Pesticides Manual” , Eighth Edition.The British Crop Protection Council Oudejans, J. H, (1991) “Agro pesticides: Properties & Functions in Intergrated Crop Protection”, Bangkok: Economic and Sosial Commission for Asia and the Pacific (ESCAP) United Nation. Loos, R. % Nesser, R. (1991) “ Analysis of atrazine, terbuthylazine and their N-dealakylated chloro and hydroxyl metabolites by solid-phase extraction and gas chromatography-mass spestometry and capillary electrophoresis-ultraviolet detection”. J. Chromatogr. 835 : 217229. Jabatan Pertanian & Industri Asas Tani Malaysia. (2003) “Racun Makhluk Perosak yang berdaftar dengan Lembaga Racun Makhluk Perosak untuk tempoh 2000 hingga 2003” (atas talian) http://www.agrolink.moa/doa/registered_pesticides ( 20 Disember 2003). Engineering Services Division, Ministry of Health Malaysia. (2000) Draft National Standard for Drinking Water Quality. EU (1998), Council Directive 98/83/EC on the quality of water intended for human consumption, Official Journal of the European Communities.

4. 5. 6.

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7. 8. 9. 10. 11.

12. 13. 14.

15. 16. 17. 18. 19. 20. 21. 22.

Pawliszyn, J. (1997) “ Solid Phase Microextraction Theory and Practice”. New York : WileyVCH, Inc. Brereton, R. J. (1990) “Chemometric: Applications of mathematics and Statistics to Laboratory Systems”, Chichester; Ellis Horwood. Miller, J. N. & Miller, J. N. (2000) “Statistic and Chemometrics for Analytical Chemistry”, 4th Edition, Edinburg, UK: Pearson Education Limited. Montgomery, D.C (2001) “Design and Analysis of Experiment”. 5th. Edition. New York, John Wiley & Sons. Inc Ferrari, R., Nilsson, T., Arena, R., Arlati, P., Bartolucci, G., Balsa, R., Cioni, F., Carlo, G., D., Dellavedova, P., Fattore, E., Fungi, M., Grote, C., Guidotti, M., Morgillo, S., Muller, L. & Volante, M. (1998). “Inter-laboratory validation of solid-phase microextraction for the determination of triazine herbicides and their degradation products at ng/l levels in water samples”. J. Chromatogr A. 795 : 371-376 Hernandez, F., Beltran, J., Lopez, J. & Gaspar, J. V. (2000). “Use of solid-Phase Microextraction for the Quantitative Determination of Herbicides in Soil and Water Samples”. Anal. Chem. 72 (10) : 2313-2322. Hu, R., Elia, D., Berthion, J. M. & Poliak, S. (2001). “Determination of Triazines and Amides in Water Using Solid Phase Microextraction Coupled with GC-MS”. Chromatographia. 53 : 306-310. Frias, S. Rodriguez, M. A. & Perez-Trujillo, J. P. (2003). “Optimisation of a solid-phase microextraction procedure for the determination of triazins in water with gas chromatography-mass spectrometry detection”. J. Chromatogr A. 1007 : 127. (atas talian) http://www.sciencedirect.com (2 Jan 2004) Baldwin, R. et al. (1997). ASMS Fall Workshop : “Limits to Confirmation, Quantitation, and Detection”. J. Am. Soc. Mass Spectrom. 8 : 1180-1190. Stein, S. E. (1999). “An Intergrated Method for Spectrum Extraction and Compound Identification from Gas Chromatography/Mass Spectrometry Data”. J. Am. Soc. Mass Spectrom. 10 : 770-781 Andre, F., Katia,K. G., Hubert, F.D.B. et al. (2001). “Trends in the identification of organic residues and contaminants : EC regulations under revision”. Trends. Anal. Chem. 20 (8) : 435-445. Bethem, R., Boison, J., Gale, J., Heller, D., Lehotay, S., Loo, J., Musser, S., Price, P. and Stein, S. (2003). “Establishing the Fitness for Purpose of Mass Spectrometric Methods”. J. Am. Soc. Mass Spectrom 14 : 528-541 Design Expert Version 6 Stat Ease, Inc. 2000 Barcelo, D dan Hennion, M.C. (1997). “Trace Determination of Pesticides and their Degradation products in Water”. Amsterdam: Elsevier Gadiner, W. P. (1997). “Statistical Analysis Methods for Chemist”. Glasgow, UK: The Royal Society of Chemistry. Calvez, N. L., Bodineau, L. & Fischer, J.C. (2002) “Application of solid-phase microextraction for the determination of organonitroen pesticiedes in aqueous samples by gas chromatography with nitrogen-phosphorus detector”. Intern. J. Environ. Anal.. Chem. 82. (10) : 691-703.

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DEVELOPMENT OF A SUBCRITICAL WATER EXTRACTION FOR THE QUANTITATIVE DETERMINATION OF POLYCHLORINATED BIPHENYLS IN SEWAGE SLUDGE

Umi K. Ahmad and A. Zamani Ab Halim Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia 81310 UTM Skudai, Johor, Malaysia. e-mail: umi@kimia.fs.utm.my Abstract. Extractions of organic contaminants such as polychlorinated biphenyls (PCBs) for sewage sludge have often involved the use of large volume of organic solvents and long extraction time. In this study, a home made subcritical water extraction (SWE) was developed with the aim of overcoming the disadvantages associated with other conventional extraction technique such as soxhlet extraction. By merely adjusting the water temperature, selectivity can be achieved for organic compounds and for polar or non-polar organic compounds. Instrumentation for subcritical water extraction was developed and modified in several ways with the aim of simplifying the procedure while maintaining good recoveries. Extracts were analyzed by GC-ECD on a HP-5MS capillary column. Recoveries of 60-90% were obtained for the seven PCB congeners (PCB 28, 52, 101, 118, 138, 153 and 180) analysed. Comparison of PCBs extracted with Soxhlet extraction revealed the SWE technique to be fast, easy, and environmentally clean technique. Keywords: Polychlorinated Biphenyls, Sewage Sludge, SWE, Soxhlet, GC-ECD Abstrak. Pengekstrakan pencemar organik seperti bifenil politerklorinat (PCBs) dalam enapcemar sering melibatkan isipadu pelarut yang banyak dan masa pengekstrakan yang lama. Dalam kajian ini, instrumen pengekstrakan sub lampau genting air (SWE) dibangunkan dan diterokai dengan tujuan untuk mengatasi kelemahan pengekstrakan konvensional seperti pengekstrakan soxhlet. Dengan hanya mengubahkan suhu air, kepilihan pengekstrakan boleh diperolehi untuk bahan organik dan untuk sebatian organik berkutub atau tak berkutub. Instrumen ini dibangunkan dan dimodifikasi dengan tujuan untuk mengekalkan peratusan pengembalian yang tinggi. Hasil ekstrak yang diperolehi dianalisis dengan gas kromatografi pengesanan penangkapan elektron menggunakan turus rerambut HP-5MS. Peratusan pengembalian sebanyak 60-90% diperolehi untuk tujuh PCB (PCB 28, 52, 101, 118, 138, 153 and 180) yang dikaji. Perbandingan ekstrak PCB daripada Soxhlet menunjukan SWE adalah teknik yang pantas, mudah dan mesra alam sekitar. Katakunci: Bifenil politerklorinat, Enapcemar, Pengekstrakan sub lampau genting air. Soxhlet, GCECD Introduction Disposal of sewage sludge poses serious problems to municipalities. In Malaysia, it has been estimated that about 3 million cubic metres of sewage sludge are annually generated and this volume is expected to rise to 7 million cubic metres by the year 2020 (Kadir and Haniffa, 1998). Agricultural use of sewage sludge has many beneficial aspects. It helps to increase crop production, maintain the organic pool in soil and reduce the use of mineral fertilizers (Hing et al., 1998). Sewage sludge contains many organic contaminants that may have an impact on soil and the food chains associated with arable land. Polychlorinated biphenyls (PCBs) have been produced worldwide or used as dielectric fluids in capacitors and transformers, hydraulic fluids, fire retardants, and plasticizers (Hutzinger et al., 1974). For several of these applications, PCBs are chosen because

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of their physical and chemical stability and their electrical insulating properties. Due to their weak solubility, PCBs are eliminated from water in the process of partitioning during sewage treatment. Traditionally, PCBs determination has been performed by Soxhlet extraction technique. This technique is often time-consuming and requires large volumes of organic solvent. The use of large volumes of extracting solvents adds additional cost because of the fee associated with purchasing and disposal of toxic solvents. For these reasons, alternative extraction strategies have been developed and applied namely subcritical water extraction (Smith, 2002). Subcritical water extraction is an emerging technique based on the use of water as extraction solvent at temperatures between 100 and 374 oC and at a pressure, which is high enough to keep it in the liquid state (Smith, 2002). At ambient temperature and pressure, water has a dielectric constant (ε) of ~ 80, making it an extremely polar solvent, and therefore the solubility of nonpolar organic in ambient water are very poor. PCBs have solubilities ranging from ~1 µg/mL (dichlorobiphenyls) to only ~0.005 µg/mL (heptachlorobiphenyls) in ambient water (Hutzinger et al., 1974). Water is inexpensive and environmentally friendly, and its polarity (measured by its dielectric constant) can be adjusted simply by changing the temperature. In this study, a home made subcritical water extractor was developed and modified in several ways with the aim of simplifying the procedure while maintaining good recoveries of seven PCB congeners (PCB 28, 52, 101, 118, 138, 153 and 180) from sewage sludge samples.

EXPERIMENTAL

Reagents Neat individual standards of PCB 28, 52, 101, 118, 138, 153 and 180 (Figure 1) at a concentration of 10 µg/mL were obtained from Dr. Ehrenstorfer Lab (Germany). A mixed standard solution was prepared in iso-octane (BDH Limited Poole England) at a final concentration of 1.0 µg/mL, which was used to spike the sludge and for calibration purposes. Dichloromethane, n-hexane and n-heptane were obtained from Fisher Scientific (USA). Deionized water was used throughout the experiment.
Cl Cl

Cl

Cl
Cl PCB 28
PCB 52

Cl Cl

Cl

Cl

Cl

Cl

Cl

PCB 118 Cl PCB 101

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Cl Cl Cl Cl PCB 153 Cl Cl
Cl

Cl

Cl

Cl

Cl

Cl Cl

PCB 138

Cl

Cl

PCB 180

Cl

Figure 1 Molecular Structures of seven PCBs under study Instrumentation and Apparatus The schematic set-up of the extraction unit is shown in Figure 2. All connecting tubings (1/16 inch) were made from stainless steel. Connections were made using Swagelok fittings. The SWE system consisted of a Waters 515 HPLC pump to pump in the water and pressurize the system. A Shimadzu gas chromatography oven (Tokyo, Japan) was used to raise the extraction temperature to the subcritical state. A preheated coil (3 m stainless steel tube, 1/16 inch) was placed in the oven and was followed by a 1.0 mL extraction vessel. A pressure gauge (0 – 6000 psi) from Omega Engineering, Inc. was needed to measure the extraction pressure of the system. A home made measuring flask was used to collect the extract. Two shut-off valves from Whitey were used. PCBs analyses were carried out using a Perkin Elmer XL GC equipped with an electron capture detector (ECD) and data processing using Chromatography Version 4.1 software. Nitrogen was used as the make up gas for ECD at a flow rate of 30 mL/min and helium as the carrier gas at pressure of 14.0 psi. An HP-5MS column (30 m x 0.25 mm ID x 0.25 µm film thickness) from Hewlett Packard, (USA) was used. Injection port temperature was set at 280oC and detector temperature at 290oC. The temperature was initially held at 80oC for 3 minutes, and then programmed from 80 oC to 230 oC at a ramp of 25 oC/min. The temperature was again held for 3 minutes at 230 oC and then increased to a further 260 oC at 2 oC/min.

5 1 3
2
4
7 7

9

6

8 10

Figure 2 Schematic diagram of the basic set up for subcritical water extraction. 1: deionized water, 2: HPLC pump, 3: Shut-off Valve A, 4: preheated coil, 5: pressure gauge, 6: oven, 7: extraction vessel, 8: cooling coil, 9: shut-off valve B, and 10: collection vial Spiked Sludge Samples

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Blank sludge samples used for analyte recovery studies were prepared in the laboratory. The sludge samples were initially subjected to exhaustive Soxhlet extraction for removal of organic contaminants, therefore enabling it to be used as blank sludge samples. 0.5 g of the blank sludge sample was accurately weighed and spiked with 100 µL of a 0.5 µg/mL PCB standard mixture. The spiked sample was left to expose at room temperature in order to evaporate off the solvent. General Analytical Procedure The spiked sludge was separately extracted according to the following procedures. Subcritical Water Extraction Spiked sludge samples (0.5 g) were weighed and loaded into the extraction vessel located inside the extractor oven. After assembling the extraction cell in the oven, the oven was brought up to the working temperature. The high-pressure pump was switched on, valve A was opened, and valve B was closed. The water was pumped into the extraction vessel until the pressure reached 50 kg/cm2. The high-pressure pump was switched off and valve A was then closed to allow for a 1-minute static extraction. The pump was again switched on, and valves A and B reopened so as to control the set pressure at 50 kg/cm2. The outlet tubing was immersed in an iced water bath and extract collected in a vial containing 5.0 mL dichloromethane as trap solvent. The water layer from each extraction was partition twice with DCM using liquid-liquid extraction, and the two organic solvents were later combined. The organic extract was evaporated to near dryness, and 100 µL iso-octane was added prior to GC-ECD analysis. Conventional Soxhlet Extraction Spiked sludge sample (0.5 g) was placed in an extraction thimble together with 0.5 g of anhydrous sodium sulfate. Soxhlet extraction was performed using 320 mL acetone-dichloromethane (1:1) mixture and the extraction carried out for 24 hours. The soxhlet extract was filtered through a Whatman filter paper. The filtrate was concentrated to about 1-2 mL using a rotary evaporator. The elimination of sulfur was carried out using copper powder (0.5 g) previously activated using concentrated HCl. The final determination by GC-ECD was then carried out. Results and Discussions The following SWE parameters were investigated for optimized analyte recoveries (i) extraction temperature, (ii) water flow rate, (iii) solvent trapping, and (iv) extraction time. Influence of Water Temperature Temperature is an important extraction parameter of PCBs (non polar organic). The spiked sample was extracted in triplicate at 50 kg/cm2 using a 1-minute static and followed by a 10 minute dynamic extraction, extractant flow rate of 1.0 mL/min and experiments conducted at five different temperatures (50, 100, 150, 200 and 250 oC). As shown in Table 1, no PCBs were extracted at 50 and 100 oC, and even at 150 oC poor recoveries of PCB congeners (7-27%) were obtained. However, as the temperature was increased to 200oC, the recoveries of most PCB congeners were generally greater than 41%. Further increase in temperature led to PCB better recoveries of between 80 – 91% with the exception of PCB 28 and 52 with only 58- 60 % recoveries. The temperature effect observed for extractions of PCBs mainly resulted by the decreased polarity of water at higher temperatures (Hutzinger et al., 1974). The dielectric constant of water is dramatically reduced by raising the water temperature. Therefore, water behaves more like an organic solvent at elevated temperatures. The decreased polarity of water at higher temperatures makes it possible to dissolve more organic species so that the solubility is enhanced (Figure 3). Since the extraction efficiency is highly dependent upon the solubility in the extraction fluid, better recovery is

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obtained at higher temperatures for analytes that have poor solubility in ambient water. Pressure is known to have a little effect on the solvent strength of subcritical water as long as the liquid state is maintained. Table 1 PCBs extraction efficiencies from spiked sample by extraction with varyingtemperatures Temp. (oC) PCB 28 52 101 118 138 153 180 50 0 0 0 0 0 0 0 100 0 0 0 0 0 0 0 % Recovery (RSD, %) 150 200 41.24 (5.44) 15.12 65.65 (4.12) (5.11) 70.22 16.23 (10.04) (4.16) 72.16 27.17 (15.24) (7.14) 63.23 (9.24) 20.31 79.26 (5.23) (13.30) 10.42 65.17 (4.32) (12.22) 15.46 (3.22) 7.62 (3.42) 250 58.24 (10.02) 66.43 (13.21) 90.11 (12.32) 85.13 (11.20) 91.16 (9.24) 89.20 (5.12) 86.02 (12.10)

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FID

0

10

20

30

Figure 3 GC-ECD Chromatograms for SWE extracts at different extraction temperature (50, 100, 150, 200 and 250 oC). Peak identity 1: iso-octane 2: PCB 28, 3: PCB 52, 4: PCB 101, 5: PCB 118, 6: PCB 138, 7: PCB 153 and 8: PCB 180. GC conditions: injection port temperature at 280 oC, detector temperature at 290 oC, oven temperature from 80oC (held for 3 min) to 230 oC (held for 3 min) at a rate of 25 oC/min and increased to 260 oC (held for 3 min) at a rate of 2 oC/min.

Influence of Water Flow Rate

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Different flow rates were tested in an attempt to improve the extraction efficiencies. Triplicate extractions were performed at 250 oC and 50 kg/cm2 using 0.5, 1.0, 1.5 and 2.0 mL/min. The extraction time used for each extraction was a 1 minute static followed by a 10 minutes dynamic extraction. The percent recoveries of PCB congeners obtained using different flow rates are shown in Figure 4. At a flow rate at 0.5 mL/min, all PCB congeners were extracted in the range of 45-77 %. However, as the water flow rate was increased to 1.0 mL/min, the removal all PCB congeners were higher than 15 % compared at a flow rate of 0.5 mL/min. When the water flow rate was further increased to 1.5 mL/min, the percentage recoveries of PCB were increased slightly. At a higher flow rate of 2.0 mL/min, the percentage recoveries of PCB congeners were poorer. At higher flow rates, the volume of water collected was also higher such that the concentration of extracted PCBs in the collection solvent became very low and thus made it difficult to quantitatively remove the organic collection solvent from the water extract. Hence, a water flow rate of 1.0 mL/min was chosen as the optimal flow rate for the SWE of PCBs.

100 90 Percentage Recovery % 80 70 60
0.5 ml/min

50 40 30 20 10 0 28 52 101 118 138 153 180 PCB isomer

1.0 ml/min
1.5 ml/min
2.0 ml/min

Figure 4 Effect of extraction water flow rate on the extraction efficiency of all PCB congeners from spiked sample using SWE at 250 oC and 50 kg/cm2, 1 minute staticfollwed by a 10 minute dynamic extraction and extract collected in 5 mL dichloromethane (DCM). Influence of solvent trapping Dichloromethane, n-hexane, iso-octane and n-heptane were tested as possible analyte trap solvents. Reasonably good collection efficiencies with dichloromethane (58-91%) were obtained for individual PCB congeners. Unfortunately, none of the non-halogenated solvents could efficiently collect the PCBs from the water extracts. Collection efficiencies were typically 30-70% for n-heptane, 35-66% for n-hexane and 23-67% for iso-octane (Figure 5).

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100 90 80
Percentage Recovery %

70 60 50 40 30 20 10 0
PCB 28 PCB 52 PCB 101 PCB 118 PCB 138 PCB 153 PCB 180 PCB Isomer

iso-octane

n-hexane n-heptane DCM

Figure 5 Effect of solvent trapping on the extraction efficiency of all PCBs congener from spiked sample using SWE at 250 oC and 50 kg/cm2 and 1 min static followed by a 10 minute dynamic extraction. Effect of Duration Time As shown in Figure 6, a 5 min extraction could only extract 30-65 % of the PCB congeners. The recoveries increased to 55-91% for a 10 min extraction. At a longer extraction time of 15 and 20 minutes, percentage recoveries were found to decrease slightly. Longer extraction time resulted in a larger volume of extraction solvent collected which in turn would cause an apparent low concentration of the extracted PCBs. Thus it became difficult for the organic solvent to trap back the PCBs from the water extract.

Percentage Recovery %

100 90 80 70 60 50 40 30 20 10 0 5 10 15 20

PCB 28 PCB 52 PCB 101 PCB 118 PCB 138 PCB 153 PCB 180

Time (min)

Figure 6 Effect of extraction time on the extraction efficiency of PCBs congeners from spiked sample using SWE at 250 oC and 50 kg/cm2 and analyte collected in dichloromethane.

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Comparison of SWE and Soxhlet Extraction

The organic solvent extracts (Soxhlet) were found to be much darker and highly turbid (blackish in colour), while the extracts from subcritical water (collected in dichloromethane) were orange and somewhat turbid. The soxhlet extract also yielded more artifact peaks in the GC–

Figure 7 Comparison of GC-ECD chromatograms for SWE and Soxhlet extraction. Peak identity 1:Iso-octane, 2: PCB 28, 3: PCB 52, 4: PCB 101, 5: PCB 118, 6: PCB 138, 7: PCB 153 and 8: PCB 180.

ECD chromatograms (Figure 7). Table 2 compares the extraction using soxhlet to that using subcritical water. Soxhlet extract would require further cleanup using solid phase extraction (SPE) while subcritical water extracts only require partitioning into the dichloromethane collection solvent. Although the recoveries of PCBs were comparable in both techniques, SWE has the added advantage of producing a cleaner extract and shorter extraction time. Table 2 Comparison between soxhlet and subcritical water extraction Soxhlet Extraction Solvent Collection Solvent Volume of organic solvent use (mL) Pressure (kg/cm2) Temperature (oC) Extraction Time Color of extract Extract clean-up Recovery of 7 PCB analytes (%) DichloromethaneAcetone 320 Ambient ~ 80 24 hour Dark Required 60-90 SWE Water Dichloromethane 5 50 250 15 minute Light orange Not required 60-90

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CONCLUSIONS This study clearly demonstrates the potential of subcritical water extraction for the analysis of PCBs from sludge samples.. Optimized SWE parameters found were extraction temperature of 250 oC, extraction pressure of 50 kg/cm2, 1 min static followed by a 10 min dynamic extraction and analyte collected in dichloromethane. SWE yielded cleaner extracts than the Soxhlet method. SWE was found to be a viable and a good alternative to conventional soxhlet extraction for the analysis of PCBs in sludge.

ACKNOWLEDGEMENT Thanks are due to the Ministry of Science, Technology and Innovation (MOSTI) for financial support (IRPA Vote 74085; 08-06-060027 EA 204) under the IRPA RM 8 mechanism. References Hing, L., D., Zenz, D., R., Tata, P., Kuchencrither, R., Malina, J., F., and Sawyer, Jr., B. (1998). “Municipal Sewage Sludge Management : A Reference Text on Processing, Ultilization and Disposal”. Volume 4. second edition. Technomic Publishing Company Inc. Lancaster, Pennsylvania, USA. Hutzinger, O., Safe, S., and Zitko, V., (1974). “The Chemistry of PCBs”. CRC Press, Inc. Kadir, A., and Haniffa, M., (1998). “The Management of Municipal Wastewater Sludge in Malaysia”. Paper Presented at IEM Talk on Sewage Sludge Management Issues, 18th August 1998. Petaling Jaya. Smith, R., M., (2002). “Extraction with Superheated Water”. J. Chromatogr. A.975. 31-46.

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ANALYSIS OF PM10 IN KUALA TERENGGANU BY INSTRUMENTAL NEUTRON ACTIVATION ANALYSIS Norhayati Mohd Tahir1*, Poh Seng Chee1, Suhaimi Hamzah2, Khalik Hj Wood2, Shamsiah Abd. Rahman2, Wee Boon Siong2, Suhaimi Elias2 and Nazaratul Ashifa Abdullah Salim2.
1

Department of Chemical Sciences, Faculty of Science and Technology Kolej Universiti Sains dan Teknologi Malaysia(KUSTEM) Mengabang Telipot, 21030 Kuala Terengganu, Terengganu e-mail: hayati@kustem.edu.my
2

Malaysian Institute for Nuclear Research (MINT), Bangi 4300 Kajang, Selangor

Abstract. Instrumental neutron activation analysis was used for the determination of trace elements in airborne particulate matter (PM10) for air pollution monitoring. For the collection of air samples, the PM10 high volume sampler unit and Whatman 41 cellulose filter papers were employed. Samples were collected at 13 selected sampling sites covering areas in the city center, inner and outer city of Kuala Terengganu during the month of March 2005. The average PM10 was 69.64 µg/m3, 83.58µg/m3 and 72.22 µg/m3 for sampling stations located in the city center, inner and outer city of Kuala Terengganu, respectively. It was found that the mass of air particles in the study area was higher compared to Bangi and Kuala Lumpur. This study also included the chemical analysis of selected elements (Al, Fe, Cu, Pb, V, Mn, Zn, Cr, As Cd), ionic species (Na+, SO42-, Cl-, NH4+,Mg2+, K+ , Ca2+) and some rare earth elements (REE). In general, most of the average concentration of trace elements in the city center sampling stations was generally higher than the inner and outer city sampling stations. The concentrations of trace elements in sampling stations follow the general trend of Al>Fe>Zn>Cu>Mn>Pb>V>Cr>As>Ni>Cd. The elements concentration ranged from 680-2119 ng/m3, 170-1132 ng/m3, 8.13-122.4 ng/m3, 8.48-77.3 ng/m3, 7.68-14.4 ng/m3, 1-90.4 ng/m3, 1.47-3.25 ng/m3, 1.43-5.03 ng/m3, 1.15-4.45 ng/m3, 0.24-3.75 ng/m3 and 0.28-1.36 ng/m3, respectively. Abstrak. Kajian kualiti udara telah dijalankan melalui penentuan paras kepekatan beberapa logam dalam habuk halus (PM10) dengan kaedah pengaktifan neutron (NAA). Pensampel isipadu tinggi PM10 yang dilengkapi kertas turas selulosa jenis Whatman 41 telah digunakan untuk menentukan kandungan zarahan terampai udara. Sebanyak 13 lokasi pensampelan telah di pilih merangkumi pusat bandar, luar pusat bandar dan pinggir bandar Kuala Terengganu. Purata kepekatan habuk halus (PM10) yang diperolehi adalah 69.64 µg/m3, 83.58µg/m3 dan 72.22 µg/m3 pada ketiga-tiga lokasi pensampelan tersebut. Kepekatan habuk halus (PM10) yang dicatat di ketiga lokasi ini adalah lebih tinggi berbanding dengan Bandar Bangi dan Kuala Lumpur. Kandungan kepekatan logam (Al, Fe, Cu, Pb, V Mn, Zn, Cr, As Cd dan REE), cation dan anion (Na+, SO42-, Cl-, NH4+,Mg2+, K+ , Ca2+) dalam habuk halus (PM10) telah dikaji. Secara amnya, turutan logam dalam habuk halus (PM10) adalah seperti berikut Al>Fe> Zn>Cu>Mn >Pb>V>Cr>As>Ni>Cd dan julat kepekatan masing-masing adalah seperti berikut 680-2119 ng/m3, 170-1132 ng/m3, 8.13-122.4 ng/m3, 8.48-77.3 ng/m3, 7.6814.4 ng/m3, 1-90.4 ng/m3, 1.47-3.25 ng/m3, 1.43-5.03 ng/m3, 1.15-4.45 ng/m3, 0.24-3.75 ng/m3 dan 0.28-1.36 ng/m3. Key words: airborne particulate matter, trace elements, PM10, INAA, Kuala Terengganu

Introduction The effects of atmospheric particulate matters on environment and human health have been of great global concern. Many epidemiological studies [1, 2, 3] have established an association between the

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particle concentration in the atmospheric and adverse effects on health; the PM10 fraction (diameter < 10 µm), and particularly the PM2.5 fraction (diameter < 2.5 µm) are known to be the primary cause of COPD (Chronic Obstructive Pulmonary Disease), asthma exacerbation, respiratory symptoms, morbidity and mortality, decrement in lung function and possible risk of lung cancer. Atmospheric aerosol found in urban areas represent a mixture of primary particles emitted from various sources (e.g. vehicles exhausts, coal-fired power plants, oil refineries, forest fires, industrial emission, sea spear, volcano eruption etc) and secondary particles from aerosols formed by chemical reactions [4,5]. The morphology and composition of these particles may change through several processes, including vapor condensation, evaporation and coagulation. The final ‘products’ usually vary according to origin, chemical composition and physical properties, leading to particular deposition patterns in the human respiratory system. For this reason, intensive efforts to control pollution sources and to examine contamination levels through the analysis of various air pollution parameters are being followed up all around the region. Mass concentrations of PM10 in the ambient air are usually less than 0.1 mg/m3 as and may contain elements at low concentration range (ng/g). In order to get good results, the methods of analyzing these elements need to be sensitive and precise, and also be able to identify multielements simultaneously because of the diversity of elements in the samples. Generally, these requirements could not be satisfied by conventional techniques (ICP-AES, and AAS) in a single chemical analysis. However, instrumental neutron activation analysis (INAA) using thermal neutrons in a nuclear reactor as an irradiation source, and high-resolution semiconductor detectors as measuring equipment, is one of the most suitable methods for satisfying the above mentioned requirements. INAA for airborne particulate matter can analyze up to µg/g ~ ng/g level of concentrations for 30~40 trace elements simultaneously [6,7,8]. Previous study conducted by Poh [9] on TSP levels in Kuala Terengganu has shown moderate particulate matter concentrations (17.2-148µg/m3) in different areas. However, to the best of the authors’ knowledge, there have been very little studies on the chemical characterization in PM10 fraction in Terengganu. The main objective of this study was to determine the distribution characteristics of trace elements in a rapidly urbanized area around Kuala Terengganu. Using appropriate statistical analysis such as correlation and enrichment factor analysis, attempt will be made to identify and apportion source(s) of these particulate matters in an effort to provide some baseline data on air quality for Kuala Terengganu for future references.

EXPERIMENTAL

Study Sites and Location Kuala Terengganu is the capital of state Terengganu and located in the east part of Penisular Malaysia. The area of capital is 605.28 km2 with a population of 360,708 people, which accounts for about 35% of the total state population. Samples were taken during March 2005, aerosol samples were collected in 13 sampling stations representing town center (4 station), inner (5 station) and outer city (4 station). Figure 1 illustrated the map off sampling location. Particles with an aerodynamic diameter smaller than 10 µm (PM10) were collected on 8’x10’ cellulose membrane filters exposed for 24 hours using PM10 high-volume samplers (Environmental Tisch, USA) at the average flow rate of 1.13 m3/min. Filters were pre-weighed and then dried in a desiccator for at least 24 hours after being exposed to air. PM10 and Elemental Concentration

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The mass of particulate matter on filters were determined by gravimetric method using a microbalance to an accuracy of ±0.0001g. The chemical analysis was done for soluble ionic species such as Cl-, SO42-, Na+, NH4+, K+, Mg2+, and Ca2+ and metallic elements such as Al, Fe, Zn, Mn, As, Cr, Cd, Pb, Co, Sb, Th, Cs, Sm, Sc and Eu. For ionic species analysis, portion of filters (5cm in diameter) were placed in 25ml pp centrifuge tube and sonicate for 60 minutes in the ultrasonic bath. The sonic bath temperatures were maintained within 27oC. After removing from the sonic bath, the tubes were continuous shake for 12 hour. The water extracted ionic species were determined using ion chromatography (DX120, Dionex). Most of the elemental concentrations in particles were determined by INAA. The filter samples were cut into a portion of 5cm in diameter then folded into clean polyethylene vials. Together with the standards vessel and filter blanks were irradiated in MINT Triga reactor. Both short and long irradiation programs were used for the analysis. In short irradiation program, samples were irradiated individually using a pneumatic transfer system (Rabbit). Samples were counted for 5 minutes and subsequently for 30 minutes on PC based gamma spectrometer systems. For measurement of elements that produced long-lived radionuclides (6 hour irradiation) after 2 weeks of cooling period, the samples were also counted for 1 hour on gamma spectrometer systems. The elemental concentrations were calculated based on comparative method. Standard references material NIST SRM1648 Urban Particulate Matter and IAEA CRM SL-1 Lake Sediment were analyses flowing same procedures in every batch of irradiation for quality assurance and quality control purposes. In this work, Pb and Cd determination were unpractical using INAA. Since Cd and Pb are key element for sources apportionment, acid digestion couple with ICP-AES method suggested by Poh [9] was used .

Figure 1. Map of Kuala Terengganu and sampling zones; (red-town center, blue-inner city, green-outer city).

Results and Discussion Recoveries of the elementals in SRM and CRM Table 1 shows the results of the recovery study using NIST 1648 (Urban Particulate Matter) and IAEA CRM (SL-1 Lake Sediment). Briefly, most of the elements recoveries were within 80-120%. In the INAA analysis of PM10 samples, some elements like Fe, Zn, Mn, As, Cr, Co, La, Sb, Th, Cs, Sm, Sc and Eu were mostly detected in samples. However it was not easy to determine Na, K, Ti, V, Mg,

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Cl and Al in most of the samples as the sensitivities for these elements were not good. Especially the essential elements (Ca, Na, K, Mg, Cl) the accuracy of recoveries test were far to further from the recommended errors, 15%. For Al, Ti and V samples always lose count in gamma detector due to very short half-life radionuclides. Besides that, some of the key elements like Pb and Cd were not determined because of unpractical determination using INAA or mostly the recoveries results was too bad in many samples. In order to improve accuracies and uncertainties, cations (NH4+, K+,Na+,Mg2+ and Ca2+), anions (Cl- and SO42-) was determined by Ion Chromatography (DX 120) and INAA unpractical elements (Pb and Cd) and othres poor recoveries elements was determined by more sensitive instruments, ICP-OES (Pb, Cd and others elements). PM10 and Elementals concentration Table 2 and Figure 2 illustrated average mass concentrations of PM10 in this study. Highest mean concentration was observes at inner city with value of 83.58 µgm-3 followed by outer city with mean value of 72.22 µgm-3. Surprisingly town center poses the lowest concentration (69.64µgm-3); the town center zone was associated with relatively high population and traffic density. It was found that the mass of air particles in the study area was higher compared to Bangi (63.6±26.4 µgm-3) and Kuala Lumpur (55.4±30.3 µgm-3)[14].The high mass concentrations of Inner city are cause by high value obtained from the fifth monitoring stations called Losong. During the sampling period, we manage to observe major soil excavation and building demolishing activities was happen about 3 km radius from Losong monitoring station. The wind blow disturbed surface soils and fines debris in particles form were increasing the mass loaded in the areas significantly. Whereas, the range of mass concentration in outer city (suburban residential areas) were relatively small compared to others. The free flows of air and totally no tall obstruction (buildings, trees and vehicles) around the monitoring stations make it ideally best criteria to collect air samples. All the PM10 values obtained are still well below the recommended Malaysia guideline for PM10 (mean of 24-hour measurement = 150 µgm-3). Table 3 presents the concentration of the measured elements and ionic species in PM10 in three zones during to entire sampling period. The concentration levels between each zone are given in term of average mean and range of the value. In general, most of the average concentration of trace elements in the city center sampling stations was generally higher than the inner and outer city sampling stations. The concentrations of trace elements in sampling stations follow the general trend of Al>Fe> Zn>Cu>Mn>Pb>V>Cr>As>Ni>Cd. The elements concentration ranged from 680-2119 ng/m3, 1701132 ng/m3, 8.13-122.4 ng/m3, 8.48-77.3 ng/m3, 7.68-14.4 ng/m3, 1-90.4 ng/m3, 1.47-3.25 ng/m3, 1.43-5.03 ng/m3, 1.15-4.45 ng/m3, 0.24-3.75 ng/m3 and 0.28-1.36 ng/m3, respectively. For outer city, the concentration of REE showed the trend of La>Sb>Th>Cs>Sm>Sc>Eu, whereas in inner city and town center followed the trends of Sb>La>Th>Cs>Sc>Sm>Eu. In general, average of concentration of ionic species followed the trends of Na+>SO42->Cl,>NH4+>Mg2+> K+ >Ca2+. High loading of Na+,Mg2+ and Cl- species in samples were founds in inner city zones. Whereas, higher concentration of NH4+, K+ and SO42- species are inhabit in outer city aerosol samples. The average and range of species ionic concentration are given in Table 3. The high loadings of Na+, Mg2+ and Cl- in inner city were most probably generated from sea breeze. Correlation analysis indicated there was no clear correlation between these species in data combined from three zones (Table 4) due to large variation in the analysis results for some ionic species in three different zones. However, the plot of Na against Mg2+ and Cl- (figure 3) within inner city samples only show some trends of better correlation (Cl-, r=0.995; Mg2+, r=0.999)which might be explained sea breeze as a major sources for Na+,Mg2+ and Cl- in inner city.

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Table 1. Recoveries results of certified/standard references material (INAA method)
SRM 1648 CRM SL 1

Element Al Mn Fe As Cr Co Zn Eu Sm Th Sc La Ce Sb

Certified Value 34200 786 39100 115 403 609 4760 0.8 4.4 7.4 7 42 55 45

Obtained Value 30882 694 36722 110 349 579 3839 0.73 3.73 6.75 6.22 39.44 53.43 40.62

Recovery (%)
90.3 88.4 93.92 95.76 86.68 95.10 80.66 92 85.5 91.31 88.91 93.92 97.16 90.28

Certified Value 89000 3460 67400 27.5 104 19.8 223 1.6 9.25 14 17.3 52.6 117 1.31

Obtained Value 77990 3699 58253 28.7 120 20.8 276.5 1.72 7.80 15.02 19.03 60.67 120.40 1.45

Recovery (%)
87.63 106.93 86.43 104.47 115.56 105.15 123.97 108 84.42 107.32 110.21 115.36 102.91 110.76

Table 2. PM10 range and average within 3 different sections Site Average (range) Town center 69.64 (58.37-83.06) Inner city 83.58 (52.91-134) Outer city 72.22 (70.02-73.25)

Figure 2. PM10 comparisons within 3 sections

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Table 3. The range of concentration and mean value of elements and ionic species in PM10
Element Na+ NH4+ K+ Mg+ Ca2+ ClSO42Al Fe As Pb Cd Ni V Cu Zn Cr Mn Cs Sb Sc La Sm Th Eu Outer City (ng/m3) Inter City (ng/m3) Town Center (ng/m3)

18366 (5708-41262) 5184 (1571-9393) 1881 (1418-2211) 2574 (727-4507) 1527 (903-2069) 5393 (2898-8678) 10960 (5115-17065) 780 (680-2119) 430 (170-650) 1.09 (1.32-4.45) 3.973 (1.00-7.35) 0.329 (0.28-0.39) 1.003 (0.24-2.05) 2.362 (1.47-2.89) 30.67 (8.48-41.0) 16.14 (8.13-42.8) 1.30 (1.43-2.20) 8.29 (7.68-8.89) 0.29 (0.28-0.30) 1.41 (0.45-2.58) 0.14 (0.04-0.20) 2.04 (1.72-4.04) 0.21 (0.18-0.23) 0.47 (0.37-0.57) 0.063 (0.055-0.086)

35094 (1063-64518) 4461 (3863-5099) 1484 (592-2594) 2243 (216-7176) 1160 (441-2671) 7717 (475-21453) 9477 (3055-15282) 630 (510-1520) 660 (360-1040) 1.53 (1.15-2.35) 25.34 (3.61-51.3) 0.563 (0.24-1.07) 0.959 (0.48-1.39) 2.391 (1.83-2.73) 39.72 (20.2-77.3) 59.34 (14.6-88.2) 1.32 (1.73-2.97) 9.89 (7.78-11.5) 0.23 (0.11-0.28) 1.79 (1.21-3.06) 0.18 (0.09-0.31) 1.67 (0.89-2.27) 0.14 (0.09-0.18) 0.51 (0.20-0.77) 0.095 (0.071-0.147)

15526 (2175-30417) 3660 (2935-4378) 1758 (944-3243) 1784 (335-3461) 1764 (731-2760) 6298 (2213-10409) 9872 (6982-13555) 1020 (950-1380) 690 (570-1132) 1.70 (1.57-2.93) 55.45 (53.6-90.4) 0.843 (0.25-1.36) 1.715 (0.49-3.75) 2.376 (1.91-3.25) 43.85 (22.4-66.5) 72.36 (7.34-122.4) 2.65 (1.67-5.03) 11.2 (8.45-14.4) 0.19 (0.13-0.44) 1.96 (0.89-3.51) 0.19 (0.06-0.32) 1.56 (1.12-2.29) 0.14 (0.10-0.12) 0.45 (0.23-0.85) 0.087 (0.089-0.153)

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Table 4. Correlation analysis of ionic species and element data combined from 3 sampling zones (n=13) * Correlation is significant at the 0.05 level (2-tailed). ** Correlation is significant at the 0.01 level (2-tailed).

Figure 3. The plot of Na against Mg2+ and Cl-

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Enrichment Factor The enrichment factor method has commonly been used as a tool to evaluate the strength of enriched, or depleted, relatively to specific sources. The enrichment factor for any element X relative to control/unpolluted references material is defined by EF,X = (X/Y)air/(X/Y)control, Where EF,X is the enrichment factor of X, Y is a reference element for crustal material and (X/Y)air is the concentration ratio of X to Y in the samples and (X/Y)control is the same ratio in control/unpolluted references material. If EFX approached unity, crustal soils are likely the predominant sources for element X[10]. In this study, the (X/Y)control, ratio calculation for each elements was based on samples collected from a rural area call Manir (one of the sampling station in outer city). Al is normally use as references element since it can be accurately measured by a number of analytical method and abundance in the earth crusts [11].

Figure 4. Enrichment factors of selected trace elements relatively to Al for the PM10 samples at three locations. Figure 4 indicated that the enrichment factors of all elements except La and Sm in town center are higher than those in inner and outer city. The rapid urbanization activities in Kuala Terengganu had a good explanation for high elements loading to ambient atmospheric. The enrichment factor for Cd, Cr, Ni, Ti, Pb and Zn in town center exhibited at least 10 times higher than others places. The strong correlation (table 4) between Pb, Zn, Ni and SO42- (r≥0.696) explained these elements may originate from traffic emission sources. Cadmium, Pb, Cr, Ti and Zn probably emitted on atmospheric as part

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of brake dust, road paint, diesel exhaust particles, road construction materials, or car catalyst materials [12]. Whereas Ni, which are frequently associated with fuel burning, this factor was easily attributed to emissions from the petrochemical combustion such as vehicle gasoline. According to Chiarenzelli et al.[13] rare earth elements may be useful as reference elements in environmental studies because of transport in the particulate phase, lack of significant anthropogenic sources, coherent group geochemistry, generally robust concentrations, and upper crustal signatures. For REE except La, most of the enrichment factor was below 15 and close to each study zones. This indicated the REE may appear to be soils origin. Conclusion Highest mean PM10 concentration was observes in inner city with value of 83.58 µgm-3 followed by outer city with mean value of 72.22 µgm-3 and town center, 69.64µgm-3. The Al, Fe, Cu, Zn, Mn, Pb, V, Cr, As, Ni, Cd, Cs, Sb, Sc, La, Sm, Th and Eu concentration ranged from 680-2119 ng/m3, 1701132 ng/m3, 8.13-122.4 ng/m3, 7.68-14.4 ng/m3, 1-90.4 ng/m3, 1.47-3.25 ng/m3, 1.43-5.03 ng/m3, 1.15-4.45 ng/m3, 0.24-3.75 ng/m3 and 0.28-1.36 ng/m3, 0.11-0.44 ng/m3, 0.0.45-3.51 ng/m3, 0.04-0.32 ng/m3, 0.89-4.40 ng/m3, 0.09-0.23 ng/m3, 0.2-0.85 ng/m3 and 0.05-0.153 ng/m3, respectively. For ionic species, all 3 location followed similar trends of Na+>SO42->Cl-,>NH4+>Mg2+> K+ >Ca2+. The high loadings of Na+, Mg2+ and Cl- in inner city were most probably generated from sea breeze. Lastly enrichment factor for Cd, Cr, Ni, Ti, Pb and Zn indicated these elements may origin from traffic emission sources and REE may appear to be soils origin. Acknowledgment Authors wishes to acknowledge the Malaysian Institute of Nuclear Technology (MINT) for the financial support to conduct this research. References 1. 2. 3. 4. 5. 6. Jalaludin B., O’Toole B.I., Leeder S.R. (2004). “Acute effects of urban ambient air pollution on respiratory symptoms, asthma medication use, and doctor visits for asthma in a cohort of Australian children” J. Environmental Research, A. 95, 34-32. Kappos A.D., Bruckmann P., Eikmann T., Englert N., Heinrich U., Höppe P., Koch E., Krause G.H.M., Kreyling W.G., Rauchfuss K. (2004). “Health effects of particles in ambient air” International Journal of Hygiene and Environmental Health A. 4, 399-407. Desqueyroux H., Pujet J.C., Prosper M., Squinazi F., Momas I. (2002). “Short-Term Effects of Low-Level Air Pollution on Respiratory Health of Adults Suffering from Moderate to Severe Asthma” J. Environmental Research A. 89, 29-37. Salma I., Chi X.G., Maenhaut W. (2004) “Elemental and organic carbon in urban canyon and background environments in Budapest, Hungary” J. Atmospheric Environment, A. 38, 27-36. Pun B.K. and Seigneur C. (1999). “Understanding particulate matter formation in the California San Joaquin Valley: conceptual model and data needs” J. Atmospheric Environment, A.33- 29, 4865-4875. Rizzio E., Giaveri G., Arginelli D., Gini L., Profumo A., Gallorini M. (1999). “Trace elements total content and particle sizes distribution in the air particulate matter of a ruralresidential area in north Italy investigated by instrumental neutron activation analysis” J. The Science of The Total Environment, A. 226-1, 47-56. Freitas M.C., Almeida S.M., Reis M.A., Oliveira O.R. (2003). “Monitoring trace elements by nuclear techniques in PM10 and PM2.5” J. Nuclear Instruments and Methods in Physics Research Section A, A. 505-1, 430-434. Alemón E., Herrera L., Ortiz E., Longoria L.C. (2004). “Instrumental nuclear activation analysis (INAA) characterization of environmental air filter samples” J. Applied Radiation and Isotopes A.60-6, 815-823. Poh S.C. (2004). “Determination of heavy metals in airborne particulate matter in Kuala Terenggu, Malaysia” Bsc Environmental Analytical Chemistry, FST, KUSTEM; 88p.

7. 8. 9.

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10.

11. 12. 13. 14.

Gao Y., Nelson E.D., Field M.P., Ding Q., Li H., Sherrell R.M., Gigliotti C.L., Van Ry D.A., Glenn T.R., Eisenreich S.J. (2002). “Characterization of atmospheric trace elements on PM2.5 particulate matter over the New York–New Jersey harbor estuary” J. Atmospheric Environment A.36, 1077–1086. Chester R., Nimmo M., Corcoran P.A. (1997). “Rain water-aerosol trace metal relationship at Cap Ferrat: A costal site in the western Mediterranean” J. Marine Chemistry A.58, 293-312. Kim K.W., Myung J.H., Ahn J.S., Chon H.T. (1998) “Heavy metal contamination in dusts and stream sediments in the Taejon area, Korea” J. Geochemical Exploration A. 64, 409- 419. Chiarenzelli J., Aspler L., Dunn C., Cousens B., Ozarko D., Powis K. (2002). “Multi-element and rare earth element composition of lichens, mosses, and vascular plants from the Central Barrenlands, Nunavut, Canada” J. Applied Geochemistry, V. 16- 2, 245-270. Suhaimi Hamzah M., Shamsiah A.R., Mohd Khalid M., Khalik W. (2001). “Characterisation of Air Particulate Matter in Klang Valley by NAA technique”. Seminar RND 2000, MINT.

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CO2 / H2 Methanation on Nickel Oxide Based Catalyst Doped with Various Elements for the Purification of Natural Gas Faridah Mohd Marsin, Nor Aziah Buang*, Wan Azelee Wan Abu Bakar, Mohd Hasmizam Razali Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia 81310, Skudai, Johor. Abstract: Nickel possess characteristics similar to noble metals, apart from being easily deactivated by carbon deposition and poisoning. In this study, the activity of prepared nickel based catalysts doped with selected elements (Mg, Zr, Mo, Mn, Co, Fe, and Cu) that were presumed to help nickel active sites has been investigated for the CO2 elimination in the presence of H2 in the hopes for a methanation reaction. With the addition of lanthanide series as co-dopant in the catalyst, the synthesized catalysts were tested for its catalytic activity and reproducibility by FTIR spectroscopy. It was found that only several elements can boost CO2 elimination, namely magnesium, cobalt and ferum, with cobalt showing the highest conversion for both ratios, Ni/Co/Pr 60:30:10 and 60:10:30. Furthermore, Ni/Co/Pr with the ratio of 60:30:10 was proven superior as it yielded highest CH4 in the lowest conversion temperature of approximately 350 °C. Further characterization on Ni/Co/Pr with the ratio of 60:30:10 showed the supremacy towards the conversion of CO2 to CH4. Single point BET analysis showed that Ni/Co/Pr did not have any changes in the surface area, as it did not adsorb CO2. This statement is in agreement with the XRD and EDX results obtained whereby there are no traces of carbon deposition. From TPD results showed CO2 desorption peaks at low and high temperature indicated intermediate bonding of CO2 on the surface of the catalyst. This shows the presence of dopant will result in the enhancement of CO2 elimination to a 100%. Keywords: nickel based catalyst, cobalt, CO2 elimination, methanation Abstrak: Nikel mempunyai ciri-ciri yang menyamai logam yang mahal (paladium, platinum). Walau bagaimanapun ia mudah terencat dengan kehadiran karbon dan keracunan mangkin. Dalam kajian ini, aktiviti pemangkinan bagi mangkin Ni yang didop dengan bahan pendop terpilih (Mg, Zr, Mo, Mn, Co, Fe, and Cu) untuk meningkatkan keupayaan pemangkinan disamping memberi ketahanan kepada keracunan mangkin dalam proses menukarkan CO2 kepada gas metana. Dengan penambahan praseodymium daripada kumpulan lantanida, mangkin tersebut telah di uji dari segi keupayaan pemangkinan dan ketahanan menggunakan analisis FTIR. Kajian telah mendapati bahawa hanya beberapa elemen sahaja yang boleh menyingkirkan CO2, iaitu magnesium, ferum dan cobalt. Cobalt menunjukkan keupayaan pemangkinan yang tinggi dalam kedua-dua nisbah Ni/Co/Pr 60:30:10 dan 60:10:30. Selain itu, Ni/Co/Pr dengan nisbah 60:30:10 merupakan mangkin yang paling efektif untuk menyingkirkan CO2 disamping menukarkannya kepada CH4 pada suhu lebih kurang 350ºC. Kajian pencirian mangkiin menunjukkan bahawa Ni/Co/Pr dengan nisbah 60:30:10 lebih berkesan menukarkan CO2 kepada CH4. Kajian luas permukaan BET menunjukkan bahawa Ni/Co/Pr tidak menunjukkan sebarang perubahan dalam luas permukaan, bermakna ia tidak menyerap CO2. Analisis XRD and EDX menunjukkan tiada perubahan struktur dan tiada karbon terbentuk di atas permukaan mangkin. Analisis TPD menunjukkan puncak penyahjerapan CO2 berada pada kedua-dua suhu yang rendah dan tinggi. Ini menunjukkan terdapat penjerapan CO2 di atas permukaan mangkin. Ini semua menunjukkan peningkatan kepada keupayaan mangkin untuk menyingkirkan CO2 100%. Kata kunci: mangkin berasaskan nikel, cobalt, penyingkiran CO2, pembentukan metana.

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1.0

Introduction

The necessity of natural gas purification into high quality products boosted both the applied and academic research activity. In recent years, renewed interest in CO2 elimination process has arisen. Noble metals such as Rh, Ru and Ir exhibit high stability and less sensitivity in catalytic process [1-2], cost and limited avaibility of these metals discourage their widespread industrial applications. Nickel oxide exhibited high activity and selectivity of methane due to the ability of NiO to undergo reduction process owing to the presence of defect sites of the surface [3]. Despite of the fast catalyst deactivation and carbon deposition, NiO catalyst was still favored for its high thermal stability [4], and its low price [5]. Therefore, it is very important to develop stable and effective nickel oxide catalyst with improved resistance to deactivation caused by coking and poisoning. A study by Valentini et al. [6] found that the catalytic properties, metal dispersion and the structural features of species depend on the method to process these materials and on the support used. The addition of dopant into nickel oxide base catalyst was found to enhance the capability of the catalyst. The usage of catalyst will depend on the dopant used in order to form a durable, sulfur tolerant, high catalytic activity catalysts. The selected dopants studied were magnesium (Mg), zirconium (Zr), molybdenum (Mo), manganese (Mn), ferum (Fe), cobalt (Co), and copper (Cu). Cobalt was mainly used for Fisher-Troposh Synthesis (FTS) and was chosen due to its reducibility of CO. Jacobs et al. [7] found that addition of Ru and Pt exhibited a similar catalytic effect on decreasing both the reduction temperatures of cobalt oxides. The hydrogenation of CO and CO2 were found to catalyze by the larger cobalt clusters formed by three incipient wetness impregnations [8]. Other research by Guczi et al. [9] found that Co–Pd samples are fully reducible and form bimetallic particles which can be reversibly oxidized/ reduced. CO hydrogenation takes place in the range of 200–300oC producing mainly alkenes on pure cobalt catalyst with short chains. Synergism on the addition of small amount of palladium to cobalt is observed and the rate of the CO hydrogenation significantly increases. In this research, Ni was doped with selected metal elements and praseodymium, which were doped in catalyst material to alter the surface structure of a catalyst so it will become polycrystalline, with different ratios and it was used to eliminate CO2 with the presence of H2 with the hopes for CO2 hydrogenation to occur. The reducibility and characteristics of the Ni based catalysts were tested by means of FTIR for catalytic activity of CO2 eliminated and methane produced, X-Ray Diffraction for phase or structural changes, Temperature Programmed Desorption for the determination of active site, and BET Surface Area for surface area of the catalyst.

2.0 2.1

Experimental Catalyst preparation

Ni/M*/Pr (M*= Mg, Zr, Mo, Mn, Fe, Co, and Cu) in both higher and lower atomic ratios of selected dopant, 60:30:10 and 60:10:30, were prepared via optimized sol gel method. This method includes the addition of its specific metal salts with its base metal, Ni(NO3)2.6H2O, and second dopant, Pr(NO3)3.6H2O. it was then aged at 75°C for 48 hours before it was calcined at 400°C for 17 hours. 2.2 Catalytic activity measurement

The prepared samples were then tested for its capability to eliminate CO2 by methanation reaction. This was done using a flow bed reactor of 10 mm inner diameter under atmospheric pressure. The reaction gas mixture of CO2 and H2 was passed continuously through the catalyst that was filled inside the sample tube and CO2 peaks from the composition of the gas that flows through was detected by FTIR. The temperature of the sample ranged from 25-500°C. The tabulated data gave percentages of CO2 eliminated and CH4 converted within the temperature range.

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2.3

Characterization 1. X-ray Diffraction Analysis : The catalyst samples were characterized by using a Philips D5000 X-Ray Diffractometer (Cu-Kα radiation) with a degree ranging from 10-80°, 2. Single Point BET Surface Area: the surface area of samples before and after exposure to CO2/H2 were measured using Micromeritics ASAP, whereby it uses nitrogen as the adsorption gas for the physical characterization of the catalyst. 3. Temperature Proggrammed Desorption Analysis: the H2 and CO2 desorption from the catalyst were taken into account using Thermofinnigan TPD/R/O 1100

3.0 3.1

Results and disscussion Catalytic activity

For the lower ratio of the selected dopant, Ni/M*/Pr (M =Mg, Zr, Mo, Mn, Co, Fe, and Cu) with the ratio of 60:10:30. The results indicated that for the lower addition of various metals as dopants, most of the catalyst exhibit CO2 elimination properties. The three highest CO2 elimination catalysts were Ni/Mg/Pr, Ni/Fe/Pr, and Ni/Co/Pr. Starting from 300°C, Ni/Mg/Pr has already eliminated over 80% of the CO2. The full elimination is at 330°C with 93.26%. For Ni/Fe/Pr and Ni/Co/Pr, the light off temperature (TLO) was both at 250°C, while complete elimination of CO2 was also both at around 427°C. Ni/Mo/Pr and Ni/Cu/Pr also showed response in the CO2 elimination whereby at 300°C, the catalyst eliminated 17.69% and 40% respectively. Hence the elimination for Ni/Cu/Pr ended at 430°C with a maximum elimination of 67%, while after 50% of elimination, the performance of Ni/Mo/Pr declined at 450°C. Ni/Mn/Pr, and Ni/Zr/Pr did not show any significant elimination throughout the testing. Figure 3.1 showed the catalytic activity of Ni/M*/Pr (M =Mg, Zr, Mo, Mn, Co, Fe, and Cu) catalyst ratio 60:30:10 for CO2 elimination that were calcined at 400°C for 17 hours and tested under stoichiometric conditions, consisting of CO2 and H2, from 300-500°C. From the results indicated that for the higher addition of various metals as dopants, most of the catalysts performances have been suppressed from eliminating CO2 in the system. Most of the catalyst, except Ni/Co/Pr, eliminated below than 20% of CO2. Only Ni/Co/Pr maintained its performance in eliminating CO2 with the TLO at 300°C was 20%, and the full elimination was at 400°C with 97.42%.

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100 80
%elimination

60 40 20 0 300 330 350 400 temperature
Mg
Zr
Mo
Mn
Co
Fe
Cu

427

450

500

Figure 3.1 Catalytic activities of Ni/M*/Pr (M =Mg, Zr, Mo, Mn, Co, Fe, and Cu) ratio 60:10:30 for CO2 elimination versus temperature

100 80
% elimination

60 40 20 0 300 330 350 400 temperature 427 450 500

Mg

Zr

Mo

Mn

Co

Fe

Cu

Figure 3.2 Catalytic activities of Ni/M*/Pr (M =Mg, Zr, Mo, Mn, Co, Fe, Cu, and Zn) ratio 60:30:10 for CO2 elimination versus temperature

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45 40 35 30 25 20 15 10 5 0 Mg Mn dopant Ni/M*/Pr 60:30:10 Ni/M*/Pr 60:30:10 Zr Mo Co Fe Cu

100 90 80 70 60 50 40 30 20 10 0

CH4 conversion (%)

Ni/M*/Pr 60:10:30 Ni/M*/Pr 60:10:30

Figure 3.3 Catalytic activities of Ni/M*/Pr (M =Mg, Zr, Mo, Mn, Co, Fe, Cu, and Zn) ratio 60:30:10 and 60:10:30 for CO2 elimination and CH4 conversion at 400ºC As we compare the dopants according to ratios shown in Figure 3.3, we can see significant difference in each ratio for each dopant. The line chart that showed the CO2 elimination for the catalyst indicated that the catalyst doped with cobalt for both higher and lower amount of dopant, were superior from the rest it was observed in Ni/Co/Pr catalyst, which has special features itself, showed that, it still does the function of eliminating CO2 and formed CH4 as the product. Further study on the cobalt doped catalyst shows that the optimized ratio of the dopant added is Ni/Co/Pr with the ratio of 60:35:5. with only little addition praseodymium of the lanthanide series, the structure has been altered to a perfect structure for methanation catalyst. 3.2 Characterization

In order to further understand the performance of the catalyst and role of dopant in the enhancement of the catalytic performance material, the best catalyst was characterized by various techniques. 3.2.1 X-Ray Diffraction

X-ray Diffraction was carried out on Ni/Co/Pr with the ratio of 60:35:5 prepared by calcination at 400oC for 17 hours. From Figure 4.1, it was found out that the catalyst formed a polycrystalline phase, a form of mixture of amorphous and crystalline phases, which indicates a good catalyst. We can clearly see formation of spinel compound of Co3O4 on the surface of catalyst. This was preferred because the spinel compound is a combination of two oxidation state that is CoO and Co2O3 which served as an active site on the surface. Many of the peaks overlapping each other with Table 4.2 showed the major peaks with their dominant references. The particle size calculated from Scherrer’s equation is 17.65nm. From the tabulated data, we presumed that spinel compound that formed in the catalyst plays a major role in enhancing the performance of the catalyst. Meanwhile, the addition of Pr has also played a part in the enhancement of the catalyst, probably affected the physical structure by making it more amorphous and also contributes in the mechanism of the conversion.

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Figure 3.4

Diffractogram for Ni/Co/Pr with the ratio of 60:35:5 calcined 400°C for 17 hours

3.2.2

BET surface area

Ni/Co/Pr 60:35:5 catalysts have been sent for surface area analysis. Table 4.1 showed the comparison between the previous studied catalyst Ni/Pr with the ratio of 60: 40 and Ni/Co/Pr 60: 35: 5. It seems that the surface areas for Ni/Pr 60:40 and Ni/Co/Pr 60:35:5 catalysts have only had a slight difference in the surface area. While Ni/Co is about half of Ni/Co/Pr, which means that, the surface area of the new modified catalyst have similarity of the previous catalyst and we can presume that the catalytic activity should be the same. This also showed that the addition of Pr change the physical properties by enlarging the surface area of the catalyst

Table 3.1

BET surface area of catalyst Catalyst Ni/Pr [11] Ni/Co/Pr Ni/Co BETsurface area (m2 /g) 75.58 59.86 38.49

3.2.3

Temperature Programmed Desorption

To determine the sorption file of CO2 and H2 on the surface of catalyst, TPD was used whereby the ability of Ni/Co/Pr with the ratio of 60: 35: 5 to absorb CO2 and H2 was studied. The TPD profile of CO2 by the catalyst is shown in Figure 3.5. Three desorption peaks at 270°C, 600°C and 750°C can be distinguished, a peak existed at a lower temperature and two remain peaks are at a higher temperature. This was probably due to the different bonding modes of CO2 with the active sites, during the adsorption on the surface.

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Figure 3.5

TPD profile of CO2for Ni/Co/Pr with the ratio of 60:35:5

A study by Cox [12], mentioned that the coordination of CO2- onto metal oxide has different energies, which will cause different desorptions in the profile. The peak in the lowest temperature is due to desorption of the weakest bonding mode of CO2. It was proposed that three bonding modes occurred. The peak area showed the amount of CO2 sorption of the species. It was clearly seen that the metal carbene (M=CO2) primarily dominated the bonding mode on the surface of the catalyst. This means that the catalyst that formed strong bonds with CO2, and needed high temperature to produce more energy for desorption the CO2. The sorption sites of H2 on the surface of the catalyst were also being studied. Two significant peaks were detected at 350°C and 780°C. It was probably due to the sorption of hydrogen between the NiO or CoO lattices to form metal hydrates (M-H). Nevertheless, H2 sorption correlates with the CO2 sorption at the higher temperature of 780°C. It can be concluded that the chance for formation of methane at lower temperature is less compared to catalyst at higher temperature. But the lower desorption peaks can still be accounted because it still can convert to methane

4.0

Conclusion

From entire screening test of all the catalyst with higher and lower incorporation of dopants (ratio 60:30:10 and 60:10:30) respectively, the testing showed that addition of some dopants resulting in high catalytic activity, and some suppress the catalytic activity of the catalyst. For the catalytic ability to eliminate CO2 and at the same time converting to methane, Ni/Co/Pr was found to be able to complete the mission successfully with almost 100% of CO2 elimination. Some of the catalysts also have the potential to eliminate CO2 such as catalyst incorporated with Mg and Fe. But it was found that the amount of methane formed was low and the catalytic ability occurred at very high temperature. Further study to determine the optimized ratio for Ni/Co/Pr, thus the optimized ratio is Ni/Co/Pr 60:35:5. Characterization of the best performance catalyst was studied. For XRD analysis it was found out that the catalyst consists of cubic NiO, and spinel compound of Co3O4 which consist of cubic CoO and Co2O3. This structure was presumed to enhance the catalytic activity. From the single point BET surface analysis, it was found out that Ni/Co/Pr 60:35:5 has a high surface area of 59.86m2/g when compared to Ni/Co 60:40 (38.4987m2/g). It was predicted that the incorporation of Pr attributed to the surface area and thus making it superior. Commencing the TPD analysis, the catalyst has three major peaks for the sorption of CO2 and two major peaks for the sorption of H2. As the lower peaks contributed to the elimination of CO2 at lower temperature (270°C) with the full formation of methane (350°C), the actual desorption of CO2 and H2 was at higher temperature. This means that this catalyst full potential lies in the temperature ranging from 600-800°C.

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From the characterization, we can conclude that the addition of Co as dopant enhanced the capability of the catalyst as it contributed to the additional active sites and change the chemical properties of the catalyst. Meanwhile the incorporation of Pr was to change the physical properties. 5.0 Acknowledgements

The writers would like to thank the Ministry Of Science Technology Education for a research grant. The financial support of MOSTE under project (vot: 74178) is recognized. References 1. Kudo, K., and Komatsu, K. (1999). Reduction of alkali metal carbonate to methane with water in the presence of Raney alloy. Journal of Molecular Catalysis A: Chemical. 145: 159–167 2. Cattenot, M., Geantet, C., Glasson, C., and Breysse, M. (2001). Promoting effect of ruthenium on NiMo/Al2O3 hydrotreating catalysts”. Applied Catalysis A: General. 213: 217–224. 3. Jose, A., R., Jonathan, C., H., Anatoly, I., F., Jae, Y., K. and Manuel, P. (2001). Experimental and Theoretical Studies on the Reaction of H2 with NiO. Role of O Vacan cies and Mechanism for Oixde Reduction. J. Am. Chem.Soc. 124, 346-354 4. Wang, L., Murata, K., and Inaba, M. (2004). Control of the Product Ratio of CO2/ (CO+CO2) and Inhibition of Catalyst Deactivation for Steam Reforming of Gasoline to Produce Hydrogen. Applied Catalysis B: Environmental. 48: 243-248 5. Hou, Z., Yokota,O., Tanaka, T., and Yashima, T. (2004). Surface Properties of a Coke-free Sn Doped Nickel Catalyst for the CO2 Reforming of Methane. Applied Surface Science. 233: 5868. 6. Valentini, A., Carreno, N.L.V., Probst, L.F.D., Lisboa-Filho, P.N., Schreiner, W.H., Leite, E.R., and Longo, E. (2003). Role of vanadium in Ni:Al2O3 catalysts for carbon dioxide reforming of methane. Applied Catalysis A:General 255: 211-220 7. Jacobs, G., Das, T.K., Zhang, Y., Li, J., Racoillet, G., and Davis, B.H. (2002). Fisher-Troposh synthesis: support, loading, and promoter effects on the reducibility of cobalt catalyst. Applied Catalysis A: General 233: 263-281. 8. Zhang, Y., Jacobs, G., Sparks, D.E., Dry, M.E., and Davis, B.H. (2002). CO and CO2 hydrogenation study on supported cobalt Fisher-Troposh synthesis catalysts. Catalysis Today 71: 411-418 9. Guczi, L., Schay, Z., Stefler, G., and Mizukami, F. (1999). Bimetallic catalysis: CO hydrogenation over palladium–cobalt catalysts prepared by sol gel method. Journal of Molecular Catalysis A: Chemical. 141: 177–185. 10. Mohd Hasmizam, R. (2005). CO2 Methanation on Nickel-Based Catalysts. MSc thesis, UTM. 11. Cox, P., A. (1995). The Elements on Earth-Inorganic Chemistry in the Environment. Oxford. Oxford University Press. 147-158.

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ESTERIFICATION OF BETULINIC ACID USING LIPASE AS CATALYST Yamin Yasina*, Faujan Hj Ahmadb and Mahiran Basrib
a

International Education Centre, Universiti Teknologi MARA, Kampus Seksyen 17, 40200 Shah Alam b Chemistry Department, , Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia email: yamin961@salam.uitm.edu.my

Abstract. Betulinic acid ester was synthesized from reaction of betulinic acid and phtalic anhydride using lipase as catalyst in an organic solvent system. The reaction was carried out at 37°C for 24 hours in chloroform. Product was identified using Infrared spectroscopy (IR), Mass Spectrum (MS) and Nuclear Magnetic Resonance (NMR). Based on spectroscopy data, product was identified as ester of betulinic acid. The product produced gave a melting point of 230oC. Keywords: Betulinic acid, betulinic acid ester, lipase

Introduction Betulinic acid, a pentacyclic triterpene, has recently been reported to possess anti-tumor activity against cultured human melanoma cells in both vitro and vivo models [1]. Furthermore, the ability of betulinic acid to induce apoptosis in melanoma [1] and other cell types [2] and the favorable therapeutic index from the lack of toxicity towards normal cells [1] suggest that betulinic acid is an attractive and promising agent. Other biological activities reported for betulinic acid include antiinflammatory activity [3] and inhibition of phorbol ester-induced epidermal ODC accumulation in the mouse ear model with subsequent inhibition of the carcinogenic response in the two-stage mouse skin mode [3]. Betulinic acid derivatives can be used more efficiently in a topically applied composition to selectively treat or prevent or inhibit a melanoma [4]. It could be absorbed more efficiently as compared to their counterpart as they are more hydrophobic and thus are more desirable. Derivatives of betulinic acid have also been investigated as specific inhibitors of HIV-1 and as potential of antiHIV drug candidates [5]. Synthesis of betulinic acid derivatives by chemical method: e.g. esterification of betulinic acid in the presence of acid and alkaline catalyst usually results in complex mixture. This alternative method of using environmentally friendly enzyme-catalyzed synthesis offers mild reactions conditions such as at atmospheric pressure, moderate temperature and pH. Enzyme catalyzed reactions also produce relatively pure product using a single procedure with high efficiency. This present paper reported the synthesis of betulinic acid ester using Lipozyme as catalyst.

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Experimental

Materials Lipase from Mucor Miehei (Lipozyme) was purchased form Novo Nordisk Inc. (Danbury, CT). Other chemicals were of analytical grade. Esterification procedure The reaction system consisted of betulinic acid (50 mg), phtalic anhydride (119 mg), chloroform (5 ml) and Lipozyme lipase (5 mg). The mixture was incubated for 24 h at 37°C with continuous shaking at 150 rpm. The product was detected using thin layer chromatography and developed in the mobile phase of chloroform. The mixture was chromatographed on a silica gel column (3 cm, i. d. and 30 cm length) using chloroform as eluting system. The product was then characterized using spectroscopic method. Results and Discussion Product identification Thin Layer Chromatography analysis The development of product from the esterification reaction between betulinic acid and phtalic anhydride catalyzed by Lipozyme was monitored by TLC analysis in a solvent system containing chloroform. Excellent separation of betulinic acid, phtalic anhydride and betulinic acid ester was achived. Individual components were well resolved from one another and migrated as tight, circular entities as indicated by its retention values (Rf) of 0.32, 0.7 and 0.62 respectively.

Fourier Transform Infrared Analysis Figure 1 shows the infrared spectrum of reaction mixture in the presence of Lipozyme after 24 h incubation. The C=O stretching for carbonyl group of betulinic acid showed absorption at vmax 1642 cm-1 while the C=O stretching for the expected ester carbonyl showed the absorption at vmax 1682 cm1 . The C=O absorption was in agreement with Silverstein et al. [6] which proposed that ester compounds have a characteristic strong absorption bands arising from C=O stretching at vmax 1740 cm-1.

Nuclear Magnetic Resonance Analysis (NMR) The structure of the product was proven as obtained from its NMR spectra. Typically, the proton magnetic resonance as shown in Figure 2 indicate the presence of six methyl groups resonated as singlets at δ 0.92, 0.95, 0.99, 1.02, 1.08, and 1.73, respectively. The presence of two hydrogen at C-29 position was observed at δ 4.78 (s, 1H, C-29 β/α) and 4.65 (s 1H, C-29 α/β). A doublet of triplet resonance at δ 3.05 assigned the hydrogen at C-19. The signals due to the five aromatic protons observed at δ 7.46, 7.58 and 8.06 respectively, confirmed the present of phtalic group attached to betulinic acid.

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OH

C-H

C-O

C-H stretch

C=O

Silverstein, R. M., Bassler, G. C. and Morill, T. C. (1991). Spectrometric Identification of Organic Compounds. (5th ed.) pp: 122-124. New York: Wiley. Figure 1: Infrared spectrum of product of reaction between betulinic acid and phtalic anhydride

H-3

H-29

H-19

Figure 2: 1H-NMR spectrum of product of reaction between betulinic acid and phtalic anhydride

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Conclusions Synthesis of betulinic acid ester was accomplished using hydrolytic enzymes as biocatalyst. From the analysis of spectroscopic data, the product that produced from the reaction of betulinic acid and phtalic anhydride was found to be ester of betulinic acid. The used of Lipozyme as biocatalyst was successfully catalyzed the esterification of betulinic acid and phtalic anhydride. References [1] Pisha, E.; Chai, H.; Lee, I.-S.; Chagwedera, T. E.; Farnsworth, N. R.; Cordell, G. A.; C. W. W.; Fong, H. H. S.; Kinghorn, A. D.; Brown, D. M.; Wani, M. C.; Wall, M. E.; Hieken, T. J.; Das Gupta, T. K.; Pezzuto (1995) “Discovery of betulinic acid as selective inhibition of human melanoma that functions by induction of apoptosis” J. M. Nature Medicine 1, 1046. Fulda, S.; Friesen, C.; Los, M.; Scaffidi, G.; Mier, W.; Benedict, M.; Nunez, M; Krammer, P. H.; Peter, M. E.; Debatin, K. M (1997) “Betulinic acid triggers CD95 (APO-1/Fas) and p53 independent apoptosis via activation of capases in neutrothermal tumors” Cancer Res. 57, 4956. Darrick S. H. L. Kim, John M. Pezzuto, Emily Pisha (1998) “Synthesis of betulinic acid derivatives with activity against human melanoma” Bioorganic & Medicinal Chemistry letters 8, 1707-1712. Pezzuto, J. M.; Dasguta, Tapas, K.; Kim.; Darrick.; S. H. L. (1999) “Use of betulinic acid derivatives for the treatment and prevention of melanoma” U. S. Patent 5,869,535, 5-68. Kashiwada, Y.; Wang, H. K.; Nagao, T.; Kitanaka, S.; Yasuda, I.; Fujioka, T.; Yamagishi, T.; Consentino, L. M.; Kozuka, M.; Okabe, H.; Ikeshiro, Y.; Hu, C. Q.; Lee, K. H. (1998) “AntiHIV activity of oleanolic acid, pomolic acid and structurally related triterpenoids” J. Nat. Pro., 1090-1095. Silverstein, R. M., Bassler, G. C. and Morill, T. C. (1991). “Spectroscopic Identification of Organic Compounds”, New York Wiley. 122-124.

[2]

[3]

[4] [5]

[6]

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RETENTION THERMODYNAMICS IN HIGH TEMPERATURE REVERSEDPHASE LIQUID CHROMATOGRAPHY USING HYDRO-ORGANIC AND SUPERHEATED WATER ELUENT Hong Heng See,* M. Marsin Sanagi, Wan Aini Wan Ibrahim, Ahmedy Abu Naim Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia e-mail: hongheng80@yahoo.com Abstract. Retention thermodynamic based on Van’t Hoff analysis in reversed-phase liquid chromatography is evaluated using various test compounds (i.e. tebuconazole, hexaconazole and αtocopherol acetate) on zirconia based stationary phases. The retention thermodynamics for tebuconazole and hexaconazole are consistently unchanged at high column temperatures (80οC150οC) using water-rich and superheated water as eluent. The results obtained are compared with our previous reports and variations of enthalpy in the range of 0.1%-6.9% were observed. Irregularity of the Van’t Hoff analysis was noted when higher organic proportions (40-50%) were used as eluent to separate α-tocopherol acetate at same range of operating temperatures. The Van’t Hoff plot tends to deviate from linearity at higher operating temperatures (140οC-150οC) with linear deviation errors of 12.7%-41.6% respectively for the mobile phase examined. Keywords: High temperature HPLC; Van’t Hoff; Retention thermodynamic; Zirconia based stationary phase.

Introduction Recently, a more general acceptance of the importance of temperature as a separation parameter in HPLC has taken place. High temperature operation in reversed-phase HPLC provides the opportunity to reduce the quantity of organic solvent used in mixed organic-water mobile phase, increases analyte mass transfer rates and decreases column back pressure and total analysis time significantly [1]. Several workers have demonstrated the use of superheated water as eluent for reversed-phase separations [2-4]. The rapid development of high temperature HPLC is due to the recent findings of high stability stationary phases. Zirconia stationary phase coated with polybutadiene (PBD) has been increasingly used as a reversed-phase (RP) stationary phase because it is much more durable substrate compared with silica, while not imparting the high retentive characteristics of the aromatic polymerbased column [5,6]. Meanwhile, carbon clad zirconia stationary phase has been found to have different selectivities compared to traditional bonded phase reversed phase LC media. This phase has properties somewhat in between those of reversed-phase and normal-phase sorbents [7]. The use of temperature in investigations of liquid chromatographic retention mechanisms is widespread. Neither a linear or quadratic extrapolation of retention as a function of temperature has a rigorous thermodynamic basis. As a theoretical basis for the Van’t Hoff plots, the retention factor is expressed in terms of standard enthalpies and entropies of transfer from mobile to stationary phase. The relation between the logarithm of the retention factor (ln k) and enthalpies and entropies equals [8]: ln k = -∆Ho/RT + ∆So/R + ln Φ (1) where k is the measured retention value, ∆Ho the enthalpy, ∆So the entropy, T the absolute temperature, R the gas constant and Φ the phase ratio of the column. ∆Ho and ∆So are the standard enthalpy and standard entropy of transfer of a solute from the mobile phase to the stationary phase. A plot of ln k versus 1/T gives a slope of -∆Ho/R and an intercept of ∆So/R + ln Φ. Such plots can therefore be used to calculate enthalpies and entropies of transfer although the latter can be difficult to determine owing to the non-trivial nature of calculating the phase ratio, especially for commercial columns and columns for which the nature of the ligand bonding is ambiguous [9]. If the enthalpy,

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entropy, and phase ratio remain constant over the temperature range studied, ln k should be a linear function of 1/T. Non-linear Van’t Hoff plots suggest a change in the nature of the interactions between the solute and the mobile phase or between the solute and the stationary phase or both. The present study was set out to investigate the effect of high column temperature on retention thermodynamic in reversed-phase HPLC using hydro-organic, water-rich and superheated water as mobile phases. The present study is also intended to compare with our research study reported recently [3,10] based on different operating temperature range.

EXPERIMENTAL

Reagents Double-distilled deionized water of at least 18 MΩ was purified by Nano ultra pure water system (Barnstead, USA). HPLC grade acetonitrile was obtained from Caledon Laboratories Ltd. (Canada). Hexaconazole and tebuconazole were obtained from Dr. Ehrenstorfer (Augsburg, Germany). αTocopherol acetate (α-TAc) was obtained from Sigma Aldrich (USA). All test portions were made up in acetonitrile. The mobile phase used was prepared by mixing double distilled deionized water with acetonitrile and the mixture was subsequently degassed using vacuum-ultrasonic method.

Chromatographic conditions The instrumental set-up used in this study has been described in our previous report [3,10]. In brief, the systems consisted of a Waters 515 HPLC pump (Milford, USA) for mobile phase delivery. A Rheodyne 7125 injection valve (Cotati, USA) fitted with a 5 µL loop was used for sample introduction. Analyte peaks were detected using a Shimadzu SPD-6A UV detector (Kyoto, Japan) and were recorded on a Hewlett Packard HP 3396 Series II integrator (USA). A 20 cm × 0.25 mm I.D. length of stainless-steel tubing was placed in the oven between the injection valve and column as a pre-heating coil. The column and the pre-heating coils were placed together in the Perkin Elmer Autosystem Gas Chromatography (USA) oven. A Jasco 880-81 (Japan) backpressure regulator was attached at the outlet of the detector to maintain a constant backpressure (~20 bar) in the detector cell. The analytical column used were: (i) 3 µm ZirChrom-PBD 300 Å (100 mm × 2.1 mm I.D.) (ZirChrom Separations, Anoka, MN, USA); (ii) 3 µm ZirChrom-CARB 300 Å (100 mm × 2.1 mm I.D.) (ZirChrom Separations, Anoka, MN, USA). Results And Discussion Thermodynamics of retention in water-rich and superheated water In this study, the retention thermodynamic for two triazole fungicides was investigated at high operating temperatures (80οC-150οC) using water-rich and superheated water as eluent. This study was aimed to compare with our previous report [3,10] where the Van’t Hoff plots were only based on three data points study using same operating conditions with temperature range from 100οC-150οC. Up to 8 data-points were examined for each analyte on different separation conditions. From the Van’t Hoff plots for the tebuconazole and hexaconazole using two zirconia based stationary phases (Fig. 1-2), it was obvious that there was no significant deviation from linearity for each line plotted. The results showed a close correlation between ln k and the reciprocal absolute temperature (1/T) with correlation coefficient values generally exceeding 0.9958. It was therefore concluded that over the temperature range, there was no significantly changes in the retention mechanism for the fungicides. The entire enthalpy values are remaining constant over the temperature range studied. Recently, Coym and Dorsey [11] reported that the retention thermodynamic for several analytes is remaining unchanged over the high temperature range studied. However, significant

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deviation from the extrapolation of the plot to ambient temperature was observed. Hence, based on he results in our present study, the extrapolation of retention data as a function of temperature will unsuitable for use to predict the retention data at lower temperature. A further study is absolutely necessary to prove that the retention mechanism is remained unchanged over the lower temperatures examined before it used for the prediction purposes.

R2 = 0.9992

2.50 2.00 1.50 ln k 1.00 0.50 0.00 2.3 -0.50 2.4 2.5 2.6

R2 = 0.9994 R2 = 0.9981 R2 = 0.9988 R2 = 0.9964 R2 = 0.9958

HX 10% MeCN HX 5% MeCN HX 0% MeCN TB 10% MeCN TB 5% MeCN TB 0% MeCN

2.7

2.8
-3

2.9
-1

1/T x 10 (K )
Fig. 1. Van’t Hoff plots for tebuconazole (TB) and hexaconazole (HX) using different proportions of acetonitrile (MeCN) in the eluent on ZirChrom-PBD column.

2.50 2.00 1.50 1.00 ln k 0.50 0.00 2.4 2.5

R2 = 0.9998

R2 = 0.9994 R2 = 0.9999

R2 = 0.9988

R2 = 0.9980 R2 = 0.9961

HX 10% MeCN HX 5% MeCN HX 1% MeCN TB 10% MeCN TB 5% MeCN TB 1% MeCN

-0.50 2.3 -1.00 -1.50 -2.00

2.6

2.7

2.8 2.9 -3 1/T x 10 (K-1)

Fig. 2. Van’t Hoff plots for tebuconazole (TB) and hexaconazole (HX) using different proportions of acetonitrile (MeCN) in the eluent on ZirChrom-CARB column. Table 1 shows the enthalpy values obtained on zirconia based stationary phases in relative with the results reported in our previous study [3,10]. It can be observed that there were no significant

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deviations of the enthalpy values between present and previous results examined. Based on the t-test calculations, the differences of these two batches of results were insignificant. In all cases, the ∆Ho values were negative under the experimental conditions, demonstrating that the retention of compounds studied is an exothermic process. It is energetically more favorable for the compounds to remain in the stationary phase than in the mobile phase. As expected, the value of ∆Ho became more negative with decreasing acetonitrile content in the eluent. This showed that a strong retention interaction between mobile and stationary phase occurred when the percentage of organic modifier in the eluent is decreased. This phenomenon is due to the water rich solvent characteristics changes when high temperature is applied. There is a significant change on the hydrogen-bond network in water when it exists in extreme high temperature. Only monomeric water with no hydrogen bonding is noted at high temperature. This fact was also supported by the observation of the decrease in the dielectric constant of water [12], as well as the shift in the polarity value at high temperature [13]. Hence, thermodynamically, as the hydrogen-bond is disrupted at high temperature, retention shifted from being entropically driven to enthalpically driven [11]. That could be the reason why a change in enthalpy with temperature, with more favorable (more negative) enthalpies at high temperature was observed. Table 1. Enthalpy data for tebuconazole and hexaconazole at high column temperatures (80οC-150οC) on zirconia based stationary phases. Enthalpy ∆Ho(kJ/mol) ZirChrom-PBD Column ZirChrom-CARB Column Compounds Mobile phase composition: acetonitrile-water (v/v) 10 – 90a 5 – 95a 0 – 100a 10 – 90a 5 – 95a 1 – 99a Tebuconazolea Tebuconazoleb Deviation (%) Hexaconazolea Hexaconazoleb Variation (%)
a b

−35.1 −34.1 2.8 −35.2 −36.5 3.7

−41.8 −41.5 0.7 −44.2 −44.2 0.0

−48.9 −50.9 4.1 −51.2 −53.0 3.5

−46.9 −48.8 4.1 −41.1 −42.3 2.9

−50.5 −52.3 3.6 −44.1 −45.8 3.9

−59.1 −56.9 3.7 −51.1 −49.2 3.7

Results obtained in present study Results obtained in the previous report ref.

Thermodynamics of retention in organic-rich eluent at high temperature A study was carried out to examine the effect of mobile phase composition on influencing the retention thermodynamic at high column temperature. Fig. 3a shows the Van’t Hoff plots for the αtocopherol acetate with temperatures up to 150oC using different proportions of organic modifier. It can be seen that for each line plotted, there was slight deviation from linearity especially at higher temperature range (140οC-150oC). The plots tend to be curved at these temperatures. In order to further investigate the influence of temperature on the separation mechanism, the graph was re-plotted by excluding the point at 140oC and 150oC. This new Van’t Hoff plot is illustrated in Fig. 3b. It can be noted that, in general, for each line plotted, good linearity with a correlation coefficient values of higher than 0.9945 was obtained. It was therefore concluded that over the temperature range, there was no changes in the retention mechanism for α-tocopherol acetate. Conversely, there was a slight amount of error in this extrapolation at the temperatures that were excluded. Based on the equation that was obtained on each set of conditions, the percentage of linear deviation error at 140oC and 150oC was calculated. It can be noted that the percentage of error increased dramatically when going from 140oC to 150oC in all conditions studied (Table 2).

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Table 2. Enthalpy data for the tocopherols and tocotrienols at high column temperatures on PBDcoated Zirconia column. Mobile Phase Composition: MeCN-water Compound: α-Tocopherol acetate Linear Equation Enthalpy 80 °C to130 °C ∆Ho(kJ/mol) (Correlation, r) y=4.5237x-10.903 y=5.5741x-12.619 y=6.0173x-12.944 -37.61 (0.9983) -46.35 (0.9988) -50.53 (0.9972)

Linear deviation error (%) at 140 °C 12.7 14.7 15.4

Linear deviation error (%) at 150 °C 39.4 41.6 37.2

50:50 45:55 40:60

A)

3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00 -0.50 2.3 -1.00

40% MeCN 2 R = 0.9896 45% MeCN R2 = 0.9890 50% MeCN 2 R = 0.9878

ln k

2.4

2.5

2.6

2.7

2.8
-3

2.9
-1

1/T x 10 (K )

B)

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3.50 3.00 2.50 ln k 2.00 1.50 1.00 0.50 0.00 -0.50 2.3 -1.00
( ) ( ) ( ) ( ) ( )

40% MeCN R2 = 0.9945 45% MeCN 2 R = 0.9977 50% MeCN 2 R = 0.9967

( )2.4

2.5

2.6

2.7

2.8 2.9 -3 1/T x 10 (K-1)

Fig. 3. Van’t Hoff plots for α-tocopherol acetate using different proportions of acetonitrile in the eluent at high column temperatures on PBD-coated zirconia column. (A) Linear extrapolation on all temperature studied. (B) Linear extrapolation with a temperature range from 80οC to 120οC. Data points in brackets ( ) represent the points that are excluded from the linearity calculations for the plots This phenomenon could be due do the significant thermodynamic change in retention mechanism at extreme high temperatures with the present of high proportion of organic modifier in eluent. The present of organic modifier in eluent will significantly affect the properties of the mobile phase especially the hydrogen-bonding interaction. The hydrogen-bond network was disrupted when high proportion of organic modifier was added in the eluent with present of high temperature effect. This phenomenon could result in retention shift in thermodynamic interaction. This could be the main reason why the Van’t Hoff plots were tend to be curved at extreme high temperature range (140oC150oC) with the present of high proportion of organic modifier in the eluent.

CONCLUSIONS Linear Van’t Hoff plots were obtained for tebuconazole and hexaconazole on both zirconia based stationary phase using water-rich and superheated water liquid chromatography. Similar to the previous results obtained, there were not significant differences observed on the enthalpy values. With the presence of high proportion of organic modifier in the eluent, the Van’t Hoff plots tend to deviate from linearity at high temperatures, indicating a small temperature dependence of enthalpy. The current work stimulates a great interest to further investigate on the retention thermodynamic at temperatures exceeding 150οC by varying different proportion of organic modifier and also the influence of phase ratio on the thermodynamic interaction.

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ACKNOWLEDGEMENT The authors would like to thank Universiti Teknologi Malaysia and the Ministry of Science, Technology and Innovation, Malaysia, for financial support through the IRPA programme and a studentship for H. H. See.

References 1. M. Djordjevic, P. W. J. Fowler, and F. Houdiere, “High Temperature and Temperature Programming in High-Performance Liquid Chromatography: Instrumental Consideration.” J. Micro. Sep. 11 (1999) 403. 2. P. K. Dasgupta, T. S. Kephart, “Superheated Water Eluent Capillary Liquid Chromatography.” Talanta 56 (2000) 977-987. 3. M. M. Sanagi, H. H. See, W. A. W. Ibrahim, A. A. Naim, “High Temperature High Performance Liquid Chromatography of Triazole Fungicides on Polybutadiene-Coated Zirconia Stationary Phase.” J. Chromatogr. A 1059 (2004) 95-101. 4. R. M. Smith, R. J. Burgess, “Superheated Water as an Eluent for Reversed-Phase HighPerformance Liquid Chromatography.” J. Chromatogr. A 785 (1997) 49-55. 5. P. W. Carr, Y. Hu, J. Li, “Fast Separation at Elevated Temperature on Polybutadiene-Coated Zirconia Reversed-Phase Material.” Anal. Chem. 69 (1997) 3884. 6. J. Sun, P. W. Carr, “Chromatography of Proteins Using Polybutadiene-Coated Zirconia.” Anal. Chem. 67 (1995) 3717-3721. 7. P. T. Jackson, P. W. Carr, “Study of Polar and Nonpolar Substituted Benzenes and Aromatic Isomers on Carbon-Coated Zirconia and Alkyl Bonded Phases.” J. Chromatogr. A 958 (2002) 121-123. 8. R. J. M. Vervoort, E. Ruyter, A. J. J. Debets, H. A. Claessens, C. A. Cramers, G. J. Jong, “Characterization of Reversed-Phase Stationary Phases for the Liquid Chromatographic Analysis of Basic Pharmaceuticals by Thermodynamic Data.” J. Chromatogr. A 964 (2002) 67. 9. C. M. Bell, L. C. Sander, S. A. Wise, J. Chromatogr. A 757 (1997) 29-39. 10. M. M. Sanagi, H. H. See, W. A. W. Ibrahim, A. A. Naim, “High Temperature Liquid Chromatography of Triazole Fungicides on Carbon-Clad Zirconia Stationary Phase.” Malaysian Journal of Chemistry, 6 (1) (2004) 55-66. 11. J. W. Coym, J. G. Dorsey, “Reversed-phase retention thermodynamics of pure-water mobile phases at ambient and elevated temperature.” J. Chromatogr. A 1035 (2004) 23. 12. R. C. Weast (ed.), CRC Handbook of Chemistry and Physics, 1st Student ed., CRC Press, Boca Raton, FL, 1988. 13. J. Lu, J. S. Brown, E. C. Boughner, C. L. Liotta, C. A. Eckert, “Solvatochromic Characterization of Near-Critical Water as a Benign Reaction Medium.” Ind. Eng. Chem. Res., 41 (2002) 2835.

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A HOME-MADE SPME FIBER COATING FOR ARSON ANALYSIS Umi K. Ahmad*, Abdul Rahim Yacob and Geetha Selvaraju Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, 81310 UTM Skudai, Johor, Malaysia. Tel: 607-5534522, Fax: 607-5566162 E-mail: umi@kimia.fs.utm.my Abstract. A number of adsorbents are available commercially as coatings for SPME fibers but some analytical methodologies might demand specific properties for the extraction of selected compounds, special coatings that have particular volume and a selectivity towards particular analytes. This paper presents a simple, fast, effective and environmental friendly methodology for the determination of accelerants in arson samples using headspace solid-phase microextraction coupled to gas chromatography. A new fiber prepared by sol-gel method, containing 1:1 molar ratio of octyltriethoxysilane (C8-TEOS): methyltrimethoxysilane (MTMOS) was employed in this technique. The efficiency of the new fiber coating prepared by sol–gel technology for the determination of accelerants was compared to that of commercial PDMS/DVB fibers. Polydimethylsiloxane divinylbenzene (PDMS/DVB) is the most common fiber coating for the extraction of hydrocarbon compounds. Compared with commercial PDMS/DVB fiber, the new homemade fiber exhibited higher extraction capability and good selectivity for accelerants. The homemade fiber was also applied for the simulated arson samples. The home-made SPME adsorbent was shown to be a good alternative to commercially available fiber for the determination of accelerants in arson cases. Abstrak. Pelbagai jenis gentian penjerap bagi teknik SPME wujud di pasaran. Namun demikian, ciriciri khas bagi pengekstrakan sebatian yang terpilih, salutan khas dengan isipadu tertentu dan keterpilihan terhadap analit tertentu diperlukan dalam beberapa kaedah analisis. Kertas kerja ini membentangkan suatu kaedah yang mudah, pantas, efektif dan mesra alam sekitar untuk menentukan bahan penggalak kebakaran dalam sampel kebakaran yang disengajakan dengan menggunakan teknik pengekstrakan fasa pepejal mikro yang dihubungkan dengan kromatografi gas. Gentian baru yang digunakan dalam teknik ini disediakan melalui kaedah “sol-gel”, di mana ia terdiri daripada C8-TEOS dan MTMOS dalam nisbah 1:1. Keberkesanan gentian penjerap baru yang disediakan melalui teknologi “sol-gel” untuk penentuan bahan penggalak kebakaran dibandingkan dengan gentian penjerap yang diperolehi secara komersil (PDMS/DVB). Gentian PDMS/DVB adalah gentian yang biasa digunakan dalam pengekstrakan sebatian hidrokarbon. Berbanding dengan gentian PDMS/DVB, gentian buatan sendiri yang baru menunjukkan kapasiti pengekstrakan yang lebih tinggi dan keterpilihan yang baik bagi bahan penggalak kebakaran. Gentian buatan sendiri ini juga diaplikasikan pada sampel simulasi kebakaran yang disengajakan. Gentian buatan sendiri untuk teknik SPME menunjukkan suatu alternatif yang baik kepada gentian komersial bagi penentuan bahan penggalak kebakaran dalam kes kebakaran yang disengajakan Keywords: Home-made SPME adsorbent, arson, accelerants, SPME-GC Introduction Arsons are particularly difficult crimes to prove due to the usual lack of physical evidence associating a suspect to the crime. The accelerants most commonly used by offenders because of their volatility, availability and flammability are petrol, kerosene and diesel. The more volatile components of an accelerant evaporate at a faster rate than the heavier components so that overall chemical profile of the accelerant will change during the fire and before sampling [1, 2]. It is difficult to conclusively determine if a sample of an accelerant was the same as that to initiate or propagate a fire, because of the universal composition of the common accelerants. The chemical components of the common accelerants are aliphatic and aromatic hydrocarbons and oxygenated hydrocarbons such as alcohols. The oxygenated hydrocarbons are to a degree water

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soluble and therefore washed away during the extinguishing of the blaze, so that little trace remains [3-6]. The amount of accelerant remaining at the fire scene available for sampling is governed by the initial loading of the accelerant, volatility of the accelerant, severity of the fire, water solubility of the accelerant, porosity of the substrate material, dryness of the area after the fire and the elapsed time between the fire and sampling [3-6].

An important aspect of an investigation of a suspected arson case involves the chemical analysis of the debris remaining after the fire. Currently, accelerant extraction and analytical techniques have been refined to improve sample turnover and to reduce the number of inconclusive findings. For this purpose, solid-phase micro extraction (SPME) have been introduced. The major advantage of SPME is that it uses no solvents and can be used for either direct sampling or sample clean-up. It is fairly economical and is a relatively simple and sensitive technique. The qualities that enable an SPME adsorbent to be successfully used for accelerant extraction and analysis are its selectivity towards accelerant components which separates and concentrates the accelerant from the headspace to yield a sample that is suitable for introduction to GC-FID [7]. Recently, many novel coatings have been developed using different techniques and technology for use in SPME. Compared with commercially available SPME adsorbents, the new materials exhibited higher thermal stability (350 °C), solvent stability (organic and inorganic) and extraction capability [8-19]. However, up to now, none of the novel fibers have been evaluated for the determination of accelerants in arson analysis. This paper presents a recent development in the forensic aspects of fire investigation. As a preliminary study, a home-made SPME adsorbent comprising of sol-gel derived C8-coating was developed and evaluated for the determination of accelerants in arson samples, with the aim of improving the quality of ignitable liquid residue analysis.

Experimental

Chemicals and Materials Samples of diesel and unleaded gasoline were purchased from a petrol station in Skudai, Johor while kerosene was obtained from a grocery shop at Taman Universiti, Skudai, Johor. Samples of carpet were purchased from a carpet retail shop in Taman Ungku Tun Aminah, Skudai, Johor. Apparatus

A glass apparatus (400 cm3) for sample preparation step of headspace SPME was specially designed [20]. A Supelco SPME holder, commercially available PDMS/DVB fibers (Bellefonte, Pennsylvania,

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U.S.A.), and home-made sol-gel derived C8-coated fibers containing (1:1, C8-TEOS:MTMOS) [21] were employed for the extraction of accelerants. Instrumentation

Gas chromatography was conducted using a Hewlett-Packard 6890 GC (Wilmington, Delaware, U.S.A.). The HP 6890 gas chromatograph was equipped with FID and a HP ChemStation for data processing. An Ultra-1 capillary column (Agilent) of dimensions 25 m x 0.20 mm x 0.11 µm film thickness was used. Helium was used as the carrier gas at a flow rate of 1.2 mL/min. The injection port temperature was set at 250 °C and FID temperature at 310 °C. SPME injections were performed using a split mode injection (1:5).

Preparation of Spiked Fire Debris sample

A sample of carpet (15 cm x 10 cm) placed on a sheet of aluminium foil was ignited with a fire starter and left to burn until about 1/3 remained on the aluminium foil. Fire was extinguished and the partially burnt carpet was exposed to the surrounding air for 30 minutes to cool down. 100 µL of accelerant (petrol, kerosene and diesel) were individually spiked on to the burnt carpet fire debris sample. The spiked sample was placed in the glass apparatus prior to headspace SPME . Procedures for Headspace SPME

The spiked fire debris sample was placed in the sample preparation apparatus that was immersed in a hot water bath and heated for 15 min at 100 °C. The C8-coated fiber was exposed in the headspace and the fiber extracts were analyzed using GC-FID. The oven temperature was initially set at 40 °C, programmed at a rate of 10 °C/min until a final temperature of 270 °C. The headspace SPME procedure was repeated using PDMS/DVB fiber for comparison. Results And Discussion Selectivity of C8-coated fiber for accelerants In order to examine selectivity of the home-made SPME fiber towards petroleum based accelerants, burnt carpet samples were individually spiked with known amount of gasoline, kerosene and diesel. The samples were subjected to headspace SPME using sol-gel derived C8-coated fiber and the GC profiles were compared with that from direct injection. As seen in Figure 1 (b), all the hydrocarbon components in gasoline spiked burnt carpet sample were recovered by using C8-coated fiber and the chromatogram was comparable with the profile of gasoline from direct injection (Figure 1 (a). The hydrocarbon components in kerosene (Figure 2) and diesel (Figure 3) spiked burnt carpet sample were also effectively extracted using the home-made fiber and similar comparisons were obtained with direct injection of the respective accelerants. This indicated that the home-made SPME fiber favored the extraction of hydrocarbons, thus providing a good selectivity towards accelerants. The GC profiles of gasoline, kerosene and diesel spiked samples obtained in this study were in good agreement with those obtained by Borusiewicz [1] using Tenax as adsorbent and Yong [20] using commercially available fibers. Extraction capability of the C8-coated fiber

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The extraction capability of the sol-gel derived C8-coated fiber was determined by comparing it with the commercially available PDMS/DVB fiber. PDMS/DVB fiber was selected for comparison because previous work done in this lab [20] proved that the fiber has the highest sensitivity towards hydrocarbon compounds and most suitable for accelerants identification. Both C8-coated fiber and PDMS/DVB fiber is capable of extracting early, middle and late eluting hydrocarbon compounds sufficiently. However, the C8-coated fiber showed a slightly higher extraction capability by contrast with conventional PDMS/DVB fiber for all the accelerants as shown in Figure 4. This result was comparable to that reported by Gbatu et al. [8]. A higher extraction capability could be due to the existence of higher surface area for the C8-coated fibers [8]. Up to now, all the headspace SPME extractions were carried out with the optimized conditions of the commercially available PDMS/DVB fiber [20]. A study is now underway to optimize the extraction and desorption time of the C8-coated fiber. The optimized conditions of the C8-coated fiber might result in faster extraction time as reported by Gbatu et al. [8].

(a)

(b)

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(c)

Figure 1. GC profiles of (a) direct injection of gasoline, (b) gasoline spiked burnt carpet sample and (d) the blank burnt carpet sample using C8-coated fibers. Peak identities: (1) methylbenzene, (2) ethylbenzene, (3) 1, 3-dimethylbenzene, (4) 1, 2- dimethylbenzene, (5) 1-ethyl-2-methylbenzene, (6) 1, 2, 4-trimethylbenzene, (7) 1, 2, 3- trimethylbenzene. (a)

(b)

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Figure 2. GC profiles of (a) direct injection of kerosene and (b) kerosene spiked burnt carpet sample using C8-coated fibers.Peak identities: (1) C9, (2) C10, (3) C11, (4) C12, (5) C13, (6) C14, (7) 2, 6-dimethylnapthalene, (8) C15

Figure 3. GC profiles of direct injection of diesel spiked burnt carpet sample using C8-coated fibers. Peak identities: (1) C9, (2) C10, (3) C11, (4) C12, (5) C13, (6) C14, C15, (7) C16

(a)
Average Peak area
30 25 20 15 10 5 0 1 2

Spiked with Gasoline

3

4

5

6

7

C8-coated fiber

PDMS/DVB

Label identities: (1) methylbenzene, (2) ethylbenzene, (3) 1, 3-dimethylbenzene,(4) 1, 2dimethylbenzene, (5) 1-ethyl-2-methylbenzene, (6) 1, 2, 4-trimethylbenzene, (7) 1, 2, 3trimethylbenzene.

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(b)
Average Peak area
20 15 10 5 0 1 2

Spiked with Kerosene

3

4

5

6

7

8

C8-coated fiber

PDMS/DVB

Label identities: (1) C9, (2) C10, (3) C11, (4) C12, (5) C13, (6) C14, (7) 2, 6-dimethylnapthalene, (8) C15

(c)
Average Peak Area
8 6 4 2 0
1 2

Spiked with diesel

3

4

5

6

7

8

C8-Coated fiber

PDMS/DVB

Label identities: (1) C9, (2) C10, (3) C11, (4) C12, (5) C13, (6) C14, C15, (7) C16 Figure 4: Comparison of sol-gel derived C8-coated fiber and commercially available PDMS/DVB fiber in extracting hydrocarbon compounds from (a) gasoline, (b) kerosene and (c) diesel spiked burnt carpet Conclusions The sol-gel derived C8-coated fiber was successfully evaluated for the determination of accelerants in arson samples. The home-made fiber was capable of extracting hydrocarbon compounds from simulated arson samples and showed a good selectivity towards accelerants. The C8-coated fiber exhibited a slightly higher extraction capability by contrast with conventional PDMS/DVB fiber. The experiments performed proved that the developed fiber could be successfully applied for arson analysis. Acknowledgements Thanks are due to Ministry of Science, Technology and Innovation, Malaysia (MOSTI) for the financial support under the IRPA RM8 mechanism. (Vote 09-02-06-0072 EA209). References

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1. R. Borusiewicz, G. Zadora and J. Z. Palus (2004). “Application of headspace analysis with passive adsorption for forensic purposes in the automated thermal desorption GC-MS system.” Chromatographia supplement. 60. S113-S142. 2. T. Ma, S. M. Olenick, M. S. Klassen and R. J. Roby (2004). “Burning rate of liquid fuel on carpet (porous media).” Fire Tech. 40. 227-246. 3. J. S. Almirall and K. G. Furton (2004). “Characterization of background and pyrolysis products that may interfere with the forensic analysis of fire debris.” J. Anal. Appl. Pyrolysis. 71. 51-67. 4. J. S. Almirall, K. G. Furton and J. C. Bruna (1996). “A novel method for the analysis of gasoline from fire debris using headspace solid-phase microextraction.” J. Forensic. Sci. 71. 12-22. 5. K. Cavanagh, E. D. Pasquier and C. Lennard (2002). “ Background interference from car carpetsthe evidential value of petrol residues in cases of suspected vehicle arson.” Forensic. Sci. Int. 125. 22-36. 6. P. M. L. Sandercock and E. D. Pasquier (2003). “Chemical fingerprinting of unevaporated automotive gasoline samples.” Forensic. Sci. Int 134. 1-10. 7. J. Dolan (2003). “ Recent advances in the applications of forensic science to fire debris analysis.” Anal. Bioanal. Chem. 376. 1168-1171. 8. T. P. Gbatu, K. L. Sutton and J.A Caruso (1999). “Development of New SPME Fibers By Sol-gel Technology for SPME-HPLC Determination of Organometals.” Anal Chim Acta. 402. 67-79 9. A. kabir, C. Hamlet, K. S. Yoo, J. R. Newkome and A. Malik (2004) “Capillary Microextraction on Sol-gel Dendrimer Coatings,” J. Chromatogr. A. 1034. 1-11. 10. C. Basheer, S. Jagadesan, S. valiyaveetil and H. K. Lee (2005). “Sol-gel coated oligomers as novel stationary phases for solid-phase microextraction.” J.Chromatogr. A. Article in Press. 11. Y. Hu, Y. Yang, J. Huang and G. Li (2005). “Preparation and application of poly(dimethylsiloxane)/B-cyclodextrin solid-phase microextraction membrane.” Anal. Chim. Acta. Article in Press. 12. L.Yun (2003). “High Extraction Efficiency Soild-phase Microextraction Fibers Coated with Open Crown ether Stationary phase using Sol-gel Technique.” Anal Chim Acta. 486. 63-72. 13. J. Yu, L. Dong, C. Wu, L. Wu and J. Xing (2002). “ Hydroxyfullerene as a Novel Coating for Solid-phase Microextraction Fiber with Sol-gel technology.” J. Chromatogr. A. 978. 37-48. 14. V. G. Zuin, A. L. Lopes, J. H. Yariwake and F. Augusto (2004). “Application of a Novel Sol-gel Polydimethylsiloxane-Poly(vinyl alcohol) SPME Fiber for Gas Chromatographic Determination of Pesticide Residues in Herbal Infusions.” J. Chromatogr. A. 1056. 21-26. 15. J. Yu, C. Wu and J. Xing (2004). “Development of New SPME Fibers By Sol-gel Technology for The Determination of Organophosphorus Pesticide Multiresidues in Food.” J. Chromatogr. A. 1036. 101-111. 16. C. Basheer, S. Jagadesan, S. valiyaveetil and H. K. Lee (2005). “Sol-gel coated oligomers as novel stationary phases for solid-phase microextraction.” J. Chromatogr. A.Article in Press. 17. D. Wang, J. Xing, J. Peng and C. Wu (2003). “ Novel benzo-15-crown-5 Sol-gel Coating for Solid-phase Microextraction.” J. Chromatogr. A. 1005. 1-12. 18. J. Wu and J. Pawliszyn (2004). “Solid-phase microextraction based on polypyrrole films with different counter ions.” Anal. Chim. Acta. 520.257-264. 19. R. Aranda, P. Kruus and R. C. Burk (2000) “Assessment of polycrystalline graphites as sorbents for solid phase microextraction of non-ionic surfactants.” J. Chromatogr. A. 888. 35-41. 20. T. Y. Yong (2004) “Development of Headspace SPME-GC Technique for the Forensic Analysis of Ignitable Liquid Residues in Fire Debris.” Universiti Teknologi Malaysia: Master of Science Thesis. 21. U. K. Ahmad, A. R. Yacob and G. Selvaraju (2005) “A New SPME Adsorbent for the Forensic Analysis of Accelerant Residues.” Ibnu Sina Institute, Universiti Teknologi Malaysia: Proceeding of the Annual Fundamental Science Seminar.

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THERMODYNAMIC OF THE BINDING OF THE THREE – WATER SOLUBLE PROPHYRINS WITH DNA AUTHORS: MORTEZA KESHAVARZ*a, ABDOL-KHALEGH BORDBAR b Addresses: a) Department of Chemistry, Islamic Azad University, Shahreza, Isfahan, Iran. b) Department of Chemistry, Isfahan University, Isfahan, 81746 – 73441, Iran. • • • • ABSTRACT In the present work , the interaction of three water soluble porphyrins, tetra (p-trimethyle) ammonium phenyl porphyrin iodide (TAPP) as a cationic prophyrin , tetra sodium meso– tetrakis (p–sulphonato phenyl) porphyrin (TSPP) as an anionic porphyrin and manganese tetrakis (p–sulphonato phenyl) porphyinato acetate (MnTSPP) as a metal porphyrin , with Gulf thymus DNA have been comprehensively studied at 1mM phosphate buffer , pH , 7.0 , and various temperatures , using UVVis absorption spectroscopy. The binding constant and stoichiometry were determined by analysis of optical absorption spectra of porphyrin at various DNA concentrations using SQUAD software. The results represents that the best fitting corresponds to 1:1 complex model between base pair of DNA and porphyrin. All of the thermodynamic parameters were calculated by van’t Hoff equation at various temperatures. The results represents that the process is essentially entropy driven. The MnTSSP has the highest affinity respect to TSPP and TAPP that can be represent the formation of axial bond between phosphate group of nucleotide and Mn in the central core of MnTSSP. However, the higher affinity of TAPP as a cationic porphyrin respect to TSPP as an anionic can be related to the role of electrostatic interactions. Keywords: DNA, Prophyrin, Thermodynamic of binding, Optical absorption, SQUAD Corresponding author Tel: +98-311-7770199 Fax: +98-3213232701

E-mail: keshavarzm2000@yahoo.com

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INTRODUCTION Introduction of DNA – cleaving functional groups into an antisepsis oligonucleotide has been shown to generate site – directed damage on the target nucleic acids through chemical or photochemical reaction (1-3). A variety of chemical molecules have been covalently linked to oligonucleotides which were hybridized to cellular DNA to affect processes such as cellular uptake (4), nuclease resistance (5), and binding affinity (3). One of the most important group of those reactive molecules is porphyrins and their metal derivatives (6-8). These compounds are widely used as probes for nucleic acid structure and dynamics and have possible medical applications. The binding strength of porphyrin to DNA is one of the important parameters on its efficacy.Thethermodynamic parameters of binding can also help as to obtain more insights into the molecular nature of interactions. This paper reports a compnehensive thermodynamic study on interaction of three water – soluble porphyrins , tetra (p– trimethyle) ammonium phenyl porphyrin iodide (TAPP) as a cationic porphyrin , tetra sodium meso – tatrakis (p-sulphonato phenyle) porphyrin (TSPP) as an anionic porphyrin and manganese tetrakis (p– sulphonato phenyle) porphinato acetate (MnTSPP) as a metal porphyrin , with DNA at various temperatures . The interaction process has been followed using UV–Vis optical absorption spectroscopy. The spectral date was analyzed in order to obtain the mode of binding and binding constant using SQUAD software.

EXPERIMENTAL SECTION MATERIALS AND METHODS DNA from calf–thymus was obtained from Sigma– Chemical Co. TSPP and TAPP were prepared by methods described previously (9, 10). TSPP was metallated according to the literature method (11). These complexes were characterized by UV–Vis spectroscopy and elemental analysis. The spectral characteristics of the isolated materials were compared to the literature values and found to be in excellent agreement. All of the chemicals, which have been used for these syntheses, were of analytical grade and purchased form Sigma Chemical Co. All solutions were prepared using double – distilled water. Porphyrin stock solution was made by dissolving the solid porphyrin in buffer solution. Phosphate buffer, 1 mM, pH, 7.0, was used as buffer. Porphyrin stock and working solutions were stored at room temperature in the dark to avoid undesired photochemical reactions. UV-vis measurements were performed on a Cary 100 Spectrophotometer using 1 cm quartz cuvettes. To prepare the DNA stock solution, about 2 mg of DNA was dissolved in 1 ml of the phosphate buffer at 4°C for 48 h.with occasional stirring to ensure the formation of a homogenous solution. The DNA concentrations were determined using molar extinction coefficients of ε258 nm = 6700 M-1 cm-1. In all experiments, the porphyrins and DNA – solutions were freshly prepared before spectral analysis and were protected from direct sun lights until they were inserted into the cell compartments. To observe

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

the salt effect on the porphyrin absorption, the titrations were made by addition of aliquots of the NaCl solution into cuvette containing the porphyrin solution of appropriate concentration. The obtained spectra were corrected with respect to dilution effect. The titration of porphyrin solution as a function of DNA concentration was performed at pH 7.0, 1 mM phosphate buffer and at 20, 25, 30, 35, 40 and 45 °C.

RESULTS AND DISCUSSION SOLUTION PROPERTIES OF TSPP, MNTSPP AND TAPP In order to identify the solution properties of these porphyrins we employed UV-vis spectroscopy. Optical absorption spectrum of MnTSPP shows 4 distinct picks at 563, 466, 400 and 379 nm. The Soret band appears at 466nm. The molar absorption of the Soret band is 1.82 × 105 M-1 cm-1. The Soret band maximum obeys Beer’s law over an extended concentration range between 1.0 ×10-5 to 2.0 ×10-4 M, in phosphate buffer 1mM, pH, 7.0.After the upper limit of this concentration range a negative deviation has been observed that corresponds the self association of this porphyrin due to increasing of concentration . In spite of MnTSPP, the spectrum of TSPP just shows a distinct Soret band at 4.13nm. The molar absorptive of this band is 1.80 × 105 M-1, cm-1. The Soret band maximum obeys Beer’s law in a concentration rang between 1.0 ×10-5 M to 1.0×10-4M in l mM phosphate buffer, pH. 7.0. The observed positive deviation from linearity that represents the self aggregation was occurred after this range. The spectral feature of TAPP at the same condition shows a Soret band at 412 nm, with a shoulder. The molar absorptivity of Soret band was 3.02 × 105 M-1 cm-1 . The Soret band obeys Beer’s law in the concentration rang from 1.50 × 10-5 to 1.10 × 10-4 M. A negative deviation was observed after this range. The results in this part represent the higher tendency of TAPP for concentration self-aggregation respect to others. However, the less tendency of MnTSPP may be related to Mn. It seems that metallation of TSPP with Mn inhibited the intermolecular interactions which responsible of self-aggregation. EFFECT OF SALTS The effect of NaCl on the absorption spectrum of Mn TSPP, TSPP and TAPP are shown in Figs 1, 2 and 3, respectively. As the concentration of NaCl increases, the absorbance at all of the spectral regions of studied porphyrins has been significantly decreased. The decreasing of the absorbance for MnTSPP spectra is accompanying with red shift and disappearing of Q-bands which represents the formation of well define aggregates. These results also represent the strong electrolyte effect on aggregation state of MnTSPP. The bath chromic shift of spectra with broading of spectral bandwidth which have been observed for TSPP and TAPP can be related to formation of ill-define aggregate in the presence of salt. However, the tendency of TSPP for formation of aggregate is more than TAPP.

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

TAPP+NaCl
0.7 0.6 0.5 0.4

Abs
0.3 0.2 0.1 0 370

390

410

430

450

wavelenght(nm)

Fig.1. Corrected absorption spectra of TAPP upon titration with NaCl in 1mM phosphate buffer, pH7.0 at 25

o

c.
TSPP+NaCl

0.8 0.7 0.6 0.5

Abs 0.4
0.3 0.2 0.1 0 350

400

450

500

wavelenght(nm)

Fig.2. Corrected absorption spectra of TSPP upon titration with NaCl in 1mM phosphate buffer, pH7.0 at 25

o

c.

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

MnTSPP+NaCl
0.8 0.7 0.6 0.5

Abs

0.4 0.3 0.2 0.1 0 350

400

450

500

550

600

650

wavelenght(nm)

Fig.3. Corrected absorption spectra of MnTSPP upon titration with NaCl in 1mM phosphate buffer, pH7.0 at 25

o

c.

INTERACTION OF PORPHYRINS WITH DNA: OPTICAL ABSORPTION STUDY With respect to our previous discussion, we can conclude that in homogenous aqueous solution at low ionic strength these studied porphyrins exist mainly as monomer. So, we conducted the titration of porphyrin solution at fixed concentration and varying [DNA] at pH, 7.0 and 1mM phosphate buffer as a low ionic strength medium. Figs 4, 5, and 6 show a representative titration spectrum of MnTSPP, TSPP and TAPP upon increasing concentration of DNA, at 25 °C. In all of the spectral regions, the intensity of Soret band decrease as DNA concentration increased. The absorption data were analyzed in order to calculate the binding parameters using SQUAD program. This program is designed to calculate the best values for the stability constants of the proposed equilibrium model by employing a non – linear least square approached. The results represent the formation of 1:1 complex model between studied porphyrins and DNA at all temperatures with sum of squares of reduced error between 10-3 – 10-4 . Hence a simple equilibrium between free porphyrin and DNA pair base is exist. The estimated equilibrium constants for the formation of base-pair porphyrin complex at various temperatures are listed in Tables 1, 2 and 3.

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

TAPP+DNA
0.9 0.8 0.7 0.6

Abs

0.5 0.4 0.3 0.2 0.1 0 370

390

410

430

450

wavelenght(nm)

Fig.4. Corrected absorption spectra of TAPP upon titration with DNA in 1mM phosphate buffer, pH7.0 0 at 25

o

c.

TSPP+DNA
0.7 0.6 0.5 0.4

Abs
0.3 0.2 0.1 0 350

400

450

500

wavelenght(nm)

Fig. 5. Corrected absorption spectra of TSPP upon titration with DNA in 1mM phosphate buffer, pH7.0 0 at 25

o

c.

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

MnTSPP+DNA
0.8 0.7 0.6 0.5

Abs 0.4
0.3 0.2 0.1 0 350

400

450

500

550

600

650

wavelenght(nm)

Fig. 6. Corrected absorption spectra of MnTSPP upon titration with DNA in 1mM phosphate buffer, pH7.0 0 at 25

o

c.

Table 1. Thermodynamic parameters for binding of TAPP to DNA in 1mM phosphate buffer, pH7.0 at various temperatures. t c 20 25 30 35 40 45

o

( K ± ∆K ) ×10 −4
5.754 ± 1.026 7.080 ± 1.023 10.715 ± 1.030 16.982 ± 1.028 24.547 ± 1.023 33.884 ± 1.030

∆G o ± ∆∆G o KJmol −1
-26.717 ± 0.061 -27.691 ± 0.057 -29.196 ± 0.076 -30.858 ± 0.072 -32.318 ± 0.060 -33.688 ± 0.079

∆H o ± ∆∆H o KJmol −1
57.777 ± 0.117 57.777 ± 0.117 57.777 ± 0.117 57.777 ± 0.117 57.777 ± 0.117 57.777 ± 0.117

∆S o ± ∆∆S o JK −1mol −1
288.228 ± 0.208 286.661 ± 0.191 286.898 ± 0.254 287.636 ± 0.117 287.706 ± 0.138 287.490 ± 0.254

Table 2. Thermodynamic parameters for binding of TSPP to DNA in 1mM phosphate buffer, pH7.0 at various temperatures. t c 20 25 30 35 40 45

o

( K ± ∆K ) ×10 −4
9.333 ± 1.023 12.302 ± 1.031 14.135 ± 1.033 21.878 ± 1.026 28.841 ± 1.028 40.738 ± 1.035

∆G o ± ∆∆G o KJmol −1
-27.897 ± 0.056 -29.057 ± 0.072 -30.071 ± 0.083 -31.507 ± 0.063 -32.666 ± 0.077 -34.175 ± 0.093

∆H o ± ∆∆H o KJmol −1
45.519 ± 0.055 45.519 ± 0.055 45.519 ± 0.055 45.519 ± 0.055 45.519 ± 0.055 45.519 ± 0.055

∆S o ± ∆∆S o JK −1mol −1
250.438 ± 0.191 250.129 ± 0.242 249.349 ± 0.274 249.963 ± 0.205 249.673 ± 0.227 250.492 ± 0.249

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

Table 3. Thermodynamic parameters for binding of MnTSPP to DNA in 1mM phosphate buffer, pH7.0 at various temperatures. t c 20 25 30 35 40 45

o

( K ± ∆K ) ×10 −4
1.023 ± 1.030 1.622 ± 1.028 3.631 ± 1.026 7.081 ± 1.023 14.125 ± 1.033 28.841 ± 1.035

∆G o ± ∆∆G o KJmol −1
-22.508 ± 0.032 -24.035 ± 0.030 -26.469 ± 0.028 -28.619 ± 0.026 -30.878 ± 0.083 -33.259 ± 0.074

∆H o ± ∆∆H o KJmol −1
105.551 ± 0.017 105.551 ± 0.017 105.551 ± 0.017 105.551 ± 0.017 105.551 ± 0.017 105.551 ± 0.017

∆S o ± ∆∆S o JK −1mol −1
436.836 ± 0.109 434.634 ± 0.101 435.494 ± 0.093 435.405 ± 0.083 435.667 ± 0.265 436.304 ± 0.193

THERMODYNAMICS OF DNA– PORPHYRIN INTERACTION The energetic of DNA–porphyrin equilibrium can be conveniently characterized by three thermodynamic parameters , standard Gibbs free energy , ∆G°, can be calculated from the equilibrium constant ,K, of the reaction using the familiar relationship, ∆G°= -RTlnK, in which R and T referring to the gas constant and the absolute temperature, respectively. The van’t Hoff equation (1)

d ln K − ∆H ° = 1 R d( ) T
Gives a linear plot of lnK versus

(1)

1 , if the heat capacity change for the reaction is essentially zero. T − ∆H ° and the standard entropy from R

The ∆H° can be calculated from the slope of the straight line, its intercept, ∆S°

R

or by equation (2) (2)

∆S ° =

∆H ° − ∆G° T

The van’ Hoff plots for binding of these porphyrins to DNA in the phosphate buffer are shown in Fig (7). All of the thermodynamic parameters with their uncertainly for Interaction of TAPP, TSPP, and MnTSPP were calculated, and reported in Tables 1,2 and 3, respectively. The negative slopes of the lines in van’t Hoff plots Fig (7) represent the endothermicity of the reaction. The high correlation coefficient of the lines indicate the little value of heat capacity change of reaction .With respect to the values of ∆G° , the binding affinity of TAPP is more thane TSPP at all studied temperatures. This can be related to the role of electrostatic interactions in the formation of complex. The strong attractive electrostatic interaction between TAPP as a cationic porphyrin with DNA chains with negative charge density, in comparison to TSPP as an anionic porphyrin, is responsible for this observation. However, MnTSPP has the highest affinity to DNA with respect to other porphyrins. This can be related to the special role of Mn in formation of complex with DNA. Probably, the axial ligation of Mn with phosphate group increases its affinity to DNA. Comparison of enthalpy and entropy values represents the essential role of entropy in reaction driven. In conclusion the formation process in essentially entropy driven and the metal in the central core of porphyrin have an important enhancing role in the interaction with DNA.

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

vant Hoff
5.7 5.5 5.3 5.1 4.9

log K
4.7 4.5 4.3 4.1 3.9 0.003141

0.003206

0.003271

0.003336

0.003401

1/T
Fig.7.The Van’t Hoff plots for binding of TAPP (▲), TSPP (■) and MnTSPP (○) To DNA in 1mM phosphate buffer, pH7.00 at various temperatures.

ACKNOWLEDGEMENT The financial support of Science and Research Campus of Islamic Azad University is gratefully acknowledged.

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

References [1] A. M. Belikova, V. F. Zarytova. and N.I. Ginieva, (1967). tetrahedron Lett., 3557 – 3567 . [2] D.G. Knorre and V.V. Vlassov, (1989). CRC Press, Boca Raton, FL. [3] D.G. Knorre, V.V. Vlassov, V.F. Zarytova, A.V.Lebedev and O.S. Fedorova, (1994). CRC Press, Boca Raton, FL. [4] E. Wickstrom, (1991). Willey – Liss. Inc., New York. [5] S.T. Crooke and B.Lebleu, (1993). GRC Press, Boca Raton, FL. [6] O.S. Fedorova, A.P. Savitskii, K.G. shoikhet and G.V. Ponomarev, (1990). FEBS Lett, 259, 335 – 337. [7] L. Mastruzzo, A. Woisard, D.D.F. Ma, Photochem. Photobiol. 60,316 –322. [8] D. Magda, M. Wright, R.A. Miller, J.L. Sessler and P.I.Sanson, (1995). J. Am. Chem. Soc. 117, 3629 - 3630 [9] E.B. Fleischer, J.M. Palmer, T.S. Srinivastava and A.J. Chaterjee. (1971). Am. Chem. Soc. 93, 3162. [10] M.Krishnamurthy (1977). Indian J. Chem. 964. [11] A. Harriman and G. porter, (1979). J. chem. soc. Faraday Trans. 75, 1532. E. Rizzarelli, A. Favre and T. Le Doan , (1994).

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BIOPENDERIA ELEKTROKIMIA UNTUK PENGESANAN KETOKSIKAN ASID 2,4-DIKLOROFENOKSIASETIK BERASASKAN ENZIM ALKALINE FOSFATASE TERPEGUN Loh Kee Shyuan1, Lee Yook Heng1, Musa Ahmad1, Salmah Abdul Aziz2 & Zamri Ishak2 1 Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains & Teknologi, Universiti Kebangsaan Malaysia (UKM), 43600 Bangi, Selangor 2 Pusat Penyedilikan Bioteknologi, Institut Penyelidikan & Kemajuan Pertanian Malaysia (MARDI), P.O. BOX 12301, GPO KL Abstrak. Biopenderia elektrokimia berasaskan pemegunan enzim alkaline fosfatase dibangunkan untuk mengesan keracunan pestisid 2,4-diklorofenoksiasetik asid (2,4-D). Biopenderia elektrokimia dibina dengan memegunkan enzim alkaline fosfatase pada elektrod bercetak skrin (SPE). Asid askorbik 2-fosfat digunakan sebagai substrak. Pemegunan enzim alkaline fosfatase dilakukan dengan pemerangkapannya dalam bahan hibrid sol-gel/kitosan dalam komposisi tertentu. Penentuan ketoksikan pestisid 2,4-D secara analisis kuantitatif dan kualitatif dapat dilakukan melalui perencatan enzim alkaline fosfatase. Keupayaan +600mV sesuai digunakan untuk pengoksidaan produk tindak balas enzim-substrak manakala pH yang optimum ialah pH 8.5. Julat rangsangan linear biopenderia untuk kepekatan substrak asid askorbik 2-fosfat ialah 10µM-80µM. Biopenderia 2,4-D menunjukkan perencatan enzim alkaline fosfatase adalah maksimum (50% perencatan) pada 80ppm 2,4-D. Kata kunci: Biopenderia eletrokimia, alkaline fosfatase, asid 2,4-diklorofenoksiasetik, sol-gel, kitosan 1. Pengenalan Pestisid asid 2,4-diklorofenoksiasetik (2,4-D) merupakan herbisid yang banyak digunakan untuk mengawal tumbuh-tumbuhan berdaun lebar [1]. Herbisid 2,4-D merupakan jenis pestisid dari kumpulan organoklorin yang sukar mengalami biodegradasi dan ia mampu menumpu dalam lipid lalu tersebar melalui rantai makanan [2]. Penggunaan 2,4-D secara meluas dalam pertanian telah mengakibatkan masalah pencemaran pada sekitaran dan ekosistem, secara langsung ini turut memberi impak yang mengancam kepada manusia [3]. Masalah pencemaran boleh dipantau dengan biopenderia untuk mengesan tahap ketoksikan pestisid dalam air dan sumber makanan. Biopenderia elektrokimia berasaskan enzim untuk pengesanan pestisid [4-6] dan pegesanan logam berat [7] telah pesat dibangunkan kerana ia dapat bertindak dengan lebih cepat berbanding teknik analisis konvensional. Walaupun kaedah kromatografi gas (GC), kromatografi cecair berprestasi tinggi (HPLC) dan kromatografi gas-spektroskopi jisim (GC-MS) dapat menganalisis sampel pestisid, tetapi cara pemonitoran ini memerlukan kos yang tinggi, masa analisis yang lama dan kepakaran dalam pengendalian instrumen. Biopenderia telah mengambil peranan sebagai peranti pelengkap untuk kaedah konvensional. Biopenderia boleh digunakan untuk menjalankan analisis secara in situ di lapangan, kebolehulangan yang baik dan melibatkan kos yang rendah. Biopenderia berasaskan enzim boleh disediakan melalui pengubahsuaian pada permukaan elektrod dengan pemegunan bahan penderia yang sesuai. Pemegunan enzim pada penyokong lebih baik berbanding enzim dalam larutan bebas kerana enzim bukan sahaja lebih senang dikendali, malahan dapat memisahkan enzim serta produk hasil secara berasingan dan enzim tersebut dapat diguna semula [8]. Dalam kajian ini, pemegunan enzim alkaline fosfatase dilakukan dengan bahan hibrid solgel/kitosan. Kedua-dua bahan ini adalah lengai terhadap biomolekul [9,10] dan enzim dapat dipegun dengan stabil dalamnya tanpa berlaku pengikatan kovalen atau interaksi kimia di antara biomolekul dengan matrik tersebut. Miao dan Tan [11] mendapati biopenderia yang dihasilkan dengan mengguna sol-gel/kitosan sebagai bahan pemegunan memberikan tindak balas yang cepat. Kajian ini menggunakan sistem bioelektrokimia untuk penentuan pestisid 2,4-D secara perencatan enzim. Biopenderia 2,4-D dapat menentukan kehadiran pestisid 2,4-D secara perencatan aktiviti ezim alkaline fosfatase yang terpegun pada elektrod bercetak skrin.(Rajah 1)

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

Asid Askorbik 2-fosfat

SPE
Alkaline fosfatase

H3PO4 + Asid Askorbik
Pengoksidaan

Dehidro asid askorbik + 2H+

2edikesan secara elektrokimia

Rajah 1: Rajah menunjukkan enzim alkaline fosfatase memangkinkan asid askorbik 2-fosfat kepada asid askorbik, satu spesies elektroaktif yang dapat teroksida pada permukaan elektrod bercetak skrin. Jika pestisid hadir, keaktifan enzim alkaline fosfatase akan terencat dan tindak balas enzim-substrak berkurang dan keamatan arus turut berkurangan.

2.Eksperimen 2.1 Reagen. Bahan-bahan yang digunakan dalam kajian ini adalah tetraetilorthosilikat (TEOS) daripada Fluka, asid hidroklorik dan asid asetik glasial daripada Merck. Kitosan, enzim alkaline fosfatase (AP), asid askorbik-2-fosfat (AA2P), 2,4-diklorofenoksiasetik asid (2,4-D) dan MgCl2 daripada Sigma, tris(hidroksilmetil) amino metana (Tris-HCl) daripada ICN Biomedical Inc, air nyahion digunakan untuk semua penyediaan larutan. 2.2 Peralatan. Ujikaji dijalankan dengan menggunakan potensiostat Model AUTOLAB dengan PGSTAT 12 (AUT 71681). Elektrod bercetak skrin (SPE) rekaan UKM diguna sebagai elekrod kerja, elektrod Ag/AgCl diguna sebagai elektrod rujukan dan elektrod platinum diguna sebagai elektrod lawan. 2.3 Pemegunan enzim alkaline fosfatase dengan hirbid sol-gel/kitosan. Enzim AP dengan kepekatan tertentu dicampurkan bersama hibrid sol-gel/kitosan untuk pemegunan. Campuran enzim dengan solgel/kitosan disalut pada permukaan elektrod bercetak skrin dan dibiarkan untuk pengeringan. Elektrod bercetak skrin terpegun enzim AP kemudian digunakan untuk kajian seterusnya. 2.4 Ujian hidrodinamik. Elektrod bercetak skrin yang terpegun degan enzim AP direndam dalam larutan penimbal Tris-HCl, yang mengandungi MgCl2, bersama-sama dengan elektrod rujukan dan elektrod lawan. Larutan penimbal bertindak sebagai elektrolit dan dikacau secara perlahan oleh

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

pengacau magnetik. Kepekatan akhir untuk substrak asid askorbik 2-fosfat ditetapkan pada 80uM. Julat keupayaan 0.2V-0.8V digunakan dalam ujikaji untuk menentukan keupayaan yang sesuai untuk tindak balas enzim AP dengan substrak AA2P. Perubahan arus sebelum dan selepas penambahan substrak AA2P dicatatkan. Ujian hidrodinamik diulangi dengan menggunakan enzim AP dalam larutan bebas untuk membandingkan aktiviti enzim dalam keadaan berlainan yang tidak terpegun. 2.5 Kesan pH. Elektrod bercetak skrin terpegun dengan enzim direndam ke dalam larutan penimbal Tris-HCl yang berlainan pH, iaitu pH 4.0 sehingga pH 10.5. Keupayaan untuk ujikaji ini ditetapkan pada +0.6V, iaitu keupayaan yang optimum berdasarkan ujian hidrodinamik dan kepekatan akhir substrak AA2P ditetapkan pada 80µM. Perubahan isyarat arus bagi setiap elektrod bercetak skrin dalam medium pH yang berbeza dibandingkan.. 2.6 Rangsangan enzim AP terhadap substrak AA2P. Aktiviti enzim AP terhadap substrak AA2P dikaji pada julat kepekatan substrak 10µM-160µM. Elektrod bercetak skrin direndamkan dalam 10.0 ml larutan penimbal Tris-HCl. Arus yang berbeza akan dijalankan dengan kepekatan substrak yang berbeza. Graf kalibrasi biopenderia diplotkan untuk mendapatkan julat rangsangan linear enzim AP terhadap substrak AA2P. 2.7 Perencatan enzim AP oleh pestisid 2,4-D. Perencatan enzim AP dijalankan dengan mendedahkan elektrod bercetak skrin terpegun enzim AP kepada larutan 2,4-D (1-200ppm) selama 15 minit. Perencatan aktiviti enzim oleh pestisid 2,4-D dapat ditentukan dengan membandingkan isyarat arus biopenderia sebelum dan selepas perencatan. Peratusan perencatan dapat dikira seperti berikut: % perencatan = Io- IA × 100 Io

Io ialah perubahan arus sebelum perencatan dan IA ialah perubahan arus selepas perencatan.

3. Keputusan dan Perbincangan Perhubungan di antara keupayaan elektrik dengan tindak balas enzim-substrak ditunjukkan dalam Rajah 2. Arus kelihatan malar pada permulaan, apabila sampai +0.6V arus terus meningkat dan mencapai maksimum bagi tindak balas enzim dalam larutan bebas dan enzim terpegun. Maka keupayaan +0.6V diguna sebagai keupayaan optima untuk pencirian rangsangan biopenderia walaupun Mazzei et al. [12] telah melaporkan keupayaan +0.4V boleh digunakan untuk biopenderianya. Jelas bahawa enzim AP pada keadaan bebas adalah lebih aktif berbanding enzim AP terpegun dalam hibrid sol-gel/kiotsan. Substrak AA2P tidak dapat bertindak secara terus dengan enzim kerana memerlukan masa untuk meresap ke dalam membran polimer [13].

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

0.14 0.12 0.10 Enzim AP terpegun Enzim AP larutan bebas

Perubahan Arus/ µΑ

0.08 0.06 0.04 0.02 0.00

0.2

0.4

0.6

0.8

Keupayaan voltan/ V

RAJAH 2: Voltammogram hidrodinamik untuk rangsangan enzim AP dengan substrak AA2P pada julat keupayaan berbeza dalam keadaan larutan bebas dan terpegun Rajah 3 menunjukkan profil rangsangan biopenderia enzim AP terhadap substrak AA2P pada julat kepekatan 10-160µM. Arus semakin meningkat dengan kepekatan substrak AA2P dan arus menjadi hampir malar pada kepekatan substrak 80µM. Julat rangsangan linear biopenderia enzim AP ialah julat 10-80µM dengan nilai R2 = 0.98.
0 .1 0

0 .0 8

Perubahan Arus (µA)

0 .0 6

0 .0 4
0 .0 6

Perbezaan Arus/ A (x10 )

-6

0 .0 5 0 .0 4 0 .0 3 0 .0 2 0 .0 1 0 .0 0 0 20 40 60 80 100

0 .0 2

y = 0 .0 0 0 5 x + 0 .0 0 3 6 R 2 = 0 .9 8 2 6

K e p e k a ta n AA2 P (x1 0 - 6M)

0 .0 0
0 20 40 60 80 100 120 140 160 180

k e p e k a ta n A A 2 p (µ Α )

RAJAH 3: Profil rangsangan biopenderia terhadap kepekatan substrak AA2P dan julat rangsangan linearnya. Ujian pH dijalankan terhadap biopenderia enzim AP kerana semua protein, termasuk enzim adalah peka terhadap perubahan parameter pH. Setiap enzim mempunyai pH optimum yang tersendiri dengan aktiviti tindak balas yang paling maksimum pada pH itu. Rajah 4 menunjukkan kesan pH ke atas rangsangan biopenderia. Aktiviti enzim AP didapati tinggi pada medium pH 8.5, enzim AP

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

dilaporkan aktif pada julat pH 8-10 [8,14]. Graf aktiviti enzim adalah berbentuk ”loceng” dan ini menerangkan ”model diprotik” yang dicadangkan dalam kerja awal Michaelis dan Davidson. Assai kinetik yang melibatkan perencatan sistem enzim yang spesifik telah banyak digunakan sebagai kaedah analisis untuk pengesanan pencemaran makanan dan persekitaran. Dalam kajian ini, asai perencatan enzim telah dilakukan dengan mengguna biopenderia yang mampu menentu sampel keracunan pestisid. Biopenderia yang dibangunkan berasaskan perencatan secara kaedah elektrokimia boleh menentukan ketoksikan 2,4-D dengan cepat dan mudah. Perencatan berlaku disebabkan enzim AP merupakan metaloenzim yang mengandungi tapak aktif Zn2+ dan Mg2+ yang bertindak balas dengan sebatian organik (misalnya pestisid) dan logam toksik yang mengakibatkan pengurangan aktiviti enzim [2]. Rajah 5 menunjukkan peratusan perencatan enzim AP selepas 15 minit terdedah kepada larutan pestisid 2,4-D. Perencatan enzim AP oleh pestisid 2,4-D mencapai maksimum dan tetap pada 50% perencatan.

0.16 0.14 0.12

Perubahan Arus (µM)

0.10
0.08 0.06 0.04 0.02 0.00
pH 4.0 pH 6.0 pH 8.5 pH 10.5

RAJAH 4: Kajian kesan ke atas pH pada rangsangan biopenderia (substrak AA2P 80µM)

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70

60

50

% Perencatan

40

30

20 AA2P 80uM AA2P 160uM AA2P 40uM

10

0 0 100 200 300

kepekatan 2,4-D (ppm)

RAJAH 5: Tahap perencatan enzim AP pestisid 2,4-D dalam tiga kepekatan substrak AA2P yang berbeza

4. Kesimpulan Biopenderia 2,4-D terpegun enzim AP telah dibangunkan berasaskan analisis aktiviti perencatan enzim AP. Interaksi enzim dengan pestisid 2,4-D telah menghasilkan satu biopenderia ringkas untuk pemonitoran pestisid dengan cepat dan mudah. Di samping itu, biopenderia 2,4-D berasaskan elektrod bercetak skrin adalah mudah disediakan dan boleh diguna secara pakai buang.

Penghargaan Penghargaan ditujukan kepada MOSTI yang membiayai projek penyelidikan melalui gran IRPA 0903-03-0184-EA001 dan Biasiswa Pascasiswazah.

Rujukan 1. Balague, C.E., Ruiz, C.S., Rey, R., Evangelista de Duffard, A.M, Elena, M. & Nader-Macias. 2002. Effect of herbisid 2,4-D dichlorophenoxyacetic acid on uropathogenic Escherichia coli virulence factors. Toxicology.177: 143-155 2. Sanchez, F.G., Diaz, A.N., Peinado, M.C.R. & Belledone, C. 2003. Free and sol-gel immobilized alkaline phosphatase-based biosensor for the determination of pesticides and inorganic compounds. Analitical. Chimica. Acta. 484: 45-51 3. Kaur, J. Singh, K.V., Schmid, A.H., Varshney, G.C., Suri, C.R. & Raje, M. 2004. Atomic force spectroscopy-based study of antibody pesticide interactions for characterization of immunosensor surface. Biosensors & Bioelectronics. 20 : 284-293

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4. Nunes, S. G., Jeanty, G. & Marty, Jean-Louis. 2004. Enzyme immobilization procedures on screen-printed electrodes used for the detection of anticholinesterase pesticides comparative study. Analytica Chimica Acta. 523: 107-115 5. Mazzei, F., Botre, F., Montilla, S., Pilloton, R., Podesta, E. & Botre, C. 2004. Alkaline phosphatase inhibition based electrochemical sensors for the detection pesticides. Journal of Electroanalytical Chemistry. 574:95-100 6. Wang, J. & Chen, L. 1999. Amperometric thick-film strip electrodes for monitoring organophosphate nerve agents based on immobilized organophophorus hydrolase. Analytical Chemistry.71: 2246-2249 7. Satoh, I. & Iijima, Y. 1995. Multi-ion biosensor with use of a hybrid-enzyme membrane. Sensors & Actuators B. 24-25: 103-106 8. Diaz, A. N., Sanchez, F. G., Ramos, M.C. & Torijas, M. C. 2002. Horseradish peroxidase solgel immobilized for chemiluminescence measurements of alkaline-phosphatase activity. Sensors & Actuators B. 82: 176-179. 9. Guo, Y. & Gudalupe, A. R. 1998. Screen-printable surfactant-induce sol-gel graphite composites for electrochemical sensors. Sensor & Actuators B. 46: 213-219. 10. Yang, Y.M., Wang, J.W., Tan, R.X. 2004. Immobilization of glucose oxidase on chitosanSiO2 gel. Enzyme and Microbial Technology. 34: 126-131. 11. Miao, Y & Tan, S.N. 2001. Amperometric hydrogen peroxide biosensor with silica solgel/chitosan film as immobolization matrix. Analytica Chimica Acta. 437: 87-93 12. Mazzei, F., Botre, F., Montilla, S., Pilloton, R., Podesta, E. & Botre, C. 2004. Alkaline phosphatase inhibition based electrochemical sensors for the detection pesticides. Journal of Electroanalytical Chemistry. 574:95-100. 13. Wang, J., Pamidi, P.V.A. & Park, D.S. 1996. Screen-printable sol-gel enzyme-containing carbon inks. Analytical Chemistry. 68: 2705-2708 14. Kokado, A., Arakawa, H. & Maeda, M. 2000. New electrochemical assay of alkaline phosphatase using ascorbic acid 2-phosphate and its application to enzyme immunoassay. Analytica Chimica Acta. 407: 119-125A

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SURFACE AND ACIDITY ANALYSIS OF SOLID SUPER ACID BASED ON HZSM-5 Wong Hon Loong1, Sugeng Triwahyono2 and Mustaffa Shamsuddin1
1

Department of Chemistry, Faculty of Science,

Universiti Teknologi Malaysia, 81310 UTM Skudai, Johor, Malaysia
2

Ibnu Sina Institute for Fundamental Science Studies, email: mustaffa@kimia.fs.utm.my

Universiti Teknologi Malaysia, 81310 UTM Skudai, Johor, Malaysia

Abstract: The acidity of HZSM-5 zeolite in the presence of tungstophosphoric acid (WP) has been studied. Tungstophosphoric acid has been immobilized onto HZSM-5 by impregnation method followed by calcinations at 823K. The physical properties of the solid acid were studied by XRD, FTIR, TG/DTA, pyridine adsorption and nitrogen adsorption analysis. The pyridine adsorption FTIR study shows the presence of both the Brønsted and Lewis acid sites in HZSM-5 samples. However, the HZSM-5 supported tungstophosphoric acid exhibited higher ratio of Brønsted acid sites over Lewis acid sites. Abstrak: Keasidan zeolit HZSM-5 dengan kehadiran asid tungstofosforik (WP) telah dikaji. Asid tunsgtofosforik telah dimmobilisasikan ke atas HZSM-5 melalui kaedah pengisitepuan diikuti dengan pengkalsinan pada 823K. Sifat fisik asid pepejal telah dikaji melalui analisis XRD, FTIR, TG/DTA, penjerapan piridina dan penjerapan nitrogen. Kajian FTIR penjerapan piridina menunjukkan kewujudan kedua-dua tapak asid Brønsted dan Lewis dalam sample HZSM-5. Sampel asid tungstofosforik asid berpenyokong HZSM-5 menunjukkan nisbah tapak asid Brønsted terhadap tapak asid Lewis yang lebih tinggi. Keywords: Solid acid, HZSM-5, tungstophosphoric acid. Introduction Heteropoly acids are widely used as catalysts due to their very strong Brønsted acidity and special structural properties. Usually, Keggin-type tungsten heteropoly acids are preferred over molybdenum heteropoly acids as acid catalysts because of their stronger acidity, higher thermal stability and lower oxidation potential [1]. Tungstophosphoric acid (WP), H3PW12O40 is the strongest acid in the heteropoly acid series. Supported heteropoly acid catalysts are important for applications, as bulk heteropoly acids have a low specific surface (< 10 m2/g). Acid or neutral substances such as SiO2, active carbon, acidic ion-exchange resins have been suggested as suitable as supports [2, 3]. In this research, a new type of solid super acid catalyst on zeolitic material has been synthesised and studied. The HZSM-5 zeolite supported tungstophosphoric acids (WZ) have been prepared in order to obtain material with enhanced acidity, higher thermal stability and higher surface area catalysts. Experimental Reagents Tungstophosphoric acid, H3PW12O40, was purchased from Sigma-Aldrich. Protonated-ZSM-5 (HZSM-5) was prepared using a commercially available ammonium form ZSM-5 (Zeolyst, CBV 3024E) with silica/alumina ratio of 30. All solvents and chemicals were used without further purifications. Sample Preparation The HZSM-5 supported tungstophosphoric acids were prepared by a conventional impregnation method [4]. The samples (WZ) were dried overnight at 373 K in air followed by calcination at 673 K.

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The supported samples are abbreviated as 1WZ, 5WZ, 10WZ and 15WZrepresenting the 1, 5, 10 and 15 wt.% WP on HZSM-5, respectively. Surface and Acidity Analysis BET surface area measurements were performed on a Surface Area Analyzer (Thermo Finnigan Qsurf Series) using nitrogen gas (99.999%) adsorption at 77 K. Samples (30 mg) were evacuated at 473 K for 30 min prior to measurements. Powder X-ray diffraction studies were conducted on a Bruker Advance D8 using Siemens 5000 diffractometer using a Cu Kα source with a wavelength of 0.15418 nm. The data were collected with a scan rate of 1 0/s and a step size of 0.05 0 at room temperature. In situ infrared spectroscopy of pyridine adsorption was recorded on a Perkin Elmer Spectrum One FTIR Spectrometer. The self-supporting wafer (10 mg) was prepared and mounted in a vacuum chamber. Sample was pretreated at 673 K under vacuum and exposed to pyridine for 5 min at room temperature. Desorption of pyridine was carried out by evacuation for 1 h at 423 and 623 K. Simultaneous TG/DTA measurements were carried out on a Perkin Elmer’s Pyris Diamond Thermogravimetric/Differential Thermal Analyzer under nitrogen atmosphere with a flow rate 200 ml/min using approximate 15.0 mg of the sample. The sample were heated in the temperature range 323 K – 1073 K with a heating rate of 10 K/min. Results and Discussion Samples characterization Table 1 shows the BET surface areas of HZSM-5 and supported samples. As expected, the surface area of the bulk WP sample was low, around 5 m2/g. The parent HZSM-5 support had a surface area of 387 m2/g. Little decrease in the surface area was observed for the supported samples. The higher loading of the 15WZ sample caused a decrease in the surface area to 294 m2/g. Table 1: Physical properties of various tungstophosphoric acid loadings samples. Sample H3PW12O40 HZSM-5 1WZ 5WZ 10WZ 15WZ WP Loaded (wt. %) 100 1 5 10 15 Surface Area (m2/g) 5 387 364 323 326 294

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The effects of different loadings of WP on HZSM-5 in the XRD patterns are illustrated in Figure 1. With increasing WP loadings, the intensities of the peaks decrease slightly, which demonstrates that the intact Keggin structure of WP occurs and starts to aggregate on the walls of HZSM-5 [6]. The lines appearing at 2θ = 7.90, 8.86 and 23.14 0 are assigned to d(011), d(200) and d(051) diffraction of the HZSM-5.

(a)

Intensity

(b) (c) (d) (e)
5 10 20 30

2-Theta

Figure 1: XRD patterns of WZ samples with various loadings of WP. (a) HZSM-5; (b) 1WZ; (c) 5WZ; (d) 10WZ and (e) 15WZ. Meanwhile, Figure 2 shows the FTIR spectra of pyridine adsorbed on HZSM-5 supported samples. The of the peak at 1540 cm-1 based on Brønsted acid sites (B) and the peak at 1450 cm-1 based on Lewis acid sites (L) were observed on the samples [7]. With higher loadings of WP, FTIR spectra show the ratio of Brønsted acid sites to Lewis acid sites in the supported samples also increased. But, in the case of, The 1WZ sample exhibited the highest Brønsted acid sites, as indicated by the height of the peak at 1540 cm-1, which may be related with the change in the acid strength by the supporting of WP.

B

L (a)

Sample
Absorbance

(b) (c) (d) (e)

(a) HZSM-5 (b) 1WZ (c) 5WZ (d) 10WZ (e) 15WZ

B 0.190 0.193 0.177 0.133 0.122

Peak Height L 0.091 0.085 0.079 0.060 0.053

B/L 2.09 2.27 2.24 2.21 2.30

1600

1560

1520

cm

-1

1480

1440

1400

Figure 2: Pyridine adsorption FTIR spectra of WZ samples. (a) HZSM-5; (b) 1WZ; (c) 5WZ; (d) 10WZ and (e) 15WZ. The thermogravimetric profiles of uncalcined samples were depicted in Figure 3. Initial weight loss of 2.5% (below 573 K) for the supported sample was probably due to dehydration of the sample. Another stage of weight loss was observed in the high temperature region (723-823 K) indicating that the decomposition of WP had occurred.

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At about 823 K, WP on support seemed to undergo an exothermic decomposition, according to the equation [6], 2H3PW12O40 → P2O5 + 24WO3 + 3H2O
100

99 1WZ 5WZ 10WZ 15WZ Weight %

98

97

96 300

500

700 Temperature (K)

900

1100

Figure 3: Thermogravimetric analysis of various WP loadings supported samples. However, TGA/DTA technique was not suitable to accurately analyze the supported samples. Instead, FTIR technique was used to study the stability of bulk WP and supported samples after a thermal treatment at 873 K (Figure 4). In agreement with the TGA/DTA results just described, the infrared spectrum of bulk WP (Figure 4a) and the supported sample (Figure 4b) show the characteristic bands of the Keggin structure at 892 and 983 cm-1. The IR spectrum of WZ after heat treated at 873 K (Figure 4c) clearly indicates that decomposition occurred above 873 K as evidenced by the disappearance of characteristic IR bands.

(a)

Transmittance

(b)

(c)

1500

1200

cm-1

800

400

Figure 4: Infrared spectra of (a) tungstophosphoric acid (WP); (b) 10WZ and (c) 10WZ, after heat treated at 873 K.

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Conclusion The HZSM-5 zeolite upon contact with a very strong heteropoly acid WP retains the structure of zeolite. The WP on support was undergoing an exothermic decomposition above 823 K. The HZSM-5 support WP samples exhibited higher ratio of Brønsted acid sites over Lewis acid sites. Acknowledgements The authors would like to acknowledge the Ministry of Science, Technology and Innovation Malaysia for the financial support through IRPA funding 09-02-06-0057-SR005/09-05 and Ibnu Sina Institute, Universiti Teknologi Malaysia for research facilities. References 1. 2. 3. 4. 5. 6. M.N. Timofeeva (2003). “Acid Catalysis by Heteropoly Acids”. Appl. Catal. A 256, 19-35. I.V. Kozhevnikov, K.R. Kloetstra, A. Sinnema, H.W. Zandbergen and H. van Bekkum (1996). “Study of Catalyst comprising Heteropolyacid H3PW12O40 supported on MCM-41 and amorphous silica”, J. Mol. Catal. A: Chem. 114, 287-298. T. Blasco, A. Corma, A. Martinez, and Martinez-Escolano (1998). “Supported Heteropolyacid (HPW) Catalyst for the Continuous Alkylation of Isobutane with 2-Butene: The Benefit of Using MCM-41 With Larger Pore Diameter”, J. Catal. 177, 306-313. H.L. Wong, S. Triwahyono and M. Shamsuddin (2004), unpublished report. A. Miyaji, T. Echizen, K. Nagata, Y. Yoshinaga and T. Okuhara (2003). “Selective Hydroisomerization of n-Pentane to Isopentane over highly dispersed Pd-H4SiW12O40/SiO2”. J. Mol. Catal. A: Chem. 201, 145–153. Q.Y. Liu, W.L. Wu, J. Wang, X.Q. Ren, Y.R. Wang (2004). “Characterization of 12Tungstophosphoric Acid Impregnated on Mesoporous Silica SBA-15 and Its Catalytic Performance in Isopropylation of Naphthalene with Isopropanol”. Micropor. Mesopor Mater. 76, 51. M.R. Basila, T.R. Kantner and K.H. Rhee (1964). “The Study of the Acidic Sites on a SilicaAlumina: Characterization by Infrared Spectroscopy of Trimtheylamaine and Pyridine Chemisorption”. J. Phys. Chem. 68 (11), 3197. DETERMINATION OF POLYCYCLIC AROMATIC HYDROCARBONS IN PALM OIL MILL EFFLUENT BY SOXHLET EXTRACTION AND GAS CHROMATOGRAPHY-FLAME IONIZATION DETECTION Nor Fairolzukry Ahmad Rasdy, M. Marsin Sanagi,* Wan Aini Wan Ibrahim, Ahmedy Abu Naim Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia 81310 UTM Skudai, Johor, Malaysia *E-mail: marsin@kimia.fs.utm.my ABSTRACT A method has been developed for the determination of polycyclic aromatic hydrocarbons (PAHs) from palm oil mill effluent based on gas chromatography-flame ionization detection. Extraction of spiked PAHs (napthalene, fluorene phenanthrene, fluoranthene and pyrene) in palm oil waste was carried out by Soxhlet extraction using hexane-dichloromethane (60:40 v/v) as the solvent. Excellent separations were achieved using temperature programmed GC on Ultra-1 fused-silica capillary column (30 m × 250 µm ID), carrier gas helium at a flow rate of 1 mL/min.

7.

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Keywords: Polycyclic aromatic hydrocarbons, GC-FID, Soxhlet extraction Introduction Polycyclic aromatic hydrocarbons (PAHs) are a class of diverse organic compounds containing two or more fused aromatic rings of the carbon and hydrocarbon atoms. They are ubiquitous pollutants formed from the combustion of fossil fuels and are always found as a mixture of individual compounds. PAHs as one of the typical persistent organic compounds (POPs) featured in regional and global cycling. PAHs are emitted mainly into the atmosphere and have been detected at long distances from their source. Because of their low vapor pressures, compounds with five or more aromatic rings exist mainly adsorbed to airborne particulate matter, such as fly ash and soot [1]. The analysis of these PAHs is of great interest because of their toxicity and persistence in the environment. PAHs are adsorbed strongly to the organic fraction of sediments and soils [2]. Therefore, it can be concluded that sediments and soils are usually considered as the main sinks for PAHs in the environment. Polycyclic aromatic hydrocarbons (PAHs) are reported to have mutagenic and/or carcinogenic effects. The ability of PAHs to induce cancer has been documented by epidemiological studies of worker in coal tar, creosote, coal gas, coke, and cutting oil industries [1]. Some analogues of these compounds, such as polycyclic aromatic sulfur heterocycles (PASHs), are also potentially mutagenic and carcinogenic. But, although they have a high bioaccumulation and have been found in some water and sediment samples, they have not been studied as extensively as PAHs. PAHs are routinely analyzed by one-dimensional capillary gas chromatography (GC). Normally, high-resolution mass spectrometry can detect the PAHs in sample [3]. This study will discuss more about analysis of PAHs using one-dimensional gas chromatography-flame ionized detection (GC-FID). This is significant for screening PAHs present in environmental sample before further analysis.

Experimental Reagents Methanol was obtained from HyperSolv, BDH Laboratory, (England). Acetonitrile, dichloromethane, hexane (all in HPLC grade) were supplied by Fisher Chemicals (USA). The polycyclic aromatic hydrocarbons (napthalene, fluorene, phenanthrene, fluoranthene and pyrene) were obtained from Fluka Chemika, Sigma-Aldrich Chemic, Steinheim, (Switzerland). The molecular weight and molecular structures of the PAHs are shown in Table 1. Double-distilled deionized water of at least 18 MΩ was purified by Nano ultra pure water system (Barnstead, USA). Table 1: Properties of four polycyclic aromatic hydrocarbons (PAHs) 3D Molecular Structure

Compound

Formula Structure

Molecular Weight

Napthalene

128

C10H8

Fluorene

166

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C13H10

Phenanthrene

178

C14H10 Fluoranthene 202

C16H10 Pyrene 202

C16H10 Chromatographic conditions The GC-FID system consist of a Hewlett Packard Model 6890GC gas chromatography (GC) equipped with a flame ionization detector (FID) and a data processor (USA). The gas chromatographic column used was Ultra-1 932530, a non-polar, fused-silica capillary column (30 m length × 250 µm inner diameter × 0.20 µm film thickness) (USA). Helium gas was used as the carrier gas at a flow rate of 1 mL/min at a pressure of 75 kpa. The injector temperature was set at 250°C and the detector temperature at 310°C. The temperature program used was; 2 minute s hold time at 250°C, a ramp to 130°C at 30°C/min followed by 3 minutes hold time, a ramp to 240°C at 7°C/min and a final ramp to 285°C at 12°C with an 8 minutes hold time.

Procedure The mixture of standard solution was prepared from the 1000 ppm stock solution. The mixed standard solution was prepared to produce the calibration graph of each PAHs to determine the limit of detection. The prepared mixture solution (80, 60, 40, 20, 10 ppm) was injected in triplicate onto the column. About 10 g of dried palm oil mill effluent sample, thoroughly mixed with anhydrous sodium sulphate (10 g) was Soxhlet extracted with dichloromethane (200 mL) for 6 hours. The solvent was concentrated to 5 mL in a rotary evaporator under reduced pressure. 0.5 M potassium hydroxide (100 mL) in methanol was added and the mixture was refluxed for 4 hours in a water bath at 80ºC. After cooling, deionized water (20 mL) was added and extraction was performed with hexane (3×50 mL). The combined organic extracts were dried over anhydrous sodium sulphate (0.5 g). The decanted extract was evaporated at 40ºC in a rotary evaporator under reduced pressure to near dryness, dissolved in isooctane (1 mL) for silica clean-up. The glass column (1.2 cm I.D.) was slurry packed with silica gel (10 g) in dichloromethane and a top layer of anhydrous sodium sulphate (0.5 g). The column was rinsed with hexane (40 mL)

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before use. The extract was transferred on to the column and sequentially eluted with hexane (25 mL) and hexane-dichloromethane, 60:40 (30 mL) to give fractions enriched in alkanes and PAHs, respectively. The second eluate was evaporated under reduced pressure to near dryness and replaced with acetonitrile (1 mL) before injection can be made. After cleaning up the sample, 1 µL was injected into GC-FID column. The temperature program used was exactly same with the temperature used for standard solution described above. For detection, peaks interfere were compared to the standard and from peak area, recovery value was calculated for spiked sample.

Results and Discussion PAHs peak was identified in the mixture standard solution based on their retention time. All of the PAHs were eluted within 25 minutes. Based on the chromatograms obtained (Figure 1), it was noted that the elution orders for the five PAHs on Ultra-1 column were strongly in order of increasing molecular weight. Napthalene (8.182 min) with the lowest molecular weight was first eluted across the column followed by fluorene (14.591 min), phenanthrene (17.917 min), fluoranthene (22.279 min) and pyrene (23.004 min). Even fluoranthene and pyrene with the same molecular weight value can be separated accordingly. The chromatograms were very clear with no interfering peaks appearing in the areas of interest.

Figure 1: Chromatogram of PAHs study (100 ppm). Peaks identification: (1) naphthalene, (2) fluorene, (3) phenanthrene, (4) fluoranthene (5) pyrene In order to examine the sensitivity of gas chromatography system, the limits of detection for PAHs are investigated. Theoretical limits of detection (LOD) were determined taking the usual definition, which that gives a peak with a height three times the background noise level. The calibration graph obtained was used to determine the limit of detection (LOD). A linear calibration graph was produced for each standard PAHs with the correlation coefficient ranging between 0.99140.9989. The value of the correlation coefficient obtained for each calibration graph shows that the correlation between relative peak area and concentration is good. The calibration graph of naphthalene, fluorene, phenanthrene, fluoranthene and pyrene are shown in Figure 2. Table 2, given the regression equation, correlation coefficient and LOD for each PAHs. Table 2: Limit of detection, regression equations and correlation coefficient of PAHs studied using GC-FID

PAHs

Regression Equation

Correlation Coefficient, r2

LOD in ppm (From calculation)

RSD (%) (n = 3)

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Naphthalene Fluorene Phenanthrene Fluoranthene Pyrene

y = 0.6486x + 6.3900 y = 1.1729x + 3.8878 y = 0.9770x + 0.5149 y = 0.6152x + 0.7479 y = 0.6904x + 3.4882

0.9989 0.9930 0.9914 0.9910 0.9927

3 (2.83) 8 (7.22) 8 (7.28) 5 (4.30) 5 (4.33)

9.0 5.2 13.1 5.3 4.9

The silica gel column clean-up provided all PAHs in second fraction together with alkylbenzenes, but these monoaromatic hydrocarbons did not interfere with GC analysis. Hence the use of silica gel for clean-up extracts appeared more suitable for PAHs determination. Saponification also improved the determination of PAHs. Associations between minor PAHs and lipid palm oil waste fraction are reduced when the raw extract is submitted to a basic treatment, and liquid-liquid partitioning allows fatty acid removal and therefore, extract clean-up is made easier. After doing the clean-up using the same procedure, the sample (1 µL) was injected into the GC-FID injection port. According to their retention times, PAHs should be identifiable in the palm oil waste if they are present above the detection limits. However, in this work, none of them were identified, probably because they are nonexistent or present in a concentration lower than the detectable limit (chromatogram not shown). The effectiveness of the method was assessed with the analysis of a palm oil waste spiked with PAHs. The chromatogram obtained (Figure 3) showed that all the peaks of analyte studied were well resolved but there were some peaks corresponding to other organic products present in sample observed but did not interfere with the PAHs peaks studied. The concentration of spike sample was obtained from peak area value from calibration graph equation. The recovery percentage was then calculated by dividing with the standard spike value, which is 100 ppm. Table 3 given the peak area value and concentration for each PAHs obtained. Recoveries of PAHs from palm oil waste sample ranged from 36.14 % to 67.57 % for Soxhlet extraction with silica gel clean-up (Figure 4). The chromatogram of spiked standard PAHs in palm oil waste is shown in Figure 4. Table 3: Peak area, concentration and percentage recovery of PAHs studied using GC-FID PAHs Naphthalene Fluorene Phenanthrene Fluoranthene Pyrene
Napthalene
90

Peak Area 17.050 53.583 37.216 31.734 43.162

Concentration (ppm) 36.14 42.37 38.62 52.80 67.57

% Recovery (RSD) 36.14 (8.36) 42.37 (5.22) 38.62 (11.47) 52.80 (6.35) 67.57 (3.13)

Phenantrene

50 40 30 20 10 0 0

80

y = 0.6486x - 6.39 R2 = 0.9989

70 60 50 40 30 20 10 0

y = 0.977x - 0.5149 R2 = 0.9914

20

40

60

80

100

0

20

40

60

80

100

Fluorene 120 100 80 60 40 20 0

Fluoranthene 60 50 40 56130 20 10 0

y = 1.1729x + 3.8878 R2 = 0.993

y = 0.6152x - 0.7479 R2 = 0.991

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Pyrene 60 50 40 30 20 10 0 0

y = 0.6904x - 3.4882 R2 = 0.9927

20

40

60

80

100

Figure 2: Calibration graph of the five PAHs studied: naphthalene, fluorene, phenanthrene, fluoranthene and pyrene

Figure 3. Chromatogram of standard PAHs (100 ppm) spiked in palm oil mill effluent using GC-FID after Soxhlet extraction. (1) naphthalene, (2) fluorene, (3) phenanthrene, (4) fluoranthene (5) pyrene

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% Recovery 80 70 60 50 40 30 20 10 0 Naphthalene Fluorene Phenanthrene Fluoranthene Pyrene PAHs

Figure 4. Percentage of extracted PAHs (100 ppm) in palm oil mill effluent after Soxhlet extraction and GC-FID analysis

Conclusions The separations of polycyclic aromatic hydrocarbons by GC-FID with temperature programming have been examined. It was observed that a good separation and linearity was achieved during the operating temperature. The LOD is still considered acceptable since EPA method indicated 50 to 1000 ppm is moderately toxic, which means method limits of detection are below the risk-based values. Therefore, Soxhlet extraction with GC-FID detection can be chosen as a preliminary and as an alternative analysis technique for PAHs detection. Acknowledgements We thank Universiti Teknologi Malaysia and The Ministry of Science, Technology and Innovation, Malaysia (MOSTI) for financial supports through IRPA Project 09-02-06-0074 EA211 (Vote No. 74255).

References [1] Sigmund F. Zakrzewski (1991). Principles of Environmental Toxicology. ACS Professional Reference Book. American Chemical Society, Washington D.C. [2] Vanina Flotron, Justin Houssou, Audrey Bosio, Corine Delteil, Alan Bermond, Valerie Camel (2003). Rapid Determination of polycyclic aromatic hydrocarbons in sewage sludges using microwave-assisted solvent extraction comparison with other extraction methods. J. Chromatogr. A, 999. 175-184 [3] P. Korytar, P.E.G. Leonards (2002). High-resolution separation of polychlorinated biphenyls by comprehensive two-dimensional gas chromatography. J. Chromatogr. A, 958. 203-218 [4] Colin S. Chen, P. Suresh C. Rao, Linda S. Lee (1996). Evaluation of Extraction and Detection Methods for Determining Polynuclear Aromatic Hydrocarbons From Coal Tar Contaminanted Soils. Pergamon. 32. 1123-1132 [5] G. Codina, M.T. Vaquero, L. Commellas, F. Broto-Puig (1994). Comparison of various extraction and clean-up methods for the determination of polycyclic aromatic hydrocarbons in sewage sludge-amended soils. J. Chromatogr. A, 673, 21-29 [6] Dadan Hermawan, M. Bachi Amran and Buchari (2002). Study of Polycyclic Aromatic Hydrocarbon (PAH) Content in Sediment by HPLC Method. Proceeding InSECT 2002. 206-212 [7] J. D. Berset, R. Holzer (1995). Organic micropollutants in Swiss agriculture: Distribution of Polycyclic Aromatic Hydrocarbons (PAHs) and Polychlorinated Biphenyls (PCBs) in Soil, Liquid Manure, Sewage Sluge, and Compost Samples; A Comparative Study. J. Environ. Anal. Chem. 59. 145-155

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[8] C. Miege, J. Dugay, M.C. Hennion (2003). Optimiztion, validation and comparison of various extraction techniques for the determination of PAH in sewage sludges by liquid chromatography coupled to diode-array and fluorescence detection. J. Chromatogr. A. 995. 87-97

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SYNTHESIS AND CHARACTERIZATION OF NANO SIZE POLY(STYRENE-DIVINYLBENZENE) USING DISPERSION COPOLYMERIZATION

Norsyariza Abd Aziz, Ahmedy Abu Naim, M. Marsin Sanagi,* Wan Aini Wan Ibrahim, Asiah Hussain Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia 81310 UTM Skudai, Johor, Malaysia. e-mail: marsin@kimia.fs.utm.my Abstract. Poly(styrene-divinylbenzene) (PS-DVB) is a widely used adsorbent for the extraction and separation of various types of compounds. In this work, a method for the synthesis and characterization of PS-DVB using dispersion polymerization was developed to produce nano particle size resins. The characterization of the synthesized PS-DVB was carried out using Fourier Transform Infrared (FTIR), Scanning Electron Microscopy (SEM) and Nitrogen Adsorption (NA). PS-DVB had an average particle size of about 800 nm with very fine pore size and high surface area. Abstrak. Kromatografi Poli(stirena-divinilbenzena)(PS-DVB) adalah bahan penjerap yang digunakan secara meluas dalam pengekstrakan dan pemisahan bagi pelbagai jenis sebatian. Dalam penyelidikan ini, sintesis dan pencirian PS-DVB menggunakan pempolimeran ampaian dijalankan bagi menghasilkan resin PS-DVB yang bersaiz nano. Pencirian PS-DVB ini dijalankan menggunakan spektroskopi Inframerah (FTIR), Mikroskopi Imbasan Elektron (SEM) and Penjerapan Nitrogen (NA). Saiz zarah resin PS-DVB yang disintesis adalah sekitar 800nm dan ia mempunyai saiz liang yang sangat kecil serta luas permukaan yang tinggi. Keywords: Poly(styrene-divinylbenzene); Dispersion Co-polymerization Introduction Poly(styrene-divinylbenzene) (PS-DVB) copolymers are the most widely used polymeric material in chromatographic analysis. Resin based on PS-DVB are stable with eluents from pH 1-13 and overcome many of the limitation of bonded silicas especially the pH stability of the silica under acidic or basic condition and the presence of residual silanol groups that can cause peak broadening or tailing by interaction with polar compound [1]. PS-DVB resin also has greater analyte retention, mainly for polar compound because their hydrophobic surface contains a relatively large number of active aromatic sites that allow π-π interaction with these aromatic analytes. However, there only few reports on the use of PS-DVB as an adsorbent due to its difficulties such as lack of selectivity and low breakthrough volumes for highly polar compound, which leads to their incomplete extraction for predominantly aqueous samples [2]. Previous work [3] has report on the synthesis of PS-DVB using suspension and emulsion polymerization between styrene as a monomer and the divinylbenzene as the crosslinker. Water used as a medium, poly(vinyl alcohol) as a stabilizer and benzoyl peroxide as an initiator and the reaction was ran at 70°C. This paper will discuss the polymerization of PS-DVB using dispersion polymerization. Dispersion polymerization generally involves the polymerization of a monomer dissolved in an organic diluent to produce insoluble polymer dispersed in the continuous phase in the presence of an amphipathic graft or block polymer as the dispersant [4]. Clearly, dispersion polymerization is quite different from an aqueous emulsion or suspension polymerization in which the monomer has only a limited solubility in the aqueous phase and also

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requires surfactant for its stabilization in the emulsified form. The polymer particles produced are larger than those obtained by emulsion polymerization in its strictest sense and tend to settle out on further dilution of the aqueous phase. The term dispersion polymer is frequently used to describe the polymer with colloidal dimension 0.01µm - 10 µm in any medium [4, 5].

EXPERIMENTAL

Reagents Styrene (99%, stabilized with 0.005% 4-tent-butylcatecol) was from Fluka Chemika (Switzerland). The styrene monomer was pre washed consecutively with 10% sodium hydroxide (50 ml × 3) and distilled water (50 ml × 3) prior to use. Technical-grade divinylbenzene (DVB) used as the crosslinker was laboratory grade (70-85%) stabilized with 0.2% 4-tert-butylcatecol obtained from Merck (Schuchardt, Germany) and Fluka Chemika. The DVB was purified with 10% sodium hydroxide (50 ml × 3) and distilled water (50 ml × 3) before use. The procedure was carried out for each PSDVB synthesis. Azobis(isobutyronitrile) (AIBN) used as initiator without any purification. Polyacrylic acid (PAA) used as stabilizer was obtained from Fluka Chemica and used without any purification. Isopropanol, ethanol and butanol are obtained from Fluka Chemica and used as dispersion medium in the composition with deionized water.

Procedure Preparation of Stock Solution of Dispersion Medium Containing Polyacrylic Acid (PAA) Isopropanol (IsoPrOH) were mixed with deionized water (DI) in various ratios. This solution is used as a dispersion medium for the polymerization reaction in the synthesis of PS-DVB. Then, polyacrylic acid (PAA) 1.0 g / 0.1 L as stabilizer was added few at the time to the medium while stirring on the hot plate until the PAA dissolved in the medium. The stock solution then was placed in the volumetric flask. The method was followed for different kinds of alcohols. Amount of dispersion medium varied as shown in the (Table1). Table 1: Dispersion medium ratio used in polymerization of PS-DVB Sample Dispersion Medium IsoPrOH DI PS-DVB-1 0 100 PS-DVB-2 70 30 PS-DVB-5 80 20 PS-DVB-6 50 50 PS-DVB-8 90 10 Synthesis of PS-DVB The oil bath temperature was controlled at 700C and the reaction vessel equipped with thermometer, nitrogen gas inlet and condenser were placed in it. The dispersion medium which contained PAA was placed in a 3-neck round-bottom flask and stirred using mechanical stirrer at 100 rpm until the temperature reached 700C. After the above solution temperature reached 700C (isothermal conditions), the reactor was purged with nitrogen gas, Styrene, DVB and AIBN were premixed and added to the reactor. The reaction mixture was mechanically stirred for 24 hours at 100 rpm. Then the bead was

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filtered off on a sintered funnel and wash with acetonitrile (50 mL × 3), acetone (50 mL × 3) and DI (50 mL × 3). For further purification, the beads were subjected to Soxhlet extraction with Methanol (MeOH) for 24 hours and washed with DI and filtered off on a sintered funnel and dried at room temperature. In all runs the recipe containing 0.01 mol AIBN, 1.0g / 0.1L PAA, monomer / dispersion ratio: 1.0 / 10 mL.

Characterization Fourier Transform Infrared (FTIR) The characterization of PS-DVB by far region IR was carried out to detect the functional groups contains in the sample and structure confirmation. FTIR used was from Shidmadzu FTIR 8300. Scanning Electron Microscopy (SEM) Samples were placed on an aluminium made stub which was covered with double-sided tape. The tape was used to eliminate any possible discharge of samples from the surface when the scanning was done. After the sample was well spread on the surface of the tape, the sample was coated with gold (aurum). This coating step was carried out to ensure that the sample was able to undergo electron bombardment without any discharge effect. Scanning was done using a Philips XL 40 and the bombardment using electron gun with tungsten filament under 30kV resolution to get the scanning image. Nitrogen Adsorption The instrument used in this analysis was Micromeritic ASAP 2010, nitrogen gas was used as the adsorbate at 77K. Before adsorption, about 0.2 g sample was outgassed for 2 hours at 110oC and under vacuum of about 10-3 Torr. Adsorption was carried out by increasing the pressure until the relative pressure reached to unity and followed the desorption process by decreasing the pressure to initial pressure. Results And Discussion Infrared Analysis PS-DVB which have been synthesized will be discuss according to their chemical characterization. Samples of PS-DVB have been prepared in various ratio of dispersion medium using Isopropanol and deionized water. The reaction medium is one of the several important parameters in the synthesis of PS-DVB phase. In suspension polymerization, water is used as the reaction medium whereas in dispersion polymerization technique, the reaction medium constitutes an organic portion, such as Isopropanol [6]. The absence of water from the medium means that fewer nuclei are stabilized, but its presence promotes a gel affect which increase the polymerization rate. The addition of solvent, however, will affect the rates of particle growth and finally the size and distribution of the particles [7]. 100% of DI has been used in preparing of PS-DVB-1, but 5 hours after stirring periods the solution flocculated. PS-DVB-2 was synthesized using the dispersion medium ratio (IsoPrOH : DI) of 70:30. Figure 1 shows an IR spectrum of PS-DVB-2 before Soxhlet extraction. IR spectrum of this sample shows an absorption peak at νmax 3002.0 cm-1 for stretching of C-H sp3 and 3025.1 cm-1 for CH2 , 3058.9 cm-1 for C-H aromatic. Sharp absorption peaks at νmax 1600.8 cm-1 and 1450.4 cm-1 refers to absorption of C=C aromatic. In a fingerprint range, there are also some absorption of monosubstitude C=C at νmax 758.0 cm-1 and 699.1 cm-1. However, it shows an absorption of C=O which strongly believe from the stabilizer, PAA at νmax 1705.9 cm-1. So, Soxhlet extraction has been done for 24 hours to isolate it from this sample as shown in Figure 2. In synthesizing PS-DVB-6, dispersion medium with a ratio 50 : 50 of IsoPrOH : DI was used. IR spectrum of this sample is shown in Figure 4 and confirmed that the sample is pure PS-DVB.

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PS-DVB-5 was synthesized using less polar reaction medium where the dispersion medium with the ratio of IsoPrOH : DI; 80 : 20 was used. The IR spectrum of this sample is shown in Figure 3. IR spectrum of PS-DVB-8 is shown in Figure 5 and it gives the true peaks of PS-DVB.

100.0 PS DVB-1 % T 75.0 2530.4 50.0 1941.2 1803.3 1869.9 1582.5 1267.1 1155.3 1716.51600.8 1492.8 697.2 758.0 1000.0 500.0 1/cm 1114.8 964.3 1068.5 1028.0 406.0

3082.0 3024.2 3001.0 2848.7 3058.9

796.5 838.0 905.5 539.1

25.0

2922.0 0.0 3500.0 2000.0

1452.3

1500.0

Figure 1. IR spectrum of PSDVB-2 beads before Soxhlet extraction
100.0 psdvb-3 % T 75.0 3003.0 50.0 2850.6 3058.9 3026.1 2922.0 25.0 1450.4 1492.8 1583.4 1600.8 758.0 1326.9 837.0 1068.5 906.5 1155.3 1028.0

542.0

698.2 0.0 3500.0 2000.0 1500.0 1000.0 500.0 1/cm

Figure 2. IR spectrum of PSDVB-2 beads after Soxhlet extraction
100.0 PS-DVB-5 % T 75.0 1944.1 1326.9 2850.6 3058.9 3026.1 2922.0 25.0 1600.8 758.0 837.0 1068.5 906.5 1155.3 1028.0 540.0 50.0

1450.4 1492.8

698.2 0.0 3500.0 2000.0 1500.0 1000.0 500.0 1/cm

Figure 3. IR spectrum of PS-DVB-5
100.0 ps-dvb-6 % T 75.0 2850.6 1600.8 50.0 3026.1 2922.0 1450.4 1492.8 758.0 1028.0 540.0

25.0

698.2

0.0 2000.0 1500.0

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Figure 4. IR spectrum of PSDVB-6

100.0 % T 75.0 3744.5 3676.1 3821.7 3628.8 3853.5 3650.0 ps-dvb-8 1701.1 1583.4 1648.1 1653.8 1508.2 1684.7 1560.3 1601.8 403.1 1154.3 1029.0 905.5 540.0

3082.0 3001.0 2849.6 3058.9 3025.1 2922.9

50.0 758.0 1452.3 1492.8

25.0 698.2 0.0 3500.0 2000.0 1500.0 1000.0 500.0 1/cm

Figure 5. IR spectrum of PSDVB-8 SEM result of synthesized PS-DVB A general view of the particles scattered on the surface of the tape was small resembling of the spherical beads as shown in Figure 6. In the micrograph magnification 10 000×, PS-DVB-2 with the average bead size of 0.81 µm (810 nm) is shown in 6A. PS-DVB-6 (Figure 6C) gives the average particle sizes of 0.67 µm (670 nm) with the magnification of 10 000×. The average bead size of 1.23 µm (Figure 6B) was given by PS-DVB-5 with the magnification of 5000× and PS-DVB-8 with the same magnification and less polar dispersion medium tend to give larger spherical bead with the average size of 1.44 µm as in Figure 6D. This phenomenon was probably due to the polarity of the reaction medium. Increasing the polarity of the dispersion medium resulted in the formation of smaller particles, so it could be consumed that this would affect on high the total surface area of the particle. As describe by Lok and Ober,[8], ethanol functioned as a steric stabilizer which prevented flocculation and aggregation of the particles being formed. The monomer and initiator diffusion rate would be higher in the presence of smaller particles due to their higher total surface area [9].

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A

B

C

D

Figure 6: SEM result of synthesized PS-DVB; (A) PS-DVB-2 with magnification 10000×; (B) PSDVB-5 with magnification 5000×; (C) PS-DVB-6 with magnification 10000×; (D) PS-DVB-8 with magnification 5000×

Nitrogen Adsorption Analysis Figure 7a shows nitrogen adsorption isotherm obtained from PS-DVB-2 which related to the combination of isotherm type II and IV. Therefore, the type of pores in the PS-DVB-2 is a mixture of mesopore and macropore. It was found that the BET surface area for this resin 3.0529 m2/g and average pore volume of 0.00458 as shown in figure 7b and in Table 2. The adsorption/desorption isotherm of PS-DVB-5 (Figure 7c) was a type II and IV with a hysteresis loop indicating the presence of slit-shaped pore. The presence of the loop in the high relative pressure region shows that the material is essentially mesoporous, with a minimal macropore contribution, and steepness of the desorption branch shows a high degree of uniformity. The BET surface area and average pore volume is given in Table 2 as 2.6082 m2/g and 0.003852 cm3/g. The PS-DVB-6 has a combination of type II and IV isotherm. The isotherm for this resin is shown in Figure 7e and with BET plot in Figure 7f. The isotherm also illustrated that PS-DVB-6 have a mixture of mesopore and macropore and a slit-shaped pore. The BET surface area for this resin is greater (4.2067 m2/g) than PS-DVB-2 and 5 due to the increasing the polarity of dispersion medium. The result in Table 2 shows that decreasing the polarity of the dispersion medium will decrease the BET surface area of the resin. It prove when the PS-DVB-8 with the most less polar combination of dispersion medium give the lowest BET surface area for 1.1649 m2/g and average pore volume for 0.001751 cm3/g. The isotherm plot for this resin shows the same pattern as others with the combination of type II and IV.

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(a)
4 3.5 3 Volum e 2.5 2 1.5 1 0.5 0 0 0.2

Graf Isoterm

(b)
1/ [VA(Po/P-1)] 0.5 0.4 0.3 0.2 0.1 0 0

Surface Graph

0.1

0.2 P/Po

0.3

0.4

0.4 P/Po

0.6

0.8

1

(c)
3.5 3 2.5 Volum e 2 1.5 1 0.5 0 0

Graf Isoterm

(d)
1/[VA(Po/P-1)] 0.6 0.4 0.2 0 0

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0.1

0.2 P/Po

0.3

0.4

0.2

0.4 P/Po

0.6

0.8

1

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5 4.5 4 3.5 Volum e 3 2.5 2 1.5 1 0.5 0 0 0.2

Graf Isoterm

(f)
1/[VA(Po/P-1)] 0.4 0.3 0.2 0.1 0 0

Surface Graph

0.1

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0.3

0.4

0.4 P/Po

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1.8 1.6 1.4 1.2 Volum e 1 0.8 0.6 0.4 0.2 0 0 0.2

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(h)
1/ [VA(Po/P-1)] 1.5 1 0.5 0 0

Surface Graph

0.1

0.2 P/Po

0.3

0.4

0.4 P/Po

0.6

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1

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Figure 7: (a) Nitrogen adsorption isotherm and (b) pore size distribution of PS-DVB-2; (c) Nitrogen adsorption isotherm and (d) pore size distribution of PS-DVB-5; (e) Nitrogen adsorption isotherm and (f) pore size distribution of PS-DVB-6; (g) Nitrogen adsorption isotherm and (h) pore size distribution of PS-DVB-8. Table 2: Pore and surface characteristic of synthesized PS-DVB BET surface area (m2/g) 4.2067 3.0529 2.6082 1.1649 Average pore volume (cm3/g) 0.005037 0.00458 0.003852 0.001751 Average pore width (Å) 477.8901 60.0087 59.071 60.1278 Type of isotherm II & IV II & IV II & IV II & IV

Sample PS-DVB-6 PS-DVB-2 PS-DVB-5 PS-DVB-8

Type of pore Meso & Macro Meso & Macro Meso & Macro Meso & Macro

CONCLUSIONS PS-DVB with the nano-sized particle can be synthesized using dispersion polymerization technique. Increasing the polarity of the dispersion medium resulted in the formation of smaller particles with higher surface area. This was evident from the SEM and nitrogen adsorption analysis.

ACKNOWLEDGEMENT We thank University Technology Malaysia and The Ministry of Science, Technology and Innovation Malaysia (MOSTI) for financial support through IRPA Project 09-02-06-0074 (Vote No. 74091). References 1. K. Hosoya and J. Frechet, “Influence of the Seed Polymer on the Chromatographic Properties and Size on Monodisperse Polymeric Separation Media Prepared by a Multi-Step Swelling And Polymerization Method”, J. Polym. Sci., 31, 1993, 2129-2141. 2. Masque, N., Galia, M. Marce, R. M. and Borull, F., J. Chromatogr. A. 803, 1998, 147-155. 3. Mohd Marsin Sanagi, “Pembangunan Dan Penggunaan Bahan Penjerap dan Fasa Pegun Dalam Analisis Kimia”, Laporan Akhir Penyelidikan IRPA Vot No.72298, 2002, 3-6. 4. K. E. J. Barret, “Dispersion Polymerization in Organic Media”, John Wiley & Sons, 1975. 5. Suboh Salawati, Mohd Marsin Sanagi. Ahmedy Abu Naim, Asiah Hussain and Hamizah Mohd Zaki, “Synthesis of Poly(Styrene-Divinylbenzene) Phases For High Performance Liquid Chromatography: Stirrer And Solvent Effect.”, Thesis, 2002, 1-9.

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6. W. Bremser and B. Reather, “A Method for Controlled Radical Polimerization and for the Synthesis of Solvent Free Dispersions”, Progress In Organic Coatings, 45, 2002, 95-99. 7. L. C. D. Santa Maria, C. A. M. L. Marcia, S. S. C. Marcos, M. S. Ribeiro Jose, F. S. Lilian and R. S. Manoel, “Characterization of Magnetic Migrospheres Based On Network Styrene and Divinylbenzene Copolymers”, 58, 2004, 3001-3006. 8. K. P. Lok and K. C. Ober, “Particle Size Control in Dispersion Polymerization of Polystyrene”, Can. J. Chem., 63, 1985, 209-216. 9. J. Liu, L. M. Gan, C. H. Chew, C. H. Quek, H. Gong, and L. H. Gan, “The Particle Size of Latexes From Dispersion Polymerization of Styrene Using Poly(ethylene oxide) Macromonomer as a Polymerizable Stabilizer” J. Polym. Sci., 35, 1997, 3575-3583.

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PRELIMINARY INVESTIGATION ON THE SEPARATION OF 2,3,7,8-TETRACHLORODIBENZODIOXIN AND 2,3,7,8-TETRACHLORODIBENZOFURAN USING MICELLAR ELECTROKINETIC CHROMATOGRAPHY Wan Aini Wan Ibrahim*, Mohd. Marsin Sanagi and Sharain Liew Yen Ling Separation Science Research Group (SSRG) Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia 81310 UTM Skudai, Johor, Malaysia. e-mail: wanaini@kimia.fs.utm.my Abstract.: Micellar electrokinetic chromatography (MEKC) was used in the preliminary investigation on the separation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) and 2,3,7,8tetrachlorodibenzo-p-furan (2,3,7,8-TCDF) by varying the percentage of acetonitrile (MeCN) organic modifier in the background solution (BGS) to aid separation. The migration time, peak area and peak height reproducibility was compared at various percentage of CAN. Excellent separations were achieved between both analytes with the selected separation conditions. We were also able to separate a third peak which appeared before the 2,3,7,8-TCDF peak that was detected at above 10% MeCN. We suspect this peak to be other tetraCDF isomers present. BGS were 20 mM sodium cholate and 20 mM di-sodium tetraborate decahydrate with separation voltage at 25 kV and temperature at 25ºC with final BGS pH range from 9.16-9.22. UV detection wavelength was at 225 nm. Abstrak. Kromatografi eletrokinetik misel (MEKC) telah digunakan dalam kajian awal pemisahan 2,3,7,8-tetraklorodibenzo-p-dioksin (2,3,7,8-TCDD) dan 2,3,7,8-tetraklorodibenzo-p-furan (2,3,7,8TCDF) dengan mempelbagaikan peratus pelarut organik asetonitiril (MeCN) dalam larutan latar belakang (BGS) untuk membantu pemisahan analit. Kebolehulangan pada pelbagai peratus MeCN dikaji dari segi masa penahanan, luas puncak dan ketinggian puncak. Pemisahan yang memuaskan tercapai untuk kedua-dua analit. Kami juga dapat memisahkan puncak ketiga yang keluar sebelum 2,3,7,8-TCDF pada peratus MeCN 10% dan ke atas. Kami mengesyaki puncak ini merupakan salah satu isomer tetraklorodibenzofuran. Larutan latarbelakang ialah 20 mM natrium kolat dan 20 mM dinatrium tetraborat dekahidrat dengan voltan pemisahan 25 kV dan suhu 25ºC. Panjan gelombang UV yang digunakan ialah 225 nm. Keywords: MEKC, polychlorinated dibenzo-p-dioxins and polychlorinated dibenzo-p-furans, organic modifier. Introduction Polychlorinated dibenzodioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) are highly toxic trace contaminants in several industrial organic chemicals (e.g. chlorinated phenols, phenoxy herbicides, PCBs) and have been identified as environmental pollutants. PCDDs and PCDFs have never been produced intentionally for industrial use but occur as unwanted trace contaminants in many industrial and thermal processes [1] or as an agrochemical. They have been identified in fly ash, flue gas condensates and other products from municipal incinerators and accidental fires. Different PCDD and PCDF isomers portray different biological side effects. PCDDs and PCDFs are two series of almost planar tricyclic aromatic compounds with very similar chemical properties. The number of chlorine atoms varies between 1 to 8 atoms. There are 75 PCDDs and 135 PCDFs congeners. Among these PCDDs, the most toxic and most extensively studied among the chlorinated dioxins is 2,3,7,8-tetrachlorodibenzo-para-dioxin (2,3,7,8-TCDD) followed by 2,3,7,8-tetrachlorodibenzo-para-furan (2,3,7,8-TCDF). The ability of PCDDs and PCDFs to partition between sediment or biota and water [2] and also due to the very low solubility in water, these compounds have the potential to accumulate in food chains as they are resistant towards metabolism or chemical degradation. The heat stability of these compounds (700°C or higher) is the

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reason these compounds survive high temperatures in incinerators and in some of accidents involving electrical systems [3]. Therefore they possess a threat to human well-being and the environment. Gas chromatographic methods have been well established and popularly used in the analysis of these chlorinated compounds. They are able to not only to quantitatively analyze these compounds but also to separate and identify substituted congeners using high-resolution gas chromatography (HRGC) [4, 5]. In the identification of the specific congeners, high-resolution gas chromatography (HRGC) coupled to mass spectrometry (MS) is mainly used. In addition to chromatographic methods, bioanalytical methods were also used in the detection of these toxic compounds [6]. Bioassay was used in the determination of TEQs based on the assumption that dioxin-related compounds all act through the Ah receptor and immunoassays (IAs) which are based on the ability of antibodies, to selectively and reversibly bind organic molecules. Micellar electrokinetic chromatography (MEKC) another branch of capillary electrophoresis (CE) is heavily used in the separation of both ionic and neutral analytes especially in environmental pollutants such as PCDDs and PCDFs. Capillary gas chromatography (cGC) is highly attractive due to its high sensitivity and high speed analysis, but due to PCDDs and PCDFs have very high boiling temperatures of 700ºC, therefore, these method cannot be applied. Due to this problem, MEKC is useful for the analysis of these samples through its high efficiency and sensitivity. Furthermore, MEKC requires much less sample amount at about a few nanoliter for each injection and less energy and solvent. In other words it is more environmentally friendly as compared to cGC. As most detection in MEKC utilizes UV detection, there is a problem with the low detection sensitivity and is still not satisfactory as compared to cGC. This is due to the short path-length through on-column detection. Due to this, various methods have been used in improving the limit of detection (LOD) such as on-line and off-line preconcentration techniques. The solubility of PCDDs and PCDFs in aqueous phase is very low due to its high hydrophobicity with solubility in water at 2x10-7 gL-1. Therefore there is an inability of the PCDD and PCDF to partition between the micelle and aqueous phase. Instead it would have a very high affinity towards the micelles giving a very poor resolution as all of them would migrate continuously in the micellar phase and therefore at the same migration time as the electroosmotic flow (EOF) [7]. Benito et al. [8] has been able to separate fourteen polychlorinated biphenyl congeners (PCBs) which are similar to PCDD and PCDF by cyclodextrin modified MEKC (CD-MEKC) due to its high hydrophobicities. Good resolution was achieved using a mixture of β-CD (beta cyclodextrin) and γCD (gamma cyclodextrin) giving a more cost-effective analysis, as γ-CD is more costly. Due to their hydrophobic internal cavity, CDs are able to form inclusion complexes with organic compounds such as PCBs [8] and PCDD/F [9] in which their stability is largely affected by the hydrophobic character, size and shape of the analyte. CD-MEKC has also been applied in the isomer identification of chlorinated dibenzo-p-dioxins in which the separation buffer consisted of borate, SDS, urea and γ-CD [10]. In this study we varied the percentages of the organic modifier acetonitrile (MeCN) in the electrophoretic buffer, which can influence the separation efficiency. The reproducibility of the data is also determined by the concentration and type of organic modifier used [11]. To the best of our knowledge this is the first MEKC study conducted using 2,3,7,8-TCDD and 2,3,7,8-TCDF. Most studies regarding this analytes were conducted using gas chromatography [2-6]. In this preliminary investigation we aim to achieve baseline separation with good resolution between the analytes of interest within the fastest time possible by varying the MeCN percentage in the BGS.

EXPERIMENTAL Reagents Sodium cholate AR Grade (Wako, Japan), di-sodium tetraborate decahydrate AR Grade (Fisher Chemicals, UK), sodium dodecyl sulphate AR Grade (Fisher Chemicals, UK) acetonitrile HPLC Grade (J.T. Baker, California, USA), 1,4-dioxan HPLC Grade (BDH Laboratory Reagents, UK). 2,3,7,8-TCDF and 2,3,7,8-TCDD as test analytes at 1 mg (AccuStandard, New Haven, Connecticut,

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USA) and their chemical structures are shown in Table 1. Double distilled deionised water (DDDW) was obtained from an UltraPure Water System purified up to 18 MΩ.

Apparatus All experiments were conducted on a capillary electrophoresis system (220V Agilent Capillary Electrophoresis System, Hanover, Germany) equipped with a diode array detector with optimum wavelength set at 225 nm. Data acquisition and system control was carried out by the 3D-CE ChemStation Software by Agilent Technologies (1990-2003). Standard bare fused silica capillaries (Agilent Technologies, Hanover, Germany) with 48.5 cm total length, 40 cm effective length and 50 µm i.d. were utilized to perform the separation. Injection offset was set at 4 mm. Polypropylene vials (Agilent Technologies, Hanover, Germany) of 1 mL were used to place samples, buffers and other solutions in the electrophoretic systems. All samples and buffer solutions were filtered through a 0.45 µm Nylon filter disc (Whatman).

Table 1: Properties of 2,3,7,8-TCDD and 2,3,7,8-TCDF. Compound 2,3,7,8-tetrachlorodibenzop-dioxin (2,3,7,8-TCDD) Chemical Structure
Cl O Cl

Molecular Weight 322 g/mol

Cl

O

Cl

2,3,7,8-tetrachlorodibenzop-furan (2,3,7,8-TCDF)

o Cl Cl

304 g/mol

Cl

Cl

Conditioning of the capillary and MEKC separation conditions

MEKC separations of the two analytes were performed with a 20 mM di-sodium tetraborate decahydrate buffer containing 20 mM of sodium cholate as the chiral surfactant. The effect of various percentages of MeCN) on the migration time and separation efficiency was investigated using 5-20% MeCN. The final buffer pH range after combining all components was from pH 9.16-9.22. At the beginning of each day, the capillary was rinsed with methanol for 5 minutes followed by 10 minutes with 1M NaOH to activate the silanol groups of the capillary. Before each sample injection, the capillary was rinsed for 5 minutes with 0.1M of NaOH, followed by 5 minutes of the running buffer. After each injection, high-pressure water was passed through the capillary for 10 minutes to flush out any impurities and to ensure a longer capillary life followed by 1 minute of the running buffer to ensure reproducibility. At the end of the day, the capillary was flushed with 10 minutes of water followed by 10 minutes of air. Both ends of the capillary were dipped in double distilled deionized water (DDDW). Standard samples of 2,3,7,8-TCDD (40 ppm) and 2,3,7,8-TCDF (40 ppm) were injected individually to obtain their individual migration time. This was then followed by a mixture of the two

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analytes (100 ppm each) for the optimization step. Both analytes were dissolved in 1,4-dioxan via sonication for 20 minutes. Hydrodynamic injections were made by applying pressure at 50 mbar to the sample vial for 1s. Separation runs were carried out at positive polarity (anodic injection) at 25 kV with a constant temperature of 25°C.

Results and Discussion Confirmation of individual peaks Prior to optimization of analyte separation, individual solutions of 2,3,7,8-TCDD and 2,3,7,8-TCDF were injected individually at 40 ppm each to determine their respective migration times before injecting them in the mixture form. For both analyses, both analytes had a slower migration time compared to the solvent peak (1,4-dioxan), which appeared before 2,3,7,8-TCDF and 2,3,7,8-TCDD (Figure 1). This was due to 1,4-dioxan having a smaller molecular weight compared to 2,3,7,8-TCDF and 2,3,7,8-TCDD and a higher polarity and solubility in water. Therefore, it will partition more in the aqueous phase than in the micelle giving a higher mobility compared to 2,3,7,8-TCDD and 2,3,7,8TCDF. 2,3,7,8-TCDF has a smaller molecular weight compared to 2,3,7,8- TCDD and the polarity is higher which explains its higher electrophoretic mobility compared to 2,3,7,8-TCDD. 1,4-dioxan appeared as very sharp and intense peak. Both compounds were separated within 8 minutes.

1,4-dioxan

TCDD

TCDF

Figure 1: Electropherogram of 2,3,7,8-TCDD and 2,3,7,8-TCDF at 40 ppm each. Run buffer: 20 mM di-sodium tetraborate decahydrate, 20 mM sodium cholate and 5% (v/v) MeCN. Hydrodynamic injection for 1 s at 50mbar. Detection wavelength: 225 nm.

Optimization of Analyte Separation

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The criteria used for the optimization of the separation of the two compounds of interest was the baseline resolution of each individual analyte together with no peak splitting in the shortest possible time. The concentration of acetonitrile as the organic modifier was varied from 0% to 20% with an increment of 5%. Each analysis was carried out in triplicates. Overall, the migration order remain unchanged throughout the whole experiment as the organic solvent content of the buffer was increased from 5-20%.

Migration Window (min) 8 7 6 5 4.642 5.015 4.482 4 3.691 3 2 1 0 10 15 0 5 % v/v acetonitrile

6.871

20

25

Figure 2: Graph illustrating the effect of percentage of acetonitrile against the migration window for the MEKC separation of 2,3,7,8-TCDF and 2,3,7,8-TCDD.(n=3).

2,3,7,8-TCDF

X 2,3,7,8-TCDF

A

D

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B

X 2,3,7,8-TCDF

E

XE 2,3,7,8-TCDF

C

X 2,3,7,8TCDF

Time/
Figure 3: Electropherogram of 2,3,7,8-TCDF with different percentages of MeCN in BGS at (A:0%), (B:5%), (C:10%), (D:15%) and (E:20%). At 20%, the TCDF peak appeared before peak X. Separation conditions are the same as in Figure 1.

The migration window increased greatly from 5% to 20% MeCN (Figure 2). The migration window gives a measure of the total time taken to separate the analyte mixture in the electropherogram [11]. This implies that in order to achieve baseline separation between the solvent peak (1,4-dioxan) and 2,3,7,8-TCDF, a percentage of acetonitrile above 10% is recommended as in figure 2. It is also useful if determination of additional analytes is required besides 2,3,7,8-TCDF and 2,3,7,8-TCDD. Baseline separation was achieved for the two analytes, 2,3,7,8-TCDF and 2,3,7,8TCDD respectively without and with acetonitrile (5-20%) in the running buffer. For 2,3,7,8-TCDF a third peak was detected before the TCDF peak at percentages of 5% MeCN onwards. We suspect that the extra peak could be due to other tetrachlorodibenzofuran isomers. Evidence of the unknown peak as peak X is illustrated in Figure 3. At 20% MeCN, there was a change in selectivity in which the 2,3,7,8-TCDF peak appeared before peak X.

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12 10 9.95 6.15 7.32 7.18 7.82

A

M igration, min

8 6 4 2 0 0

2.72

3.00

3.02

3.12

3.40

5

10

15

20

25

%v/v acetonitrile
2,3,7,8-TCDF
2,3,7,8-TCDD

B
N(efficiency in plate numbers)
Thousands 90 80 83.65133 70 60 50 40 30 20 10 0 0 13.324 5 84.64367 67.00433

85.59733

33.015 20.20233 16.01733 15.03067 10 15 20 25 29.479

%v /v tonitrile 2,3,7,8-TCDF ace2,3,7,8-TCDD

Figure 4: effect of different percentages of acetonitrile in running buffer on (a) migration time and (b) efficiency of the separation of 2,3,7,8-tcdf and 2,3,7,8-tcdd.

In general, the migration time of both analytes increased with increasing fraction of the acetonitrile in the running buffer as illustrated in Figure 4. Generally, the effect of increasing the fraction of acetonitrile was more significant for 2,3,7,8-TCDD compared to 2,3,7,8-TCDF. Acetonitrile has greater effect on the solubility of 2,3,7,8-TCDD, which is more hydrophobic, compared to 2,3,7,8-TCDF in the aqueous buffer. The organic modifier greatly lengthened the migration time of both analytes from 10% MeCN onwards. While for 2,3,7,8-TCDF, the change in migration time was less significant. Increasing the percentage of acetonitrile improved the separation efficiency of 2,3,7,8-TCDF from 10% acetonitrile onwards. While for 2,3,7,8-TCDD, the separation efficiency decreased from 10% acetonitrile onwards. At 20% MeCN, the symmetry of the 2,3,7,8TCDD peak worsened and peak broadening was obvious. If one were to comment on the best fraction of acetonitrile suitable for a mixture of these two analytes, then 5% acetonitrile would be best as it was the maximum for both analytes. Use of organic modifiers is often avoided due to reproducibility problems as evaporation of the organic solvent from the electrolyte [11] buffer can occur. Adsorption onto the capillary during in

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between runs can also cause reproducibility problems, which can be reduced by flushing the capillary with running buffer for a longer time. This experiment was repeated for the second time with longer flushing time of the capillary with the run buffer compared to the previous experiment and the reproducibility for migration time and peak area and height improved. The reproducibility for both migration times, peak area and peak height is shown in table 2 for both analytes. 20% acetonitrile gave the highest rsd for both peak area and peak height. Therefore for quantification purposes, it is not recommended to use concentration of acetonitrile above 20%. At higher concentrations of acetonitrile, destabilization of the micelle may occur as its critical micelle concentration is altered. In general, 10% acetonitrile gave the best reproducibility in terms of peak area and peak height while for the migration time, 15% acetonitrile gave the best reproducibility. Yet at concentrations below 20% the analysis time was reasonable at less than 8 minutes. While for 20%, the analysis time was 10 minutes. Therefore we would settle for 15% mecn for further optimization.

Table 2: Relative standard deviations, RSDs (n=3) for 2,3,7,8-TCDF and 2,3,7,8-TCDD under different fractions of acetonitrile in running buffer. Separation conditions remain the same as before. % acetonitrile 0 5 10 15 20 RSD, % Migration time Peak area Peak height Migration time Peak area Peak height Migration time Peak area Peak height Migration time Peak area Peak height Migration time Peak area Peak height 2,3,7,8-TCDF 0.149 19.914 4.807 0.727 10.243 1.828 0.249 37.419 8.992 0.551 12.968 11.007 0.331 41.503 36.541 2,3,7,8-TCDD 1.039 21.178 23.559 1.712 3.129 6.277 0.802 19.255 22.147 0.064 13.916 13.607 1.569 36.126 32.433

CONCLUSIONS Baseline separation was achieved in the separation of 2,3,7,8-TCDF and 2,3,7,8-TCDD with the running buffer consisting of 20 mM sodium cholate, 20 mM di-sodium tetraborate decahydrate and acetonitrile as the organic modifier with percentages of 5-20%. Even without the organic modifier, both 2,3,7,8-TCDF and 2,3,7,8-TCDD were baseline separated but the addition of acetonitrile managed to separate an unknown peak, which appeared next to the 2,3,7,8-TCDF peak, which we suspect to be another tetrachlorodibenzofuran isomer. The addition of acetonitrile lengthens the migration time generally and widened the migration window from 10% MeCN onwards. In conclusion, 15% MeCN was considered for further optimization as it gave a reasonable reproducibility for both peak areas, peak height and migration time for both analytes. The usage of acetonitrile is therefore recommended where it has not been possible to achieve an analytical separation using unmodified MEKC buffers.

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ACKNOWLEDGEMENT We thank Universiti Teknologi Malaysia and the Ministry of Science and Technology and Innovation, Malaysia (MOSTI) for financial support through the Fundamental Grant Vote No. 75149. References 1. 2. Gough, M. (1986) “Dioxin, Agent Orange: The Fact” , Plenum Press, 28-29. Kimbrough R. D. and Jensen, A. A. (1989) “Halogenated Biphenyls, Terphenyls, Naphthalenes, Dibenzodioxins and Related Producs” Topics in Environmental Health. 4. 71102. 3. Choudhary, G. Keith, L. H and Rappe, C. (1983) “Chlorinated Dioxins and Dibenzofurans in the Total Environment”, Butterworth Publishers, 355-363. 4. Gardinali, P. R., Wade, T. L., Chambers, L. and Brooke, J.M. (1996). “A Complete Method For The Quantitative Analysis of Planar, Mono, and Diortho PCB’s, Polychlorinated Dibenzop-dioxins and Furans in Environmental Samples”. Chemosphere. 32. 1-11. Tiernan, T. O., Taylor, M. L., Garrett, J. H., VanNess, G. F., Solch, J. G., Deis, D. A. and Wagel, D. (1983).“Chlorodibenzodioxins, chlorodibenzofurans and Related Compounds in the Effluents from Combustion Processes”. Chemosphere. 12. 595-606. Singh, S. A. and Kulshrestha, G. (1997). “Gas Chromatographic Analysis of Polychlorinated Dibenzo-p-dioxins and Dibenzofurans”. J. Chromatogr. A. 774. 97-109. Benito, I., Saz, J. M., Marina, M. L., Jiménez-Barbero, J., González, M. J., and Diez-Masa, J. C. (1997). “Micellar Electrokinetic Capillary Electrochromatographic Separation of Polychlorinated Biphenyl Congeners”. J. Chromatogr. A. 778. 77-85. Benito, I., Saz, J. M., Marina, M. L., Jiménez-Barbero, J., González, M. J., and Diez-Masa, J. C. (1996). “Separation of Chiral Polychlorinated Biphenyls by Micellar Electrokinetic Chromatography Using β- and γ- Cyclodextrin Mixtures In The Separation Buffer”. J. Chromatogr. A. 752. 265-270. Otsuka, K., Hayashibara, H., Yamauchi, S., Quirino, J. P., Terabe, S. (1999). “Highly-sensitive Micellar Electrokinetic Chromatographic Analysis of Dioxin-related Compounds Using On-line Concentration”. J. Chromatogr. A. 853. 413-420. Grainger, J., McClure, P. C., Liu, Z., Botero, B., Sirimane, S., Patterson, D. G., Sewer, M., Gillyard, C., Kimata, K., Hosoya, K., Araki, T., Tanaka, N. and Terabe, S. (1996). “Isomer Identification of Chlorinated Dibenzo-p-dioxin by Orthagonal Spectroscopic and Chromatographic Techniques” Chemosphere. 32. 13-23. Bretnall, A. E. and Clarke, G. S. (1995). “Investigation and Optimization of the Use of Organic Modifiers in Micellar Electrokinetic Chromatography” J. Chromatogr. A. 716. 49-55.

5.

6. 7.

8.

9.

10.

11.

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SEPARATION OF PROPICONAZOLE ENANTIOMERS BY MICELLAR ELECTROKINETIC CHROMATOGRAPHY WITH DIFFERENT BUFFER pHs Wan Aini Wan Ibrahim*, Dadan Hermawan, Mohd Marsin Sanagi Separation Science Research Group (SSRG) Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia 81310 UTM Skudai, Johor, Malaysia e-mail: wanaini@kimia.fs.utm.my Abstract. Micellar electrokinetic chromatographic separation of four propiconazole enantiomers using hydroxypropyl-γ-cyclodextrin (HP-γ-CD) as chiral selector were investigated in the phosphate buffer pH ranges of 2.0-4.0 (acidic pH) and 7.0-9.40 (neutral and basic pH). Complete separations of the four enantiomeric peaks of propiconazole were achieved using 30 mM HP-γ-CD, 50 mM sodium dodecyl sulphate (SDS), 15% methanol, 25 mM phosphate buffer at all pH range, except at pH 2.0. The reversal of the migration order of propiconazole enantiomers occurs with acidic buffer pH. The effect of buffer pH on the resolution, efficiency and selectivity is evaluated in this study. Abstrak. Pemisahan empat enantiomer propikonazol menggunakan kromatografi elektrokinetik misel dengan hidroksipropil-γ-siklodekstrin (HP-γ-CD) sebagai pemilih kiral telah dikaji dalam penimbal fosfat dalam julat pH 2.0-4.0 (pH asid) dan 7.0-9.4 (pH neutral dan bes). Pemisahan lengkap empat puncak enantiomer propikonazol dapat diperoleh menggunakan 30 mM HP-γ-CD, 50 mM natrium dodesil sulfat (SDS), 15% methanol, 25 mM penimbal fosfat pada semua pH, kecuali pada pH 2.0. Pembalikan urutan migrasi enantiomer propikonazol terjadi dengan pH penimbal berasid. Pengaruh pH penimbal terhadap resolusi, kecekapan, dan kepilihan ditentukan dalam kajian ini. Keywords: Propiconazole, micellar electrokinetic chromatography, hydroxypropyl-γ-cyclodextrin, buffer pH

Introduction The separation of enantiomers has become one of the most important fields of analytical chemistry especially for pharmaceutical or agrochemical products since the stereochemistry has a significant influence on the biological activity. Currently, enantiomer separations are primarily perfomed by gas chromatography (GC) and high-performance liquid chromatography (HPLC), where chiral stationary phases or chiral columns are often used to achieve optical recognitions. These analyses can be complicated and difficult to optimize. In addition, chiral stationary phase are usually expensive [1-2]. Micellar electrokinetic chromatography (MEKC) is a hybrid of electrophoresis and chromatography. Introduced in 1984 by Terabe et al. [3], MEKC has shown to be a powerful separation technique for separation of enantiomers [4-10]. The inherent ability of MEKC to provide high separation efficiencies combined with rapid method development and minimal use of expensive chiral reagents makes it an ideal technique for enantiomer separations. In MEKC, two modes of enantiomer separations are mainly used: (1) MEKC using chiral surfactants, and (2) MEKC using cyclodextrin (CD) as chiral selector (CD-MEKC). In CD-MEKC separation mode, the migration behaviors of individual enantiomers are determined by their competitive distributions into the three “phases” (water, CD and micelles). The addition of CD to the buffer displace the distribution of the

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analytes from the micellar to the water phase as a function of the possible interaction between the water soluble CD and the analytes. Inclusion complexes can be formed if the cavity of the CD is large enough leading in the best cases to chiral recognition of the analytes and the separation of their enantiomers [4-10]. Propiconazole is one of the chiral triazole fungicides and it contains 2 stereogenic centers (Figure 1). Thus, there are four enantiomers of propiconazole. Cyclodextrin-modified MEKC (CDMEKC) was applied to the enantiomeric and isomeric separation of seven commonly used pesticides. However, only two isomers of propiconazole (a chiral triazole fungicide) peaks were observed [11]. In this paper, we report the utilization of MEKC using hydroxypropyl-γ-cyclodextrin (HP-γ-CD), as chiral selector with different phosphate buffer pH (acidic and neutral/basic pH), for separation of four propiconazole enantiomers. The effect of buffer pH on the resolution, peak efficiency and selectivity is evaluated.
Cl Cl N N

CH2 O O

N

CH2CH2CH3

Figure 1. Structure of propiconazole

Experimental Apparatus The experiments were perfomed with an Agilent Capillary Electrophoresis System (Agilent Technologies, Germany), equipped with temperature control and diode array UV detection. Data were collected and processed on computer using Agilent ChemStation software supplied with the instrument. The separation capillary used was an uncoated fused-silica capillary of 50 µm i.d. × 64.5 cm (with an effective length of 56 cm to the detector window).

Reagents Propiconazole was obtained from Dr. Ehrenstorfer GmbH (Augsburg, Germany), 2Hydroxypropyl-γ-cyclodextrin from Sigma (St. Louis, MO, USA), sodium dodecyl sulfate (SDS) from Fisher Scientific (Loughborough, UK), sodium hydroxide pellets and di-sodium hydrogen phosphate 12-hydrate from Riedel-de Haen (Seelze, Germany). Stock propiconazole solution was prepared by diluting propiconazole with HPLC grade methanol from J.T. Baker (Phillipsburg, NJ, USA). All the stock buffers and surfactants were prepared in distilled deionized (DD, 18 MΩ) water. All buffers were filtered through 0.45 µm Whatman nylon filters.

Procedure

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The capillary was conditioned by passing 1 M NaOH solution for 10 min and then washing with deionized water for 10 min and finally equilibrating with an appropriate running buffer for 10 min. Between runs the capillary was washed with 0.1 M NaOH and water for 2 min each, and with run buffer for 5 min. The applied separation voltage was -30 kV (for acidic pH) and +30 kV (for neutral and basic pH) with the detection wavelength set at 200 nm and the capillary temperature was maintained at 20 ºC. Sample introduction was performed hydrodynamically at 50 mbar for 1 s. The EOF was determined using methanol as the neutral marker. Enantioseparations were evaluated with resolution (Rs), peak efficiency (N), and separation factor or selectivity (α) [12]. The Rs, N, and α value were calculated using the equation via ChemStation software as follows. Rs = (2.35/2) (tR(b)-tR(a))/(w50(b)+w50(a)) N = 5.54 (tR /w50)2 α = tR(b) / tR(a) where tR is the retention time of the analyte/enantiomer, w50 is the peak width at half-height (min). The repeatability (R.S.D.) of the resolution, peak efficiency, and selectivity was determined over three replicate injections.

Results And Discussion In this study, effect of buffer pH on the chiral separation of propiconazole was investigated at different pH, ranging from 9.4 to 7.0 (basic and neutral pH) and from 4.0 to 2.0 (acidic pH) with MEKC separation buffer containing 30 mM HP-γ-CD, 50 mM SDS, 15% methanol in 25 mM phosphate buffer. These conditions were identified from our previous study [13]. pH 9.4 was selected as maximum basic pH in order to increase the life-time of the capillary since higher pH degraded the silica inner wall of the capillary rapidly. The resulting electropherograms of propiconazole enantiomers at basic/neutral and acidic buffer pH are shown in Figure 2 and Figure 3, respectively.

pH 9.4

pH 8.0

pH 7.0

Time/min

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Figure 2. Electropherogram of propiconazole enantiomers in basic and neutral buffer pHs. Sample:200 ppm propiconazole (in methanol), hydrodynamic injection (HDI) for 1 s at 50 mbar; Separation solution: 30 mM HP-γ-CD, 50 mM SDS, and 15 % methanol in 25 mM phosphate buffer; capillary, 50 µm i.d. × 64.5 cm (effective length, 56 cm); applied voltage, +30 kV; detection wavelength, 200 nm. At basic/neutral pH, complete resolution of the four propiconazole enantiomers was observed where detection was carried out at the cathodic end of the capillary and all enantiomers migrated towards the negative electrode due to the strong electroosmotic flow (EOF). The migration times of propiconazole enantiomers decreased with a decrease in the buffer pH from 9.4 to 7.0 (Figure 2). In strongly acidic conditions, where detection point was placed at the anodic end, four propiconazole enantiomers were observed at pH 4.0 and 3.0, while at pH 2.0, incomplete separation of the four propiconazole enantiomers was observed. At pH 5, broad peaks were observed (data not shown) and separation at pH 6 was not carried out, as it would take a long time for the peaks to appear (more than an hour). A decrease in pH from 4.0 to 2.0 resulted in a decrease of the migration times of all the propiconazole enantiomers (Figure 3). A reason behind this observation is that lower pH tended to suppress the EOF inside the separation capillary. In addition, the reversal of the enantiomer migration order of propiconazole occurs with acidic buffer pH, because of the reversal of the migration direction (enantiomers migrated toward the positive electrode due to the suppressed EOF).

pH 2.0

pH 3.0

pH 4.0

Time/min

Figure 3. Electropherogram of propiconazole enantiomers at acidic buffer pH. Conditions as in Figure 2, except applied voltage, -30 kV. Table 1. Electrophoretic data of propiconazole enantiomers at different buffer pH

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Rs Basic/neutral P1 P2 P3 P4 P1-P2 P2-P3 P3-P4 22.34 22.8 23.47 23.78 2.03 2.79 1.21 9.4 (1.65) (1.66) (1.62) (1.63) (2.89) (4.90) (4.97) 20.11 20.5 21.03 21.29 1.88 2.54 1.22 8.0 (2.87) (2.92) (2.98) (3.02) (3.72) (2.76) (2.50) 19.77 20.13 20.65 20.89 2.59 3.59 1.58 7.0 (0.35) (0.39) (0.39) (0.40) (3.65) (2.74) (1.67) Acidic P4 P3 P2 P1 P4-P3 P3-P2 P2-P1 4.0 32.67 33.19 34.44 35.40 1.13 2.61 2.02 (5.62) (5.76) (5.76) (5.90) (34.29) (23.47) (23.73) 26.69 27.03 28.22 28.87 1.30 4.34 2.33 3.0 (4.36) (4.36) (4.61) (4.73) (10.85) (7.42) (3.01) 2.0 ns ns ns ns ns ns ns • At basic/neutral pH; P1, P2, P3, and P4 are first, second, third, and fourth-migrating enantiomers of propiconazole, respectively. • At acidic pH; P4, P3, P2, and P1 are first, second, third, and fourth-migrating enantiomers of propiconazole, respectively. (reversal of the enantiomer migration order). • P1-P2, P2-P3, and P3-P4 are resolution between first and second, second and third, and third and fourth-migrating enantiomers, respectively. • ns: no separation • values in brackets ( ) are migration time RSD of triplicate runs • Analysis condition as in Figure 2 (basic/neutral pH) and Figure 3 (acidic pH) Electrophoretic data of propiconazole enantiomers is presented in Table 1. In basic/neutral pH, resolution value (Rs) of all propiconazole enantiomers were found to be greater than 1.20. The best resolution was found to be in pH 7.0 (minimum Rs between enantiomers = 1.50). While in acidic pH, the best resolutions for all enantiomers were obtained at pH 3.0 (minimum Rs = 1.30). Resolution was lower in pH 4.0 because of increased migration times of enantiomers. The effect of buffer pH on the peak efficiency (N) of propiconazole enantiomers is illustrated in Figure 4. Peak efficiency was found greater than 100,000 at pH 3.0 and pH 7.0-9.4, and 80,000 at pH 4.0. These data show that MEKC method provide high separation efficiencies for the separation of the propiconazole enantiomers. A graph of propiconazole enantiomers selectivity as a function of buffer pH is shown in Figure 4. Selectivity of propiconazole enantiomers at pH range 7.0-9.4 was constant (α P1-P2 = 1.02; α P2-P3 = 1.03; and α P3-P4 = 1.01). These data show that buffer pH does not significantly affect the selectivity of all propiconazole enantiomers. At acidic pH, the highest selectivity is between first and second migrating enantiomers (α P1-P2 = 1.02), between third and fourth-migrating enantiomers (α P3-P4 = 1.03) were found to be at pH 4.0. While between the second and third-migrating enantiomers, selectivity was constant at pH range 3.0-4.0 (α P2-P3 = 1.04).

Buffer pH

tm (min)

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Efficiency (N x 10,000)

35 30 25 20 15 10 5 0 0 2 P1 4 Buffer pH P2 P3 P4 6 8 10

Figure 4. Effect of buffer pH on the peak efficiency (N)

1.1 Selectivity

1

0.9 2 3 P1-P2 4 Buffer pH P2-P3 P3-P4 7 8 9.4

Figure 5. Graph of selectivity as a function of phosphate buffer pH. One general problem in CE is relatively poor detectability in terms of concentration sensitivity. To apply on-line sample preconcentration technique, acidic conditions are generally more preferable than neutral ones in terms of the achievable concentration efficiency when sweeping or stacking methods are employed in MEKC. Further enantioseparation of propiconazole study is undertaken in our laboratory where acidic conditions are used to enhance the detection sensitivity using on-line sample preconcentration technique [14].

CONCLUSIONS Enantiomeric separations of propiconazole were successfully achieved by MEKC using 30 mM HP-γCD, 50 mM SDS, and 15 % methanol in 25 mM phosphate buffer at pH range 7.0-9.0 (neutral and basic condition) and pH 3.0-4.0 (acidic condition). Peak efficiency was highest at pH 7 for all the enantiomers and lowest at pH 4 for all the enantiomers. Selectivity was not significantly affected by buffer pH for the range of pH studied for propiconazole enantiomers. Resolution of P1-P2 and P3-P4

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were best at pH 7 (Rs=2.59 and 1.58, respectively). Resolution of P2-P3 was best at pH 3 (Rs= 4.34). Overall, the best resolution, selectivity and peak efficiency was obtained at pH 7. To the best of our knowledge, no literature has ever reported the complete separations of the four enantiomeric peaks of propiconazole using CD-MEKC at basic/neutral and acidic pH.

ACKNOWLEDGEMENT The authors would like to thank the Ministry of Science, Technology and Innovation (MOSTI), Malaysia, for financial support through the IRPA grant project number 09-02-06-0035 EA158.

References 1. Maier, N. M., Franco, P., Lindner, W. (2001) “Separation of enantiomers: needs, challenges, perspectives”, J. Chromatogr. A. 906. 3-33. 2. Heiger, D. (2000) “An introduction High performance capillary electrophoresis”, Germany: Agilent Technologies. 3. Terabe, S., Otsuka, K., Ichikawa, K., Tsuchiya, A., Ando, T., (1984) "Electrokinetic separations with micellar solutions and open-tubular capillaries", Anal. Chem. 56. 111-113. 4. Otsuka, K., Terabe, S., (2000) "Enantiomer separation of drugs by micellar electrokinetic chromatography using chiral surfactants", J. Chromatogr. A. 875. 163-178. 5. Schmitt, Ph, Garrisonb, A.W., Freitaga, D., Kettrupa, A. (1997) “Application of cyclodextrinmodified micellar electrokinetic chromatography to the separations of selected neutral pesticides and their enantiomers”, J. Chromatogr. A. 792. 163-178. 6. Edwards, S.H., Shamsi, S.A., (2002) “Chiral separation of polychlorinated biphenyls using a combination of hydroxypropyl-γ-cyclodextrin and a polymeric chiral surfactant”, Electrophoresis. 23. 1320-1327. 7. Wu, Y.S., Lee, H.K., Li, S.F.Y. (2000) “Simultaneous chiral separation of triadimefon and triadimenol by sulfated β-cyclodextrin-mediated capillary electrophoresis”, Electrophoresis. 21. 1611-1619. 8. Ingelse, B.A. (1997) “Chiral separations using capillary electrophoresis” PhD Thesis, Eindhoven University of Technology, Eindhoven. 9. Chankvetadze, B., (1997) “Separation selectivity in chiral capillary electrophoresis with charged selectors”, J. Chromatogr. A. 792. 269-295. 10. Wu, Y.S., Lee, H.K., Li, S.F.Y. (2001) "High-performance chiral separation of fourteen triazole fungicides by sulfated β-cyclodextrin-mediated capillary electrophoresis". J. Chromatogr. A. 912. 171–179 11. Penmetsa, K.V., (1997) "Applications of capillary electrophoresis in pesticide analysis and toxicology studies” PhD Dissertation, Department of Toxicology, North Carolina State University, USA. UMI Number: 9804241. 12. Chankvetadze, B., Saito, M., Yashima, E., Okamoto, Y. (1997) “ Enantioseparation using selected polysaccharides as chiral buffer additives in capillary electrophoresis”, J. Chromatogr. A. 773. 331-338. 13. Wan Aini Wan Ibrahim, Hermawan, D., Sanagi, M.M., (2005) "Evaluation of parameters affecting separation of propiconazole enantiomers by micellar electrokinetic chromatography", Paper presented at the Annual Fundamental Science Seminar (AFSS) 2005, Institut Ibnu Sina, UTM, Johor Bahru, Malaysia, 4-5 July 2005. 14. Otsuka, K., Matsumura, M., Kim, J.B., Terabe, S., (2003) “On-line preconcentration and enantioselective separation of triadimenol by electrokinetic chromatography using cyclodextrins as chiral selector” J. Pharm. and Biomed. Anal. 30. 1861-1867.

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EFFICIENT DECHLORINATION OF CHLOROAROMATIC COMPOUNDS BY USING ELECTROCHEMICAL METHOD
1 1

Murni Sundang and 1Aishah Abdul Jalil

Faculty of Chemical and Natural Resources Engineering, Universiti Teknologi Malaysia, 81310 Skudai Johor. Email: annznik03@yahoo.com

Abstract Dechlorination of chloroaromatic compounds has been performed by using electrochemical method in a one-compartment cell fitted with a platinum cathode and a zinc anode. The electrolysis of chlorobenzene, 1,3-dichlorobenze and 1,2,4-trichlorobenzene were carried out in an acetonitrille solution containing tetraethylammonium perchlorate as a supporting electrolyte at 0oC and a constant current of 60mA/cm2 under an air atmosphere, respectively. Complete dechlorination of chlorobenzene was achieved at 5 F/mol of current passed. The highest total conversion of dechlorination is 93 % for 1,3-dichlorobenzene while 88 % for 1,2,4-trichlorobenzene. It was obtained at 20 F/mol and 26 F/mol respectively. This method could be applied to dechlorinate chloroaromatic compounds in wastewater in order to solve environmental problem.

Abstrak Penyahklorinan sebatian kloroaromatic telah dilakukan dengan menggunakan kaedah elektrokimia di dalam satu cell-compartment yang dilengkapi dengan katod platinum dan anod zink. Elektrolisis terhadap klorobenzena, 1,3-diklorobenzene dan 1,2,4-triklorobenzene telah dijalankan di dalam larutan asetonitril yang mengandungi tetraetilammonium perklorat sabagai elektrolit penyokong pada 0oC dan arus malar iaitu 60mA/cm2 di bawah atmosfera. Penyahklorinan yang lengkap untuk klorobenzena dicapai pada 5 F/mol arus dilalukan. Prtukaran keseluruhan tertinggi untuk penyahklorinan ialah 93% untuk 1,3-diklorobenzena manakala 88% untuk 1,2,4triklorobenzena. Pertukaran tersubut dicapai masing-masing pada 20 F/mol dan 26 F/mol. Kaedah ini boleh dilakukan untuk penyahklorinan sebatian kloroaromatik dalam rawatan air sisa untuk menyelesaikan masalah alam sekitar. Keywords: Dechlorination, electrochemical, chloroaromatics 1.0 Introduction Chloroaromatics have a variety of natural and synthetic sources. They are naturally produced by bacteria, insects, plants, animals, and volcanic eruptions. They are also synthetically manufactured for use as chlorinated solvents, herbicides, insecticides, preservatives, heat exchanger and insulating fluids. For example, 1,4-dichlorobenzene is an air freshener, pentachlorophenol is a wood preservative and herbicide, 2,4,5-trichlorophenoxyacetic acid is a herbicide in agent orange, and PCBs are insulators in transformer oils. Chloroaromatics are also unwanted waste by-products and contaminants in the manufacture of other chemicals. After chloroaromatics production in the 1930s, some chloroaromatics were classified as environmental and biological pollutants because they were toxic, bioaccumulated, and resisted degradation. The widespread application of these chloaromatics in industry and agriculture has led to their contamination in the atmosphere, land, ocean, and biological life. Chloroaromatics are insoluble in water and accumulate in fats. Consequently, chloroaromatics have concentrated in animal fatty

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tissues and at the higher end of the food chain. Chlorinated dioxins and furans not only alter endocrine, reproductive, and immune system functions, and effect the developing embryo, fetus, and infant, but may also cause cancer (Sidhu, 2000). Significant environmental challenges accompany the safe disposal of chlorinated aromatic compounds. The toxicity of these aromatic compounds is connected with the chlorine content and their biodegradability is strongly affected by chlorine substitution in the molecule. Nowadays, there are four type of dechlorination methods that has been studied and some of it has been commercialized. Incineration for example is claimed to convert chloroaromatics into carbon dioxide, water, hydrochloric acid, and salt under controlled conditions. However, incineration below the extreme conditions may produce toxic by-product such as chlorinated dioxins, furans and carbon monoxide. Other methods are chemical reduction and chemical oxidation. Chemical reduction requires high reductant to organochlorine ratio, high temperatures and high pressures. Meanwhile, chemical oxidation also requires high oxidant-to-organochlorine ratio. Thus, both of these methods are not favourable. Chlorinated aromatic compounds also can be biodegraded but since the ring cleavage may occur before dechlorination, it can form more complex intermediates of varying toxicity. By using a simple electrochemical method, chlorine molecules are removed from these aromatic compounds in order to reduce their toxicity. This is an innovative approach to making electrochemical treatment of organochlorine waste a practical possibility. Since 1970s, the electrochemical reductive approach has been suggested as a promising method for detoxification of chlorine-containing chloroaromatic hydrocarbons. This method ensures the selective removal of chlorine atoms from various chloroaromatic under mild experimental conditions without using a highly reactive reducing agent (Tsyganok, 1999). The electrochemical reaction is a unit process occurring on the working electrode where either oxidation or reduction takes place. The electrolytic process requires that an electrolyte, an ionized solution or molten metallic salt complete an electric circuit between two electrodes. This reaction is a heterogeneous chemical process involving the transfer of charge from an electrode. Thus, electrochemical reduction is considered in this study to remove the chlorinated compound. Objective of this study is to determine the efficiency of dechlorination process from electrolysis of various chloroaromatic compounds. Comparative studies of electrolysis to remove chlorinated aromatic compound has been performed with Chlorobenzene, 1,3-Dichlorobenzene, 1,2,4Trichlorobenzene. A number of techniques have been explored for the elimination of chlorinated aromatic compound by electrolysis. Schmal et al. (1991) have examined the electrochemical dechlorination using a MPCell (Electro Cell System AB) and cathodes of bundles of carbon fibres and carbon felt. Experiments showed that after 20 minutes of electrolysis at a current of 10 A, the initial concentration of PCP (50 ppm) fell to about 10 ppm. Zimmer et al. (1994) dehalogenated chloroform, 5-chlorosalicyclic acid, 2methoxy-5-chlorobenzoic acid and 4-chloroaniline (each in 1 g/L concentreation) in aqueous sulfuric acid solution, using Cu, Pb and Ni cathodes in a Micro-Flow Cell (Electro Cell AB) and Eniko, et al. (1999) reported dechlorination of chlorinated hydrocarbons in a monopolar packed bed electrochemical reaction. Tsyaganok et al. (1999) dechlorinated various type of chloroaromatics in water-MeOH medium containing triflouroacetic acid and tetraalkylammonium salt by electrocatalytic reduction over palldium-loaded carbon felt at room temperature.

2.0 Materials and Methods Reagents that were used are chlorobenzene, 1,3-dichlorobenzene and 1,2,4-trichlorobenzene. A platinum plate (2×2 cm2) was used as cathode and a zinc plate (2×2 cm2) as anode. N, N-

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dimethylformamide (DMF) and acetonitrille ware used as solvent and tetraethylammonium perchlorate (Et4NClO4) as a supporting electrolyte in order to complete the dechlorination process of chloroaromatic compound. The electrolysis was carried out at 0oC at a constant current 60 mA/cm2 under air atmosphere in one-compartment cell.

3.0 Result and Discussions 3.1 Dechlorination of Chlorobenzene The result of the electrolysis is shown in Figure 1. From the figure, conversion of benzene increases by the increasing of chlorobenzene and increasing of the current passed. The complete conversion to benzene is achieved after 5 F/mol current passes through the electrolyte.

3.2 Dechlorination of 1,3-dichlorobenzene Figure 2 shows the conversion and recovery of 1,3-dichlorobenzene under the optimum condition that has been performed on chlorobenzene. Based on the figure, recovery of DCB was decreased by increasing the current passing through the electrolyte. This is because of the usage of DCB in the formation of CB and benzene. Besides, the conversion to benzene is increase by increasing the current passes until it reach the end point where the current cannot get through the electrolyte. Complete conversion of DCB to CB is achieved after 16 F/mol current passes through the electrolyte. Recovery of CB was increased by increasing the current passed until the current reaches 5 F/mol and afterward it starts to decrease until it reaches approximately zero. The increased of recovery of CB is because of the active dechclorination process of DCB which convert it to CB before it is followed by the second stage dechlorination process which converts CB to benzene. The conversion to benzene causes the decreasing amount of CB because of the third stage dechlorination process which converts CB to benzene.

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Figure1: Conversion and recovery of of chlorobenzene

Figure 2: Conversion and recovery for electrolysis 1,3-dichlorobenzene

3.3 Dechlorination of 1,2,4-trichlorobenzene Figure 3 shows the recovery and conversion of 1,2,4-trichlorobenzene for each sample that was taken after several current passed to the electrolyte. From the graph, the recovery of TCB decreases exponentially as increasing of current passing to the electrolyte until it totally convert to other compound. The graph shows that TCB as a reagent has been undergone electroreduction process that converts the TCB to another compound which is DCB, CB, or benzene. Complete conversion from TCB to DCB is achieved after 12 F/mol current passed through the electrolyte. Furthermore, the recovery of DCB increases until 3 F/mol of current passed and after that it starts to decreases as the current passing to the electrolyte is increased. This trend shows the formation of CB or benzene from DCB is more rapid compared to the formation of DCB from TCB. The reaction rate of conversion from TCB to DCB is lower than the reaction rate conversion from DCB to CB or benzene. This is the main reason for the decreasing of recovery of DCB exponentially. The complete conversion from DCB to CB was achieved after 26 F/mol of current was passed. The graph for CB does not show any trend. From that graph, recovery of CB was decreased as the current passing to the electrolyte was increased until the current passed is 10 F/mol, the graph starts to go up and the recovery of CB increases slowly. The decreasing of CB recoveries is because after 10 F/mol current has passed, more TCB has been converted to DCB so that the formation of CB is much easier after the complete conversion of TCB. After 15 F/mol, the recovery of CB starts to decrease because of conversion from CB to form benzene. The graph for benzene shows that it increases

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throughout the experiment. This result shows that this experiment achieved the target to convert the chloroaromatic aromatic compound to benzene which does not contain any chlorine atom. The final conversion of CB to Benzene is 88.1% which is after 26 F/mol current passes, and only CB and benzene remain in the sample. The highest total conversion of TCB to benzene is 88.1 %. These explanations justify the dechlorination of chloroaromatic compounds by using electrochemical methods has been successfully achieve due to the high conversion of TCB.

Figure 3: Conversion and recovery for electrolysis of 1,2,4-trichlorobenzene The supposed mechanism of electroreduction of chloroaromatics for this study is shown in figure 4. The electrolysis of chloroaromatics in an acetonitrille solution with a platinum cathode and a zinc anode results in anodic dissolution of zinc metal to give zinc ions. (Anode) Zn → Zn2++ 2e-

The two electrons released from this oxidation are transferred to the organic substrate for each halogen atom that will be expelled as halide ion. One-electron reduction of TCB occurs to give radical anion of TCB. Another one-electron reduction anion of TCB will remove chlorine atom from TCB. DCB formed by protonation of DCB anion by means of solvent. By continuing the current passed, more electrons will be produced to encourage further dechlorination of DCB to form CB. This process continuous until complete dechlorination was achieved.

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Cl
Cl

Cl Cl Cl

e-

e-Cl +H-

Cl

Cl

Cl

eCl Cl Cl

e-

e-Cl +HCl

e- +H-

-Cl

Figure 4: Mechanism of electroreduction of chloroaromatics

4.0 Conclusion Total conversion of dechlorination of 1,3-dichlorobenzene was 92.47% at 0 F/mol of current passes. While, electrolysis of 1,2,4-trichlorobenzene achieved 88.1% of total conversion of dechlorination at 26 F/mol of current passes. This high total conversion shows that this simple and cheap method will be applied to wastewater treatment. Moreover, this technique is environmentally friendly compared to other dechlorination methods since no harmful residue was produced at the end of the electrolysis.

References Eniko, S. G., Edit, G. P., Gabor, B., and Erno, P. (1999), “Dechlorination of Chlorinated Hydrocarbons in a Monopolar Packed Bed Electrochemical Reactor.” Peiodica Polytechnica. 6576 Sidhu, J. K., (2000) “Kinetics and Mechanisms of Methoxide Substitution and Electroreduction of Hexachlorobenzene” Macarthur: University of Western Sydney. Schmal, D., Duin V. P. J., Jong, D., (1991)“Dechema Monographien -English Version” Netherland: TNO Delft Tsyganok, I. A., Yamanaka, I., Otsuka, K. (1999), “Dechlorination of Chloroaromatic by Electrocatalytic Reduction over Palladium-Loaded carbon Felt at RoomTemperature.” Chemosphere. 39. 1819-1831.

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Zimmer, A., Franke, L., Mahlow, K. (1994) “Dechlorination of Chlorinated Organic Compound” Dresden: Technical University Dresden.

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PRELIMINARY WORK ON THE APPLICATION OF FTIR AND CHEMOMETRICS IN HONEY ANALYSIS

Mohamed Noor Hasan and Mohamed Nuruddin M. Nasir
Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia 81310 UTM Skudai, Johor, Malaysia. e-mail: mnoor@kimia.fs.utm.my Abstract. Honey is a popular food and sweetener and is widely consumed all over the world. Since honey is considered a high-value food product, its quality must be maintained even after processing, packaging, storage and transport. There are various analytical methods currently utilised to monitor the composition and quality of commercially-produced honey. However, most of these techniques are time-consuming and destructive to the honey sample being analysed. Fourier Transform Infrared Spectrometry (FTIR) is a common analytical technique that can be performed quickly, cheaply and without destruction of the sample. In this study, FTIR scanning method was combined with chemometrics analysis to determine the viability of FTIR as an analytical method for determining the quality of honey. A total of 20 honey samples from various sources were scanned to establish a data set for the chemometrics analysis. After converting the spectra to digital form, the 20 by 361 data matrix was analysed with Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) methods. Results from the PCA method indicated that the variation in the 20 samples can be adequately described by four principal components and most of the wavelengths that cause the most variation fall between 1200 cm-1 and 400 cm-1. The observed clustering of the samples can be attributed to the different countries of origin. There were also several samples not belonging to any group, which could be considered as outliers. Results of the clustering analysis appeared to reaffirm the PCA patterns by showing similar groupings.

Abstrak. Madu ialah sejenis makanan dan pemanis yang digunakan dengan meluas di seluruh dunia. Oleh kerana madu boleh dianggap produk bernilai tinggi, mutunya mesti dikekalkan walaupun selepas pemprosesan, pembungkusan, penyimpanan dan pengangkutan. Terdapat berbagai kaedah analisis yang kini digunakan untuk mengawal komposisidan mutu madu yang dihasilkan secara komersial. Bagaimanapun, kebanyakan teknik ini mengambil masa yang lama dan memusnahkan sampel yang dianalisis. Spektrometri Inframerah Jelmaan Fourier (FTIR) ialah kaedah analisis yang boleh dilakukan dengan cepat, dengan harga yang murah dan tidak memusnahkan sampel. Dalam kajian ini, ikaedah imbasan FTIR telah digabungkan dengan analisis kimometrik untuk menentukan kesesuaian FTIR sebagai kaedah analisis untuk menentukan mutu madu. Sejumlah 20 sampel madu daripada berbagai-bagai sumber telah diimbas untuk membentuk set data bagi analisis kimometrik. Selepas menukar spektrum kepada format digital, matik data 20 x 361 telah dianalisis menggunakan kaedah analisis komponen utama (PCA) dan Analisis Gugusan berhirarki (HCA). Hasil daripada kaedah PCA menunjukkan bahawa variasi dalam sampel boleh di terangkan oleh empat komponen utama dan kebanyakan panjang gelombang yang menyebabkan variasi berada antara 1200 cm-1 dan 400 cm-1 . Kumpulan yang dapat dilihat boleh dikaitkan dengan negara asal sampel itu diperolehi. Terdapat juga beberapa sampel yang tidak termasuk dalam mana-mana kumpulan yang boleh dikira sebagai outlier. Hasil analisis gugusan menyokong corak PCA dan menunjukkan pengumpulan yang sama. Keywords: Honey analysis, FTIR, principal component analysis, hierarchical cluster analysis

Introduction Honey is a supersaturated sugar solution manufactured by bees for use as food. It is a popular food and sweetener and is widely consumed all over the world. Since honey is considered a high-value food product, its quality must be maintained so that it remains high even after processing, packaging, storage and transport. Also, the high value of honey compared with other sweeteners makes it extremely susceptible to adulteration with other, cheaper sweeteners. There are various analytical methods currently utilized to monitor the composition and quality of commercially-produced honey. Among them are NMR spectroscopy, HPLC, GC, and carbon isotope ratio analysis [1]. These techniques, while reported to be successful, are costly and require considerable analytical skill. With the exception of NMR spectroscopy, these techniques are also time-consuming and will destroy samples under test. A need therefore exists for a rapid, nondestructive, and less expensive method suitable at least for screening honey samples for authenticity confirmation. Fourier-transform infrared (FTIR) spectroscopy is a scanning method that is rapidly gaining prominence as an alternative in food analysis. It is a nondestructive and fast scanning method with

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relatively easy sample preparation method. The mid IR region (4000 – 400 cm-1) is the most widely used range for organic components and functional groups identification. The main problem with FTIR is it is not a selective method. There may be pertinent information contained in the spectrum that is indecipherable to the human eye because of a complex spectrum. This is caused by a number of factors including overlapping peaks, interference bands due to water or carbon dioxide, and instrumental artifacts due to measurement conditions [2]. To extract useful information from the whole spectra, multivariate data analysis is needed. Different multivariate strategies may be used to accomplish classification tasks on the basis of infrared spectra, but, in general, given the highdimensionality and the highly correlated nature of the spectral variables, often coupled with a low number of samples, data reduction is almost a mandatory preliminary step [3]. In this study, two exploratory pattern recognition methods, namely Principal Component Analysis (PCA) and Hierarchical Clustering Analysis (HCA) were used to analyse FTIR spectra of honey samples taken from various sources. The whole spectral data were analysed without using any class information, e.g. absorption wavelength of certain functional groups; to see which variables (wavelengths) contribute the most to the variation.. This preliminary work foccused on the analysis of spectral data to see if there is natural groupings in the samples and to determine whether it will conform to any known class information. The main objective was to see whether FTIR is a viable method for determining the quality of honeys. Experimental Samples A total of twenty honey samples were collected from various sources in Johor. Fifteen of the samples were commercial honeys obtained from supermarkets while five samples (non-branded) were obtained directly from honeybee growers (Table 1). All samples were stored in air-tight jars at 4°C. Prior to spectroscopic analysis, honey samples were incubated in a water bath at about 50°C until all the sugar crystals were melted. The samples were then mixed at room temperature to ensure uniform distribution of suspended materials before subsamples were taken for analysis.

Table 1: Honey Samples and Countries of Origin
Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Sample Code TAWARI CED CHINNAT NB01 NB02 NB03 NB04 NB05 SONGSHAN WHITE WINGS MANUKA KAMAHI CAPILANO ALLOWRIE WESCOBEE EVA LEABROOK AL-MANSOOR POLLENEY COUNTRY Country of Origin New Zealand Malaysia China Malaysia (non-branded) Malaysia (non-branded) Malaysia (non-branded) Malaysia (non-branded) Malaysia (non-branded) China China New Zealand New Zealand Australia Australia Australia Malaysia Australia Malaysia Australia Australia

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Instrumentation FTIR scanning was done by Perkin-Elmer Model 1600 spectrometer (Perkin-Elmer). During scanning, the samples were held in an ATR cell containing a 45o ZnSe crystal with eleven internal reflections. The spectra were then truncated to a range of 4000 cm-1 to 400 cm-1 before being saved to a disk. Next, the spectra were digitised prior to data set development. Data Set Development Spectral files in JCAMP-DX format were pre-processed using Microsoft Excel. Absorbance values from 4000 cm-1 to 400 cm-1 at the interval of 10 cm-1 were obtained for every spectrum. These data were used to construct a 20 by 361 data matrix with rows representing samples (20 samples) and columns representing absorbance values at 361 wavelengths. The data matrix was then imported into MATLAB for use in chemometrics analysis. Chemometrics Analysis The software used to run the analysis on the spectral data matrix was MATLAB (version 6.1; Mathworks Inc.) with PLS_Toolbox (version 2.1; Eigenvector Inc.) installed. The data matrix was first loaded for Principal Component Analysis (PCA). Based on the eigenvalues generated, four PCs (principal components) were selected for further analysis. The loadings scores were examined to determine whether there are variables that contribute more variations to the PCs [2]. From the observations on the loadings plot, a new data matrix was constructed. The truncated data matrix represented absorbance values from 1200 cm-1 to 400 cm-1. The scores plot, which shows groupings of the samples, was also examined for this data matrix. The PCA tool was used once again to analyse the newly created data matrix. The loadings plot for each PC was examined to determine which variables contribute the most to the variations. The scores plot for this dataset was also examined to see whether the groupings remains the same or becomes different. Then, the groups were examined to see whether there are any characteristics shared by each member of the group. Samples not belonging to any groups were also examined to determine whether they are outliers of the existing groups or are instead the sole member of a new group. After the PCA, Hierarchical Cluster Analysis (HCA) was then performed on both data matrices to see whether the groupings obtained in the PCA were similar or different from the groupings obtained in HCA. Several dendrograms obtained using various similarity criteria (k-nearest neighbour vs k-means, Euclidean vs Mahalanobis distances) were constructed and compared. Results and Discussion An example of the IR spectrum of honey is shown in Figure 1. The IR spectrum of honey is dominated by sugar absorptions. The bands appearing between 1470 cm-1 and 1150 cm-1 are due to bending modes of C-C-H, C-O-H, and O-C-H groups. The more intense peaks in the region around 1000 - 1150 cm-1 arise mainly from C-O and C-C stretching modes, with a peak around 1020 - 1420 cm-1 due to O-H vibrations. At higher wavenumbers, bands due to C-H and O-H bending vibrations are also useful for discrimination and quantification purposes [4]. The spectra’s peaks and absorption intensities were found to be quite similar in regions from 4000 cm-1 to 1200 cm-1. The noticeable differences in the spectra was observed in regions known as the ‘fingerprint’ region, which are from 1200 cm-1 to 600 cm-1 [5].

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Figure 1: A typical spectrum of honey obtained from the IR scanning The Principal Component Analysis done on the data matrix revealed several interesting information. The loadings plots for all PCs (PC1 loadings plot is shown in Figure 2) were shown to have similar patterns in magnitude. The wave numbers that have the larger magnitude (either positive or negative) were those from 1200 cm-1 to 400 cm-1. This range of wave numbers includes the ‘fingerprint’ region which consists of complex absorption patterns and bands with interactional vibration modes. The ability of PCA to discern differences in this region demonstrates its usefulness in interpreting complex spectra. Since the wave numbers that contribute to the variation in the sample have been identified to a more narrow range, a new data matrix can be constructed. This will prevent data overloading that often caused pertinent information to be lost.

Figure 2: A loadings plot for the PC1 of the original spectral data matrix (4000 cm-1 to 400 cm-1)

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The second piece of information from the PCA is gained by looking at the scores plot of the PCA. The scores plot shows the relationship of the samples to each other with respect to the measurement variables. The two-dimensional scores plot with PC1 (45.65% variation explained) and PC2 (19.25% variation explained) as axes showed the major groupings as illustrated in Figure 3. Basically there were there main groups. When the samples in each group were examined, it was found that the groupings can be related with the samples’ countries of origin. As shown in Table 1, honey samples used in this study originated from four countries, i.e. Malaysia, China, Australia and New Zealand. The largest group with eight members is located in the bottom left part of the scores plot and is composed mainly of honeys originating from Australia. The second largest group is located on the right-hand side of the plot and its members are honeys from Malaysia and China. The third group with only three members is located near the top-left of the plot. The honeys in this group all came from New Zealand. Besides the samples in groups, there were also several samples that probably do not belong to any of the groups. The most notable example is sample 17 (LEABROOK), a honey sample originating from Australia. This sample is possibly a new group of honey altogether since it has about the same distance from the three major groups. Further class information about the group requires more advanced sample analysis and chemometrics techniques and is beyond the scope of this project. Another sample that apparently not included in any group was sample 7 (NB04). However, since it was quite close to group 2, it is safe to assume that it probably belongs to the same group.

Figure 3: Scores plot (PC2 vs PC1) of the original spectral data matrix The other unsupervised method, HCA yields largely the same results as the PCA method, with some variations. This is because HCA represents all the variations in the original data matrix. Thus, it is inevitable that some samples will be placed at different distances from each other because of the variations not considered in PCA (The PCA method with 4 PCs selected includes 80.04 percent

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of the variation). Nonetheless, the information and the groupings provided are similar to the PCA method and reaffirm the PCA’s validity as an analytical tool. Figure 4 shows a dendrogram plotted using the original set of wavelength (4000 cm-1 to 400 cm-1) with K-Nearest Neighbor (KNN) and Euclidean distance used as distance measurements between samples.

Figure 4: A dendrogram obtained by using KNN and Euclidean distance. Conclusions In this preliminary work, it has been shown that it is possible to extract useful information from the FTIR spectra of honeys by utilizing chemometrics methods. Two complementary pattern recognition techniques, PCA and HCA, were successfully used to classify the honey samples according to country of origin. PCA had shown the ability to discern pertinent information from the broad range of data contained in the spectrum and to use the information in grouping the samples. The HCA method was used as a verification tool for the PCA and provided an alternative view to the row space plot grouping method in PCA FTIR spectroscopy has the potential to be used as a rapid screening technique for the analysis of honey and, with more powerful analytical software and more sensitive chemometrics methods, is likely to be able to detect possible adulterants or quality deterioration in samples. References 1. Cordella C, Moussa I, Martel AC, Sbirrazzuoli N, Lizzani-Cuvelier L (2002). Recent Developments in Food Characterization and Adulteration Detection: Technique-Oriented Perspectives. J. Agric. Food Chem. 50, 1751-1764 2. Beebe KR, Pell RJ, Seasholtz MB (1998). Chemometrics: A Practical Guide; New York: John Wiley and Sons,. p 100-101, 186. 3. Cocchi M, Foca G, Lucisano M et al. (2004). Classification of Cereal Flours by Chemometric Analysis of MIR Spectra. J. Agric. Food Chem. 52, 1062-1067 4. Kelly JFD, Downey G, Fouratier V (2004). Initial Study of Honey Adulteration by Sugar Solutions Using Midinfrared (MIR) Spectroscopy and Chemometrics. J. Agric. Food Chem. 52, 33-39 5. Silverstein RM, Bassler GC, Morrill TC (1981). Spectrometric Identification of Organic Compounds, 4th Edition. New York: John Wiley and Sons,. p 95-105

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THE EFFECT OF MECHANOCHEMICAL TREATMENT ON THE STRUCTURE AND CATALYTIC PERFORMANCE OF VANADIUM PHOSPHATE CATALYSTS Yun Hin Taufiq-Yap1*, Chee Keong Goh1 and Graham Hutchings2
1

Department of Chemistry, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. 2 School of Chemistry, Cardiff University, Cardiff CF10 3AT, UK *Email: yap@fsas.upm.edu.my

Abstract. The precursor of VOHPO4·½H2O was synthesised by using reduction of VOPO4·2H2O with isobutanol under reflux for 16 h. The blue solids of VOHPO4·½H2O was subjected to mechanochemical treatment. Then, the unmilled and milled materials were calcined under the flow of n-butane/air at 673 K for 75 h. Only (VO)2P2O7 phase was observed after calcination. The surface area of catalysts was increasing with the milling duration. The mechanochemical treatment also increased the mobility of the lattice oxygen of the catalyst as evidenced by H2-TPR analysis and influences the catalytic performance. Abstrak. Prekursor VOHPO4·½H2O telah disintesiskan melalui penurunan VOPO4·2H2O oleh isobutanol di bawah refluks selama 16 j. Pepejal biruVOHPO4·½H2O telah dilakukan rawatan mekanokimia. Bahan yang tidak dan telah dikisar dikalsinkan di bawah aliran n-butana/udara pada 673 K selama 75 j. Fasa VOHPO4·½H2O masih dikekalkan selepas proses kisaran. Hanya fasa (VO)2P2O7 diperhatikan selepas kalsin. Luas permukaan mangkin telah meningkat dengan penambahan masa kisaran. Rawatan mekanokimia telah menambahkan mobiliti kekisi oksigen mangkin yang telah dibuktikan oleh analisis H2-TPR dan juga mempengaruhi prestasi mangkin. Keywords: vanadium phosphate, ball milling, selective oxidation, butane Introduction Maleic anhydride (MA) is very important for manufacture of unsaturated polyester resins, which is used in the production of fibreglass composites for boats, cars, construction, electrical industries and pipeline [1]. In 2001, total capacity of worldwide maleic anhydride was at ~1,329 thousand metric tons, which 35 % was in Asia, 36 % was in Western Europe, and 19 % was in North America. From 2001 to 2006, the maleic anhydride market is anticipated to grow at an average annual rate of about 2.8 % in the United States and 3.5 % in Western Europe. Total worldwide consumption is expected to grow at a rate of approximately 6–7 % annually during this time, reaching a level of 1.5 million metric tons in 2006 [2]. Maleic anhydride was originally produced almost exclusively by oxidation of benzene [3]. Later, the process for the synthesis of maleic anhydride from n-butane over vanadyl pyrophosphate, (VO)2P2O7 catalysts was developed around 1974 by Monsanto [4]. Nowadays, this process has been almost completely replaced the process based on benzene due to low price and environmental neutrality of alkane [5]. Nevertheless, the conversion of n-butane over (VO)2P2O7 catalysts is not satisfactory, about 75 % [6]. The mechanochemical method using ball mill can improve the catalytic performance of the (VO)2P2O7 catalysts [7]. Mechanochemical treatment of the VOHPO4·½H2O impairs a specific real structure that enhances the catalytic properties of the final catalysts, (VO)2P2O7 [8]. The enhanced catalytic performance are due to (i) an increase in surface area and the generation of fresh and reactive surfaces [9], (ii) an enhanced exposure of the basal planes [10], and (iii) a decreased particle size and an increased microstrain in the VOHPO4·½H2O [8]. The aim of this work is to study the effect of mechanochemical treatment on the structure and reactivity of the vanadium phosphate catalysts. Experimental The VOPO4·2H2O material was prepared by reacting V2O5 (20.0 g from Fluka) with H3PO4 (120 ml, 85 % from Merck) in water (480 ml) under reflux with continuous stirring for 24 h. Then the yellow solid was filtered and dried at 385 K for 16 h. This material obtained was refluxed with 160 ml of

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isobutanol (from BDH) for 21 h and the solid product was recovered by vacuum filtration and washed with acetone and water. The resulting solid was dried in air at 385 K overnight. The ball mill experiment was carried out by using planetary ball mill (model Pulverisette 4 from Fritsch) with an agate bowl having 250 ml volume together with fifty 10 mm diameter agate balls. About 18 g of the VOHPO4·½H2O and 50 ml of ethanol as solvent were put together inside the bowl. The bowl spins around its own axis and around a second axis outside its center at 1400 rpm for 30 and 60 min. All prepared precursors were then undergone calcination in a flow of n-butane/air mixture (0.75 % nbutane in air) for 75 h at 673 K to generate the active catalysts. The prepared catalysts were characterised by using a Shimadzu Diffractometer model XRD-6000 for X-ray diffraction (XRD) analyses, a ThermoFinnigan Sorptomatic 1990 for B.E.T. surface area measurements, and a ThermoFinnigan TPDRO 1100 for temperature-programmed reduction in hydrogen (H2-TPR). The oxidation of n-butane to maleic anhydride was carried out in a microreactor with a standard mass of catalyst (250 mg) with online gas chromatography. Results and Discussion Only VOHPO4·½H2O phase was observed for precursors (Figure 1), indicating that the milling process did not affect the primary phase of the precursors. The XRD patterns of all unmilled and milled catalysts exhibited only (VO)2P2O7 phase as shown in Figure 2. However, the ball milling process significantly decreased the intensity of the peaks. Furthermore, the catalyst’s crystallite size was reduced (calculated by using Debye-Scherrer equation) from 12.4 to 9.9 nm. It was found that the milling process has increased the surface area of the catalysts from 16.0 m2 g-1 (unmilled) to 18.6 and 24.6 m2 g-1 for milling time of 30 and 60 min, respectively (Figure 3). The H2-TPR experiments were used to investigate effect of mechanochemical treatment on the reducibility of the catalysts, as shown in Figure 4.
2000

1500

Milled = 60 min

Milled = 60 min

1500

1000
Milled = 30 min
Intensity / a.u.

Milled = 30 min

1000

Intensity / a.u.

Unmilled

500
Unmilled

500

0 10

20

30
2θ / degree

40

50

60

0 10

20

30
2θ / degree

40

50

60

Figure 1. Diffractograms of the unmilled and milled precursors and milled catalysts

Figure 2. Diffractograms of the unmilled

30

25

Surface Area, m 2 g -1

20

15

10

5

0 0 30 Ball Milling Time / min 60

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Figure 3. The surface area of the catalysts Figure 4. H2-TPR profiles of the catalysts as a function of the milling time The unmilled catalyst gave a characteristic of two reduction peaks at the region from 5001100 K. These peaks occurred at 779 and 990 K, where the first peak is associated to the reduction of V5+ phase whereas the latter peak is assigned to the removal of lattice oxygen from V4+ phase [11]. The amount of oxygen removed from both peaks is 3.85 x 1020 and 1.20 x 1021 atom g-1, respectively, which is shown in Table 1. The ratio of V5+/V4+ is about 0.32. Table 1. Characteristic of catalysts by H2-TPR Reduction activation energy, Er (kJ mol-1) 130 166 Total amount of oxygen removed / mol g-1 6.39 x 10-4 2.00 x 10-3 2.64 x 10-3 9.45 x 10-4 1.55 x 10-3 2.50 x 10-3 5.47 x 10-4 2.38 x 10-3 2.93 x 10-3 Total amount of oxygen removed / atom g-1 3.85 x 1020 1.20 x 1021 1.59 x 1021 5.69 x 1020 9.35 x 1020 1.50 x 1021 3.29 x 1020 1.43 x 1021 1.76 x 1021

Milling Time / min 0

Peak

Tmax / K 779 990

Coverage / atom cm-2 2.41 x 1015 7.52 x 1015 9.93 x 1015 3.06 x 1015 5.03 x 1015 8.09 x 1015 1.34 x 1015 5.82 x 1015 7.16 x 1015

1 2 Total 1 2 Total 1 2 Total

30

799 976

134 163

60

760 953

127 159

The 30 min milled catalysts shows the same reduction profile with the unmilled material with two reduction peaks appeared at 799 and 976 K. Nevertheless, further milling to 60 min shifted both peaks to a lower temperature, i.e. 760 and 953 K, as compared to unmilled material and milled for 30 min. The amount of oxygen related to V5+ increased to 5.69 x 1020 atom g-1 for 30 min milled catalyst, but further milling to 60 min reduced the amount to 3.29 x 1020 atom g-1. The amount for oxygen removed for the second reduction peak, which is attributed to the reduction of V4+ reduced to 9.35 x 1020 atom g-1 for 30 min of milling, whereas 60 min milled catalyst increased this amount to 1.43 x 1021 atom g-1, about 19.2% of increment. The V5+/V4+ ratio for milled catalysts is 0.61 and 0.23 for milling time of 30 and 60 min, respectively. A suitable V5+/V4+ ratio is around 0.25 which was suggested by [12] for the best catalyst performance and the 60 min milled catalyst exhibited the value near to this optimal ratio. Figure 5 shows the catalytic performance of the catalysts at 673 K and gas hourly space velocity (GHSV) of 2400 h-1. Catalyst milled for 30 min gave 63% of n-butane conversion (reduced of 20% as compared to unmilled catalyst). This may due to the increment of the first reduction peak temperature as observed in first peak of H2-TPR profiles which is associated to the increment of ~ 47.8% oxygen species from V5+ phase. Nevertheless, it increased the MA selectivity from 63 (unmilled) to 68%. On the other hand, the 60 min milled catalyst enhanced the conversion to 88% as compared to unmilled catalyst (83%), which is caused by an significant increase of surface area (about 53.8% of increment) as well as higher amount of removal of lattice oxygen from the active V4+ phase. Unfortunately, the MA selectivity decreased to 59%.

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Butane Conversion 90 80 70 60 Percentage / % 50 40 30 20 10 0 0 30 Milling Time / min

MA Selectivity

60

Figure 5. Catalytic performance of vanadium phosphate (VPO) for the oxidation of n-butane.

Conclusions The mechanochemical treatment has reduced the crystallite size and increased the surface area of the catalysts. Our finding also shows that mechanochemical treatment also plays an important role on the distribution of V4+ and V5+ phases. The 60 min milled catalyst gave a higher conversion (88%) compared to the unmilled material (83%). This may due to three salient observations, firstly, a surface area, secondly, all reduction peaks were shifted to lower temperature with higher total amount of oxygen removed, indicating a higher mobility and reactivity of the lattice oxygen and thirdly, it gave a slightly lower V5+/V4+ ratio (0.23) compared to the optimal value (0.25). Acknowledgement Financial assistance from Malaysian Ministry of Science, Technology and Innovation is gratefully acknowledged.

References 1. 2. 3. 4. 5. 6. Culbertson, B. M. (1987) "Maleic anhydride" Catal. Today 1. 609-629. Felthouse, T. P., Burnett, J. C., Mitchell, S. F. and Mummey, M. J. (1995) "Kirk-Othmer Encyclopaedia of Chemical Technology", New York: Wiley. Contractor, R. M. and Sleight, A. W. (1987) "Maleic anhydride from C-4 feedstocks using fluidized bed reactors" Catal. Today 1. 587-607. Emig, G. and Martin, F. (1987) "Economics of maleic anhydride production from C-4 feedstocks" Catal. Today 1. 477-498. Centi, G. (1993) "Vanadyl pyrophosphate- a critical overview" Catal. Today 16. 5-26. Hodnett, B. K. (1985) "Vanadium-phosphorus oxide catalysts for the selective oxidation of C4 hydrocarbons to maleic anhydride" Catal. Rev. -Sci. Eng. 27. 373-424.

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7. 8. 9. 10. 11. 12.

Hutchings, G. J. (2004) "Vanadium phosphate: a new look at the active components of catalysts for the oxidation of butane to maleic anhydride" J. Mater. Chem. 14. 3385-3395. Fait, M., Kubias, B., Eberle, H.-J., Estenfelder, M., Steinike, U. and Schneider, M. (2000) "Tribomechanical pretreatment of vanadium phosphates: structural and catalytic effects" Catal. Lett. 68. 13-18. Hutchings, G. J. and Higgins, R. (1997) "Selective oxidation of n-butane to maleic anhydride with vanadium phosphorus catalysts prepared by comminution in the presence of dispersants" Appl. Catal. A: General 154. 103-115. Zazhigalov, V. A., Haber, J., Stoch, J., Kharlamov, A., Bogutskaya, L. V., Bacherikova, I. V. and Kowal, A. (1997) "Influence of the mechanochemical treatment on the reactivity of Vcontaining oxide systems" Solid State Ionics 101-103. 1257-1262. Cornaglia, L. M., Carrara, C. R., Petunchi, J. O. and Lombardo, E. A. (1999) "The role of cobalt as promoter of equilibrated vanadium-phosphorus-oxygen" Appl. Catal. A: General 183. 177-187. Aït-Lachgar, K., Abon, M. and Volta, J. C. (1997) "Selective oxidation of n-butane to maleic anhydride on vanadyl pyrophosphate" J. Catal. 171. 383-390.

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CHARACTERIZATION OF CELLULOSE AND PULP OBTAINED FROM RICE STRAW PLANTS OF DIFFERENT PULPING PROCESS USING SCANNING ELECTRON MICROSCOPY (SEM). Suhardy Daud1*, Mustaffa Nawawi2, Wan Su Haron3, Farizul Hafiz Kasim1 and Saiful Azhar Saad1
1

School of Materials Engineering, Northern Malaysia University College of Engineering (KUKUM), Jejawi 02600 Arau, Perlis.

2

Chemistry Department, Faculty of Science, University Technology of Malaysia (UTM) 81310 Skudai, Johor Bahru, Johor.
3

Research Cluster Unit, Chancellery Department, Northern Malaysia University College of Engineering (KUKUM), Jejawi 02600 Arau, Perlis. *Tel : 04-9798379, Fax : 04-9798178, email : suhardy@kukum.edu.my

Abstract. Non-wood raw materials account for 5-7 % of the total pulp and paper production worldwide. Production of pulp from non-wood resources has many advantages such as easy pulping capability. In this study, the formic acid and NaOH pulping procedures for isolation of cellulose and pulp from rice straw were studied. The pulping processes are aim to remove as much lignin and pentosanes as possible and avoid the decomposition of cellulose during the process. The most common pulping in industries is NaOH pulping. Chemically, cellulose is a linear natural polymer anhydroglucose units and paper is composed of cellulose fibers. Lignin, which are the combined glues that hold plant cells together, are undesirable in a finished paper product. Results showed that formic acid pulping yielded 22.38 % w/w cellulose and 4.06 % w/w lignin. Besides that, with using NaOH pulping, the cellulose and lignin contents are 30.26 % w/w and 4.72 % w/w. The isolated cellulose and pulp samples were further characterized by scanning electron microscope (SEM) to investigate their fiber dimensions. For formic acid pulping, the fibrous structures of isolated pulp samples display stronger binding fibres compared to the NaOH pulping. Keywords: Rice straw, cellulose, scanning electron microscopy (SEM) and fiber dimension. Introduction Rapid increase in population and rapid economic expansion have led to an increasing demand for high quality value–added forest products, such as pulp and paper products. The amount of straw obtained from the waste of rice industry is abundant in Malaysia but the utilization of straw is still limited. Annual world rice (Oryzae sativa) production was about 577 million tons for 1997 – 98. More than 50 countries contributed to this sum with the production of at least 100, 000 tons of rice annually. In Malaysia, there is not only a shortage of wood fibre, but the abundant agricultural residues such as rice straw and sugarcane bagasse available for their use in papermaking. Among these large quantities of agricultural residues, only a minor portion of the residues is reserved as animal feed. However, a huge quantity of the remaining straw is not used as industrial raw materials and is burnt in the fields. The air pollution, therefore is a serious problem by burning these residues in this area. Therefore, the use of these straws in pulping or papermaking has many advantages including reducing the need for disposal and environmental deterioration through pollution and fires [1]. In view of the shortage of conventional raw materials for pulping and the increasing demand for paper products worldwide, non-wood plants and agricultural residues attracted renewed interest [2]. Various agricultural residues such as straw, stalk, bagasse and cereal have taken a significant place in supplementing the dwindling supply of conventional forest raw material for catering the everincreasing demand on the pulp and paper industry [3]. In some countries, their researchers found that the agricultural residues like sugarcane bagasse, leaves and rice straws are suitable raw material for the production of pulp [4].

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Non-wood raw materials account for 5-7 % of the total pulp and paper production worldwide. Production of pulp from non-wood resources has many advantages such as easy pulping capability [5]. The main reasons for the decreased use of rice straw in the paper industry is due to the presence of large amount of pentosanes in the pulp and black liquors, which also contain silica. The need to add alkali to reduce or prevent silicate deposites to remove silica from the black liquors currently limiting the use of rice straws in paper mills [6]. Cellulose, the major constituent of all plant materials, forms about half to one third of plant tissues and is constantly replenished by photosynthesis. In particular, cellulose is the main constituent of higher plants, including sugarcane bagasse and rice straw. Chemically, cellulose is a linear natural polymer anhydroglucose units. Paper is composed of cellulose fibers. The source of the cellulose fibers, and the degree to which that source is refined, determine the nature and quality of the paper produced. The two most important factors that affect the quality of paper are the presence of impurities and an acidic pH. Finished papers may contain natural impurities, such as lignins that have not been removed during processing, unnatural impurities, such as residual chemicals, like sulfites, not washed out during final processing, or such chemicals as alum that have been added during final processing. Lignins, which are the combined glues that hold plant cells together, are undesirable in a finished paper product. They age poorly, turn brown, become acidic over time, are waterproof, and resist the natural bonding of cellulose fibers to each other. If lignins are not removed and are left in contact with the surrounding cellulose fibers in paper, their acidity will break down the cellulose and the paper will become brittle [7]. In this study, the formic acid and NaOH pulping procedures for isolation of cellulose and pulp from rice straw were studied. The pulping processes are aim to remove as much lignin and pentosanes as possible and avoid the decomposition of cellulose during the process. There are various pulping processes that have been used in industries. The most common process is chemical pulping using sulfate which is known as kraft pulping. This process has many advantages compare to other processes but the waste is difficult to be treated. Many researchers have been conducted using various chemicals to replace sulfate pulping. The objective of this study is to investigate the cellulose and lignin content of the pulp from rice straw of different pulping processes. The isolated pulp samples are characterized by scanning electron microscope (SEM) to investigate their fiber dimensions. Experimental Rice straw were obtained from a local farmers in Perlis. The samples were first dried in sunlight and then cut into small pieces (1-3 cm). The cut straw were ground to pass a 1.0 mm size screen. For pulping process, the rice straw is soaked in the cooking liquor (95 % w/w) at 80 0C for 2 h. The powder then were extracted with toluene-ethanol (2:1, v/v) in a Soxhlet apparatus for 6 h and the dewaxed meal was allowed to dry in an oven at 60 0C for 16 h. All weights and calculations were made on an oven-dried (60 0C, 16 h) basis.

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Pulping process (NaOH or formic acid) Rice Straw (10 gram) Extraction with toluene-ethanol (2:1, v/v) for 6 h Dewaxed straw Treatment with NaCl for delignification Filtrate

Cellulose

Lignin

Figure 1. Scheme for isolation of cellulose from delignified rice straw. The resulting pulp was then washed with acidified NaCl (1.3 % w/w, pH 3.5 – 4.0, adjusted with acetic acid) for delignification. To reduce errors and confirm the results, each experiment was repeated in triplicate under the same conditions and the yield of cellulose was given as the average of these three replicates. The fiber dimensions analysis were performed using scanning electron microscope (SEM), JEOL (JSM/6460LA). Results and Discussion Paper is composed of cellulose fibers. Cellulose is a polymer of the sugar glucose and is used by plants to produce cell walls. Plant matter that has been processed to create a solution consisting of cellulose filaments suspended in water can be made into paper. The result of Soxhlet extraction of cellulose and lignin content in rice straw from the different pulping process can be seen in Figure 2. From the figure it can be seen that the percentage of cellulose in the rice straw are 22.38 % w/w (formic acid pulping) and 30.26 % w/w (NaOH pulping). The yield of cellulose for NaOH pulping is higher than formic acid pulping.

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35% 30.26% 30% Percentage (%) 25% 20% 15% 10% 5% 0% Formic Acid Pulping NaOH Pulping 4.06% 4.72% 22.38% Cellulose Lignin

Figure 2. The yield of cellulose and lignin were extracted from rice straw of different pulping process. Lignins, which are the combined glues that hold plant cells together, are undesirable in a finished paper product. If lignins are not removed and are left in contact with the surrounding cellulose fibers in paper, their acidity will break down the cellulose and the paper will become brittle. In the present study, delignification of the rice straw after the formic acid and NaOH pulping will yielded 4.06 % w/w and 4.73 % w/w lignin content. It showed that the formic acid pulping is more effective to remove the lignin content in the pulp compare to NaOH pulping. However, the result also shown that pulping with formic acid not only can remove the lignin content, but it also reducing the cellulose yield. To investigate the quality of the cellulose that were pulping by formic acid and NaOH, the isolated cellulose are characterized by scanning electron microscope (SEM). Temperature also has a stronger effect on cellulose yield, when cooking conditions are less efficient, particularly when pulping is performed at relatively low cooking liquor concentrations. Too low a temperature means increased contact time and too high a temperature means reducing contact time to avoid carbohydrate degradation. A 95 % w/w formic acid and NaOH concentration was shown to be the most efficient. Scanning electron microscope (SEM) micrographs of the surface of the cellulose that were pulped by both pulping processes are shown in Figure 3 (formic acid) and 4 (NaOH). For formic acid pulping, the cellulose structures are presented in drier form compare to NaOH pulping. It is because, NaOH pulping cannot remove the lignin content completely. Lignin is the “glue” that holds straw fibres together [1].

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Figure 3. SEM micrograph of cellulose were pulped by formic acid pulping.

Figure 4. SEM micrographs of cellulose were pulped by NaOH pulping.

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Figure 5. SEM micrographs of paper was produced by formic acid pulping.

Figure 6. SEM micrographs of paper was produced by NaOH pulping. Figure 5 and 6 show the scanning electron microscopy (SEM) micrographs of papers that were produced by formic acid and NaOH pulping. From the Figure 5 (formic acid pulping), it fibrous

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structures display stronger binding fibres compared to the paper that was produced by NaOH pulping (Figure 6). It showed an interlocking of the fibres and construction of fibrous bridging. References 1. 2. 3. 4. 5. 6. 7. Xiao, B., Sun, X.F. and Sun, R.C. (2003) “Chemical, Structural and Thermal Characterization of Alkali-Soluble Lignins and Hemicelluloses and Cellulose from Maize Stems, Rye Straw and Rice Straw.” Polymer Degradation and Stability. 74. 307-319. Ververis, C., Georghiou, K., Christodoulakis, N., Santas, P. and Santas, R. (2004) “Fiber Dimensions, Lignin and Cellulose Content of Various Plant Materials and Their Suitability for Paper Production.” Industrial Crops and Products. 19. 245-254. Bhaduri, S.K., Ghosh, I.N. and Ded Sarkar, N.L. (1995) “Ramie Hemicellulose as Beater Additive in Paper Making from Jute-Stick Pulp.” Industrial Crops and Products. 4. 79-84. Fragante, L.R. and Cruz, L.V. (1998) “Hand Made Paper From Bagasse and Sugarcane Leaves.” Proceedings of 45th Annual Convention, Philippine Sugar Technologist Association, Cebu, Philippine. Ardeh, S.N., Rovshandeh, J.M. and Pourjoozi, M. (2004) “Influence of Rice Straw Cooking Conditions in the Soda-Ethanol-Water Pulping on the Mechanical Properties of Produced Paper Sheets.” Bioresource Technology. 92. 65-69. Hoang, Q.L., Yves, L.B., Michel, D. and Gerard, A. (2001) “Formic Acid Pulping of Rice Straw.” Industrial Crops and Products. 14. 65-71. Sun, J.X., Sun, X.F., Zhao, H. and Sun, R.C. (2004) “Isolation and Characterization of Cellulose from Sugarcane Bagasse.” Polymer Degradation and Stability. 84. 331-339.

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HARNESSING ELECTRO DRIVEN SEPARATION TECHNIQUE FOR THE SEPARATION OF SELECTED AGROCHEMICALS Wan Aini Wan Ibrahim1, S. M. Monjurul Alam2 and Azli Sulaiman1 Separation Science Research Group (SSRG) Chemistry Department, Faculty of Science, University Teknologi Malaysia, 81310 Skudai, Johor 2 Chemistry Department, Rajshahi University, Rajshahi-6205, Bangladesh e-mail: wanaini@kimia.fs.utm.my Abstract. Electro driven separation techniques offer a different approach to the analysis of complex mixtures than do traditional pressure-driven chromatographic system; it may rely on electrophoresis, the transport of charged species through a medium by an applied field or may rely on electro driven mobile phase to provide a true chromatographic separation. In the current work the potential of an electro driven separation technique viz. micellar electrokinetic chromatography (MEKC), is harnessed for the separation of selected agrochemicals (organophosphorus pesticides, OPPs) widely used in the agriculture sector in Malaysia. The current study compares the use of MEKC in normal mode (NM) and reverse mode (RM) for the separation of the selected OPPS. This study also highlights the difference in separations produced by performing separations in normal mode-MEKC (NM-MEKC) and reverse mode-MEKC (RM-MEKC) for the selected OPPs. In RMMEKC, separation is conducted at acidic pH (pH 2.5 in the current work) where the electroosmotic flow (EOF) is weak whereas in NM-MEKC, the separation is carried out under basic pH (9.3 in this work) where the EOF is strong. A reverse migration order of the OPPs was observed under RMMEKC. Separation under NM-MEKC was found to be superior to those of RM-MEKC. A comparison is also made between separations performed under sweeping-NM-MEKC and sweepingRM-MEKC. In sweeping, the OPPs are prepared in the same background solution (BGS) minus the micelles and is adjusted to the same conductivity as the BGS. The study showed that NM-MEKC is more sensitive than RM-MEKC but sweeping-RM-MEKC is superior to sweeping-NM-MEKC. However, sweeping-RM-MEKC only separates two of the OPPs in a single run whereas sweepingNM-MEKC separates four OPPs in a single run. The better choice of separation mode would be sweeping-NM-MEKC for more OPPs separation in a single run. Abstrak. Teknik pemisahan pacuan elektro menawarkan satu pendekatan berlainan analisis suatu campuran kompleks berbanding dengan sistem kromatografi pacuan tekanan; ia boleh bergantung kepada elektroforesis, pengangkutan spesis bercas melalui media oleh medan yang dikenakan atau bergantung kepada fasa bergerak pacuan elektro untuk memberikan pemisahan kromatografi sejati. Dalam kajian ini keupayaan teknik pemisahan pacuan elektro iaitu kromatografi elektrokinetik misel (MEKC) digunakan untuk pemisahan bahan kimia agro terpilih (pestisid organofosforus) yang banyak digunakan dalam sektor pertanian di Malaysia. Kajan ini membandingkan penggunaan MEKC dalam mod normal (NM) dan mod terbalik (RM) untuk pemisahan OPPs terpilih ini. Kajian ini juga menonjolkan perbezaan pemisahan yang dihasilkan dalam mod NM-MEKC dan mod RM-MEKC. Dalam mod NM-MEKC, pemisahan dijalankan di dalam keadaan berbes (pH 9.3 dalam kajian ini) di mana daya electroosmosis (EOF) adalah kuat sementara dalam mod RM-MEKC, pemisahan dijalankan dalam keadaan berasid (pH 2.5) di mana EOF adalah lemah. Tertib migrasi yang dicerap dalam RM-MEKC adalah berlawanan dengan tertib migrasi dalam NM-MEKC. Perbandingan juga dibuat antara pemisahan secara sapuan-NM-MEKC dan sapuan-RM-MEKC. Dalam mod sapuan, OPPs disediakan dalam larutan latarbelakang (BGS) tanpa misel dan kekonduksian diubahsuai supaya sama dengan kekonduksian BGS. Kajian ini menunjukkan bahawa NM-MEKC lebih sensitif berbanding RM-MEKC tetapi sapuan-RM-MEKC lebih baik berbanding sapuan-NM-MEKC. Walau bagaimanapun, sapuan-RM-MEKC hanya mampu memisahkan dua OPPs dalam satu larian berbanding dengn sapuan-NM-MEKC yang dapat memisahkan empat OPPs dalam satu larian. Pemilihan mod pemisahan yang lebih baik adalah sapuan-NM-MEKC untuk pemisahan lebih banyak OPPs dalam satu larian.
1

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Keywords: Micellar electrokinetic chromatography (MEKC), organophosphorus pesticides, normal mode MEKC, reverse mode MEKC, sweeping

Introduction

Capillary electrophoresis (CE) has become a powerful separation technique and has been applied to the analysis of a wide range of molecules. Electrokinetic chromatography (EKC) is a mode of CE and was first introduced by Terabe and co-workers [1, 2] in 1984. Since the first introduction of EKC, it has become widely popular as a powerful separation technique for both neutral and ionic compounds [3-5]. In EKC, the use of charged pseudostationary phases (PSP) like sodium dodecyl sulphate (SDS) is by far the most famous experimental form of EKC and called micellar electrokinetic chromatography (MEKC). Species having the same charge as the micelles do not interact with the micelle, while those having opposite charge strongly interact with the micelles. The separations of charged species depend on the species difference in electrophoretic mobility. The formation of micelles provides a unique chromatographic process for the separation of neutral molecules where partitioning between the micellar PSP and the electroosmotically pumped aqueous phase take place. The use of untreated silica capillaries and separation in basic buffers with positive potential were considered as the standard features in most of the MEKC works [6-8]. In these particular conditions, the bulk electroosmotic flow (EOF) is dominant and that ultimately drives the anionic sodium dodecyl sulphate (SDS) micelles toward the cathode end. However, in few works, especially those described in the newer online concentration techniques [9-12] have studied the effect of buffers pH in relation with the overall separation performances. In the current work the potential of an electro driven separation technique, micellar electrokinetic chromatography (MEKC), is harnessed for the separation of selected agrochemicals (organophosphorus pesticides, OPPs) widely used in the agriculture sector in Malaysia. Electro driven separation techniques offer a different approach to the analysis of complex mixtures than do traditional pressure-driven chromatographic system; it may rely on electrophoresis, the transport of charged species through a medium by an applied field or may rely on electro driven mobile phase to provide a true chromatographic separation. The current study compares the use of MEKC in normal mode (NM) and reverse mode (RM) for the separation of the selected OPPS. This study also highlights the difference in separations produced by performing separations in normal mode-MEKC (NM-MEKC) and reverse mode-MEKC (RM-MEKC) for the selected OPPs. In RM-MEKC, separation is conducted at acidic pH (pH 2.5 in the current work) where the anionic micelles (sodium dodecyl sulphate, SDS) move faster than the electro-osmotic flow (EOF), which is weak; thus positive potential is applied at detector end in order to detect the analytes. In NM-MEKC, the separation is carried out under basic pH (pH 9.3 in this work) where the EOF is strong. In CE, the injected sample volumes are typically limited to 1% of the total capillary volumes in order to maintain efficiency. Therefore to increase detection sensitivity, various concentration techniques can be used. In this study, sweeping is used and a comparison of sweeping in NM-MEKC and RM-MEKC is also made. Sweeping is one of the on-line concentration techniques in MEKC and is based on the accumulation of analyte molecules by an additive in the background solution (BGS), which they have a considerable affinity for [10, 13].

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Experimental

Reagents All pesticides were analytical standards purchased from Dr. Ehrenstorfers GmbH laboratory (Augsburg, Germany). SDS was purchased from Fisher Scientific (Loughborough, UK), methanol from BDH (Poole, England), acetonitrile and disodium tetraborate 10-hydrate (B4Na2O7 10H2O) from MERCK (Germany), hydrochloric acid (37.5%) from Sigma (St. Louis, MO, USA), sodium hydroxide pellets and disodium hydrogen phosphate 12-hydrate (Na2HPO4 12H2O) from Riedel-de Haen (Seelze, Germany). Instrument Capillary electrophoresis was performed on a CE – L1 instrument (CE Resources Pte. Ltd. Singapore) fitted with a UV-Visible detector (SPD – 10A VP) from Shimadzu (Kyoto, Japan). Electropherograms and data were recorded by Chromatography Station software (CSW) for Windows at 202 nm. Uncoated fused-silica capillaries (total and effective lengths are 82 and 42 cm, respectively) from SGE (Victoria, Australia) of 50 µm ID were used. Procedures Standard solutions of about 1 mg mL-1 of each pesticide were prepared in methanol. Working standard solutions were prepared by diluting the corresponding stock solutions in buffer, surfactant and modifiers, and the ionic strengths of sample matrix and running buffers were maintained at same level. Dilutions in sweeping procedures, corresponding pH of sample and buffer matrices were mentioned in respective places. All the stock buffers and surfactants were prepared in distilled deionized (DD, 18 MΩ) water. Concentrations of buffers, SDS, methanol and acetonitrile are reported based on the final volume in the running buffer. All running buffers were filtered through 0.45 µm nylon filter disc from Whatman (Clifton, New Jersey, USA). Separation voltage of 25 kV (either + or − mode) was employed depending on the requirements. Sample injections were performed electrokinetically at 15 kV in all separations where basic running buffer were involved, while samples were injected hydrodynamically at 2.8 kPa for various times where separations were performed in RM-MEKC. All experiments were conducted at an ambient temperature of 25°C. At the beginning of each day, the capillary was rinsed for at least 10 min with 0.1 M NaOH solution followed by DD water for 10 min and conditioned with running buffer for 10 min. In between runs, the capillary was also conditioned by running buffer for 5 min. At the end of the day, the capillary was rinsed with DD water for 30 minutes followed by passing air. Results and Discussions

Separation in NM- MEKC The five OPPs chosen for the study are hydrophobic compounds (Figure 1) and were baseline separated in normal mode MEKC (NM-MEKC) within 20 minutes run. A representative electropherogram is shown in Figure 2. The elution order is consistent with their water solubility (Table 1). Methidathion elutes first and the elution continues in the order of decreasing water solubility or in the order of increasing hydrophobicities (taking the upper value in the range). The limit of detection for separation in NM MEKC is in the range 1.6-6.4 ppm and migration time relative standard deviation (RSD) is less than 1%.

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H3C

N N S
Methidathion

S CH2S P

H3C

COCH3
N O N (H3C)2HC

S P

OCH2CH3

COCH3

OCH2CH3
Diazinon

O

O O P

OCH 2 CH 3

Br

SCH 2 CH 2 CH 3 Cl
Profenofos
Cl S Cl O N Cl P OCH2CH3 OCH2CH3

N O N

S P

OCH2CH3

Quinalphos

OCH2CH3

Chlorpyrifos

Figure 1. Names and structures of OPPs used in the current work Table 1.

Selected properties of investigated OPPs [14-15]. Name (mp/bp, oC) Mol. weight Sw (g/L, 20oC) Log Kow Methidathion (mp 39-40) 302.31 0.25 1.58-2.42 Diazinon (bp 83-84) 304.35 3.02-3.86 5.35×10-2 Quinalphos(bp 142) 298.3 3.04-4.44 2.4×10-2 Profenofos (bp 110) 373.64 4.68-4.8 2.0×10-2 -3 Chlorpyrifos (bp decom. 350.59 4.68-5.3 0.73×10 160) Sw = Solubility in water; Log Kow = log value of octanol, water partition coefficient
1 0.1 mAU 23 4 5

EOF

Time (min) Figure 2. OPPs separation in NM-MEKC. Conditions: separation buffer contained 10 mM 1:1 Na2HPO4-Na2B4O7 (pH 9.3), 10 mM SDS and 10 % 1:1 methanol-acetonitrile; applied potential 25 kV; sample prepared in 10 mM 1:1 Na2HPO4-Na2B4O7 (pH 9.3), 10 mM SDS and 10 % 1:1 methanolacetonitrile; sample injection: 10 s at 15 kV. Peaks: EOF electroosmotic flow marker (methanol); 1,

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methidathion (20 ppm); 2, diazinon (20 ppm); 3, quinalphos (5 ppm); 4, chlorpyrifos (5 ppm); 5, profenofos (5 ppm).

Separation in RM-MEKC RM-MEKC was investigated for the possibility of better sensitive detection. In acidic condition the use of borate in running buffer is limited and the 20 mM phosphate buffer is found optimal in the range of 5-80 mM. The presence of organic modifier (especially methanol) in buffer and sample matrix is also found crucial but only 5-10% methanol is necessary. The concentration of SDS remained at 10 mM as before. Sample injections were performed hydrodynamically at 2.8 kPa for 10 s, as electrokinetic sample injection (here by negative potentials) causes peak disappearances. Diazinon, profenofos and chlorpyrifos co-eluted and the possible optimisation schemes could not resolve these three peaks. Runs by single compound have shown that the sensitivity of diazinon is very weak and the resolution of profenofos and quinalphos peak is poorer than the chlorpyrifos and quinalphos peaks. Therefore, only metidathion, quinalphos and chlorpyrifos were separated in acidic phosphate buffer at pH 2.5. A typical electropherogram of the separation is shown in Figure 3. The concentration of quinalphos and chlorpyrifos are the same as in Figure 2 except for methidathion where the concentration is half. In RM-MEKC, the migration order is reverse the migration order in NM-MEKC. Chlorpyrifos eluted first followed by quinalphos and finally methidathion. The three OPPs were separated in less than 18 minutes with baseline resolution. The limit of detection (LOD) in RM-MEKC is in the range of 0.63-6.7 ppm and a RSD migration time of 0.69-1.4%. Sweeping-NM- MEKC Sweeping is a technique for on-column sample concentration of non-polar molecules based on the analytes ability to partition into the pseudo-stationary phase (PSP) in MEKC. The effectiveness of sweeping is closely related to the analyte/s affinity for the micellar phase. The greater the affinity of the analyte toward the micelle or the higher the retention factor of the analyte, the greater the concentrating effect. Sweeping requires low EOFs and thus is often constrained to separations performed in acidic conditions. Sweeping is defined as the picking and accumulation of analytes by the PSP that fills the sample zone that bears no micelles in it during application of voltage [10]. Samples are prepared in buffer of equivalent ionic strength to the running buffer but without any micelles, and a longer plug is injected in sweeping. Utility of sweeping in various micellar systems has been tried and recently reviewed [16]. Figure 4 shows the sweeping of five selected OPPs used in this study. It is to be noted that the concentration range in sweeping is five times lower and sample injection time is five times higher than the NM-MEKC run (Figure 2 and Figure 4).

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3

0.1 m AU
4 1

Time/min

Figure 3. OPPs separation in RM-MEKC. Conditions: separation buffer contained 20 mM Na2HPO4 (pH 2.5), 10 mM SDS and 5 % methanol. Sample prepared in 20 mM Na2HPO4 (pH 9.3), 10 mM SDS and 5 % methanol; sample injection: 10 s at 2.8 kPa; Peaks: 1, methidathion (10 ppm); 3, quinalphos (5 ppm); 4, chlorpyrifos (5 ppm).

The presence of 10% organic solvents in sample matrices has actually increased the solubility of pesticides, especially for profenofos and chlorpyrifos. The question of non-suitability of sweeping phenomenon for methidathion can be explained by the theories that were proposed initially that characteristically sweeping depends on the retention factor, therefore is effective to strongly retained analytes [10, 17]. The LOD in sweeping-NM-MEKC is in the range 0.15-3.0 ppm with a migration time RSD less than 0.5%.

3
0.25mAU

45

2

1

Figure 4. OPPs separation by sweeping-NM-MEKC. Conditions: separation buffer contained 10 mM 1:1 Na2HPO4-Na2B4O7 (pH 9.3), 10 mM SDS and 10 % 1:1 methanol-acetonitrile; applied potential 25 kV; sample prepared in 10 mM 1:1 Na2HPO4-Na2B4O7 (pH 9.3) and 10 % 1:1 methanolacetonitrile. Sample injection: 50 s at 15 kV; Peaks: 1, methidathion (4 ppm); 2, diazinon (4 ppm); 3, quinalphos (1 ppm); 4, chlorpyriphos (1 ppm); 5, profenofos (1 ppm).

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Sweeping-RM-MEKC Sweeping can offer better results where EOF is weak when neutral hydrophobic analytes were used [10]. Therefore, sweeping in RM-MEKC was tried as an effort to further increase the sensitivity. However, in sweeping-RM-MEKC, mixture of only quinalphos and chlorpyrifos was used, as the methidathion peak cannot be uniquely focused. As the profenofos and chlorpyrifos co-eluted, therefore, another mixture of quinalphos and profenofos was also swept to compare the peak sensitivity of quinalphos with respect to both of chlorpyrifos and profenofos. Here, significantly longer sample plug (ca. 10 cm that corresponds to 400s injection at 2.8 kPa) can be injected. Figure 5A & 5B shows the respective electropherogram. Both peaks were eluted within 10 minutes. The LODs for the quinalphos-chlorpyrifos and quinalphos-profenofos pair is in the range of 0.27-0.86 ppm and 0.19-0.29 ppm respectively. Table 2 summarises the LODs of the various MEKC modes used in the study. As can be seen from the table, sweeping-RM-MEKC gives the lowest LOD but the separation is only limited to two of the selected OPPS in a single run. Sweeping in NM-MEKC separates all the five OPPs but the separation of methidathion in this mode is not suitable as the peak is broad and short. A better way of online concentration for methidathion would be stacking [18, 19]. Sweeping-NM-MEKC also produces low migration time RSD (less than 0.5%). Table 2 summarises the LODs for the various modes of MEKC used in the study. Table 2. Limit of detection of various MEKC modes Mode NM-MEKC Sweeping-NM-MEKC RM-MEKC Sweeping-RM-MEKC LOD, ppm 1.6-10.4 0.15-3 0.63-6.7 0.19-0.86

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A

3 0.25 mAU 4

B

3 0.25 mAU

5

Time/min

Figure 5. OPPs separation in sweeping-RM-MEKC. Conditions: separation buffer contained 20 mM Na2HPO4 (pH 2.5), 10 mM SDS and 5 % methanol; sample prepared in 20 mM Na2HPO4 (pH 9.3) and 5 % methanol; sample injection: 400 s at 2.8 kPa; Peaks: 3, quinalphos (0.25 ppm); 4, chlorpyrifos (3 ppm). (B) Conditions: As in (A); sample prepared in 20 mM Na2HPO4 (pH 9.3) and 5 % methanol; sample injection: 400 s at 2.8 kPa. Peaks: 3, quinalphos (0.25 ppm); 5, profenofos (1 ppm)

CONCLUSIONS The study carried out showed that NM-MEKC is more sensitive than RM-MEKC for the selected OPPs. In both normal MEKC and RM-MEKC, sensitivity enhancements were found to be at least one order higher but sweeping-RM-MEKC is superior to sweeping-NM-MEKC. However, separation in acidic buffer is limited, as the short optimisation scheme could not resolve the co-elution of few OPPs. Instead of anionic SDS surfactant, the use of cationic surfactant and the use of coated capillaries where EOF is absent could be worth trying in further investigation of sweeping for this particular set of hydrophobic OPPs. Even though sweeping-RM-MEKC gave lowest LOD but it only separates 2 OPPs in a single run. The better mode of separation for the selected OPPS would be sweeping-NM-MEKC as it separates 4 OPPs in a single run with LOD in the sub-ppm level. Off-line extraction such as liquid-liquid extraction (LLE) or solid phase extraction (SPE) in combination with sweeping-NM-MEKC would achieve ppb levels needed for pesticide analysis.

ACKNOWLEDGEMENT We are grateful to Ministry of Science, Technology & Innovation (MOSTI), Malaysia for the financial support provided through IRPA grant project no: 08-02-06-0060 EA 158. References 1. 2. Terabe, S., Otsuka, K., Ichikawa K., Tsuchiya, A. and Ando T., (1984) “Electrokinetic Separations with Micellar Solutions and Open-Tubular Capillaries” Anal. Chem. 56. 113. Terabe, S., Otsuka, K. and Ando T., (1985) “Electrokinetic Chromatography with Micellar Solution and Open-Tubular Capillary” Anal. Chem. 57. 834.

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3. 4. 5. 6. 7. 8. 9.

10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Wan Ibrahim, Wan Aini, Monjurul Alam, S. M., Sulaiman, A. B., (2005) “Comparative Study of Two Common Surfactants in Micellar Electrokinetic Chromatography for the Separation of Organophosphorus Pesticides”, ACGC Chemical Research Communications. 18. 43. Wan Ibrahim, Wan Aini, Monjurul Alam, S. M., Sulaiman, A. B., (2005), “Organic modifier and Effect of Sample Matrix in the Separation of Organophosphorus Pesticides”, Mal. J. Chem. 7(1). 26. Zakaria, P., Macka, M. and Haddad, P. (2003) “Mixed-mode electrokinetic chromatography of aromatic bases with two pseudo-stationary phases and pH control” J. Chromatogr. A, 997. 207. Farran, A., Ruiz, A., Serra, C., Aguilar, M., (1996) “Comparative study of high-performance liquid chromatography and micellar electrokinetic capillary chromatography applied to the analysis of different mixtures of pesticides” J. Chromatogr. A. 737, 109. Penmetsa, K. V., Leidy, R. B, Shea, D., (1996) “Herbicide Analysis by Micellar Electrokinetic Chromatography” J. Chromatogr. A. 745. 201. Hinsmann, P., Arce, A., Rios, A., Valcarcel, M. (2000) “Determination of pesticides in waters by automatic on-line solid-phase extraction–capillary electrophoresis” J. Chromatogr. A. 866. 137. Molina, M., Wiedmer, S. K., Jussila, M., Silva, M., Riekkola, M.L. (2001) “Use of a partial filling technique and reverse migrating micelles in the study of N-methylcarbamate pesticides by micellar electrokinetic chromatography–electrospray ionization mass spectrometry” J. Chromatogr. A. 927. 191. Quirino, J. P., Terabe, S. (1998) “Exceeding 5000-fold Concentration of Dilute Analytes in Micellar Electrokinetic Chromatography” Science. 282.465. Quirino, J. P., Terabe, S. (1998) “On-line Concentration of Neutral Analytes for Micellar Electrokinetic Chromatography. 3. Stacking with Reverse Migrating Micelles” Anal. Chem. 70. 149. Quirino, J. P., Otsuka, K., Terabe, S. (1998) “On-line concentration of neutral analytes for micellar electrokinetic chromatography. VI. Stacking using reverse migrating micelles and a water plug” J. Chromatogr. B. 714. 29. Quirino, J. P., Kim, J.B., Terabe, S., (2002) ‘Sweeping: Concentration Mechanism and Applications to High-Sensitivity Analysis in Capillary Electrophoresis” J. Chromatogr. A. 965. 357. Montgomery, J. H. (1997) “Agrochemicals Desk Reference”, Boca Raton: Florida, USA, CRC Press, Lewis Publisher. Verschueren, K. (1996) Handbook of Environmental Data on Organic Chemicals”, USA: Von Nostrand, Reinhold. Kim, J.B., Terabe, S., (2003) “On-line sample preconcentration techniques in micellar electrokinetic chromatography”. J. Pharm. Biomed. Anal. 30. 1625. van Zomeren, P. V., Hilhorst, M. J., Coenegracht, P. M. J., de Jong, G. J. (2000) “Resolution optimisation in micellar electrokinetic chromatography using empirical models” J. Chromatogr. A. 867. 247. Beckers, J. L and Bocek, P. (2000) “Sample Stacking in Capillary Zone Electrophoresis: Principles, Advantages and Limitation” Electrophoresis. 21. 2747. Wan Ibrahim, Wan Aini, Monjurul Alam, S. M., Sulaiman, A. B., (2004) “Stacking as an Online Concentration of Neutral Organophosphorus Pesticides using Micellar Electrokinetic Chromatography” Buletin Kimia, 20. 23.

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KANDUNGAN LOGAM BERAT DI DALAM BEBERAPA SIRI TANAH OKSISOL DI SEKITAR TASIK CHINI, PAHANG Sahibin Abd. Rahim, Muhd. Barzani Gasim, Mohd. Nizam Mohd Said, Wan Mohd Razi Idris, Azman Hashim, Sharilnizam Yusof dan Masniyana Jamil Program Sains Sekitaran, Pusat Pengajian Sains Sekitaran dan Sumber Alam Fakulti Sains dan Teknologi, UKM 43600 Bangi Selangor, Malaysia e-mail: haiyan@pkrisc.cc.ukm.my ABSTRACT. This study was carried out to determine heavy metal content and physico-chemical properties of soil’s influencing heavy metal accumulation in some series surrounding the Chini Lakes. A total of 15 topsoil sample were collected randomly from 6 stations. The physical properties that were analyzed include particle size distribution and soil organic matter. Meanwhile, the chemical characteristics determined were pH, electrical conductivity and cation exchange capacity. It was found that heavy metal content of Cd, Cr, Cu, Co, Pb, Zn and Mn were low whereas Fe content was high. The textures of soil studied were clay, loamy sand, sandy loam, clay loam and silty clay loam. The mean of organic matter ranged from 2.68 to 11.46%. The soil pH showed that the soil studied was acidic with values ranged between 3.36 to 3.72. The range of electrical conductivity mean was between 2150 µScm-1 to 2403 µScm-1. Cation exchange capacity mean ranged from 2.85 hingga 8.59 cmolc/kg. Correlation analysis showed that there were positive and negative significant correlations between soils parameters heavy metal concentration. Analysis of variance (ANOVA) showed that there were significant differences in organic matter percentage, pH, cation exchange capacity and heavy metals except cadmium between sampling station. ABSTRAK. Kajian ini dijalankan untuk menentukan kandungan logam berat dan ciri fiziko-kimia tanah yang mempengaruhi pengayaan logam berat dalam beberapa siri tanah di sekitar Tasik Chini, Pahang. Sebanyak 15 sampel tanah daripada 6 stesen telah diambil berdasarkan jenis tanah. Ciri fizik yang ditentukan termasuklah taburan saiz partikel dan kandungan bahan organik tanah. Manakala ciri kimia yang ditentukan pula adalah pH, kekonduksian elektrik dan kapasiti pertukaran kation. Didapati bahawa kandungan logam berat Cd, Cr, Cu, Co, Pb, Zn dan Mn adalah rendah manakala kandungan Fe adalah tinggi. Tanah kawasan kajian terdiri daripada tekstur lempung, pasir berlom, lom berpasir, lom lempung dan lom lempung berkelodak. Kandungan bahan organik berjulat di antara 2.68 hingga 11.46%. Nilai pH menunjukkan tanah di kawasan kajian adalah berasid iaitu pada julat antara 3.36 hingga 3.72. Manakala julat kekonduksian elektrik pula berada di antara 2150 µScm-1 hingga 2403 µScm-1. Purata kapasiti pertukaran kation berjulat di antara 2.85 hingga 8.59 cmolc/kg tanah. Analisis korelasi menunjukkan terdapat perhubungan yang signifikan positif dan negatif di antara parameter tanah dengan kandungan logam berat. Analisis varians (ANOVA) pula menunjukkan terdapat perbezaan yang signifikan di antara stesen bagi kandungan bahan organik, kapasiti pertukaran kation serta logam berat kecuali logam kadmium. Katakunci: logam berat, pengayaan, tanah oksisol, tasik chini Pendahuluan Luluhawa dan pedogenesis merupakan dua proses utama yang menyumbang dalam pembentukan tanah. Luluhawa fizik adalah proses di mana batuan dipecahkan dan dihancurkan oleh agen iklim seperti haba, air yang mengalir, graviti dan angin melalui proses pengembangan dan penguncupan yang berterusan. Luluhawa kimia pula melibatkan proses seperti pengoksidaan, penurunan, hidrasi, hidrolisis dan pengkarbonan sementara luluhawa biologi pula melibatkan peranan benda-benda hidup seperti sesetengah hidupan seni, akar tumbuhan primitif seperti liken dan lumut [8]. Saling tindakan iklim, topografi dan juga masa ke atas bahan induk mengakibatkan pembentukan pelbagai jenis tanah di Malaysia. Sebagai contoh, tanah yang terbentuk daripada batuan granit berbeza dengan tanah yang terbentuk daripada batuan basalt. Perbezaan tersebut bukan sahaja dari segi minerologinya, malahan juga morfologinya [10]. Tanah di kawasan kajian didasari oleh batuan

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metasedimen daripada batupasir, batu lodak dan batu lumpur yang telah mengalami canggaan hebat. Komposisi batuan adalah berbagai dengan sebahagiannya mempunyai kandungan ferum yang tinggi. Ini menunjukkan tanaih di sini adalah tanah oksisol. Perbezaan komposisi batuan induk menyebabkan terbentuk beberapa siri tanah di sekitar Tasik Chini. Antara siri tanah yang ditemui di sini adalah siri Melaka, Bungor dan Rasau. Siri Melaka dikenali dengan warna merah gelap berkomposisi laterit yang tinggi. Horizon pada profil tidak begitu jelas. Siri Bungor berwarna kekuningan, manakala siri Rasau berkomposisi pasir dengan dicirikan oleh warna cerah. Kajian ini cuba melihat kepada ciri fizikokimia tanah yang terdapat di kawasan sekitar Tasik Chini. Bahan Dan Kaedah Lokasi dan stesen kajian Kawasan kajian iaitu Tasik Chini adalah terletak di mukim Penyur, Pahang. Kedudukan lokasi kajian adalah merangkumi garis lintang 3°24’ hingga 3º28’ utara dan garis bujur di antara 102°55’ hingga 102°60’ timur. Tasik Chini terletak kira-kira 100 km dari Kuantan terbahagi kepada 12 cabang saliran yang dikenali sebagai laut. Stesen 1 merupakan tanah siri Melaka yang terdiri daripada tanah laterit yang mengandungi logam ferum yang tinggi. Kawasan persampelan meliputi tanah hutan dan tanah pertaniah kelapa sawit tua. Stesen 2 pula terdiri daripada tanah siri Rasau berwarna cerah putih meliputi kawasan pertanian kelapa sawit yang baru ditanam dan hutan sekunder. Tanah di stesen ini mempunyai struktur yang halus dan peroi. Stesen 3 terletak di Kampung Gumum yang terdiri daripada kawasan terbuka dan tanah hutan yang kecerunannya lebih kurang 4º. Tanah di stesen ini dikategorikan sebagai tanah siri Bungor. Stesen 4 terletak di Bukit Jerangking yang mempunyai kecerunan lebih kurang 20 hingga 25° dengan litupan sebanyak 60 hingga 70%. Tanah di stesen 4 juga dikategorikan sebagai tanah siri Bungor. Stesen 5 pula adalah lokasi Kampung Chenahan yang terdiri daripada kanopi sebanyak 30% tetapi tiada litupan pada lantai hutan serta mempunyai kecerunan lebih kurang 5º. Tanah di stesen 5 ini pula dikategorikan sebagai Siri Melaka. Stesen 6 merupakan kawasan hutan yang mempunyai litupan kanopi lebih kurang 90% dan litupan sebanyak 70% pada lantai hutan. Stesen ini terletak di Pulau Babi. Tanah di sini adalah siri Melaka. Selain itu di stesen keenam ini terdapat banyak daun dan sampah-sarap pada lantai hutan yang menyumbang kepada pembentukan humus. Kaedah Kajian Kaedah yang digunakan dalam kajian ini dikelaskan kepada 4 peringkat yang terdiri daripada cerapan di lapangan, persampelan tanah, analisis makmal dan analisis data.

Persampelan Tanah Persampelan tanah dijalankan dengan mengambil secara rawak kira-kira 1kg berat tanah permukaan atas (0-20cm) dengan menggunakan dutch auger. Sampel tanah tersebut dimasukkan ke dalam beg plastik dan dilabelkan mengikut lokasi masing-masing untuk analisis makmal. Penyediaan Sampel Analisis makmal merangkumi proses penyediaan sampel di mana semua sampel tanah dibiarkan kering udara pada suhu bilik. Semasa proses pengeringan yang dijalankan, gumpalan sampel tanah dipecahkan kepada bahagian yang lebih kecil untuk mempercepatkan proses pengeringan. Sampel tanah kering udara kemudian ditumbuk dengan menggunakan penumbuk lesung kayu. Pecahan-

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

pecahan tanah diayak dengan menggunakan ayak bersaiz 2mm. Hasil ayakan tanah yang mempunyai saiz yang kurang daripada 2mm dibahagikan di mana satu bahagian tanah digunakan untuk analisis parameter fiziko-kimia manakala bahagian yang lain disimpan untuk kajian ulangan. Sebanyak 3 replikasi digunakan untuk setiap penentuan. Analisis Makmal Penentuan saiz partikel dilakukan mengikut kaedah pipet berserta ayakan kering [1]. Kandungan bahan organik ditentukan secara pembakaran [3]. Pengukuran pH tanah dilakukan di dalam nisbah 1:2.5 bagi tanah:air suling [6] menggunakan meter pH berelektrod kaca Model WTW INOLAB Level 1. Kekonduksian elektrik ditentukan daripada ekstrak CaSO4.2H2O tepu [5] menggunakan alat meter kekonduksian Model H 18819 Hanna. Kation asid boleh tukar ganti Al3+ dan H+ diekstrak dengan larutan 1M KCl dan kemudian ditentukan secara titratan. Kepekatan kation boleh tukar K+, Na+, Ca2+ dan Mg2+ daripada ekstrak 1M Ammonium Asetat [7] ditentukan menggunakan alat Spektrometer Serapan Atom Nyalaan (FAAS). Kandungan logam berat di dalam tanah diekstrak dengan menggunakan kaedah Archer and Hodgson (1987) [2], kemudian kepekatannya di dalam larutan ekstrak ditentukan dengan Spektrometer Serapan Atom Nyalaan (FAAS).

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

THAILAND KEDAH 0 50 100 km

PERAK

KELANTAN TERENGGANU LAUT CHINA SELATAN

PAHANG

SELANGOR

JOHOR SUMATERA

Rajah 1. Peta menunjukkan kawasan kajian dan persampelan

Hasil Dan Perbincangan Kandungan Logam Berat Dalam Tanah

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

Purata dan sisihan piawai bagi setiap logam yang dikaji di kesemua stesen cerapan ditunjukkan pada Rajah 1 dan 2. Julat purata kepekatan bagi logam kadmium (Cd) di dalam tanah adalah di antara 1.03 hingga 2.03 mg/kg. Kepekatan tertinggi dicatatkan pada stesen 5 dan yang terendah dicatatkan pada stesen 6 dengan nilai masing-masing ialah 2.03 ± 0.93 mg/kg dan 1.03 ± 0.77 mg/kg. Analisis varians menunjukkan tidak ada perbezaan signifikan di antara purata kandungan Cd di dalam tanah di stesen yang dikaji. Ini menunjukkan kandungan Cd dalam siri tanah yang berbeza adalah lebih kurang sama sahaja. Purata kepekatan logam kobalt (Co) pula adalah di antara 7.44 hingga 19.85 mg/kg. Kandungan logam berat Co yang paling tinggi dicatatkan oleh stesen 6 iaitu 19.85 ±1.71 mg/kg dan yang paling rendah adalah stesen 2 iaitu 7.44 ± 0.81 mg/kg. Analisis varians menunjukkan terdapat perbezaan signifikan di antara purata kandungan Co di antara stesen 2 dengan stesen-stesen yang lain. Stesen 2 adalah tanah siri Rasau yang didominasi oleh pasir.
Cd 3.5 Kepekatan (mg/kg)
Kepekatan (mg/kg) 25 20 15 10 5 0 Co

3 2.5 2 1.5 1 0.5 0 1 2 3 Ste se n 4 5 6

1

2

3 Ste se n

4

5

6

A

B
Cr 50 45 40 35 30 25 20 15 10 5 0 1 2 3 Ste se n 4 5 6

Cu 18 16 14 12 10 8 6 4 2 0 1 2 3 Ste se n 4 5 6

Kepekatan (mg/kg)

C

D

Rajah 2. Kandungan logam berat (Cd, Co, Cr, Cu) dalam sampel tanah di kawasan kajian Bagi kandungan unsur kromium (Cr) di dalam tanah pula purata kepekatannya berada pada julat 0.57 hingga 31.63 mg/kg yang mana stesen 5 mencatatkan nilai kepekatan tertinggi iaitu 31.63 ± 13.34 mg/kg. Manakala stesen 2 mencatatkan nilai kepekatan Cr yang terendah iaitu 0.57 ± 0.32 mg/kg. Analisis varians menunjukkan terdapat perbezaan signifikan di antara purata kandungan Cr di dalam tanah di stesen 1, 5, 6 dengan stesen 2, 3 dan 4. Tanah stesen 1, 5 dan 6 merupakan tanah siri Melaka. Purata kepekatan logam kuprum (Cu) pula terletak pada julat di antara 1.04 hingga 10.65 mg/kg iaitu kepekatan logam ini dicatatkan paling tinggi pada stesen 6 iaitu 10.65 ± 4.87 mg/kg dan paling rendah pada stesen 2 iaitu 1.04 ± 0.70 mg/kg. Analisis varians menunjukkan terdapat perbezaan signifikan di antara purata kandungan C di dalam tanah di stesen 2, 3 dengan stesen 1, 5 dan 6. Tanah stesen 1, 5 dan 6 merupakan tanah siri Melaka

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Kepekatan (mg/kg)

Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

Secara keseluruhannya, logam ferum (Fe) mencatatkan nilai purata kepekatan yang paling tinggi di kawasan kajian dengan nilainya berada pada julat 1156.80 hingga 2898.40 mg/kg. Stesen 1 mencatatkan nilai purata tertinggi iaitu 2898.40 ± 98.10 mg/kg manaakala stesen 2 mencatatkan nilai purata kepekatan logam Fe yang paling rendah iaitu 1156.80 ± 511.60 mg/kg. Analisis varians menunjukkan terdapat perbezaan signifikan di antara purata kandungan Fe di dalam tanah di antara stesen 2, 3 dan 4 dengan stesen 1, 5 dan 6. Tanah stesen 1, 5 dan 6 merupakan tanah siri Melaka
Fe 3500 3000 2500 2000 1500 1000 500 0 1 2 3 4 5 6
250 Kepekatan (mg/kg) 200 150 100 50 0 1 2 3 S tesen 4 5 6 Mn

Kepekatan (mg/kg)

S tesen

E
Pb 100 80 60 40 20 0 1 2 3 4 5 6

F
Zn
Kepekatan (mg/kg)

Kepekatan (mg/kg)

100 80 60 40 20 0 1 2 3 S tesen 4 5 6

S tesen

G
Rajah 3. Kandungan logam berat (Fe, Mn, Pb, Zn) dalam sampel tanah di kawasan kajian Purata kepekatan unsur mangan (Mn) berada pada julat di antara 4.58 hingga 173.60 mg/kg. Stesen 1 mencatatkan kepekatan logam Mn yang paling tinggi iaitu sebanyak 173.60 ± 59.68 mg/kg dan stesen 2 pula mencatatkan kepekatan yang paling rendah iaitu 4.58 ± 1.82 mg/kg. Analisis varians menunjukkan perbezaan signifikan di antara purata kandungan Mn di dalam tanah di stesen 2, 3, 4 dan 6 dengan stesen 1 dan 5. Purata kepekatan logam plumbum (Pb) pula berada pada julat di antara 15.30 hingga 69.59 mg/kg di mana kepekatan Pb yang paling tinggi dicatatkan pada stesen 5 iaitu 69.59 ± 25.35 mg/kg, sementara stesen 2 mencatatkan kepekatan logam Pb yang terendah iaitu 15.30 ± 5.19 mg/kg. Analisis varians menunjukkan perbezaan signifikan di antara purata kandungan Pb di dalam tanah di stesen 2 dan 4 dengan stesen 5 dan 6. Bagi logam zink (Zn) pula, purata kepekatannya berada pada julat 12.10 hingga 52.20 mg/kg. Secara puratanya, stesen 5 mencatatkan kepekatan logam Zn yang paling tinggi berbanding stesen persampelan yang lain iaitu 52.20 ± 35.55 mg/kg, sementara stesen 4 mencatatkan kepekatan yang paling rendah iaitu 12.10 ± 6.06 mg/kg. Terdapat perbezaan signifikan di antara purata kandungan Zn di dalam tanah di stesen 1 dan 6 dengan stesen 2 dan 4. Berdasarkan analisis varians yang dijalankan, didapati terdapat perbezaan yang signifikan di antara keenam-enam stesen persampelan bagi setiap unsur logam berat kecuali Cd. Secara umumnya

H

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

kandungan logam berat dalam tanah siri Melaka adalah lebih tinggi daripada kandungan logam berat di dalam siri Bungor dan Rasau. Kandungan logam berat dalam siri Rasau adalah paling rendah. Kebiasaannya, kuprum di dalam tanah adalah berbentuk ion Cu2+ yang akan dijerap oleh tanah lempung atau bersebati dengan bahan organik. Penahanan kuprum di dalam tanah bertambah dengan peningkatan bahan organik [12]. Keterdapatan kuprum juga akan berkurangan dengan peningkatan pH. Zink pada tanah berasid kebiasaannya mudah diserap oleh tumbuhan, manakala dalam keadaan tanah beralkali, ia tidak mudah diserap [12]. Secara umum, dalam keadaan tanah berasid Fe2+ dan Mn2+ hadir dalam larutan tanah untuk keperluan tumbuhan. Dalam tanah yang terlalu berasid, kehadiran ferum dan mangan adalah pada kepekatan yang toksik. Sementara itu, kerendahan nilai pH juga bertindak mempengaruhi penjerapan logam berat iaitu melalui jerapan proton-proton asid lemah atau jerapan spesifik hasil hidrolisis atau pembentukan mendakan permukaan dari hidroksid atau silikat [4]. Ciri Fiziko-kimia Tanah Ciri fizik tanah Nilai purata dan sisihan piawai parameter fizik tanah seperti peratusan pasir, kelodak, lempung, kandungan bahan organik dan tekstur tanah ditunjukkan dalam Jadual 1. Peratusan lempung, kelodak dan pasir serta tekstur tanah Purata peratusan lempung yang diperoleh daripada kajian ini adalah berjulat di antara 7.55% hingga 70.96%. Purata peratusan lempung yang tertinggi dicatatkan ialah pada stesen 1 iaitu sebanyak 70.96 ± 19.35%, manakala stesen 2 pula mencatatkan purata peratusan kandungan lempung yang terendah iaitu sebanyak 7.55 ± 1.64%. Bagi purata peratusan kelodak di keenam-enam stesen pula adalah berjulat di antara 8.59% hingga 47.94% di mana stesen 5 menunjukkan nilai peratusan tertinggi iaitu 47.94 ± 10.47%, manakala nilai purata peratusan yang paling rendah dicatatkan oleh stesen 1 iaitu sebanyak 8.59 ± 10.02%. Purata bagi peratusan kandungan pasir pula adalah berjulat di antara 7.87% hingga 80.43%. Nilai tertinggi bagi purata peratusan kandungan pasir ini dicatatkan oleh stesen 2 iaitu sebanyak 80.43 ± 3.57% dan nilai terendahnya pula dicatatkan oleh stesen 6 iaitu sebanyak 7.87 ± 1.99%. Hasil yang diperoleh daripada data yang telah dianalisis menunjukkan bahawa stesen 1 dan stesen 6 terdiri daripada tekstur lempung, stesen 2 terdiri daripada tekstur pasir berlom sementara stesen 3 bertekstur lom berpasir. Manakala didapati stesen 4 dan 5 masing–masing mempunyai tekstur lom lempung dan lom lempung berkelodak. Jadual 1. Purata dan sisihan piawai bagi kandungan bahan organik, peratus pasir, kelodak, lempung dan tekstur tanah Stesen 1 (Melaka) 2 (Rasau) 3 (Bungor) 4 (Bungor) 5 (Melaka) 6 (Melaka) Ciri kimia tanah % Bahan organik 11.46 ± 2.23 2.68 ± 1.08 3.41 ± 0.14 3.70 ± 0.31 9.50 ± 2.10 9.81 ± 3.10 % Pasir 20.30 ± 9.79 80.43 ± 3.57 54.80 ± 6.28 35.39 ± 9.80 12.98 ± 1.94 7.87 ± 1.99 % Kelodak 8.59 ± 10.02 12.02 ± 1.97 27.21 ± 7.06 45.10 ± 8.33 47.94 ±10.47 33.32 ± 1.66 % Lempung 70.96 ± 19.35 7.55 ± 1.64 17.93 ± 0.73 19.52 ± 1.51 39.08 ± 10.43 58.81 ± 3.06

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

Nilai purata berserta sisihan piawai bagi setiap parameter kimia tanah di setiap stesen persampelan yang telah dianalisis ditunjukkan di dalam pada lampiran A, B dan C. Hasil yang didapati menunjukkan bahawa tanah di kawasan kajian adalah bersifat asid memandangkan nilai pH yang diperoleh bagi semua stesen persampelan adalah rendah. pH tanah Purata pH serta sisihan piawai bagi setiap stesen persampelan ditunjukkan di dalam Rajah 4. Nilai purata pH yang diperoleh adalah berada pada julat 3.36 hingga 3.72. Secara puratanya, tanah di stesen 1 mencatatkan bacaan bagi nilai pH yang tertinggi berbanding stesen cerapan yang lain iaitu 3.72 dengan sisihan piawainya 0.45. Tanah di stesen 4 pula mencatatkan nilai pH paling rendah iaitu 3.36 ± 0.18. Secara keseluruhannya, didapati bahawa kesemua tanah yang dikaji bagi setiap stesen persampelan adalah bersifat asid kerana purata pH yang rendah. Terdapat perbezaan yang signifikan bagi nilai pH di antara stesen 1 dan stesen 4. Keasidan tanah berpunca daripada humus atau bahan organik, tanah lempung alumino-silikat, hidrus oksida ferum dan aluminium, aluminium tukarganti, garam larut dan karbon dioksida [12]. Jadual 2. Purata dan sisihan piawai bagi pH, kekonduksian elektrik (EC) (µScm-1) dan KPK (cmolc/kg). Stesen 1 (Melaka) 2 (Rasau) 3 (Bungor) 4 (Bungor) 5 (Melaka) 6 (Melaka) pH 3.72± 0.45 3.57± 0.25 3.56 ± 0.05 3.36±0.18 3.60± 0.14 3.44 ± 0.08 Kekonduksian elektrik 2403 ± 122.8 2282.5 ± 9.6 2287.5 ± 63.4 2150±59.4 2262.5± 5.0 2267.5 ± 65 Kapasiti Pertukaran Kation 3.85 ± 0.81 2.85 ± 1.48 6.21 ± 1.67 7.91±0.26 7.67 ± 0.28 8.59 ± 1.11

Nilai purata pH yang didapati rendah bagi semua stesen kerana kebanyakannya terdiri daripada kawasan hutan dengan litupan 100%. Oleh itu, kawasan ini mengandungi banyak bahan organik dan humus hasil pereputan daun yang menyumbang keasidan tanah. Ini adalah kerana pereputan bahan organik terutamanya humus akan mengeluarkan asid organik dan asid mineral. Bahan organik tanah atau humus mengandungi kumpulan reaktif karboksil, fenolik dan amino yang mana ia boleh mengikat ion H+. Kumpulan yang tepu H+ tersebut bersifat seperti asid lemah dan H+ yang terikat secara kovalen akan terurai bergantung kepada penguraian asid yang terbentuk [12]. Keasidan tanah juga berlaku disebabkan oleh titisan air hujan yang lebat yang mengakibatkan kebanyakan garam yang terkandung di dalam tanah mengalami larut lesap. Kehilangan bes Na+, K+,Ca2+ dan Mg2+ ini digantikan oleh ion H+ dan Al3+. Kehadiran H+ dan Al3+ di tapak pertukaran kation inilah yang bertanggungjawab terhadap keasidan tanah [8]. Di samping itu, kawasan kajian yang juga terdiri daripada kawasan pertanian mengaplikasikan penggunaan baja sebagai sumber nutrien kepada tanaman. Kandungan nitrogen dan sulfur di dalam baja akan mengalami tindakbalas mikrobiologi. Nitrogen dioksidakan kepada ammonium, manakala sulfur dioksidakan kepada sulfur trioksida. Gas-gas ini masing-masing larut di dalam air untuk membentuk asid nitrik dan asid sulfat. yang kemudiannya terjerap ke dalam tanah [8]. Kekonduksian elektrik tanah Purata kekonduksian elektrik tanah yang diperoleh daripada setiap stesen adalah berada pada julat 2150.0 µScm-1 hingga 2403.0 µScm-1. Berdasarkan Rajah 4.5, didapati stesen 1 mencatatkan nilai kekonduksian elektrik yang tertinggi iaitu 2403.0 ± 122.8 µScm-1. Manakala stesen yang mencatatkan nilai kekonduksian yang paling rendah ialah stesen 4 iaitu 2150.0 ± 59.4 µScm-1. Analisis ANOVA yang dijalankan menunjukkan terdapat perbezaan yang bererti

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

pada aras keertian 5% bagi stesen 1 dengan kelima-lima stesen yang lain iaitu stesen 2, stesen 3, stesen 4, stesen 5 dan stesen 6. Manakala stesen 2 dan 3 menunjukkan perbezaan yang signifikan dengan stesen 4. Secara keseluruhannya, nilai kekonduksian elektrik yang dicatatkan oleh semua stesen cerapan adalah rendah. Ini menunjukkan kepekatan garam dalam tanah adalah rendah. Jadi, ia tidak menyekat pengambilan air oleh tumbuhan dan tumbuhan juga tidak menunjukkan simptom-simptom kekeringan. Oleh itu, tanah di kawasan kajian adalah sesuai untuk aktiviti pertanian berdasarkan indisis bagi kekonduksian elektrik tanah yang digunakan oleh MAFF (1988) [13] yang mana nilai kekonduksian yang diperoleh berada di bawah indeks 3 iaitu tidak merosakkan tumbuhan. Kapasiti pertukaran kation (KPK) Purata kapasiti pertukaran kation (KPK) bagi keenam-enam stesen ditunjukkan dalam Rajah 4.12 adalah berjulat di antara 2.85 hingga 8.59 cmolc/kg. Stesen 6 mencatatkan nilai KPK tertinggi berbanding stesen yang lain iaitu 8.59 ± 1.11 cmolc/kg, manakala stesen 2 pula mencatatkan nilai KPK yang paling rendah iaitu 2.85 ±1.48 cmolc/kg. Daripada ujian ANOVA dapat dinyatakan bahawa stesen 1 dan stesen 2 berbeza secara signifikan dengan keempat-empat stesen yang lain. Selain itu, stesen 3 menunjukkan perbezaan yang signifikan dengan stesen 1, stesen 2, stesen 4 dan stesen 6. Stesen 4 dan 6 pula mempunyai perbezaan yang signifikan dengan stesen 1, stesen 2 dan stesen 3, sementara stesen 5 berbeza secara bererti dengan stesen 1 dan stesen 2. Kapasiti pertukaran kation adalah paling tinggi di stesen 6 mungkin kerana tanahnya yang bertekstur lempung. Ini kerana lempung merupakan konstituen tanah yang paling halus serta mempunyai luas permukaan spesifik yang besar. Lebih-lebih lagi, lempung tanah terdiri daripada cas elektronegatif yang aktif secara kimia serta berupaya menarik kation yang bercas positif. Penentuan KPK ini adalah merupakan penggantian semua atau sebahagian daripada kation tukarganti pada kompleks pertukaran. Tanah lempung atau tanah organik mempunyai KPK yang tinggi berbanding tanah pasir atau tanah yang tidak begitu organik memandangkan keupayaannya yang kecil serta mudah hilang menerusi larut lesap. Koloid lempung yang bermuatan negatif membolehkan kation tertarik oleh partikel lempung dan diikat secara elektrostatik [11]. Kation bes tukarganti Ca2+, Mg2+, Na+ dan K+ boleh terlekat pada koloid tanah oleh cas elektrik dan boleh tertukarganti sesama sendiri atau dengan H+. Nilai purata KPK bagi stesen 6 adalah paling tinggi kerana kepekatan kation Al3+ tukarganti yang tinggi serta menyumbang kepada peningkatan KPK tanah. Korelasi antara parameter tanah dengan logam berat Jadual 2 menunjukkan terdapat perhubungan signifikan secara positif pada aras keertian 5% antara pH dengan logam kromium (Cr) dan korelasi signifikan positif yang bererti dengan logam mangan (Mn). Kandungan bahan organik tanah juga mempunyai perhubungan signifikan yang kuat dengan logam Cr, Fe dan Mn, sementara terdapat perhubungan signifikan yang lemah dengan logam Co dan Cu. Selain itu, peratusan kandungan pasir juga mempunyai perhubungan dengan logam yang dikaji walaupun perhubungan yang negatif. Peratusan pasir menunjukkan terdapatnya korelasi signifikan negatif yang kuat pada aras keertian 5% dengan logam Cr, Co, Cu and Fe. Sementara itu, ia juga mempunyai korelasi signifikan negatif yang lemah dengan logam Pb dan Zn. Jadual 3. Nilai korelasi di antara parameter tanah dengan kandungan logam berat (mg/kg) Cr Co Cu Cd Pb Fe Mn Zn pH 0.397* 0.186 0.016 -0.194 0.347 0.298 0.614* 0.441 % BOT 0.674* 0.404* 0.392* -0.178 0.304 0.698* 0.697* 0.543 % Pasir -0.650* -0.618* -0.698* 0.105 -0.450* -0.798* -0.371 -0.393* % Kelodak -0.058 0.267 0.261 0.056 0.486* 0.046 -0.327 0.219 %Lempung 0.620* 0.386* 0.461* -0.128 0.098 0.686* 0.540* 0.215 KE 0.308 -0.025 0.069 -0.039 0.012 0.278 0.596* 0.220 KPK -0.091 0.416* 0.422* 0.017 0.221 0.150 -0.461* -0.094

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

Aras keertian = 5% (*), KE, kekonduksian elektrik; KPK, kapasiti pertukaran kation. Bagi peratusan kandungan kelodak dalam tanah pula menunjukkan korelasi signifikan positif dengan logam Pb tetapi dalam perhubungan yang lemah. Sementara bagi peratusan kandungan lempung pula menunjukkan korelasi signifikan positif yang kuat pada aras keertian 5% dengan logam Cr, Fe dan Mn. Perhubungannya dengan logam Co dan Cu pula, menunjukkan korelasi signifikan positif yang lemah. Nilai kekonduksian elektrik pula hanya menunjukkan korelasi signifikan yang agak kuat dengan logam Mn. Kapasiti pertukaran kation (KPK) pula menunjukkan perhubungan signifikan yang lemah dengan logam Co dan Cu, manakala ia menunjukkan korelasi signifikan negatif yang lemah dengan logam Mn. Ini menunjukkan bahawa kandungan bahan organik, pasir, kelodak, lempung, pH, kekonduksian elektrik dan kapasiti [pertukaran kation mempunyai pengaruh ke atas pengayaan dan pengurangan jumlah logam berat di dalm tanah. Korelasi di antara logam berat dengan logam berat dalam tanah Berdasarkan Jadual 4, didapati Co mempunyai perhubungan signifikan yang positif dengan semua logam kecuali Mn dan Cd. Logam Co menunjukkan korelasi signifikan positif yang kuat dengan Cu (p<0.05, r = 0.5339), Cr (p<0.05, r = 0.6026), Fe (p< 0.05, r = 0.6316), Pb (p< 0.05, r = 0.6187), sementara ia mempunyai korelasi signifikan positif yang kurang kuat dengan Zn (p<0.05, r = 0.4135).

Jadual 4. Korelasi di antara logam berat dengan logam berat dalam tanah(µg/g). Co Cu Co 1.000 Cu 0.5339* 1.000 Cr 0.6026* 0.6182* Fe 0.6316* 0.5244* Mn 0.2675 0.1699 Pb 0.6187* 0.5058* Zn 0.4135* 0.2880 Cd -0.3695 -0.0306 Aras keertian = 0.05 (*) Cr 1.000 0.7014* 0.6858* 0.5977* 0.7365* -0.1316 Fe Mn Pb Zn Cd

1.000 0.6031* 0.4877* 0.5650* -0.1370

1.000 0.2120 0.5413* -0.2254

1.000 0.7339* -0.0002

1.000 0.1903

1.000

Logam Cu pula menunjukkan korelasi signifikan positif yang agak kuat dengan logam Cr (p<0.05, r = 0.6182), Fe (p<0.05, r = 0.5244) dan Pb (p<0.05, r = 0.5058). Selain itu, didapati logam Cr menunjukkan perhubungan signifikan positif yang kuat dengan logam Fe, Mn, Pb dan Zn yang mana nilai r masing-masing ialah 0.7014, 0.6858, 0.5977 dan 0.7365 pada aras keertian 5%. Logam Fe juga mempunyai perhubungan signifikan positif yang kuat dengan logam Mn dan Zn yang mana nilai r masing-masing ialah 0.6031 dan 0.5650. Manakala perhubungannya dengan logam Pb (p<0.05, r =0.4877) adalah signifikan positif yang lemah pada aras keertian 5%. Logam Mn pula mempunyai korelasi signifikan positif yang kuat dengan logam Zn (p<0.05, r = 0.5413). Sementara itu logam Pb menunjukkan korelasi signifikan positif yang kuat dengan Zn (p<0.05, r =0.7339). Manakala logam Cd pula tidak menunjukkan korelasi dengan mana-mana logam sama ada secara positif atau negatif pada aras keertian 5%. Kesimpulan Secara keseluruhannya, tanah di sekitar Tasik Chini boleh dikategorikan sebagai berasid berdasarkan nilai pH yang rendah bagi kesemua stesen persampelan. Ini kerana kawasan yang dikaji kebanyakannya terdiri daripada kawasan hutan dan pertanian yang mengandungi banyak bahan organik dan humus yang menyumbang kepada keasidan tanah. Dari segi tekstur pula, tanah di

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kawasan kajian boleh dikelaskan kepada beberapa kategori iaitu lempung, pasir berlom, lom berpasir, lom lempung dan lom lempung berkelodak. Selain itu, nilai kekonduksian elektrik yang diperoleh bagi setiap stesen persampelan adalah rendah iaitu di bawah indeks 3 yang tidak merosakkan tumbuhan (MAFF 1998). Oleh kerana kepekatannya yang rendah, maka ia tidak akan menyekat pengambilan air oleh tumbuhan dan tahap kesuburannya adalah tinggi. Kepekatan logam ferum adalah paling tinggi di dalam tanah yang dikaji berbanding logam berat yang lain memandangkan ia merupakan salah satu juzuk major di litosfera. Selain ferum, kepekatan logam mangan yang diperoleh juga adalah tinggi. Kepekatan kedua-dua jenis logam ini tinggi kerana tanah di kawasan kajian adalah bersifat asidik. Logam-logam berat ini berkemungkinan mempunyai potensi yang besar untuk mencemar tasik sekiranya langkah kawalan yang berkesan tidak dilaksanakan. Penghargaan Kajian ini dibiayai oleh gran penyelidikan IRPA no 09-02-02-0083-EA217 dan 09-02-02-0117EA294. Analisis XRF dijalankan di Makmal X-Ray, Program Geology, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia di bawah seliaan En. Abd. Hamid Othman. Rujukan 1. Abdulla, H. H. (1966). A study of the development of podzol profiles in Dovey forest. Tesis Ph.D Aberystwyth: University of Wales. 2. Archer, F. C., Hodgson, I. H. (1987). Total and extracatable trace element content of soils in England and Wales. Journal of Soil Science 38, 421-432. 3. Avery, B. W., Bascomb, C.L. (1982). Soil Survey Laboratory Methods. Soil Survey Technical Monograph No. 6. Harpenden. 4. Khairiah Jusoh (1987). Kajian Pencemaran Logam Berat Di Dalam Sistem Sungai Dengan Rujukan Khas Kepada Kawasan Perbandaran Kuala Lumpur. Tesis Sarjana Sains. Bangi: Universiti Kebangsaan Malaysia. 5. Massey, D.M., Windsor, G.W. (1967). Replication Glasshouse Crops Response Inst., hlm. 72. 6. Metson, A.J. (1956). Method of Chemical Analysis for Soil Survey samples. N.Z.D.S.I.R. Soil Bureau Bulletin no. 12 7. McLean, E.O. (1965). In Methods of Soil Analysis Pt 2, ed. C.A. Black, 978-997 8. Othman Yaacob (1982). Sains Tanah. Kuala Lumpur : Dewan Bahasa dan Pustaka. 9. Rowell, D.L. (1994). Soil science: methods and applications. London: Longman Rowell, Group UK Limited. 10. Shamshuddin, J. (1981). Asas Sains Tanah. Kuala Lumpur: Dewan Bahasa dan Pustaka. 11. Tan, K.H. (1994). Environmental Soil Science. Marcel Dekker, Inc., New York. 12. Havlin, J. L., Tisdale, S. L., Nelson, W. L., Beaton, J.D. (1999). Soil Fertility and Fertilizers. An Introduction to Nutrient Management. Prentice Hall, NY. 13. MAFF (1988). Agricultural Land Classification of England and Wales. Revised guidelines and criteria for grading the quality of agricultural land.

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ANALYTICAL APPLICATION OF FUNCTIONALIZED ZnS AS FLUORESCENCE LABEL FOR THE DETERMINATION OF PROTEINS Mustaffa Nawawi , Shemalah a/p Ramasundram* and Tee Shiau Foon Jabatan Kimia, Fakulti Sains, Universiti Teknologi Malaysia, 81310 UTM Skudai, Johor. E-mail address: musna@kimia.fs.utm.my.

Abstract The quantitative analysis of protein is essential in biochemistry and clinical medicine. The most sensitive quantitation of protein at this present is generally based on fluorescence enhancement on organic dyes determination. However, this organic fluorophores often suffer from photobleaching and low signal intensity. In order to overcome such problem, the study was carried out to investigate the possibility of employing the luminescent particle ZnS for quantitative analysis of protein. In this study, ZnS was capped with cysteine (functionalized ZnS), which renders the particles water soluble and biocompatible. Fluorescence studies showed at excitation wavelength λexc=233 nm, the maximum of functionalized ZnS peak occurred at 350 nm at pH 7. Further, general optimization procedure such as the effect of pH, temperature, concentration, reaction time of the functionalized ZnS binding with BSA (Bovine Serum Albumin) was conducted. A positive correlation with R2=0.987 was obtained between ZnS capped with cysteine binding with was obtained. The effect of interfering various metal ions and surfactant was subsequently performed in order to obtain the selectivity of the developed assay on the determination of BSA. Limit of detection of functionalized ZnS binding with BSA was 0.09 ppm. This developed method was successfully applied to the several types of protein such as egg albumin, lysozyme and amylase. The developed novel assay is simple, inexpensive, rapid and sensitive. Key words: Fluorescence, ZnS, protein

INTRODUCTION Proteins have long been recognized as biologically fundamental and medically important substance. International and industrial competition provided a healthy impetus to basic research and produced an explosive growth in fundamental understanding about protein molecules, which constituted the ‘stuff of life’ [1]. The development of novel assays for proteins is a basic requisite in both clinical and laboratory tests. The most frequently used approaches for the determination of protein is the ultraviolet and visible absorption spectroscopy, Lowry method [2], dye binding method like Bradford [3], Bromocresol green procedures [4], Bromophenol blue [5],. However, they all have some limitation in terms of sensitivity, selectivity, stability and simplicity. Disadvantages of the Lowry method include low sensitivity, poor selectivity and complexity. Due to this, fluorescence spectroscopy has become widely accepted as a modern method for the study of proteins structure, proteins biosynthesis and other biochemical problems. The characteristics of proteins fluorescence are dependent on structure of protein. The fluorescence of peptides is caused by the presence of the amino acids tyrosine, tryptophan and phenylalanine [6]. Labeling of biological molecules using fluorescent compounds is a common and very useful practice in biological science and biomedical science. The method of labeling proteins was introduced by Coon in 1941 when they demonstrated the use of antibody labeling with fluorescein [7]. Reviews

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on the application of fluorescent compound covalently bounded with protein have been reported by several studies [8, 9, 10]. Although most of the reported protein assays are based on the binding of the dyes or organic compounds with protein has been proven the most sensitive assay and very useful practice in biological science and biomedical science, however this organic fluorophores have characteristics that limit their effectiveness for such applications. These limitations included narrow excitation bands and broad emission bands with red spectral tails, which can cause simultaneous evaluation of several light-emitting probes problematic due to spectral overlap [11]. Moreover, the organic fluorophores often suffer from photobleaching, low signal intensities and random on/off light emission (blinking) [12]. Due to this, findings strongly suggested that colloidal semiconductor have the potential to overcome problems encountered by organic fluorophores in certain fluorescent labeling application by combining the advantages of high photobleaching threshold, good chemical stability and readily tunable spectral properties. Besides that, their resistance to photobleaching and high quantum yield in aqueous solution make them attractive for labeling functionalized biomolecules for fluorescent applications [11, 12, 13]. Recent advances in materials research have produced a new class of fluorescent labels by conjugating semiconductor with biorecognition molecules. These fluorophores will have key applications in biotechnology and bioengineering [14, 15, 16]. In this study, the new direction for the synthesis of ZnS capped with cysteine was employed for the binding with protein. This functionalized ZnS will be covalently link to proteins (BSA) for further studies.

EXPERIMENTAL Apparatus Perkin-Elmer Models LS-50B Luminescence Spectrometer was used for all fluorescence determination. The LS-50B employs a pulsed xenon source that produce a high output using a low voltage, 9.9 watts resulting in longer lamp life with minimal ozone and heat production. Photomultiplier tubes are employed as detection devices and LS-50B was connected to a computer for data processing. A pH meter Cyber-scan model equipped with a glass electrode combined was employed for the pH measurements. Reagents All the glassware used in this experimental work was acid washed. All the reagents were of analytical reagent grade without further purification. Doubly distilled water was used for the preparation of all solution and for all determinations. A stock Bovine Serum Albumin (BSA) purchased from Amersham Pharmacia Biotechnological solution was directly dissolved in 0.5 % of NaCl solution to prepare stock solution at a final concentration of 100 µg ml-1 and stored at 0-5oC. Procedure The simple procedure of synthesis ZnS capped cysteine was described by Kho et al. [78]. 1.970 g of L-cysteine was added to 50 mL of thoroughly degassed and N2 saturated 1.0 mol L-1 Tris buffer (tris hydroxymethyl amino methane). Next, 6.25 mL of ZnSO4 was added to the solution. Then, 25.0 mL of 0.4 mol L-1 Na2S.9H2O was dropped into the solution slowly follow by the mixed solution was stirred for 30 min. Finally, the colloidal solution was sealed and incubated at 47 oC bath water, followed 10 min of N2 to remove the excess sulfide. The purified ZnS capped cysteine powders were obtained through ethanol precipitation. In this procedure, 100 % cold ethanol (0 oC) was added drop wise in the presence of about 10 % (v/v) 1.0 M NaC2H3O2 (natrium acetate) until a white precipitate ZnS was formed. After the centrifugation for 15 min, the pellet was redissolved in 1.0 M Tris buffer. At least two rounds of ethanol precipitation were necessary to remove most of the excess

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components. To produce the final powder, the ethanol precipitated and redissolved sample was dried overnight. Finally, these powder products were stored at 4 oC. The functionalized ZnS were used to detect the BSA (protein) .The following procedure was adopted. Into a 5 ml volumetric flask, 100 µL of buffer solution 1500 µL of functionalized ZnS appropriates of protein sample were added and diluted to 5.0 mL of distilled water. Then, fluorescence excitation spectrum was recorded from 200 nm to 600 nm and fluorescence emission spectrum was scanned with a fixed excitation at λex =233 nm. Wavelength of emission spectrum at λem= 350 nm and the intensity of maximum peaks were recorded.

RESULTS AND DISCUSSIONS

Standard Calibration of Absorbance Properties on Functionalized ZnS Binding with Bovine Serum Albumin (BSA)

Figure 1 . UV absorbance spectra of functionalized ZnS binding with BSA concentration 0 ppm to 35 ppm. [Functionalized ZnS colloids] = 4x10-4 mol L-1. The UV absorbance spectra of ZnS capped L-cysteine (functionalized ZnS) and BSA was obtained under the room temperature using the Shimadzu UV-visble spectrophotometer-1601PC. The UV spectra of functionalized ZnS in the presences of various concentration of BSA are shown in Figure 1. In this study, λmax around 280 nm was used to quantify the interaction of functionalized ZnS in presences of BSA. Based on the As seen in Figure 1, the absorbance of functionalized ZnS increased with increasing amount of concentration of BSA from 5 ppm to 35 ppm. Moreover, based on the data obtained from the series of different concentration of BSA, a plot of absorbance versus concentration was obtained with R2 value 0.9755 and y-intercept with y = 0.0009x + 0.0105.

Fluorescence Properties of ZnS Capped L–Cysteine (Functionalized ZnS) binding with Bovine Serum Albumin (BSA)

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Figure 2: The emission spectra of functionalized ZnS in absences and presences of BSA under the room temperature. [BSA] = 15 ppm, [Functionalized ZnS colloids] = 4x10-4 mol L-1. The fluorescence properties of the interaction between ZnS capped L-cysteine (functionalized ZnS) and BSA was conducted under the room temperature. Figure 2 showed the behavior of emission spectra of functionalized ZnS binding with BSA. The fluorescence properties of emission wavelength were at 357 nm wavelength when the excitation peak was at 233 nm wavelength as shown in Figure 2. In this study, the fluorescence intensity of functionalized ZnS was significantly enhanced from fluorescence intensity of 394.00 to 720.37 in the presences of BSA 15 ppm. The emission spectra above indicated that BSA has the best enhancing effect on functionalized ZnS. Therefore, the enhancement of emission intensity of the mixture of functionalized ZnS and BSA suggested that interaction of functionalized ZnS and BSA had taken place. Various previous studies have been proposed to explain these phenomena. According to Kho et al. [17], it can be deduced that the L-cysteine has been covalently linked to the surface of ZnS and formed the L-cysteine capped ZnS particles [17] as shown in Figure 3 (a). Further L-cysteine binds to a Zn atom, the polar carboxyl group is available for covalent coupling to various biomolecules such protein and nucleic acids by cross-linking to active amine group (Figure 3 (b) [18].
C H 2 C H (N H 2 )C O O H
s
O
s

CH 2 CH(NH 2)COOH
s

s

ZnS
s

CH2CH(NH2)COOH

Figure 3: Schematic of (a) ZnS capped cysteine and (b) functionalized ZnS conjugated to protein

Optimizations Procedures on the Fluorescence Properties of Functionalized ZnS Binding BSA Chemical variables such as effect of pH, buffer, concentration, temperature and reaction time were performed and optimized to obtain the optimum, best measurement conditions and stable

638

CH2CH(NH2)COOH

s

ZnS
s

s

CH2CH(NH2)CN
H

Protein

CH2CH(NH2)COOH

C H 2 C H (N H 2 )C O O H

CH 2 CH(NH 2 )COOH

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fluorescent signal. The effect of the pH on the fluorescence intensity of functionalized ZnS in absence and presence of BSA was studied in the pH range of 2.0 to 11.0. In an attempt to see the relationship between fluorescence intensity and pH, the fluorescence intensity was plotted as a function of pH value in Figure 4. It was observed that in the lower pH range pH 2 to 5 the fluorescence intensity increased with an increase of pH. In the higher value range of pH 9-11, the fluorescence intensity is decreased linearly. Hence, it is widely accepted that the changes of pH values affects the sulfhydrylcontaining amino acid, cysteine coordinating to Zn2+ ions, which cause the different pH growth process of ZnS capped cysteine [19].In this study, the optimum pH of functionalized ZnS binding with BSA determination was selected at pH 7. Labeling protein should be carried out in a well-buffered system at a pH that is optimal for the reaction. Additionally, the effect of buffer nature on the fluorescence intensity was examined. In this study, there are several buffer solutions (Tris–HCl, NaAc–HCl, BRB–HCl) were selected. The result indicated that the sensitivity of the protein determination is higher in Tris–HCl (0.1 mol L−1) buffer solution compared to NaAc and BRB buffer.
800 700 Fluorescence Intensity 600 500 400 300 200 100 0 0 2 4 6 pH 8 10 12

ZnS

ZnS-BSA

Figure 4: Effect of pH for functionalized ZnS in absences and presences of BSA at room temperature.[BSA] = 15 ppm, [Functionalized ZnS colloids] = 4x10-4 mol L-1. The emission fluorescence intensity was measured over the concentration of functionalized ZnS in the range of 1x10-4 mol L-1 to 8x10-4 mol L-1 to investigate the effect of concentration for BSA determination. Based on the result obtained, graph fluorescence intensity versus concentration of functionalized ZnS in presences of BSA was plotted (Figure 5). The graph showed that the fluorescence intensity of functionalized ZnS increased linearly with the concentration and reached the optimum range at 4x10-4 to 6x10-4 mol L-1 in presences of BSA. However, further increase in the concentration of functionalized ZnS caused the fluorescence intensity decreased. This may due to functionalized ZnS tends to precipitated when the concentration of colloidal was more than 7x10-4 mol L-1. Based on result obtained it is appeared that the concentration of functionalized ZnS colloidal 4x10-4 mol L-1 was selected as optimum concentration for the BSA determination. Optimization of the temperature effect is one of the important tools in protein analysis. The binding properties between functionalized ZnS with BSA were determined at different temperatures (10, 15, 20, 25, 30, 35, 40 0C). The data appeared that the increasing temperature effect showed a minimal effect on the fluorescence intensity. It was illustrated that the value of the binding constant was the greatest at 25 0C. The effect of reaction time on functionalized ZnS binding with BSA was conducted under the room temperature. It was found that the binding between functionalized ZnS with BSA proceeds rapidly at room temperature and the intensity reached the constant value within 20 min and remained constant for 5 hours. Due to this, the reaction of functionalized ZnS and BSA was carried out for 20 min and all the measurement were made within 5 hours for further studies.

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800 700 Fluorescence Intensity 600 500 400 300 200 100 0 0 2 4
4

6

8

10

Concentration (10 - mol/L )

Figure 5: Effect of functionalized ZnS concentration on the fluorescence intensity in the presences of BSA. pH 6, [BSA] = 15 ppm, [Functionalized ZnS colloids] = 4x10-4 mol L-1. Standard Calibration Curves for the Quantitative Analysis of Functionalized ZnS Binding with BSA

1200 1000

y = 21.886x + 456.05 R2 = 0.9805

F lu o re s c e n c e In t e n s ity

800 600 400 200 0 0 5 10 15 20 25 Concentration (ppm)

(a) (b) Figure 6: (a)Emission spectra of functionalized ZnS with increasing concentration of BSA from 2.5 ppm to 20 ppm and (b) calibration curves for functionalized ZnS binding with various concentration of BSA under optimum condition pH 7, [Functionalized ZnS colloids] = 4x10-4 mol L-1. Under optimum parameters, a study was made of the relationship between emission spectra and various concentration of BSA for the quantitative analysis of BSA. Figure 4.11 showed the emission spectra of functionalized ZnS with various concentration of BSA. As a result, it was found out that the emission maxima of the fluorescence spectra of functionalized ZnS binding with BSA system are similar to that of functionalized ZnS but the intensities are significantly enhanced (Figure 6a). Thus, a small blue shift of the emission spectra was observed from the wavelength of functionalized ZnS at 357 nm to 350 nm for functionalized ZnS binding with BSA. In brief, the data indicated that BSA has the best enhancing effect on functionalized ZnS system for the quantitative analysis of proteins. Based on data obtained, the calibration graphs for BSA were constructed from

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the result obtained under the optimal condition. Figure 6b showed the plot of intensity versus concentration (ppm) which is linear graph with (R2 = 0.9805, n = 12). It shows that the range of linearity was found to be from 0.2 ppm to 22.5 ppm of BSA.

Method Validation for the Quantitative Analysis of BSA Linearity, Precision, Limit of Detection The calibration graph of fluorescence intensity functionalized ZnS with increasing of BSA concentration expressed in ppm was found to be linear in the range 0.4-22.5 ppm with R2 = 0.9805 and y-intercept is y = 21.866x + 456.05. The reproducibility of the developed method calculated from six independent runs of 15 ppm of the BSA produced the relative standard deviation (RSD) of the system is 1.7 %. In order to identify the sensitivity of the develop method, the study of limit of detection was carried out under optimum condition. The 3σ of limit detection was measured from blank measurement for BSA was 0.09 ppm. Therefore, it can be concluded that, this method have high sensitivity, high accuracy and a wider linear range. Salt Dependence Studies on Functionalized ZnS Binding with BSA Small molecules are bound to macromolecule by four binding modes such as H-bond, van der Waals, electrostatic and hydrophobic interactions. The study of the effect of salt dependence of functionalized ZnS binding with BSA on fluorescence intensity of emission has led to some interesting results. As shown in Figure 4.14 it is obvious that in the absence of BSA, the addition of NaCl to colloidal had an insignificant effect on the fluorescence intensity. However, in the presence of BSA the fluorescence intensity increased with increasing of salt concentration (Figure 7). This result indicated that the binding modes between the reagent and BSA might be due to hydrophobic binding because of the presences of the nonpolar group ZnS capped cysteine.

1000 900 800 Fluorescence Intensity 700 600 500 400 300 200 100 0 0 0.1 0.2 0.3 0.4 0.5

1 2

0.6

Concentration of NaCl (mol/L)

Figure 7: Effect of the concentration of NaCl solution on the fluorescence intensity in the absence (curve 1) and presence (curve 2) of BSA and functionalized colloidal ZnS. [BSA] = 15 ppm, [Functionalized ZnS colloids] = 4x10-4 mol L-1.

Effect of the Foreign Substances on Functionalized ZnS Binding with BSA Table 1: Effect of the foreign substances on the fluorescence intensity of functionalized ZnS binding with BSA 15 ppm. Substances Concentration (ppm) Quenching/Enhancement (%)

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Cys Gly Ala Fe (II) Fe (III) Ca2+ Cu2+ NH4+ Mg2+ Ni2+ Cd2+ Glucose Sucrose Starch Urea EDTA

10 30 20 10 10 30 10 30 30 40 10 50 50 50 40 30

-8.9 -9.1 -7.7 -8.6 -66.6 -7.8 -26.5 -8.2 -7.6 -7.0 -7.4 -7.0 -3.7 -5.8 -9.8 -6.4

In order to test the selectivity of the method for the determination of BSA, the effect of several nonprotein compounds and metals are successfully treated with functionalized ZnS binding with BSA 15 ppm to reduce their concentration below the maximum allowable limits for the respective non-protein substances. The results are given in terms of the percentage-measured value in the presences of each non-protein compound in the Table 1. Based on Table 1, we can see that interference of ions such as Fe3+, Fe2+ and Cu2+ only can be allowed at very low concentrations. However , the concentration of these interference ions are very lower in real biological samples. Futher, ion NH4+, Mg2+, Ni2+, Ca2+, EDTA and urea did not interfere when present at low concentration but the emission intensity was suppressed slightly by about 7.0-9.4 % when ion concentration was increased to 40 ppm. Besides, glucose, sucrose and starch can be allowed at higher concentration because of minimal effect on functionalized ZnS binding with BSA system.

Standard Calibration of Proteins Binding with Functionalized ZnS Fluorescence studies were carried out to observe the behaviour between functionalized ZnS and different type of proteins such as BSA, egg albumin, lysozyme and amylase under the optimum condition. Fluorescence studies were carried out to observe the behaviour between functionalized ZnS and different type of proteins such as BSA, egg albumin, lysozyme and amylase under the optimum condition. The equations of fluorescence intensity against the concentration of several proteins, linear range and correlation coefficients are listed in Table 2. A satisfactory linear relationship with high regression coefficient and a wide linear range are obtained. Table 2 summarized the results obtained from the experiments carried out on the various compound of proteins. The result indicated that various possibilities: either different proteins have different isoelectric points or the weight, size and shape of the molecules are also different, so the fluorescence intensity for various proteins is different [20]. Table 2: Analytical parameter of various proteins binding with functionalized ZnS under optimum condition.

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Proteins

Linear range Regression equation (ppm) 0.2-22.5 0.2-20 0.2-10 0.2-7.5 y = 21.886x+456.05 y =21.911x+479.13 y = 45.04x+501.21 y = 51.04x+495.42

pI

R2

BSA Egg albumin Lysozyme Amylase

4.8-4.9 4.6-4.7 11.0-11.2 8.0-9.0

0.9805 0.9367 0.9851 0.9877

CONCLUSIONS The fluorescence method of functionalized ZnS binding with BSA has been successfully developed. The results have lead to some interesting phenomena. An optimum pH 7, temperature 25 0 C and reaction time 20 min was obtained for BSA determination. Besides, under optimum condition, the fluorescence intensity is proportional to the concentration of BSA in the linear range of 0.4 ppm to 22.5 ppm. The corresponding detection limit is 0.09 ppm. In brief, the application of functionalized ZnS binding with BSA as fluorescence probe for protein leads to a particularly sensitive, stable, simple and selective method. ACKNOWLEDGEMENTS This work was supported by the Chemistry Department Faculty of Science and PTP (School of Postgraduate) OFUniversity Teknologi Malaysia, Skudai Johor Bahru for the financial support.

REFERENCES 1. Robson, B. and Garnier, J. Introduction to Proteins and Proteins Engineering. U.S.A.: Elsevier Science Publisher. 1-5; 1986. 2. Lowry,O.H., N.J.Rosebrough, A.L. Farr and R.J.Randall.(1951), “Protien Mesurement with Folin Phenol Reagent”, J.BioChem,193.265-275. 3. Braford, M.M.(1976), “A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein Binding”, Anal Biochem.72.248-254. 4. Basil T.Doumas, W. Ardwatson and Homer G. Biggs(1971) “Application Standard and Measurement of Serum Albumin With Bromocresol Green” Clin.Chim. Acta 31,87. 5. Klaus Jung, Erika Nickel, Monika Dergande (1990) “A Microalbumiria Assay using Bromophenol Blue” Clin. Chim. Acta 187,163-172. 6. White, C. E. and Argauer, R. J. Fluorescence Analysis. New York: Marcel Dekker Inc. 176178; 1970. 7. Lakowicz, J. R., Principles of Fluorescence Spectroscopy. New York: Plenum Press. 342,347; 1990. 8. Jiang, C. Q., Gao, M. X. and He, Z. X. (2002), “ Study of the Interaction Terazosin and Serum Albumin Synchronous Fluorescence Determination of Terazosin” Anal. Chim. Acta., 452. 185-189. 9. Li, D. H., Yang, H. H., Zhu, Q. Z. and Xu, J. G. (1999), “Fluorimetric Determination of Albumin and Globulin in Human Serum Using Tetra-Substituted Sulphonated Aluminium Phthalocyanine”, Anal. Chim. Acta., 401, 185-189.

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Jiang, C. Q. and Li, L.Y. (2004), “Spectrofluorimetric Determination of Human Serum Albumin Using a Doxycycline –Europium Probe”, Anal. Chim. Acta. 506, 171-175. 11. Bruchez, M. J, Moronne, M., Gin, P., Weiss, S. and Alivisatos, A. P. (1998), “Semiconductor Nanocrystals as Fluorescent Biological Labels” Science 281, 2013-2016. 12. Wang, L. Y., Bian, G. R., Xia, T. T. and Chen, H. Q. (2005) “Direct Fluorimetric Determination of γ-Globulin in Human Serum with Organic Nanoparticles Biosensor” Spectrochim. Acta A., 61 129-133. 13. Dannhauser, T., O’Neil, M., Johansson, K., Whitten, D. and McLendon, G. (1986), “Photophysics of Quantized Colloidal Semiconductor Dramatic Luminescence Enhancement by Binding of Simple Amines”, J. Phys. Chem. ,90, 6074-6076. 14. Mattoussi H., Mauro J. M., Goldman E. R., Anderson G. P., Sundar V. C., Mikulec F. V. and Bawendi M. G. (2000), “Self-assembly of CdSe-ZnS Quantum Dot Bioconjugates Using an Engineered Recombinant Protein. J. Am. Chem. Soc., 122, 12142-12150. 15. Gerion, D., Pinaud, F., Williams, S. C., Parak, W. J., Zanchet, D., Weiss, S. and Alivisatos, A. P. (2001), “Synthesis and Properties of Biocompatible Water-Soluble Silica-Coated CdSe/ZnS Semiconductor Quantum Dots”, J. Phys. Chem. B., 105, 8861-8871. 16. Parak, W. J., Gerion, D., Zanchet, D., Woerz, A. S., Pellegrino, T., Micheel, C., Williams, S. C., Seitz, M., Bruehl, R. E., Bryant, Z., Bustamante, C., Bertozzi, C. R. and Alivisatos, A. P. (2002), “Conjugation of DNA to Silanized Colloidal Semiconductor Nanocrystalline Quantum Dots” Chem. Mater., 14, 2113-2119. 17. Kho, R., Claudia, L., Mart´ýnez, T. and Mehra, R. K. (2004), “A Simple Colloidal Synthesis for Gram-Quantity Production of Water-Soluble ZnS Nanocrystal Powders” J. Colloid Interface Sci., 227 ,561–566. 18. Li, Y. Z., Chen, J. L., Zhu, C. Q., Wang, L., Zhao, D. H., Zhuo, S. J. and Wu, Y. Q. (2004), “Preparation and Application of Cysteine-Capped ZnS Nanoparticles as Fluorescence Probe in the Determination of Nucleic Acids” Spectrochim. Acta A., 60, 1719-1724. 19. Hao, E. C., Sun, Y. P., Yang, B., Zhang, X., Liu, J. M. and Shen, J. C., (1998), “Synthesis and Photophysical Properties of ZnS Colloidal Particles Doped with Silver” J .Colloidal Interface Sci., 204, 369-373. 20. Zhong, H., Wang, K. and Chen, H. Y. (2004), “ Protein Analysis with Tetra Substituted Sulfonated Cobalt Phthalocyanine by the Technique of Rayleigh Light Scattering”Anal siochem. 330, 37-42.

10.

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MINERALIZATION STUDIES ON PARAQUAT AND MALATHION USING TiO2/ZnO BASED PHOTOCATALYST Rusmidah Ali and Siti Habsah Hassan Chemistry Department, Faculty of Science, Universiti Teknologi Malaysia, 81310 Skudai, Johor Bahru, Malaysia. Abstract− Paraquat is an herbicide and malathion is an insecticide. Both pesticides always pollute our water system. A lot of efforts are carried out to treat of the polluted water. The newest technology proposed is being using photocatalyst. In this study, ZnO and TiO2 are used as photocatalysts to degrade the pesticide in the presence of UV light (λ=354 nm). The photodegradation rate was measured by using UV-Visible spectrophotometer and TOC analyzer. In this experiment, malathion showed the absorption peak at λ=210 nm while for paraquat is at λ=258 nm. The best coupled photocatalyst for degrading malathion solution is ZnO/TiO2 with % weight ratio 1:0.05 and the best coupled photocatalyst in degrading paraquat solution is TiO2/ZnO with % weight ratio 1:0.03. The result shows that Fe2+ ion present in the solution is performing better than Fe3+ ion present as a dopant. The optimum photocatalyst calcinations temperature degrading paraquat and malathion are 550°C for TiO2 and 500°C for ZnO. The physical properties of the best catalyst were characterized using SEM, XRD, UV-Vis-NIR spectrophotometer and elipsometer. By increasing calcinations temperature up to 600°C, the transformation from TiO2 anatase to rutile phase occurs while the increment of the intensity of ZnO catalyst was observed indicates that, the quality of ZnO wurtzite crystal improved. The thicknesses for ZnO, ZnO/TiO2 00.05 and ZnO/TiO2 doping with Fe3+ thin film are 130.57 nm, 150.68 nm and 153.84 nm respectively. The band gap energy values measured using UV-Vis NIR are in the range of 2.95 – 3.09 eV. Abstrak – Parakuat adalah sejenis racun rumpai manakala malation pula adalah racun serangga. Kedua-dua pesitisid ini kian mencemarkan system akuatik. Banyak usaha telah dijalankan masa kini untuk merawat air tercemar. Antara kaedah yang terbaru adalah teknik pemangkin foto. Dalam kajian ini, mangkinfoto TiO2 dan ZnO telah digunakan untuk mendegradasi dan memineralisasikan larutan racun perosak dengan kehadiran sinaran ultralembayung (λ=354 nm). Kadar fotodegradasi diukur dengan menggunakan Spektrofotometer Ultralembayung-Nampak dan analisis TOC. Malation menunjukkan serapan pada λ=210 nm manakala parakuat pada λ=258 nm. Pasangan mangkin yang terbaik mendegradasi larutan malation adalah TiO2/ZnO dengan nisbah % berat 1:0.05 manakala mangkin yang terbaik untuk mendegradasi larutan parakuat adalah pasangan mangkin TiO2/ZnO dengan nisbah % berat 1:0.03. Kehadiran dopan merencatkan tindak balas fotodegradasi manakala penambahan ion logam meningkatkan peratus fotodegradasi. Mangkin TiO2 yang dikalsin pada suhu 550°C dan mangkin ZnO yang dikalsinkan pada suhu 500°C memberikan peratus degradasi yang paling tinggi. Pencirian sifat fizik mangkin dikaji dengan menggunakan SEM, XRD, UV-Vis-NIR spektrofotometer dan elipsometer. Apabila suhu pengkalsinan dinaikkan ke 600°C, fasa anatas TiO2 telah bertukar kepada fasa rutil. Sebaliknya % intensiti puncak ZnO didapati bertambah dengan pertambahan suhu pengkalsinan. Ini menunjukkan kualiti kristal wurzit ZnO bertambah baik. Ketebalan saput tipis bagi ZnO, ZnO/TiO2 00.05 dan ZnO/TiO2 didop dengan Fe3+ ialah masingmasing 130.57 nm, 150.68 nm 153.84 nm. Nilai Ebg saput tipis ZnO adalah dalam lingkungan 2.95 – 3.09 eV. Keywords: Paraquat, Malathion, Photodegradation, Mineralization, ZnO, TiO2

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Introduction Paraquat and malathion can be introduced into aquatic environments especially in the river through intentional application (controlling of aquatic weeds, algae, fish or unwanted invertebrates), aerial drift, runoff from agricultural applications or runoff from accidental release. Rivers are directly linked to estuaries, coasts and open seas where the pollutants can be transported by the flow, and contact with the atmosphere through the volatilization [1]. Several methods were used in treating wastewater such as coagulation, photodegradation, ozonation, biological treatment, filteration, etc. The advantages of using photodegradation are no sludge is produced and foul odors are greatly reduced, safety application of the reactions being performed at atmospheric pressure and near ambient temperature and requires only dissolved oxygen in a water [2].The molecular orbital of semiconductors has a band structure that consult highest occupied valence band and lowest unoccupied valence band separated by band gap energy, Ebg. When the semiconductor is illuminated with ultra violet light with photon energy greater or equal to the band gap energy of the semiconductor, valence band electrons are excited to the conduction band leaving a hole behind (refer to Equation 1). TiO2 anatase band gap energy is 3.2 eV while. ZnO band gap energy is 3.17 eV. Superoxide anion radicals (•O2-) and hydroxyl radicals (•OH) are being generated (Equation 2-3) form in aqueous medium that responsible to accelerate the oxidation of pollutants [3, 4]. The efficiency of the photocatalysts activity can be enhanced by coupled ZnO /TiO2 semiconductor [5]. Semiconductor + hν e- + h+ (1) O2 + e•O2(2) H2O + h+ •OH + H+ (3) OH- + h+ •OH (4) •OH + H+ •OH2 (5) This research will cover the degradation reaction of paraquat and malathion by using single and couple semiconductor. The best ratio of TiO2/ZnO coupled photocatalyst for both pesticides will be doped with Fe3+ ions. The photocatalyst will be characterized further using X-Ray Diffraction (XRD) and Scanning Electron Microscopy (SEM) technique. The photodegradation will be carried out in the presence of UV-light and monitored by UV-Visible spectroscopy and TOC analyzer. Experimental Reagents and Chemicals The commercial paraquat dichloride “Gramoxone PP910” with active ingredients is 25 % w/w was purchased from CCMB Agrochemical Sdn. Bhd. Shah Alam. The standard paraquat (1,1’-dimethyl4,4’-bipyridylium dichloride) was purchased from Reference Material for Residue Analysis, Germany, malathion (active ingredients about 57 % w/w), ferric nitrate nonahydrate (Fe(NO3)3.9H2O and ferrous sulfate heptahydrate (FeSO4.7H2O) were obtained from GCE (Goodrich Chemical and Emory). TiO2 powder (99 % anatase, sigma), titanium tetraisopropoxide (TTIP), ethanol (absolute ≥ 99 %) and polyethylene glycol 2000 (PEG 2000), were obtained from Fluka Chemika while diethanolamine (DEA) from Riedel de Haen AG. Zinc acetate (Zn(OAc)2) is obtained from Analar (BDH), and sodium hydroxide (NaOH) from Ashland Chemical and 2-propanol from J.T Baker. Apparatus Photocatalytic tests were performed in a batch photoreactor of 1000 mL made of pyrex glass cylinder. The calcination of the photocatalyst was carried out in the furnace Nabertherm L3/S17 model. Ultra violet lamp (Yamato, λ=354 nm, 6 W, 100 V) was used as a UV light sources with the support of Teletron step down transformer (Model TSD 100 W). The instrumentations for monitoring and characterizing the samples are Perkin Elmer UV-Visible spectrophotometer, Shimadzu (TOC 500) Total Organic Carbon Analyzer, Siemens D5000 X-Ray diffractometer (XRD) equipped with Cu-Kα

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(λ=1.54 Å) radiation, Scanning Electron Microscopy (SEM Phillips XL 40), Ellipsometer Geatner and Shimadzu UV-3101PC UV-Vis-NIR Spectrophotometer. Preparation of TiO2 sol-gel Polyethylene glycol (PEG 2000) (6 g) was dissolved in ethanol (600 mL) in a 1 L volumetric flask. The solution was stirred continuously until PEG was completely dissolved before 31.8 g diethanolamine (DEA) was added to the solution followed by 85.2 g of titanium(IV) isopropoxide (TTIP). The solution was left for a few minutes before 5.4 mL deionized water was added. Preparation of ZnO sol-gel Zinc acetate (0.5 g) was dissolved in 2-propanol (100 mL) in a volumetric flask at 50-60oC and stirred continuously. At the same time, sodium hydroxide (NaOH) (10 mg) was dissolved in 2-propanol (100 mL) and stirred at the same temperature.. This two solution were mix together and continuously stirred in ice bath (at 0oC), then the solution was kept in water bath at room temperature for an hour. Thin Film Preparation The thin film was coated on support using the dip-withdraw method by repeating for five times. The support used in this study is a microscope slide glass (76 x 26 x 2 mm). The slides were calcined in the furnace whereby the temperature was elevated at 1°C min-1 up to 550°C for TiO2 photocatalyst and 2°C min-1 up to 500°C for ZnO and kept at this temperature for an hour. Photocatalytic measurement Photocatalytic study of TiO2 and ZnO thin film was examined in degrading 200 mL pesticide solutions (1 x 10-4 M paraquat and 2 x 10-4 M malathion) under various experimental conditions. This photocatalytic reaction was carried out in a glass reactor and irradiated with UV lamp for 4 hours in the presence of photocatalyst. An aliquot (4 mL) was taken at specific time intervals. These samples were analyzed using UV-Vis spectrophotometer and Total Organic Carbon Analyzer in order to measure the percentage of degradation and mineralization respectively. Photodegradation using TiO2, ZnO and a mixture of TiO2/ZnO thin film Seventeen microscopes slide glasses containing TiO2, ZnO and a mixture of TiO2/ZnO (ratio 1:≤ 0.08) thin film as shown in Table 1 were used in paraquat and malathion degradation. Table 1: Mixture ratios between TiO2: ZnO (≤ 5%) and ZnO: TiO2 (≤ 8%) ZnO sol- gel Total sol-gel volume % weight ratio TiO2 sol-gel (mL) (mL) (mL) TiO2:ZnO (g) 1:0.05 30 43.75 73.75 1:0.03 40 35 75 1:0.01 60 17.5 77.5 0.08:1 80 0.22 80.22 0.05:1 80 0.14 80.14 0.03:1 80 0.07 80.07 0.01:1 80 0.03 80.03 Photodegradation using the best photocatalyst doped with Fe3+ Two best photocatalyst each for paraquat and malathion was doped with Fe3+ (ratio 1:0.005). 0.05062 g and 0.0035 g Fe(NO3)3.9H2O containing Fe3+ was weighted and added into 75 mL of the best

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TiO2/ZnO and ZnO/TiO2 sol-gel respectively and stirred. A clean microscope slides were dipped into the sol-gel and withdrew gently in a constant speed for five times. Photodegradation using the best photocatalyst with Fe2+ ion dissolved into working solution 20 mL of 55.84 ppm Fe2+ (9.998 x 10-5 M) ion solution was pipetted into 500 mL volumetric flask together with 200 mL pesticides and diluted with deionized water to make working solution. The 200 mL working solution was place in a pyrex reaction cell and was treated as stated earlier in photocatalytic measurement. Effect of photocatalyst calcination temperature on photodegradation process Two best photocatalyst for each paraquat and malathion were calcined at different temperatures as shown in a Table 2. The photocatalytic measurement was carried out as stated earlier. Table 2: Calcination temperatures of the best photocatalyst Calcination temperature (°C) Number of slide ZT03 (paraquat) TZ05 (malathion) 2 600 2 200 TZ03: The best photocatalyst for paraquat, TiO2 : 3 % ZnO ZT05: The best photocatalyst for malathion, ZnO : 5 % TiO2 Effect of support on photodegradation process Prepared photocatalyst powder was weighted with the same amount of thin film coated onto the slide glass. Photocatalytic measurement was carried out as stated before using thin film and prepared powder of the best photocatalyst. Results and Discussions Calibration of total organic carbon A linear calibration curve was matched with UV absorption value in the same range in order to determine the suitable concentration working solution for further degradation process. The suitable concentration of formulated paraquat is 1 x 10-4 M (29.52 ppm) with UV absorption value, 1.363 and for formulated malathion, the suitable concentration is 2 x 10-4 M (59.89 ppm) with UV absorption value, 2.135. Effect of TiO2 thin film and commercial TiO2 on paraquat and malathion photodegradation Degradation and mineralization of paraquat with commercial TiO2 was higher than TiO2 thin film as shown in Figure 1 and Figure 2. 77.67 % paraquat was mineralized by using commercial TiO2 while only 42.21 % was mineralized using prepared thin film. Commercial TiO2 was performed better in which 58.57 % malathion was degraded compared to 50.09 % using TiO2 thin film after 4 hour reaction. Mineralization process using commercial TiO2 was performed better than TiO2 thin film through out the reaction time due to the fact that commercial TiO2 had a pure anatase crystal structure with high active surface area [6]. While the thin film catalysts prepared from sol-gel method does not have a perfect lattice like the commercial one, gave detrimental affect on their photocatalytic activity [7]. Even though the photocatalytic activity of commercial TiO2 is better than prepared thin film, the 600 200

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entire research was focused on the thin film photocatalyst in order to study the synergy effect of ZnO and TiO2 semiconductor in degrading and mineralizing paraquat and malathion solution.
100 % degradation 80 60 40 20 0 0 50 100 150 200 250 UV Illuminated time (min)

PT1

PT2

MT1

MT2

Figure 1: The % degradation of paraquat (P) and malathion (M) (PT1: commercial TiO2 in paraquat, MT1: commercial TiO2 in malathion, PT2: TiO2 thin film in paraquat and MT2: TiO2 thin film in malathion)
100 80 60 40 20 0 0 50 100 150 200 250 UV illuminated time (min) PT 1 PT 2 MT 1 MT 2

Figure 2: The % mineralization of paraquat (P) and malathion (M) (PT1: commercial TiO2 in paraquat, MT1: commercial TiO2 in malathion, PT2: TiO2 thin film in paraquat and MT2: TiO2 thin film in malathion) Effect of single TiO2, ZnO and coupled TiO2/ZnO semiconductor on paraquat and malathion photodegradation The degradation and mineralization of paraquat were higher in the presence of TiO2 photocatalyst than ZnO as shown in Figure 3. By modifying the surface properties of TiO2 with ≤ 5% ZnO, significantly enhances the rate of photocatalytic degradation of this cationic pollutant. TiO2/ZnO (TZ0.3; 1:0.03) couple semiconductor, containing 97 % TiO2 and 3 % ZnO was chosen as the best photocatalyst in degrading paraquat solution (% degradation :80.12 %, % mineralization: 52.32 %). In contrast, the photodegradation of malathion was higher in the presence of ZnO as shown in Figure 4. The best photocatalyst in degrading malathion solution is zinc oxide couple with 0.05 titanium dioxide (95 % ZnO and 5 % TiO2). The optimum degradation of malathion is 84.92 % while the total mineralization is 58.66 % after 4 hour reaction.

% mineralization

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100 Percentage (%)
70.30

76.22 42.21 52.40

80.12 52.32 66.69 52.77 61.05 35.32 67.22 50.12 65.70 49.10 66.21 44.58

50 0

T2

T Z05

T Z03

T Z01

Z2

ZT 05

ZT 03

ZT 01

Photocatalyst
%de gra da t ion %mine ra liz a t ion

Figure 3: The percentage of photodegradation of paraquat with single and couple semiconductor photocatalyst TiO2 and ZnO after 4 hours UV rediation.
100 80 60 40 20 0 T2 T Z05 T Z03 T Z01 Z2 ZT 08 ZT 05 ZT 03 ZT 01 Photocatalyst % degradation % mineralization
50.09 45.57 77.59 51.69 84.92 72.34 50.62 70.5 48.33 38.52 65.73 73.54 49.53 58.66 74.34 57.08 76.64 48.76

Figure 4: The percentage of photodegradation of malathion with single and couple semiconductor photocatalyst TiO2 and ZnO under UV light after within 4 hours reaction.

Effect of Fe3+ as a dopant (co-catalyst) and the addition of Fe2+ ions into a working solution. Theoretically, the presence of metal ions can delay the recombination process of generated electrons and holes as shown in Equation 6 and 7. Therefore the results are expected to be higher. 92.71 % total degradation and 77.07 % total mineralization for paraquat while 94.74 % degradation and 81.17 % mineralization were observed when Fe2+ ion was added into malathion solution after 4 hours illumination as shown in Figure 5 and Figure 6. Mn+ + e → M(n-1)+ (6) (n-1)+ + n+ M +h →M (7) In contrast, the use of Fe3+ as a photocatalyst dopant gave detrimental effect because when the metal is deposited on the photocatalyst, it was scattered in the bulk of the catalyst therefore it increased the recombination process of e-h+ pairs.
100.00 92.71 77.07 70.30 52.32 59.87 51.66 80.00 60.00 40.00 20.00 0.00 T Z03 + Fe2+ T Z03 Photocatalyst T Z03/Fe3+

Percentage (%)

Percentage (%)

TZ03 + Fe2+

TZ03/Fe3+

% degradation

% mineralization

Figure 5: The % photodegradation on paraquat with the best of photocatalyst doping with Fe3+ (TZ03/Fe3+) and the addition of Fe2+ into paraquat solution (TZ03 + Fe2+)

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100.00

94.74

81.17

84.92 58.66 65.42 52.81

Percentage (%)

80.00 60.00 40.00 20.00 0.00 ZT 05 + Fe2+ ZT05 + Fe2+

ZT 05 Photocatalyst

ZT 05/Fe3+ 3+ ZT05 + Fe

% degradation

% mineralization

Figure 6: The % photodegradation on malathion with the best of photocatalyst doping with Fe3+ (ZT05/Fe3+) and the addition of Fe2+ into malathion solution (ZT05 + Fe2+) Effect of photocatalyst calcinations temperature of the photocatalyst. The optimum degradation was observed with catalyst calcined at 550°C, 80.12 % paraquat degradation compared to 600°C (48.01 %) and 200°C (21.36 %) as shown in Figure 7. Similar pattern ZT05 + Fe ZT05/Fe3+ of mineralization was 2+observed. Catalyst prepared at 550°C gave the highest percent of mineralization followed by 600°C and 200°C. The amorphous titania is known to have very low photocatalytic activity compared to that of the anatase or rutile phase due to an increased of electron – hole pairs recombination rate [8]. At 550°C the anatase phase is fully formed while as the temperature reaching 600°C, the rutile phase is starting to appear. Anatase phase is performing better because it has more surface hydroxyl group that can react with holes to generate hydroxyl radicals. Therefore the recombination process was delayed by the presence of hydroxyl radical.
100.00 80.00 60.00 40.00 20.00 0.00 ZT 03 600 ZT 03 550 Photocatalyst ZT 03 200 48.01 42.70 80.12 52.32 21.36 25.00

Percentage (%)

% degradation

% mineralization

Figure 7: The % photodegradation on paraquat with the best of photocatalyst in various calcinations temperature (TZ03 600: 600°C, TZ03 550: 550°C, TZ03 200: 200°C) From Figure 8, the optimum degradation (80.12 %) and mineralization (52.32 %) was observed using catalyst calcined at 500°C. At this temperature, the ZnO wurtzite crystal was fully formed [9]. At 600°C, the percentage of the degradation and mineralization is decreased. Agglomeration of the photocatalyst will occur that attribute to the low surface area. This condition will lower down the photocatalytic activity of the photocatalyst. While at 200°C, the ZnO crystalline is incomplete.

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100.00 80.00 60.00 40.00 20.00 0.00

84.92 58.66 37.63 23.46 37.20 27.52

Percentage (%)

T Z05 600

ZT 05 500 Photocatalyst

ZT 05 200

% degradation

% mineralization

Figure 8: The % photodegradation on malathion with the best of photocatalyst in various calcinations temperature (ZT05 600: 600°C, ZT05 500: 500°C, ZT05 200: 200°C) Supported photocatalyst The catalyst coated on the support was performed better than the powder (unsupported) as shown in Figure 9 and 10. The faster substrate degradation and mineralization is due to the availability of surface adsorption site in prepared thin film resulted. Catalyst activity of the unsupported photocatalyst was slower because the catalyst particles were trapped inside the agglomeration particle where the radiation cannot penetrate. This will avoid the maximum generation of electron and hole pairs of the photocatalyst.
100 80 60 40 20 0 0 50 100 150 200 250 UV illuminated time (min) T Z03_P T Z03 ZT 05_P ZT 05

Figure 9: The % degradation with the best photocatalyst thin film (TZ03) and powder form (TZ03_P) for paraquat and the best photocatalyst thin film (ZT05) and powder form (ZT05_P) for malathion
% mineralization
80 60 40 20 0 0 50 100 150 200 250 UV illuminated time (min) T Z03_P T Z03 ZT 05_P ZT 05

Figure 10: The % mineralization with the best photocatalyst thin film (TZ03) and powder form (TZ03_P) for paraquat and the best photocatalyst thin film (ZT05) and powder form (ZT05_P) for malathion SEM analysis

% degradation

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As shown in Figure 11, TiO2 thin films were mainly composed of small spherical particles monodispersed on the surface of the slide glass in calcinations temperature 550°C. The surface of the films exhibited a certain degree of roughness and the thin film become rougher when coupled with zinc oxide. The particles are quite uniform in overall morphology.

(a) (b) Figure 11: SEM micrographs of a) TiO2 thin film b) TiO2/ZnO 0.03 couple semiconductor thin film calcined at 550°C (magnification factor: 10000x) The SEM micrographs for ZnO photocatalyst calcined in the same temperature 500°C is shown in Figure 12. It’s indicating that the particle formed irregular shape. The second thin film, which was couple with 5%, TiO2 shows smooth surface with a fine microstructure without cracks and voids. The grains of the film become larger and denser if couple with TiO2

(b) (a) Figure 12: SEM micrographs of a) ZnO thin film b) ZnO/TiO2 0.05 couple semiconductor thin film calcined at 500°C (magnification factor: 10000x) XRD analysis Diffractograms for catalyst calcined at 200°C indicated that is was an amorphous phase as shown in Figure 13. By increasing the temperature up to 450°C, transformation of phase was occurred whereby amorphous phase was transformed to anatase phase. At 600°C calcinations temperature, the rutile phase is starting to appear, therefore forming a mixture of anatase and rutile photocatalyst. The presence of rutile phase will decrease the performance of catalyst due to lack of hydroxyl ion surface species. Therefore it increased (e- - h+) pairs recombination process.

R

R

R

R

R 600° 550° 450° 200°

Figure 13: Diffractograms of TiO2/ZnO 0.03 prepared using sol-gel method and calcined at a) 450°C b) 550°C c) 600°C.

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600° 500° 200°
Figure 14: Diffractograms of ZnO/TiO2 0.05 prepared using sol-gel method and calcined at a) 200°C b) 500°C c) 600°C By increasing the calcinations temperature of ZnO photocatalyst to 500°C, the increment of intensity become stronger, which indicated that the quality of ZnO catalyst in thin film was improved as shown in Figure 14. At 200°C, the formation of metastable ZnO phase occurred. This was supported by Yang et. al. [10] who reported that low temperature portion of the ZnO-TiO2 phase diagram is difficult to study due to the sluggishness of the reactions and the similarities of the XRD patterns. Ellipsometer (Thickness measurement) The values of polarizer (P), analyzer (A), thickness and refractive index of the thin film photocatalyst are shown in Table 3. The thickness of the thin film were increased as it coupled with different semiconductor and doped with Fe3+ ions. As we add the second semiconductor and metal ion, the viscosity of the sol-gel might be increased. Table 3: The angle value of polarizer (P), analyzer (A) and the value of thickness and refractive index of the thin film photocatalyst. Parameter A1 P1 A2 P2 Thickness (nm) Refractive Index ZnO 22.9 130.4 149.2 199.8 130.57 2.4946 Angle value for thin film (°) ZnO/TiO2 0.05/Fe3+ ZnO/TiO2 0.05 18.8 24.3 128.0 127.3 158.5 154.7 214.4 218.2 150.68 153.84 2.1984 2.2042

UV-Vis-NIR analysis (Band gap Energy measurement) The band gap energy, Ebg values for all thin films were determined by plotting [αhc/λ]1/2 versus the equivalent energy at wavelength λ (h = Planck constant, c = light velocity). The α (absorption coefficient) was derived from the measuring transmittance (T) and reflectance (R) factors in a wavelength ranging from 300-500 nm. The α can be obtained by using the following relationship: T = (1-R2) exp (-αd) (8) Where d is the thickness of the thin film. The resulting diagram is called Tauc’s plot (refer to Figure 15) and the respective band gap energy was obtained by extrapolation of the Tauc plot dataset to [αhc/λ]1/2 = 0. Coupled TiO2 have the lowest bandgap energy (2.95 eV) than single ZnO photocatalyst (3.04 eV). Band gap energy for doped catalyst was higher (3.09 eV) than coupled TiO2. These data were in a good agreement with the results obtained in previous photodegradation reaction. The higher value of Ebg will reduce the formation of e- h+ pairs, therefore will reduced the performance of the photocatalyst.

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[(αhc/λ x 10 -4) cm -1] 1/2

0.0800 0.0600 0.0400 0.0200 0.0000 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 Photon Energy (eV)

Figure 15: Tauc’s plot for indirect band gap of ZnO (Ebg: 3.04 eV) Conclusion

Zinc oxide is more efficient catalyst degrading malathion solution while titanium dioxide is found to be more efficient in degrading paraquat solution The best coupled photocatalyst degrading malathion solution is ZnO:TiO2 in a ratio of 1:0.05. In contrast the best coupled photocatalyst degrading paraquat solution is TiO2:ZnO in a ratio of 1:0.03. Dissolved Fe2+ ions have showed the highest photocatalytic activity in degrading paraquat and malathion solution. Coupled photocatalyst doped with Fe3+ ion exhibited lower photodegradation compared to undoped coupled photocatalyst. The best calcination temperature for TiO2 based photocatalyst is 550°C while for ZnO based photocatalyst is 500°C. This result was supported by XRD diffractograms and SEM micrographs. Supported catalyst plays an important role in order to give the high dispersion rate and enhanced the photodegradation process of paraquat and malathion rather than the unsupported one. The thickness of ZnO, ZnO/TiO2 0.05 and ZnO/TiO2 0.05 doped with Fe3+ thin film are 130.57 nm, 150.68 nm and 153.84 nm. The band gap energy values measured using UV-Vis NIR are in the range of 2.95 – 3.09 eV.

References 1. Petit, V., Cabridenc, R., Swannell, R.P.J. and Sokhi R.S. (1995) “ Review strategies for modelling the environmental fate of pesticides discharged into riverine systems.” Environ. International. 21 (2). 167-176 2. Robertson, P.K.J. (1996) “Semiconductor photocatalysis: an environmentally acceptable alternative production technique and effluent treatment process” J. Cleaner Prod. 4 (3-4). 203-212. 3. Mills, A. and Le Hunte, S. (1997). “An overview of semiconductor photocatalysis” J. of Photochem. and Photobiol. A: Chemistry, 108. 1-35. 4. Vidal, A., Dinya, Z., Mogyorodi Jr., F. and Mogyorodi, F. (1999). “Photocatalytic of thiocarbamate herbicide active ingredients in water.” Applied Catalyst B: Environ.. 21. 259–267. 5. Yang, J. and Swisher, J.H. (1996). “The phase stabilization of Zn2Ti3O8.” Material Characterization. 37. 153-159. 6. Florencio, M.H., Pires, E., Castro, A.L., Nunes, M.R., Borges, C. and Costa, F.M. (2004). “Photodegradation of Diquat and Paraquat in aqueous solutions by titanium dioxide: evolution of degradation reactions and characterization of intermediates.” Chemosphere. 55. 345-355. 7. Araña, J., González, D.O., Doña, R.J.M., Herrera M.J.A., Cabo, C.G., Pérez P.J., Hidalgo, M.C. and Navio-Santos, J.A. (2003). “Role of Fe3+/Fe2+ as TiO2 dopant ions in photocatalytic degradation of carboxylic acids.” J. of Molecular Catalysis A: Chemical. 197. 157-171. 8. Su, C., Hong, B.-Y. and Tseng, C.-M. (2004). “Sol-gel preparation and photocatalysis of titanium dioxide.” Catalysis Today. 96. 119-126 9. Li, H., Wang, J., Liu, H., Yang, C., Xu, H., Li, X. and Cui, H. (2004). “Sol-gel preparation of transparent zinc oxide films with highly preferential crystal orientation.

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10. Yang, J. and Swisher, J.H. (1996). “The phase stabilization of Zn2Ti3O8.” Material Characterization. 37. 153-159.

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STUDIES OF POTATO PEELS EXTRACT AS NATURAL ANTIOXIDANT IN EDIBLE OIL Wan Sani Wan Nik1,*, Chek Yin Sez2, Ku Halim Ku Bulat2, Md. Mukhlesur Rahman2, Farid Nasir Ani3 Department of Engineering Science, 2 Department of Chemical Sciences, Faculty of Science and Technology, Kolej Universiti Sains dan Teknologi Malaysia, Mengabang Telipot, 21030 Kuala Terengganu, Terengganu. 3 Mechanical Engineering Faculty, Universiti Teknologi Malaysia 81310 UTM Skudai, Johor, Malaysia. e-mail: niksani@kustem.edu.my Abstract. The oxidation properties of edible oil were studied. The effects of ethanolic extract subfractions on the oxidative stability of palm olein were evaluated. The potato peels were extracted as a new source of natural antioxidant and added to palm olein in order to prevent the deterioration of palm olein as well as to extend the shelf life of the palm olein by inhibiting the auto-oxidation process. The potato peels were extracted using ethanol and then fractionated to yield four different subfractions which were dichloromethane, ethyl acetate, buthanol and water. By fourier transform infrared spectroscopic analysis (FTIR), the presence of phenolic compounds had been ensured. The extent of oil deterioration was evaluated using peroxide value test, acid value test and iodine value test and further confirmed using FTIR. Results from this study showed that the palm oil blended with 3% water subfraction was more thermally stable towards oxidation compared to 100% palm olein. Abstrak. Pengoksidaan minyak masak diselidiki. Kesan ekstrak etonolik ke atas kestabilan oksidasi minyak sawit dikaji. Selaput ubi kentang diekstrak sebagai punca baru bahan tambah antiosida dan ditambah kepada minyak sawit untuk mengelakkan kerosakannya. Selaput ubi kentang diekstrak menggunakan etanol dan dipisahkan kepada empat sub komponen. Kehadiran komponen fenolik dipastikan dengan menggunakan FTIR. Tahap kerosakan minyak adalah berpandukan kepada ujian nilai peroksida, ujian nilai asid dan ujian nilai iodin dan disokong oleh keputusan FTIR. Keputusan menunjukkan bahawa minyak sawit bersama dengan 3% komponen air adalah lebih stabil dari segi pengoksidaannya jika dibandingkan dengan minyak sawit 100%. Keywords: Sawit, kentang, minyak, pengoksidaan Introduction Atmospheric oxidation causes deterioration in lipids and reduction of the shelf life of many products. The oxidative rancidity of lipid is accelerated by exposures to heat, light, humidity and the presence of trace transition metals. Particularly, the intense frying of oils causes an oxidizing thermal degradation with the formation of decomposition products in oils. Furthermore, the oxidative deterioration can result in alterations of organoleptic characteristics, such as taste and aroma, making the oils unacceptable to the consumers. To make matters worse, oxidized oils may have undesirable effects on the human being. Thus, due to health protection and economic reasons, many investigations have been undertaken with the aim to enhance the stability of oils [1]. Antioxidants are a group of chemicals which effective in extending the shelf life of a wide variety of food products. Previously, the synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are used frequently to alleviate the autoxidation process. Recently, a number of scientific reports have discovered that the synthetic antioxidants have toxic and carcinogenic effects. As the result, the safer and natural antioxidants are required to substitute for the synthetic ones [2]. In the past, the potato peels are not effectively utilized and discarded as a waste,
1

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but now there are a large number of scientific on the possibility of the potato peels to be considered as an ingredient in health or functional food to alleviate the oxidation. The reports have confirmed that the extract of the potato peel contains phenolic compounds which are regarded as a type of antioxidants. The dietary phenolic compounds are included flavonoids, phenolic acids and polyphenols. It was found that potato peel contains phenolic acids such as chlorogenic acid, ferulic acid, caffeic acid, gallic acid and so on [3]. These compounds have been proven can be used in preventing autoxidation by their free radical scavenging activity [4]. Palm oil is classified as vegetable oil which can be used extensively as edible oil especially in cooking and baking. Therefore this study was designed and carried out to evaluate the effects of the potato peel extract on the oxidative stability of palm olein.

EXPERIMENTAL Materials and apparatus The chemicals used in this project were palm olein, ethanol, hexane, dichloromenthane, ethyl acetate, n-buthanol, starch indicator, acetic acid, chloroform, sodium thiosulfate, diethyl ether, 1% phenolphthalein indicator, potassium iodide, potassium hydroxide, cyclohexane, Wijs reagent. The equipments and apparatus used in this project were Attenuated Total Reflectance (ATR), rotary evaporator (Rotavapor R-200, BUCHI) and Perkin Elmer Fourier Transform Infrared Spectrophotometer (FTIR). Procedure The dried potato peels powder was extracted using ethanol in Soxhlet and then followed by solvent fractionation using four different solvents such as dichloromethane, ethyl acetate, n-buthanol and water. After removing the solvent, the subfraction was then analysed using FTIR to identify the functional groups which was then to be used to classify which family of organic compounds they belongs to. In order to examine the ability of each subfraction in inhibiting the oxidation process of palm olein under heat treatment, the different pecentage of subfraction by 1%, 3% and 5% (w/w) were added to palm olein. The best subfraction was then chosen to be tested with palm olein to see their ability to reduce oxidation under prolonged exposure to heat at 1350C for 200 hours. After that each sample was collected for 0, 25, 50, 100, 150 and 200 hours of exposure. The extent of oil deterioration during the heating process was evaluated using peroxide value test (PV), acid value test (TAN) and iodine value test (IV). TGA was used to estimate the oxidative stability of palm olein, using the degradation temperature and onset temperature of the oil samples. FTIR was used to identify the microstructural changes of triacylglycerol. Results And Discussion The functional groups of all the subfractions were identified using FTIR. The presence of the phenolic compounds in ethyl acetate, buthanol and water subfractions has been confirmed. By using TGA, it was found that the 3% water subfraction of the ethanolic potato peel extract was the most suitable additive to be blended with palm olein in order to determine the thermal oxidative stability of the oil [5]. From the results obtained, the rancidity of palm olein could be inhibited in short term by the distribution of 3% water subfraction although the PV of the blended palm olein was higher than the PV of the 100% palm olein (Table 1). The TAN of the 100% palm olein was always higher than the blended palm olein which was probably due to the presence of phenolic acid compounds in the subfraction which can act as a free radical scavenger [3, 6-7].

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Based on the iodine value test, the IV of blended palm olein was always higher when compared to the 100% palm olein. The results showed that the oxidation process at the C=C double bond site was inhibited in the presence of subfraction (Table 3). Furthermore, the significance of adding subfraction can be seen by comparing the IR spectra of the 100% palm olein to the blended palm olein after 200 hours of heating at a temperature of 1300C. In Figure 1, the peak at 3005 which presenting the stretching of C-H alkane decreased significantly for the sample of the 100% palm olein [1,8]. The TGA studies of the onset and maximum degradation temperatures showed that the blended palm olein was more stable towards oxidation compared to the 100% palm olein. However, the maximum degradation temperature seems to be not very much affected by the presence of subfraction. Table 1.The results of PV analysis for 100% palm olein and blended palm olein in different heating hours. Heating Hours Oil Samples Oil Oil + W 0 16.9492 1.9920 25 17.9447 1.9935 50 21.8792 5.9815 100 13.9412 31.6537 150 15.9359 9.9870 200 21.8905 21.9482

Table 2. The results of TAN analysis for 100% palm olein and blended palm olein in different heating hours. Heating Hours Oil Samples Oil Oil + W 0 0.6461 1.3437 25 0.6497 0.5374 50 0.6567 1.2367 100 1.2891 1.2923 150 3.8769 1.400 200 4.6224 1.4966

Table 3. The results of IV analysis for 100% palm olein and blended palm olein in different heating hours. Heating Hours Oil Samples Oil Oil + W 0 50.5148 51.6232 25 51.5716 50.3370 50 45.8955 49.4745 100 32.5601 46.9374 150 18.6120 26.2523 200 5.4990 20.7339

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100 .0 90
3476.36 583.88

80 70 60
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1746.01 2854.99 2925.25

1237.18 1117.49 1164.37

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(b) Figure 1. Infrared spectra of the (a) 100% palm olein and (b) blended palm olein after 200 hours of heating.

ACKNOWLEDGEMENT We greatly acknowledge the financial support from KUSTEM through vot 54045 and MOSTE (0102-12-003-EA10311). Assistance from Mr. Tarimizi, Jamal, Razman, Asrul and Pn. Asbah is also acknowledged. References 1. 2. Moya, M.M.C.M., Olivares, D.M., Lopez, F.J.A., Adelantado, J.V.G., Reig, F.B. (1999). “Analytical Evaluation of Polyunsaturated Fatty Acids Degradation During Thermal Oxidation of Edible Oils by Fourier Transform Infrared Spectroscopy”. Journal of Talanta. 50: 269-275. Tomaino, A., Cimino, F., Zimbalatti, V., Venuti, V., Sulfaro, V., De Pasquale, A., Saija, A. (2005). “Influence of Heating on Antioxidant Activity and the Chemical Composition of Some Spice Essential Oils”. Food Chemistry. 89(4): 549-554.

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3. 4. 5. 6. 7.

8.

Zia-ur-Rehman, Farzana, H., Shah, W.H. (2004). “Utilization of Potato Peels Extract as a Natural Antioxidant in Soy Bean Oil. Journal of Food Chemistry. 85: 215-220. Nandita, S., Rajini, P.S. (2004). “Free Radical Scavenging Activity of an Aqueous Extract of Potato Peel. Journal of Food Chemistry. 85: 611-616. Coni, E., Podesta, E., Catone, T. (2004). “Oxidizability of Different Vegetables Oils Evaluated by Thermogravimetric Analysis”. Thermochimica Acta. 418: 11-15. Rudnik, E., Szczucinska, A., Szulc, A., Winiarska, A. (2001). “Comparative Studies of Oxidative Stability of Linseed Oil”. Thermochimica Acta. 370: 135-140. Sharma, O. P., Bhat, T. K., Singh, B. (1998). “Thin-layer Chromatography of Gallic Acid, Methyl Gallate, Pyrogallol, Phoroglucinol, Catechol, Resorcinol, Hydroquinone, Catechin, Epicatechin, Cinnamic Acid, p-coumaric Acid, Ferulic Acid and Tannic Acid”. Journal of Chromatography A. 822: 167-171. Guillen, M.D., Cabo, N. (2002). “Fourier Transform Infrared Spectra Data Versus Peroxide and Anisidine Values to Determine Oxidative Stability of Edible Oils”. Journal of Food Chemistry. 77: 503-510.

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THE APPLICATION OF SOL GEL IMMOBILIZED FLUORESCEIN-MANGANESE COMPLEX AS FLUORESCENT CARBON DIOXIDE SENSING MATERIAL Mustaffa Nawawi*, Tee Shiau Foon, Shemalah d/o Ramasundram
Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, 81300 UTM Skudai, Johor, Malaysia. email: musna@kimia.fs.utm.my Abstract. Fluorescein is fluorescent material exhibits fluorescence with excitation peak at 483.0 nm and emission 510.2 nm. In this study, the excitation and emission spectra of fluorescein immobilized in sol gel in the presence of manganese was investigated. It was shown that the emission spectrum of fluorescein remain unchanged but the intensity of emission. The fluorescein-manganese complex changed linearly with an equation y = -7.861 x + 6.227 and R2 = 0.9613. The observed fluorescence intensity of fluorescein-manganese complex showed a quadratic changes with the increased in carbon dioxide concentration. Keywords: Fluorescence; fluorescein; metal complex; sol gel; carbon dioxide.

Abstrak: Fluoresin merupakan bahan berpendarfluor pada puncak pemancaran λem 483 nm (λex = 510.2 nm). Dalam kajian ini, puncak penyerapan dan puncak pemancaran bagi fluoresin yang terperangkap dalam sol gel dengan kehadiran mangan telah dikaji. Keputusan menunjukkan bahawa puncak pemancaran bagi fluoresin tidak berubah tetapi perubahan dari segi keamatan. Kompleks fluoresin-mangan berubah secara linear dengan persamaan y = -7.861 x + 6.227 dan R2 = 0.9613. Kajian juga mendapati bahawa keamatan bagi kompleks fluoresin-mangan menunjukkan perubahan secara kuadratik ketika kepekatan gas karbon dioksida bertambah.

Introduction The sol gel process is attractive for making porous membranes for optical sensors due to its relatively simple chemistry and because of the low polymerisation temperature which allows encapsulated or attachment of organic probe molecules that are unstable at higher temperature [1]. Recently, various studies on the development of carbon dioxide sensor based on the absorbance and emission changes of fluorescence materials [2-4] were carried out. Fluorescein (Figure 1) was the first fluorescent material used for water tracing work [5] and is still used for qualitative (visual) studies of underground contamination of wells and in biological field. The chemical sensor for pH measurement had been investigated based on the fluorescence property of FITC [6]. A sensitive pH sensor using phospholipids coating the particles labelled with fluorescein was used for intra-cellular pH measurement in murine macrophages [6]. In this study, we described a carbon dioxide sensor based on the fluorescence properties of the fluorescein-manganese complex.

Experimental Sample preparation The selected fluorescent reagent fluorescein was obtained from BDH chemical. Fluorescein (0.0332 g) was prepared by adding the powder in 100 mL ethanol 95% in volumetric flask to produce stock solution of approximately 1 x 10-3 mol L-1. Stock solution was freshly prepared for experimental work to avoid oxidation and degradation. Dilution was conducted for concentration effect, temperature effect, pH effect and solvent effect. Manganese (II) chloride-2-hydrate from Merck (MW: 161.88 g mol-1) was used throught this study. Sol gel silicate precursors tetraethoxysilane (TEOS) was from Fluka Chemika. Water purified in a Nano Pure Ultrapure water system. The metals solutions were prepared by adding its powder in volumetric flask 100 mL to prepare a concentration of 0.1 mol L-1 in ethanol. The basic sol gel was prepared following a method described by Deshpande A. V [7]. The doped silica gel matrices were

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prepared by hydrolysis and polycondensation of tetraethyorthosilicate (TEOS) in water and ethanol solution under acidic condition and stirring for 17 hours until the solution became homogeneous. After that, the solution was cooled at room temperature for 30 minutes. Nitric acid was used as a catalyst. The organic dyes were previously dissolved in ethanol will be coated on the glass slides. The glass slides were washed with nitric acid for 1 day and rinsed with deionised water and later with ethanol. Characterization The excitation and emission spectra were measured using a Perkin Elmer Models LS-50B Luminescence spectrophotometer. All measurements were performed at room temperature using 1 cm x 1 cm quartz cell. Results and Discussion Luminescence emission The fluorescence response for fluorescein in ethanol was recorded giving excitation and emission wavelengths at 483.0 nm and 510.2 nm, as can be seen in Figure 2.
804.4 750 700 650 600
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300 250 200 150 100 50 0.0 200.0 250 300 350 400 450 500 nm 550 600 650 700 750 800.0

λem = 510.2 nm

Wavelength (nm)

Figure 2. Fluorescence spectra of 1x10-4 M of fluorescein derivative, which showing excitation wavelength at 483.0 nm and emission at 510.2 nm.

As showed in Figure 3, it was observed that fluorescence emission of fluorescein in solution is strongly dependent on pH changes. A blue shift for the emission band can be observed from acidic condition to base condition. Moreover, the intensity of the emission peak was different for each other. The lowest intensity was achieved at pH 2.64. However, the optimum pH for fluorescein occurred at pH 8.90. This could be due to the different ionized chemical species formed by fluorescein. According to literature, the existence of fluorescence materials in a definite molecular form depends mainly on solvent polarity and proticity (pH) [3, 6]. In these media hydrogen bonding with the solvent moleculars may play an important role in the stabilization of the ionic forms. However, according to Carvell [8], Sanchez-Barragan and co-workers [9], the emission intensity of fluorescein is increased from pH 3 to pH 13.

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1000.0

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Figure 3. Emission spectra showed the pH effect of fluorescein in ethanol (λex = 483.0nm). (a = 2.64, b = 9.61, c = 8.17, d = 8.90, e = 7.38)

The Effect of Manganese on the Fluorescence of Fluorescein in Ethanol
The emission intensity of manganese complex decreased the intensity of fluorescein with increasing concentration of metal. The emission spectra does not show any significant change in the spectra shape. This indicated a quenching phenomenon [10-11] with an equation of y = -7.861x + 6.227. Figure 4 indicate that good linear correlation still obtainable between the emission intensity and the quantities of manganese added (R2 = 0.9613).
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vo m o mn a e (m) lu e f a g n se L

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Figure 4. The effect of manganese on the fluorescence of fluorescein which the concentration of fluorescein 1x10-4 M (5mL) (a=1mL, b=2mL, c=3mL, d=4mL, e= 5mL).

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The Effect Of Carbon Dioxide On The Emission Intensity Of Fluorescein-Manganese Complexes Solution Strong fluorescence quenching is observed when fluorescein-manganese complexes excited at 483.0 nm, with emission maximum of 514.04 nm. As shown in Figure 5, it was found that the bubbled of carbon dioxide in the solution of fluorescein-manganese complex give a linear relationship for emission intensity versus amount of carbon dioxide (y = -7.2252 x + 271.01 with correlation is 0.9234). It could be due to the reaction of fluorescein-manganese complexe with carbon dioxide forming fluorescein-manganese-CO2. The amount of CO2 influenced the formation of carbonate and affects the fluorescence property of fluorescein-manganese complexes. Increasing [CO2] causes a decreased in fluorescence intensity. The presence of water in ethanol is also crucial for response to CO2 is based on the hydrolysis of the dyes as shown in the following equations and phenomenon quenching [3]: CO2 (g) + H2O (aq) H2CO3 (aq) + H2O (aq) HCO3- (aq) + H2O (aq) DH (aq) + H2O (aq) (colour A) H2CO3 (aq) HCO3- (aq) + H3O+ (aq) CO3 2- (aq) + H3O+ (aq) D(aq) + H3O+ (aq) (colour B)

300

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intensity of emission

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100 y = -7.2252x + 271.01 R2 = 0.9234

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0 0 5 10 15 volume of carbon dioxide (mL) 20

Figure 5. Effect of carbon dioxide on the emission intensity of fluorescein-manganese complex.

Sol Gel Immobilized Complex Fluorescein-manganese for The Detection of Carbon Dioxide
The encapsulated fluorescein-manganese complex in sol gel and the properties of fluorescence were observed in the following experiments shows an effect of carbon dioxide (2-20mL) on the emission of fluorescein-manganese complexes. Figure 6 shows the emission spectra of immobilised fluoresceinmanganese complex. It was found that the emission wavelength of fluorescein-manganese-CO2 was at 482.58 nm when excited at 483.0 nm. The intensity of the emission wavelength decreased with the increases of the amount of gases carbon dioxide. However, the emission peak remains unchanged.

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Prosiding Simposium Kimia Analisis Malaysia Kelapan Belas, Johor Bahru

666.9

600 550 500 450 400 350 INT 300 250 200 150 100 50 0.0 450.0 460 470 480

a

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490 nm

500

510

520

531.5

Figure 6. Emission spectra show fluorescein-manganese complexes which dissolved carbon dioxide gases in sol gel (λex =483 nm, λem = 482.52 nm) (a= 2mL, b= 4mL, c= 6mL, d= 16mL, e=10mL).
600 50 0
intensity of emiss ion

400 300 200 100 0
0 5 10 15 20 25 volume of carbon dioxide (mL)

Figure 7. Effect of carbon dioxide gases on the emission intensity of fluorescein manganese complexes which encapsulated in sol gel

Figure 7 shows aplot of emission intensity versus with volume of carbon dioxide (mL). The emission intensity decreased as the carbon dioxide increased in the first 2-10mL. This could be due to the addition of carbon dioxide in fluorescein-manganese complex solution causing the aggregation of the sol gel resulting in the quenching phenomenon. However, this phenomenon changed and the emission intensity increased again in the presence of amount (12mL) of carbon dioxide. This will be most probably due to the diffusion of carbon dioxide through the aerogel pore network and the entrapped carbon dioxide achieved an optimum condition and exhibit fluorescence in fluoresceinmanganese-CO2 form [1, 12].

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Conclusion
In conclusion, our study has shown that fluorescein-manganese complex in sol gel has the potential to be used as carbon dioxide gas sensors. The pH value is responsible for the formation a stable form of fluorescein. Various metals affected the emission intensity of fluorescein. The effect of carbon dioxide on the complex was found that the emission intensity of complex fluorescein-manganese decreased with a quadratic curve after exposures to CO2 in sol gel. These results leads to the possibility that encapsulation of samples on sol gel can be used as active materials in solid state dye sensors.
B B

Acknowledgements The financial support of PTP-UTM (Universiti Teknologi Malaysia) is acknowledged.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Collinson M. M.and Howells A. R. (2000) “Sol Gel and Electrochemistry: Research at The Intersection”, Anal. Chem. 72:702A-709A. Walt D. R, Gabor G. and Goyet C. (1993) “Multiple Indicator Fiber-Optic Sensor for High Resolution pCO2 Seawater Measurement”. Anal. Chim. Acta, 1993, 274: 47-52. Choi M.F and Hawkins P. (1995) “A Novel Oxygen and / or Carbon Dioxide Sensitive Optical Transducer” Talanta, 42(3): 483-492. Bültzingslöwen C., McEvoy A. K., McDonagh C. and MacCraith B. D. (2003) “Lifetime-Based Optical Sensor For High-Llevel pCO2 Detection Employing Fluorescence Resonance Energy Transfer” Anal. Chim. Acta,. 480: 275–283. Dole R. B. (1906) “Use of Fluorescein in the Study of Underground Waters” USGS Water Supply Paper. 160: 73-85. Li Y. M, Huai Y.W., Hui X., Li X. X. (2004) “A Long Lifetime Chemical Sensor: Study On Fluorescence Property of Fluorescein Isothiocyanate and Preparation of pH Chemical Sensor” Spectrochimica Acta Part A. 60: 1865-1872. Deshpande A. V. and Kumar U.(2002). “Effect of Method Preparation on Photophysical Properties of Rh-B Impregnated Sol-gel Host” J. of Non-Crystalline Solid 306: 149-159. Carvell M., Robb I. D. and Small P. W. (1998) “The Influence of Labelling Mechanisms On the Fluorescence Behaviour of Polymers Bearing Fluorescein Labels”. Polymer 39 (2): 393-398. Mchedlov-Petrossyan N. O., Rubtosov M. I. and Lukatskaya L.L. (1992). “Dyes Pigments” 18, 179. Bowen E.J. and Wokes F. (1953) “Fluorescence of Solution”. London: Longman Green and Co. Ltd. White C. E. (1970) “Fluorescence Analysis. A Practical Approach” New York: Marcel Dekker, INC. Faizatul S. M., Rusli D. and Musa A. (2003) “The Potential Application of poly (1,4-pheylene diphenyvinylene), p-PDV for Oxygen Detection Based On Fluorescence Quenching”. Sens. nd Act B, 96: 537-540.
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