LI M U

C l ai trong chng ta cng tng ung sa v cng bit c nhng li ch m vic ung sa mang li cho sc khe ca con ngi. Sa p ng nhu cu ca c th v acid amin khng thay th, acid bo khng no, khong (c bit l canxi v pht pho) v vitamin. Sa khng nhng b dng m cn c tc dng gii c. Trong s cc thc n t nhin ca con ngi, khng c sn phm no m cc cht cn thit cho c th li c phi hp mt cch hiu qu nh sa. Chnh nh gi tr dinh dng cng vi s tin li m sa c hu nh mi ngi a chung v s dng hng ngy. Nhng gn y, d lun trong v ngoi nc xn xao v nhiu loi sa bt c ngun gc t Trung Quc c cht melamine gy c hi cho c th. Vy melamine l cht g? Tc hi ca n nh th no? V lm sao xc nh c s c mt ca melamine trong sa? Chng ti s gii p nhng thc mc cng nh gii thiu n mi ngi mt phng php xc nh melamine trong sa s dng ph bin nht hin nay l phng php sc k lng hiu nng cao (HPLC).

MC LC
LI M U .......................................................................................................... 1 Chng 1: Tng quan v cht Melamine ............................................................ 3 1.1. Melamine l g? .......................................................................................... 3 1.2. S ra i v phng php tng hp melamine........................................... 5 1.3. Ti sao melamine li c cho vo sa ti v sa bt tr em? .......... 5 1.4. nh hng ca melamine n sc khe? ............................................... 6 1.5. Triu chng ca nhim c melamine? .................................................. 8 1.6. Liu lng dung np melamine an ton cho mt ngi ............................. 8 1.7. Nng an ton cho sa v thc phm...................................................... 9 CHNG 2: Phng php xc nh melamine trong sa bng sc k lng cao p HPLC ......................................................................................................... 10 2.1. Cc phng php xc nh melamine trong sa ..................................... 10 2.2 S lc v h thng HPLC ..................................................................... 10 2.3 Xc nh melamine trong sa bng sc k lng cao p HPLC ................. 15 2.3.1 Ha cht v thuc th ........................................................................... 15 2.3.2 H thng sc k - detector..................................................................... 15 2.3.3 Pha dung dch ........................................................................................ 15 2.3.4 Qu trnh tin hnh ................................................................................ 17 Chng 3: Cc bin php khc phc khi sa b nhim melamine .................. 19 Kt lun ................................................................................................................. 20 TI LIU THAM KHO ................................................................................... 21

Chng 1: Tng quan v cht Melamine

1.1. Melamine l g?
Melamine c tng hp ln u tin nm 1834, Melamine (tn y 1,3,5 -triazine-2,4,6-triamine) l mt baz hu c c cng thc ha hc C3H6N6 to thnh t 3 phn t cyanamide (CN2H2). Nu tnh t l cc nguyn t ccbon (C), nit (N) v hydro (H) th nit chim ti 66% theo khi lng. Melamine tan t trong nc, c kh nng gii phng N khi gp nhit cao v c s dng lm cht chng chy. Melamine cn l dn cht ca thuc tr su cyromazine v c th c hnh thnh trong c th ng vt c v t cyromazine. C nghin cu cho rng melamine cng c hnh thnh t cyromazine khi cyromazine c mt trong m thc vt. Melamine c tng hp t cht ur - mt cht c thnh phn nitrogen cao - l cht rn mu trng, chu nhit cao n 350OC. Melamine l thnh phn chnh ca cng ngh sn xut cc vt dng gia nh (chn a), formica (dng trong cng ngh bn gh, tm vch...), bao b, nha, melamine dng lm cht keo dn...

Hnh 1.1: melamine c s dng trong sn xut nha Melamine c c tnh qua th nghim: gy ng c cp trn chut vi liu cht (LD50), qua ng ung l trn 3.000mg cho mi kg.

Mt nghin cu Lin X trc y, vo nhng nm 1980, cho rng cht mui sinh ra khi melamine kt hp vi acid cyanuric l cyanurat melamine, dng sn xut cht chng chy, c th c hi hn nhiu ln so vi melamine dng n c hay acid cyanuric dng n c. Mt nghin cu khc v ng c hc nu ln rng trong thc n ca cc vt nui trong nh (ch, mo) c nhim hn hp hai cht melamine v acid cyanuric s gy ra suy thn cp.

Hnh 1.2: cng thc cu to ca melamine C3H6N6

Hnh 1.3: cng thc khai trin ca melamine

1.2. S ra i v phng php tng hp melamine

Nh ha hc ngi c tn Justus von Liebig l ngi u tin tng hp melamine vo nm 1834. Trong phng php tng hp ca Justus von Liebig, calcium cyanamide (CaCN2) c chuyn thnh dicyandiamide sau c nung nng to melamine. Ngy nay ur c s dng trong cng nghip sn xut melamine. Phng trnh ha hc nh sau: 6(NH2)2CO C3H6N6 + 6NH3 + 3CO2 Trc ht, ur c phn tch to axit cyanic (HNCO) (phn ng cn nhit cao : (NH2)2CO HCNO + NH3 Tip theo l phn ng polyme ha axit cyanic to dioxit ccbon v melamine (phn ng ta nhit): 6HCNO C3H6N6 + 3CO2 Xt ton b qu trnh, phn ng to melamine l phn ng cn nhit cao. Chnh v vy, melamine cng l tp cht trong sn xut ur. Quy trnh kt tinh v ra melamine lm sn sinh mt lng ln nc thi c hi nu chng c x trc tip vo mi trng. Thng thng nc thi c c thnh dng rn (c th cha n 75% melamine) nhng thun tin v an ton hn trong cho qun l v x l.

1.3. Ti sao melamine li c cho vo sa ti v sa bt tr em?

i vi nh sn xut, melamine v c vi acid cyanuric, l hai cht gip lm tng nng m trong sn phm d nng m tht trong ch c bao nhiu!

V li nhun, nh sn xut thc phm nh la nh kim nh bng cch cho melamine vo thc phm. Do lng tham khng y, mt s nh sn xut cn cho vo c hai cht melamine v acid cyanuric. Th l gy ra thm ha ng c hng lot. Acid cyanuric r hn melamine nhiu, cng l l do c th c s hin din c i trong thc phm dn ti gy c m ngi ta khng ng n.

Hnh 1.4: sa nhim melamine b tiu hy

1.4. nh hng ca melamine n sc khe?

Cha c nghin cu no v tc ng ca melamine i vi sc khe. Th nhng, tnh trng ca tr em Trung Quc dng sa nhim melamine cho thy tc ng ca ha cht ny ln con ngi gn ging vi kt qu th nghim trn sc vt. c bit, Bn thn melamine khng c tnh c liu thp nhng khi kt hp vi axit cyanuric (cng c trong sa bt) s to thnh mui tinh th ng trong cc ng thn nh gy bt tc, si thn, nh hng n s to nc tiu v gy suy thn, t vong. Mt s nghin cu trn sc vt cho thy melamine c kh nng gy ung th. Nghin cu cn cho thy melamine c kh nng hy hoi h thng min dch ca con ngi.

Hnh 1.5: Melamine (mu xanh) d dng kt hp vi axt cyanuric (mu ) qua lin kt hydro to kiu lin kt phn t hnh mi ngi, lng ng v gy si thn.

Hnh 1.6: Melamine gy si thn

Melamine thng gy ng c mn tnh do kh tan trong nc v cn thi gian di thi tr. Tn thng h sinh sn, si bng quang, si thn v ung th bng quang l nhng hu qu ca vic s dng melamine trong thi gian di.

Hnh 1.7: melamine nh hng n sc khe tr nh

1.5. Triu chng ca nhim c melamine?

B kch thch da, mt, h hp, tiu ra mu, tiu t, tiu kh hoc khng tiu c, c du hiu nhim trng tiu (st, au lng, tiu gt, tiu git), huyt p tng l cc du hiu cn cnh gic sau khi s dng sn phm.

1.6. Liu lng dung np melamine an ton cho mt ngi

Nhiu hng ha lu hnh trn th trng u hm cha mt t melamine. iu ny cng d hiu, bi v ha cht ny c s dng trong cc quy trnh sn xut giy v bao b cho nhiu loi hng ha gia dng. Do , khng c g ng ngc nhin khi tm thy melamine trong cc vt gia dng ny. Vn l liu lng melamine c no c th gy tc hi n sc khe cho con ngi. Cu hi ny kh c cu tr li dt khot, bi v phn ln d liu v tc hi ca melamine trong qu kh c thu thp qua th nghim trn chut, ch v mo, ch khng phi trn ngi (v cng chng c nh khoa hc no dm lm th nghim trn ngi).

Tuy nhin, sau khi cc chuyn gia v an ton thc phm trn th gii tho lun, h nht tr rng nng melamine dung np hng ngy c th chp nhn c (ting Anh l tolerable daily intake hay TDI) l 0.5 mg/kg cn nng. M, Cc qun l thc phm v dc phm (FDA) ngh TDI 0.63 mg/kg cn nng. Khi qut ha t tiu chun trn, chng ta c th ni c mi kg cn nng, mt ngi c th hp thu 0.5 mg melamine mi ngy. Chng hn nh mt em b cn nng 20 kg th liu lng melamine an ton l 10 mg/ngy; hay mt ngi ln nng 60 kg th c th dng 30 mg/ngy m khng gy tc hi n sc khe.

1.7. Nng an ton cho sa v thc phm

Theo tiu chun mi v an ton thc phm, hm lng an ton melamine ti a l 0,15 mg/kg vi sa nc dnh cho tr em, 1 mg/kg vi sa bt dnh cho tr em v 2,5 mg/kg vi thc n cho ng vt v cc thc phm khc. l tiu chun mi do y ban lut quc t v cht lng v an ton thc phm ca LHQ (CAC) cng b ngy 5/7/2012.

CHNG 2: Phng php xc nh melamine trong sa bng sc k lng cao p HPLC

2.1. Cc phng php xc nh melamine trong sa
Dng my sc k kh khi ph GC/MS kim nghim melamine Phng php Dumas Phng php Kjedal Phng php sc k lng hiu nng cao HPLC

Ti sao li chn Phng php sc k lng hiu nng cao HPLC? V sc lng k hiu nng cao l phng php rt thun li nh lng mt cht trong hn hp v cht tch ra khi cht khc. HPLC c s dng rng ri hn c trong phn tch lng thc thc phm nh xc nh cc ha cht b cm, cc cht tn d v kim sot cht lng, n mang li chnh xc cao cng nh thi gian tin hnh ngn v tha mn cc tiu ch: Not labor intensive khng tn lao ng Efficient hiu qu Cheap gi r Low detection limit - Gii hn pht hin thp Simple method phng php n gin

2.2

S lc v h thng HPLC
H thng sc k lng hiu nng cao gm c cc b phn c bn nh sau:

Hnh 2.1: S h thng HPLC Trong : 1: Bnh cha pha ng. 2: B phn kh kh 3: Bm cao p 4: B phn tim mu 5: C sc k (pha tnh) 6: u d detector 7: H thng my tnh c phn mm ghi nhn tn hiu, x l d liu v iu khin h thng. 8: In d liu.

Bnh cha pha ng

My HPLC thng c 4 ng dung mi vo u bm cao p cho php chng ta s dng 4 bnh cha dung mi cng mt ln ra gii theo t l mong mun v tng t l ca 4 ng l 100%. Tuy nhin, theo kinh nghim, t khi s dng 4 ng dung mi cng mt lc m thng s dng 2 hoc 3 ng cho h pha ng lun c pha trn ng nht hn, h pha ng n gin hn gip n nh qu trnh ra gii.

Lu : Tt c dung mi dng cho HPLC u phi l dung mi tinh khit s dng cho HPLC. Tt c cc ha cht dng chun b mu v pha h m u phi l ha cht tch khit dng cho phn tch. Vic s dng ha cht tinh khit nhm trnh hng ct sc k hay nhiu ng nn, to nn cc peak tp trong qu trnh phn tch.

B kh kh Degases
Mc ch s dng b kh kh nhm lai tr cc bt nh cn st li trong dung mi pha ng, trnh xy ra mt s hin tng c th c nh sau: T l pha ng ca cc ng dung mi khng ng lm cho thi gian lu ca peak thay i. Trong trng hp bt qu nhiu, b kh kh khng th loi tr ht c th bm cao p c th khng ht c dung mi, khi nh hng n p sut v hot ng ca c h thng HPLC. Trong cc trng hp trn u dn n sai kt qu phn tch.

Bm cao p
Mc ch bm pha ng vo ct thc hin qu trnh chia tch sc k. Bm phi t c p sut cao khong 250-600bar v to dng lin tc. Lu lng bm t 0.1 n 10ml/pht.

B phn tim mu
a mu vo ct phn tch theo vi th tch bm c th thay i. C 2 cch a mu vo ct: bng tim mu th cng v tim mu t ng (autosamper).

Ct sc k
Ct cha pha tnh c coi l tri tim ca ca h thng sc k lng hiu nng cao. Ct pha tnh thng thng lm bng thp khng r, chiu di ct thay i t 5-25cm ng knh trong 1-10mm, ht nhi c 0.3-5m, Cht nhi ct ph thuc vo loi ct v kiu sc k.

Detector - u d

L b phn pht hin cc cht khi chng ra khi ct v cho cc tn hiu ghi trn sc k c th nh tnh v nh lng. Ty theo tnh cht ca cc cht phn tch m ngi ta la chn lai u d ph hp. Tn hiu u d thu c c th l: hp th quang, cng pht x, cng in th, dn in, dn nhit, chit sut, Trn c s , ngi ta sn xut cc loi u d sau: - u d quang ph t ngai 190-360nm pht hin UV - u d quang ph t ngoi kh kin (UV-VIS) (190-900nm) pht hin cc cht hp th quang. y l lai u d thng dng nht. - u d hynh quang (RF) pht hin cc cht hu c cha hunh quang t nhin v cc dn sut c hunh quang. - u d DAD (Diod Array Detector) c kh nng qut chng ph nh tnh cc cht theo hp th cc i ca cc cht. - u d khc x (chit sut vi sai) thng dng cc loi ng. - u d in ha: o dng, cc ph, dn. - u d o dn nhit, hiu ng nhit, C mt s cch pht hin khi mt cht i qua ct. Mt phng php ph bin l d dng gii thch s dng u d t ngoi. Nhiu hp cht hu c hp th nh sng tia cc tm bc sng khc nhau. Nu bn c mt chm nh sng tia cc tm chiu sng qua cc dng cht lng i ra khi ct, v mt u d t ngoi pha i din ca dng, bn c th c trc tip nh sng c hp th. Lng nh sng hp th ph thuc vo s lng ca mt hp cht c bit i qua chm tia vo thi im .

Bn c th t hi ti sao cc dung mi c s dng khng hp th nh sng UV. Chng c, tuy nhin, cc hp cht khc nhau hp th mnh m nht cc b phn khc nhau ca quang ph tia cc tm. V d, Methanol hp th bc sng di 205 nm, v nc di 190 nm. Nu bn ang s dng mt hn hp methanol-nc l dung mi, do bn s phi s dng mt bc sng ln hn 205 nm trnh c sai t cc dung mi.

B phn ghi nhn tn hiu

B phn ny ghi tn hiu do u d pht hin. i vi cc h thng HPLC hin i, phn ny c phn mm trong h thng ghi nhn, lu cc thng s, sc k , cc thng s lin quan n peak nh tnh i xng, h s phn gii, ng thi tnh tan, x l cc thng s lin quan n kt qu phn tch.

In d liu
Sau khi phn tch xong, d liu s c in ra qua my in kt ni vi my tnh c ci phn mm iu khin.

Hnh 2.2: Hnh nh thc t ca my sc k lng hiu nng cao (HPLC)

K m hiu: LC 20AD Hng sn xut: Shimazhu Nht Bn

2.3

Xc nh melamine trong sa bng sc k lng cao

p HPLC 2.3.1 Ha cht v thuc th

Cht chun: Melamine (99,0%) Dung mi: Methanol Dung mi ra gii: Trifluoroacetic v Methanol Mu th (sa dng bt hoc lng) Nc s dng cho vic chun b pha ng v pha long c lc bng h thng Qpod Millipore.

2.3.2 H thng sc k - detector

u d UV, ct C18 (150mm x 3.2mm ID, kch thc ht 5 m)

2.3.3 Pha dung dch

Dung mi
Dung mi 50% methanol (v th tch) c chun b bng cch pha th tch bng nhau ca methanol v nc.

Dung dch chun Melamine

Cn chnh xc 100mg melamine vo bnh nh mc 100mL, ha tan vi Methanol 50% bng cch rung trong b rung siu m 30 pht. Ln th tch 100 mL thu c dung dch c nng melamine 1000 gmL-1. Pha long 5mL melamine trn (1000 gmL-1) bng 50 ml 50% methanol c nng melamine 100 gmL-1.

Pha long tip 5mL melamine 100 gmL-1 bng 50 ml methanol 50% c nng melamine 10 gmL-1.

Chun b mu
Cho 10 ml (mu dng lng) hoc 1,0 g (mu kh) vo bnh nh mc 100 ml. Mu c pha vi 10 ml dung dch melamine ca 1000 gmL-1, c pha long vi 50 ml dung dch methanol 50% v rung siu m 30 pht. Sau khi lm lnh v nhit phng, ln th tch 100 ml bng dung dch methanol 50% v thu c dung dch hn hp vi nng melamine l 100 gmL-1 Cho dung dch mu thu c vo ng ly tm v ly tm ( 3200 rpm) ri ly phn dung dch pha trn ng (5 ml) chuyn sang bnh nh mc 50ml, v ln th tch 50 ml bng dung dch methanol 50%. Mu c lc qua b lc 0.45 m (mng Nylon). Nghin cu v dch lc ch ra rng cc b lc hp th melamine ti cc mc sau: 22% (cho 1ml ban u), 10% (2 mL), <1% (3, 5 v 7ml). Nh vy, 3ml ban u ca dch lc b loi b phn lc cn li c b sung vo mt l HPLC phn tch.

Cc thng s ci t
HPLC l phng php xt nghim melamine trong thc phm c s dng ph bin trn th gii t kh lu, cc trung tm xt nghim melamine Vit Nam cng ang s dng phng php ny. Nng c th pht hin: theo cc bo co phng php ny pht hin melamine vi nng khong vi chc ppb (parts per billion - 1 phn t). Cc tham s khi dng HPLC (Theo tiu chun ca FDA): + Ct hp ph: VenusilASBC8 kch thc: 4.6 x 250 mm; + Pha ng: buffer: acetonitrile (ACN) = 85:15; 10mM citric acid; 10mM sodium octane, pH=3.0; +tc chy: 1.0 mL/min +Bc sng tia UV: 240nm

+Nhit : 40oC +Detector: DAD

2.3.4 Qu trnh tin hnh

Nguyn tc hot ng : pha ng sau khi bi kh c bm a vo h
thng sc k, h thng s t ng bm mu vo ct. Pha ng khi i ngang qua s cun theo mu cn phn tch i theo. Khi vo trong ct sc k, cht no c i lc mnh vi pha ng s theo pha ng i ra trc, cht no c i lc yu hn s i ra sau. u d detector pht hin cc cu t i ra khi ct chuyn tn hiu cho my ghi kt qu

Hnh 2.3: S l thuyt qu trnh tin hnh Trong qu trnh ny ta cn lu mt s im sau: a mu vo: bc ny kh phc tp v p sut u vo kh cao Thi gian lu mu: l khong thi gian a mu vo n khi nh (peak) hin th r nt. B chi phi bi cc yu t: p sut u vo Tnh cht ca pha tnh ( hp cht, kch thc ht ) Thnh phn dung mi Nhit ct

My d (detector ): cc hp cht hu c thng hp thu tia UV, mi cht hp th mnh nht i vi mt bc sng nht nh. Chiu tia UV xuyn qua dng hn hp u ra, pha i din t mt my d ta c th o c mc hp th tia UV. T c th tnh c nng cc cht. Nn nh dung mi cng hp th tia UV nn la chn cc bc sng thch hp Dch kt qu t my d: Kt qu thng gm mt dy peak, mi peak i din cho mt hp cht trong hn hp i qua ct hp ph v hp th tia UV. Ta c th iu khin iu kin ca ct hp ph v thi gian lu xc nh s c mt ca cc hp cht. Nhng tham s iu khin ny c o trc vi cc mu th. Tuy nhin, t peak ny ta c th nh lng c cc hp cht. Gi s c mt hp cht X. Chng ta a mt dung dch c mt lng hp cht X bit trc vo my. T c th xc nh c thi gian lu cng nh s tng ng khi lng cht X v hnh nh ca peak. Din tch bn di peak tng ng vi s lng cht X i qua my d v din tch ny c th tnh c nh my tnh ni vi mn hnh:

Nu nng cht X nh hn trong mu, nu ta vn gi nguyn cc tham s my ta s thu c mt peak nh hn:

Kt qu ny l s so snh tng i vi mu th nn cng c ngha ta c th iu chnh my o c th nhn ra cc cht c hm lng rt nh. Kt hp vi mt my ghi ph khi: mt my ghi ph khi s bin i cc peak thnh cc vch. T so snh vi d liu c sn, chng ta s bit c s c mt ca nhiu hp cht m khng cn quan tm n thi gian lu ca cht khi i qua ct hp ph v pht hin c cht cn xc nh c vt qu gii hn cho php so vi mu chun hay khng.

Chng 3: Cc bin php khc phc khi sa b nhim melamine

Ti Vit Nam hin nay, vic x l sa nhim melamine c thc hin bng phng php t, cn ti Trung Quc x l bng cch ng gch tiu tn 100$/tn (t r hn 70%) Ngoi ra c th x l sa nhim melamine thnh phn bn hu c bng cng ngh sinh hc, qui trnh tng t nh sn xut phn vi sinh, tuy nhin im khc bit duy nht l phi b sung thm mt s chng vi sinh vt chn lc chng vi sinh vt c enzyme c kh nng phn hy sa hon ton.

Kt lun
Pht hin nh lng melamine c th c thc hin nhanh chng v d dng bng cch s dng HPLC / UV. Phng php ny c th c thng c s dng nh mt bi kim tra tiu chun melamine trnh sn phm sa cht ngi i vo th trng. Th trng sa hin nay chuyn ng khng ngng, cc sn phm sa ni ln ngoi c t tung ra th trng. Cc cng ty sa u t mnh vo cc chin dch qung co trn bo i, ngy cng xut hin nhiu cc sn phm sa vi nhiu loi, nhiu gi c, mu m v li ch khc nhau, do la chn mt loi sa ph hp an ton khng phi l iu d dng. Chng ta cn phi cn trng, sng sut trong vic la chn mua v s dng sa, m bo nhu cu dinh dng cng nh an ton cho sc khe, hy l ngi tiu dung thng thi bo v chnh bn thn mnh v nhng ngi thn yu.

TI LIU THAM KHO

[1] Gopalakrishnan Venkatasami, John R. Sowa Jr. Department of Chemistry and Biochemistry, Seton Hall University, 400 South Orange Ave., South Orange, NJ 07079, USA [2] Nguyn Thanh Nam (2012), Bi ging thit b phn tch thc phm, Khoa Cng ngh thc phm, Trng i hc Cng nghip thc phm TP.HCM [3] Nguyn Thanh Nam (2012), Gio trnh Phn tch thc phm, Khoa Cng ngh thc phm, trng i hc Cng nghip thc phm TP.HCM [4] http://www.chemguide.co.uk/analysis/chromatography/hplc.html [5]http://phantichmoitruong.com/detail/ky-thuat-phan-tich-sac-ky-long-cao-aphplc.html [6] http://www.voer.edu.vn/module/melamine-ung-dung-va-tac-hai [7] http://www.case.vn/vi-VN/87/88/117/details.case [8]http://www.ykhoa.net/binhluan/nguyenvantuan/081001_nguyenvantuan_lieulu ongmelamime.htm