Forensic Chemistry

Laboratory Manual Revision 7.1

Dr. Bruce McCord Department of Chemistry

Florida International niversity

!prin" #$$%

&his document is dedicated to the memory of 'nthony (. 'ndre)s *1+,%-#$$1. /rofessor of Forensic Chemistry0 1hio niversity.

Laboratory Schedule 2ee3 1 2ee3 # 2ee3 4 2ee3 7 2ee3 % 2ee3 , 2ee3 7 2ee3 ; 2ee3 + 2ee3 1$ 2ee3 11 2ee3 1# 2ee3 14 2ee3 17 2ee3 1% Footprints and Fin"erprints 1 Footprints and Fin"erprints # !ei5ed Dru"s6 !pot tests !ei5ed Dru"s6 microcrystal tests !ei5ed Dru"s6 F&IR8 98:C8 Documents and &race6 In3s by &LC Documents and &race6 Fibers by /LM Documents and &race6 :lass <=plosives6 Lo) <=plosives 1 <=plosives6 Lo) <=plosives # &o=icolo"y6:C8M! 1 &o=icolo"y6 :C8M! # 'rson 1 'rson # /resentation of Results *Rotation Order 1a 1b #a #b #c 4a 4b 4c 7 7 % % , , 7

Gr
1 2 3 4

1 1a 1a 1a 1a

2 1b 1b 1b 1b

3 #a #a #b #b

4 #b #b #a #a

5 #c #c #c #c

6 4a 4a 4b 4b 4b 4b 4a 4a

! 4c 4c 4c 4c

" 7 % ,

1# 7 % ,

11 % , 7

12 % , 7

13 , 7 %

14 , 7 %

15 mc mc mc mc

&able of Contents

Laboratory /rocedures and :eneral Information 1. Footprints and Fin"erprints #. Dru" analysis6 microscopic tests0 color tests0 and instrumental 4. &race <vidence6 in3s0 paints and fibers 7. :lass 'nalysis %. <=plosives analysis6 !pot tests0 F&IR0 Capillary electrophoresis ,. Dru" e=traction in urine6 :C8M! 7. 'rson analysis6 :C-FID 'ppendi= '0 color test rea"ents >>>>>>>>>>>>..>>>...7,

'ppendi= B0 !ample Lab Report>>>>>>>>>>>>>>>>>., ,%

?otes 1. <ach student )ill have one laboratory report for )hich the score )ill be thro)n out #. Removal from the labs or in"estion of controlled substances by any route0 removal of e@uipment0 "lass)are0 and8or chemicals0 cheatin"0 dishonesty0 pla"iarism0 or deception in fulfillin" re@uirements )ill not be tolerated. /enalties include failin" the course0 and criminal prosecution.

L$%OR$&OR' (O&)%OO* +ROC),-R)S 1. &he first pa"e of the noteboo3 should have your name and the course name. #. &he second pa"e of the noteboo3 should be a table of contents. <ach time an e=periment be"ins0 a pa"e number entry for the e=periment should be entered in the table of contents. 4. 2hen you enter the laboratory to start an e=periment0 the follo)in" should already be included in your noteboo3. a. <=periment name. b. 2ritten statement of purpose. c. !ummary of procedure to be performed condensed from the e=periment handout. d. !pace set aside and labelled for the e=perimental data to be ta3en. 7. Data collected in the laboratory should be entered in the allocated space in the noteboo3. %. 's you conduct the e=periment0 note observations in your noteboo3. For e=ample0 note any une=pected problems )ith the e@uipment or procedures. ,. 'll noteboo3 entries should be in in3. 7. <ach pa"e should be labelled )ith the date. ;. .m/ortant0 't the end of each period0 have the instructor or &' initial your noteboo3 pa"es.

G)()R$L .(FOR1$&.O( !tudents )ill )or3 in assi"ned "roups. <ach student is re@uired to )rite a formal report on each e=periment. &he formal report is to conform to the "uidelines of the 'C! style "uide and is due 7 days after the completion of that e=periment. 4 points )ill be deducted for each day that a laboratory report is late. <ach report M !& be typed. Aand )ritten reports )ill have 1% points deducted. &he report should follo) the outline belo) and point values are a "uideline. Introduction ' brief *1$$ )ords ma=.. description of the purpose of the e=periment. <=perimental Description of e@uipment and chemicals used. 'ppropriate information on the sample analysed *sample number0 physical description0 etc.. Results ' summary of the results that you obtained. 'ns)er any @uestions "iven in each e=periment. Conclusions 2hat you learned from the e=periment. 'n e=ample )ould be B&he tests performed *name the tests. indicated that sample CDE contained cocaineB. Dra) the correct conclusions from the results that you obtain. Include sources of error. References Include this document and any other resources you used. &' 'ssessment &ypos0 proofreadin"0 "eneral flo) of lab report0 lab behaviour includin" team)or30 cleanin" of "lass)are0 etc. 'ns)ers to Fuestions 7

:radin"6 Labs )ill be "raded on a 1$$ point scale. :radin" )ill be based on the follo)in" criterion 1. <=planation and understandin" of the science *1$ points. - Does the report sho) sufficient bac3"round information for the reader to follo) and understand the e=perimentsG #. :rammar0 or"ani5ation0 and references>>. *1$ points. - Is the document clearly )ritten0 )ell or"ani5ed and properly referencedG 4. Fi"ures and photo"raphs >>>>>>>>*#$ points. - 're there sufficient photo"raphs0 tables and "raphs necessary to support the results. 're they properly labelled and referenced in the te=tG 7. <=perimental procedures >>>>>>>.. *1$ points. - Is sufficient information available to allo) the reader to repeat the e=perimentG 2as the laboratory e=periment properly recorded in the noteboo3G *References to specific laboratory procedures in this handout are 1H. %. Results >>>>>>>>>>>>>>>*#$ points. - Do the results of the e=periment match the 3no)n and are sources of variance properly noted and e=plainedG 're all the points noted in the discussion section coveredG ,. 'ns)ers to @uestions and evaluation of paper...*4$ points.

?ote6 Certain @uestions )ill re@uire access to the library0 to your supplemental readin"s0 or to your class notes. !ee your instructor if you are havin" trouble findin" source materials. Dou are responsible for )ashin" "lass)are you used accordin" to the instructions and to the satisfaction of the instructor before you leave the laboratory. 'l)ays rinse used "lass)are thorou"hly )ith tap )ater to remove the chemicals and other contaminants before placin" them in a deter"ent solution or other cleanin" solution. se a distilled )ater final rinse. Dou are e=pected to 3eep the laboratories clean and orderly. Many of the compounds and e=periments )e )ill perform re@uire attention to detail. Read e=periments carefully prior to comin" to lab and follo) all safety precautions- "loves )ith controlled substances0 "o""les and lab coats at all times. Follo) the laboratory safety rules and the instructions on each e=periment on the disposal of )aste materials and chemicals.

1. Footprints and fingerprints Scenario 2 't the scene of the crime0 a footprint )as found in the mud. 'lso0 several items )ere collected that may have the suspectIs fin"erprints on them. sin" the supplies and procedures belo)0 determine )hich suspect matches the footprint and fin"erprints found at the scene. For the fin"erprint analysis0 determine )hich suspect the print belon"s to by hi"hli"htin" the patterns of reco"nition provided in class notes and supplemental material. Dou may )ant to practice all of the techni@ues on your o)n fin"erprints and determine )hich method )or3s best0 before analy5in" the suspect prints. (o s/eci3ic 3oot/rint /recautions or ha4ards5 Foot/rint .ntroduction Calcium sulfate dehydrate or "ypsum0 dehydrates upon heatin" to form calcium sulfate hemihydrate or plaster of /aris. #Ca!17#A#1*s. J heat *Ca!o7.#A#1*s. J 4A#1*". "ypsum plaster of /aris 2hen enou"h )ater is added to plaster of /aris to ma3e a paste0 it @uic3ly hardens as it reverts to "ypsum. &he mi=ture also e=pands as it hardens and forms a sharp impression of the obKect in contact )ith the mi=ture. &he rate of hardenin" decreases as the amount of )ater used increases. &he FBI no) recommends the use of Dental !tone or Die !tone as they re@uire less )ater and have "reater hardness. &his ma3es it unnecessary to reinforce the cast. Less material is re@uired as )ell. Su//lies and e6ui/ment 2ooden bo=0 earth and ba" of die stone cast *about 1 3".. +rocedure 1. &o hold the particles of earth in place to preserve the fine structure0 allo) a mist of lac@uer to settle onto the print by sprayin" the lac@uer hori5ontally above the shoeprint. Caution6 Do not spray directly onto the shoeprint. #. /hoto"raph the shoeprint in earth )ith obli@ue li"htin" )ith the film plane parallel to the shoeprint. /lace a , to 1#B ri"id ruler ne=t to the impression or at the bottom of the impressionLs surface. 4. 'dd appro=imately #;$ " of )ater to the ba" of Die !tone. Massa"e the mi=ture throu"h the closed ba". &he mi=ture should pour easily li3e a panca3e batter but not too )atery0 it should be runny0 but not too dry. 'dd more Die !tone or )ater if needed. 7. ?ote the time you start to add the mi=ture to the shoeprint. Carefully pour the mi=ture onto the shoeprint *from "round level.. Fill the impression completely so that the mi=ture overflo)s out of the impression. %. Inscribe identifyin" information *your initials and date. on the surface of the cast )hile it is dryin". Include any other relevant information. se strai"ht lines instead of cursive )ritin". se the dryin" time to note the characteristics of the shoes collected from the suspects. ,. ?ote the time )hen the upper crust of the cast is hard. &his )ill be at least 4$ minutes. Carefully lift the cast from the bo= of earth. Do not attempt to clean the cast0 allo) it to air dry for 7; hours. Measure the cast of the shoeprint and the shoe used0 and compare the ,

measurements. Record the measurements. Compare them )ith the measurements made on the photo"raph. 7. Carefully e=amine the cast for any uni@ue mar3s such as cuts0 tears0 )ears0 and8or imbedded materials. Record such mar3s. Dou can )ash the cast )ith )ater0 it )ill not dissolve or crumble. ,is/osal ?o specific disposal instructions. Fin7er/rint +recautions (inhydrin is rube3acient and a /oison8 use the hood5 ,o not loo9 at the -: li7ht5 Fin7er/rint .ntroduction Fin"erprints on paper0 cardboard0 unpainted )ood0 and other absorbent surfaces are hard to obtain by dustin" )ith fin"erprint po)der and are much better developed by chemical methods. <ach of the follo)in" chemicals reacts )ith a different substance0 )hich may be present in the latent print a5 iodine reacts )ith the double bonds in unsaturated fatty acids. b5 ninhydrin reacts )ith the free amino and carbo=yl "roups in proteins and peptides. c5 silver nitrate reacts )ith chloride ions. 'ny one or all three of the chemicals may be used on most absorbent surfaces. 2hen all three chemical methods are used0 they must be used in the above se@uence *a0 b0 and c.. If the chemical methods are to be used0 fin"erprint po)ders should not be applied to the articles because po)ders cannot be removed from paper and may interfere )ith some types of document e=amination. Fin"erprints on plastic ba""ies and rubber obKects cannot be obtained by po)der or the above chemicals but can be obtained by fumin" )ith the vapour of cyanoacrylate in Bsuper"luesB. &he ima"e may be seen )ith or )ithout dustin" )ith po)der or stainin" )ith dyes. Su//lies and e6ui/ment a :eneral supplies6 &)ee5ers0 disposable "loves0 scissors0 electric steam iron b c d Iodine fumin" chamber method6 Iodine0 &LC tan3 and lid0 blan3 "lass &LC plate0 scotch tape0 and "lass tray for )armin" &LC tan3 )ith hot )ater. ?ihydrin method6 ?inhydrin solution6 ninhydrin *% ". in methanol *1$$ mL.0 nylon brush0 steam iron and distilled )ater0 and cardboard and blotter to protect table top from steam iron. !ilver nitrate method6 !ilver nitrate *4 ". in distilled )ater *1$$ mL.. !tore in a bro)n bottle. :lass tray0 nylon brush0 and blotters0 hi"h intensity ultraviolet lamp and 9 protective "o""les. Cyanoacrylate method6 !uper"lue0 sodium hydro=ide *1$ M a@ueous solution.0 &LC tan30 # bea3ers *1%$ mL.0 strin"0 scotch tape0 paper clips0 aluminium )ei"hin" cups0 cotton "au5e pad.

+rocedure /reparation of fin"erprint samples /repare the follo)in" samples for your o)n e=periments. /rint your initials )ith a felt-tip mar3er on the upper ri"ht hand corner of each sample. Be careful to handle the items )hile

)earin" "loves. In dry )eather0 you can improve the fin"erprints by )ipin" your fin"ers on s)eaty parts of your body or puttin" moisturisin" lotion or cream on your hand. <ach "roup may )ant to prepare a stoc3 of the items listed belo) and use as re@uired. a5 1ne plastic ba""ie for the super"lue method. b5 1ne aluminium can or rubber obKect. c5 &hree 4=% inde= cards. d5 &hree 4=% pieces of bro)n )rappin" paper. e5 &hree 4=% pieces of )hite letter paper. Recordin" a /hoto"raphy. b Record in )ords6 *1. e=perimental conditions *such as time0 temperature0 etc.0 *#. observations and results *the speed of development0 colour and @uality of the developed print0 problems encountered and remedies proposed or attempted0 etc.0 and *4. discussion *relative merits of the different methods0 the effects of the different surfaces have on the results0 etc.. c From your results compare all the chemical methods of development of latent fin"erprints in all aspects0 includin" speed0 reliability0 clarity of prints0 effects of the materials on )hich the prints )ere made and ease of application. Iodine method &he ima"e formed by the iodine method is not permanent and should be photo"raphed as soon as the print is le"ible. a /repare an iodine-fumin" chamber by placin" sublimated iodine crystals *N teaspoon.on the bottom of a &LC development tan3 )ith cover. &ape the samples on a piece of blan3 &LC "lass and place the "lass )ith the samples facin" the bottom of the tan3. /lace the tan3 in a shallo) pan of hot )ater to speed up the iodine vaporisation. Do not heat by any other method as this )ill brea3 the &LC tan3. ?inhydrin method Brush the solution on your sample usin" a nylon brush or spray the solution. Aeat the brushed or sprayed sample )ith steam from a steam iron held about 1 inch above the paper. Record the duration of heatin" re@uired to brin" out the print. !ilver nitrate method /lace the silver nitrate solution in a "lass tray in an illuminated room but not in direct sunli"ht. Immerse the specimen in the solution until the surfaces are completely moistened. Remove it0 place it bet)een blotters to remove the e=cess solution0 and dry it. *sample can be sli"htly damp.. <=pose the treated specimen to sunli"ht0 a 1 $$$ )att photoflood lamp0 or a hi"h intensity ultraviolet lamp. 's soon as the rid"e details of the prints are clearly visible0 remove the paper from the li"ht and photo"raph it promptly. !ilver nitrate treated prints become ille"ible in a fe) hours )hen e=posed to li"ht but )ill 3eep for a lon" time in total dar3ness. Lar"er obKects may be treated by brushin" )ith the solutions. Cyanoacrylate method 'ttach a piece of strin" across the openin" of the &LC tan3 to suspend other obKects )ith paper clips. se either of the follo)in" methods and determine the conditions of the development. a5 /lace a small aluminium dish of super"lue ne=t to or above a bea3er of hot )ater in the &LC tan3 )ith cover. *It may be necessary to use a small heatin" mantle or hot plate to )arm the super"lue.. b5 !oa3 cotton "au5e pads )ith ?a1A *1$ M. and han" them up to dry in the hood. 2hen ready to fume obKects0 place a pad in an aluminium dish and drop super"lue on the pad and close the tan3. 9ie) the developed latent prints in obli@ue li"ht0 photo"raph the resultin" print0 and record in )ords. Dust the print before photo"raphin" if necessary. &o improve the print0 spray or dip the ;

print area in 'rdro= stain *1M in isopropanol.. 'fter the print area is dry0 )ear 9 protective "lasses and vie) it under a 1$$ )att mercury vapour lamp.1 ,iscussion 1. For each un3no)n fin"er and foot print attach a fi"ure and point our note % distin"uishin" features. 2hich li"htin" and developin" techni@ues )ere able to best reproduce these featuresG #. For each techni@ue0 e=plain usin" full chemical reactions0 the mechanism for developin" the latent prints. 4. Discus ho) you )ould convince a Kury that your results uni@uely identifies a particular shoe or fin"erprint.

,is/osal ?inhydrin and silver )aste should be placed in the containers provided for this )aste. ;uestions 1. 2hat are the advanta"es and disadvanta"es of plaster of /aris as a castin" materialG #. In your opinion0 )hat minimum number of identifyin" features constitutes an identityG 2hyG 4. Ao) )ould you validate the ninhydrin test for fin"er prints0 ie )hat tests )ould you perform to sho) that the tests al)ays "ive the same results for a "iven sample and that the rea"ents al)ays )or3G 7. :o to the library. Find and copy one or more current articles on the use of laser induced fluorescence in the analysis of fin"erprints or some other aspect of latent fin"erprint analysis. 2rite a brief *1-pa"e. revie) of the article )hich needs to be a summary and criti@ue of the article in your o)n )ords. &he article must be from a peer-revie)ed Kournal. Internet and company articles are not acceptable. /lease contact your instructor if you have trouble findin" an article. 'ttach the article to your lab report.

(.F. Fallano0 Hoda3 &echbits 1++#.

2. Drug analysis: microscopic tests, color tests, and instrumental analysis Scenario 2 ' raid )as performed on a dru" dealers home. !everal solid dru"s )ere found0 ho)ever it is believed the dealer )as also mi=in" the dru"s )ith other materials to improve his profits *herbs mi=ed )ith mariKuana0 aspirin mi=ed )ith cocaine0 etc.. Dou have been "iven a variety of samples to identify. sin" morpholo"y0 spot tests0 microcrystal tests and instrumental techni@ues0 determine )hich dru"s and8or diluents are present in your samples. Micro-chemical crystal tests0 IR0 M! and 9-9I! spectroscopy can be useful techni@ues in forensic chemistry. &his e=periment should "ive some indication of the usefulness and limitations of these different methods and illustrate their application in @uantitative analysis

+recautions $s detailed in $//endi< $ 3or the a//ro/riate color tests5 Chloral hydrate is harm3ul =hen in7ested5 +etroleum ether is e<tremely 3lammable5 .ntroduction Cannabis sativa *Cannabinaceae.0 marihuana0 is an annual plant of 4 to 1, ft in hei"ht. &he BresinB )hich contains the psychoactive chemicals *principal OH component is +-tetrahydrocannabinol0 C#4A4$170 M2 O 4%;.%0 C'! #4+7;-;%-$. occurs mainly in the H3C flo)erin" area0 the leaves and the stem0 particularly at O CH3 the top of the plants0 )ith the hi"hest amount found in H3C the flo)erin" area. 1nly the roots and the lo)er parts of the stal3s are usually free from the resin. <ven tiny seedlin"s that have Kust developed their first true leaves contain some resin. &he male and female plants produce resin nearly e@ually up to the time of flo)erin". &he male plants die shortly after sheddin" their pollen. &he maKor metabolites are 11-hydro=y-+-tetahydrocannabinol and 11-nor-+-carbo=y-+-tetahydrocannabinol. Marihuana is a schedule 1 dru". CH3 &he leaves of marihuana are compound and consist of % to 11 separate leaflets. <ach leaflet has characteristic hairs0 veins0 and serrated ed"es. &he most characteristic feature is the hairs. &here are t)o types of hairs 1. Cystolith hairs have deposits of calcium carbonate at their base. &hese hairs are mostly onecelled. #. :ladular hairs contain and secrete the resin. &hey are short and may be unicelluar or multicellular. Lar"er "landular hairs have a multicellular stal3 )ith heads containin" , to ; cells. /roperties Form + -&AC 15+t5 > C viscous oil ?2O Insoluble Solubility ethanol chloro3orm 1 in 1 Readily soluble ether

$m/hetamines 1$

'mphetamine0 *1-phenyl-#-aminopropane0 C+A14?0 M2 O 14%.#0 C'! CH2CHCH3 4$$-,#-+.0 is commonly available as the racemic mi=ture or the pure disomer )hich are both used as a central nervous system *C?!. stimulant NH2 and in treatin" obesity0 nacrolepsy0 and hypertension. &he d-isomer is 4 to 7 times more effective than the 1-isomer. &he metabolites are norephedrine0 phenylacetone0 and ben5oic acid. 'mphetamines are schedule II dru"s. Methamphetamine0 *1-phenyl-#-methylaminopropane0 C1$A1%?0 M2 O 17+.#0 C'! %47-7,-#.0 is anore=ic and is used in treatin" attention deficit disorder )ith hyperactivity. &he hydrochloride of the d-isomer is used in the treatment of obesity. &he 1-isomer0 )hich is purported to have )ea3er C?! stimulant activity and "reater peripheral sympathomimetic activity0 is used as a decon"estant in certain over-the-counter *1&C. inhalers. &he principal metabolite is amphetamine. /roperties Form 'mphetamine Methamphetamine 15+t5 > C li@uid Li@uid ?2O 1 in %$ !li"htly soluble Solubility ethanol very soluble miscible chloro3orm very soluble miscible ether very soluble miscible

<phedrine0 l-isomer of 1-phenyl-l-hydro=y-#-methylaminopropane0 occurs naturally in the Chinese dru" mahuan" *<phedra vul"aris0 <. !inica.. It is a sympathomimetic amine0 )hich has lar"e peripheral stimulant activity and mild C?! stimulant effect. Its chloride or sulphate is used as a bronchodilator. &he metabolite is norephedrine. /seudoephedrine *!udafed.0 d-isomer of ephedrine0 is used as a nasal decon"estant and is found in 1&C cold and aller"y dru"s in combination )ith antihistamines and anal"esics. &he metabolite is norpseudoephedrine.

Heroin Aeroin *40,-1-diacetylmorphine0 C#1A#4?1%0 M.2 O 4,+.70 C'! %,1-#7-4. is a semi-synthetic opiate produced from morphine *a naturally occurrin" substance in the opium poppy Papaver somniferum.. &he maKor metabolites are ,-monacteylmorphine O then morphine *and "lucuronides. and codeine. Morphine is a NCH3 potent narcotic anal"esic0 and its primary clinical use is in the mana"ement of moderately severe and severe pain. 'fter heroin0 morphine has the "reatest dependence liability of the narcotic CH3COO anal"esics in common use. 1ther semi-synthetic analo"ues of morphine are also available as pain3illers in the !. Aeroin is a schedule II dru". CH3COO /roperties Form base hydrochloride 15+t5 > C 17$ ##+-#44 ?2O 1 in 17$$ 1 in 1., Solubility ethanol chloro3orm ether 1 in 41 1 in 1.% 1 in 1$$ 1 in 1# 1 in 1., Insoluble

CH3 COOCH3

Cocaine *methyl ben5oylec"onine0 C17A#1?170 M2 O 4$4.70 C'! %$4,-#. is a tropane al3aloid obtained from coca0 the dried leaves of Erythroxylum coca and other species of Erythroxylum. &he !outh 11

OOCC6H5 H

'merican species contains more cocaine than the 'sian species. It can be synthesised from eco"nine or simpler startin" materials. &he natural product is optically active. &he maKor metabolites are ec"onine0 ec"onine methyl ester0 and ben5oylec"onine. &)o maKor forms are found in the !0 the hydrochloride salt *co3e. and the free base *crac3.. Cocaine is a schedule II dru". /roperties Form base hydrochloride 15+t5 > C +,-+; 1+7 *decomp. ?2O 1 in ,$$ 1 in $.% Solubility ethanol chloro3orm 1 in 7 1 in $.% 1 in 7 1 in 1% ether 1 in 7 insoluble

H2C CH H

HO CH3O

H H

!treet dru"s are commonly mi=ed )ith other materials to increase bul3 and dilute the active in"redients. Cocaine and heroin are the dru"s to )hich diluents are most commonly added. Ao)ever0 diluents may be added to any dru" that they possess similar physical appearance to. &he most common diluents are su"ars0 inositol *an isomer of "lucose.0 talcum po)der0 starch0 caffeine and @uinine *sho)n.. It is important to be able to distin"uish the diluents from the dru" compound.

Su//lies and e6ui/ment !everal samples of plant material provided by the &'. 'ppropriate solutions prepared as in 'ppendi= '. +rocedure Microscopic tests Record your observations0 dra) dia"rams0 and photo"raph the characteristic hairs and adKacent structures so that you 3no) )here to find the hairs. a /lace a small portion of plant material on a microscopic slide. 'dd one to t)o drops of distilled )ater and flatten the material )ith a cover "lass. Inspect at 4$= to 1$$= ma"nification. 'dd one to t)o drops ACl *1 M. to the materials under the cover "lass and )atch for carbon dio=ide bubbles formed at the base of the cystolith hairs. Do not leave the slide )ith ACl on the microscope sta"e. b /repare a similar sample and treat it )ith 4 drops of saturated a@ueous chloral hydrate solution. Cover )ith a cover "lass and )arm "ently over a hot plate in hood. Chloral hydrate removes coloured materials. <=amine at ,$= to #$$= for more detailed information on the structure of the plant tissues Repeat a and b )ith other plant materials. Carry out color tests on the follo)in" compounds as described in the attached article by 1I?eal.
Du@uenois-Levine Mar@uiss C C C C Mandelin C C C Cobalt isothiocyanate ?itric 'cid Mec3e C C C C C

Methamphetamine Marihuana ?utme"

1#

'spirin Codeine Cocaine 'cetaminophen <pehdrine &ea Fuinine !u"ar Caffeine n3no)n

C C C C C C C C C

C C C C C C

C C C C C C C C C C C C C C

C C C C

C C C

Su//lies and e6ui/ment

.ntroduction For absorption in the IR re"ion there must be chan"e in dipole moment *polarity. of molecule. Diatomics must have permanent dipole. !o ?#8Cl# do ?1& absorb. 1ther molecules need not have permanent dipole0 they can e=hibit a dipole by vibration6 IR spectroscopy is very useful in forensic chemistry because each molecule has a uni@ue absorption spectrum0 hence a Lfin"erprintL pattern is obtained. IR is )idely used for @ualitative purposes0 thou"h @uantitation is difficult. &o perform @uantitative IR one must remember that beers la) holds here and that the thic3ness of your HBr pellet must be constant *not really possible.. Instead an internal standard such as calcium carbonate0 napthalene or sodium nitrite is usually added to the HBR mi=ture to perform @uantitative analysis. 'lternatively0 if a mi=ture of t)o substances is present0 the ratio of t)o pea3s0 one from component ' and one from component B can be utili5ed to determine M concentration )ith a calibration curve. ?ote that you must dra) a baseline across the bottom of the pea3 and use the pea3 hei"ht in your measurements. +recautions @ Sul/huric acid is corrosiAe5 Sodium hydro<ide is corrosiAe5 1icrochemical tests introduction 'n older but still viable method for determination of un3no)n dru"s is the microchemical crystal test. In the procedure a drop of test rea"ent is added to a solution containin" a small amount of the un3no)n dru". &he procedure is performed by placin" a drop of the test solution and a drop of the rea"ent solution on a microscope slide. Ma"nification is set at 1$$- 7$$C &he t)o drops are brou"ht to"ether usin" a small "lass rod or spatula. !pecific crystals are formed by the reaction and can be characteri5ed usin" a microscope. )</erimental +rocedure /repare certain rea"ents from the list belo) *as3 &' for availability of rea"ents.. &est your samples by addin" the dru" directly to the rea"ent or by dissolvin" a small amount of the compound in the test solution minus the rea"ent and placin" a drop of each solution onto the microscope slide. se a spatula to move the t)o drops to"ether. 1bserve crystal formation at the interface bet)een the t)o solutions. .R +rocedure /repare dis3s of the test compounds *# m". in HBr *1$$ m".. /repare a HBr blan3 as )ell. *if available you may use the '&R system instead.. 1btain an IR spectrum for each compound 14

bet)een the )ave numbers 7$$ and 7$$$. Collect at least ,7 one second scans *7$+, data points8scan.. Compare the obtained spectra )ith the provided reference spectra. 1. /repare and print spectra of the blan30 dru"s and diluent samples *blan3 may contain an internal standard.. #. <=amine the spectra of the un3no)n and usin" spectral subtraction0 determine its identity and the identity of the diluent it is mi=ed )ith. 4. /repare a calibration curve consistin" of mi=tures of dru" and diluent. 7. Determine the M dru" in your un3no)n0 usin" the ratio of specific pea3s of each component. %. Determine the total @uantity of dru" in your un3no)n. ,is/osal @ Dissolve solid )aste in dilute acid and dispose of do)n the drain )ith copious amounts of )ater. -:@:is +rocedure /erform 9 spectral analysis of a set of pure dru"s and your un3no)n dru" in methanol. Does the un3no)n 3no)n appear pureG !amples should be very dilute *appro=. 1 m"8L.. Collect 9 spectra over the )avelen"ths ##$ nm to 47$ nm. Be very careful to avoid contamination or pea3 overload. se blan3s and perform several dilutions to ma3e sure your data is on scale and not overloaded. If your sample appears pure0 perform a @uantitation usin" BeerIs La). !ho) your results to your instructor. Be a)are that some diluents )ill not absorb in the 9. Ao) )ill this affect your resultsG ,iscussion 1. Ma3e a table listin" each dru" you analy5ed and the specific results from each test you employed. #. List si"nificant interferences for each test and e=plain )hy they occur 4. In your table0 compare the utility of each techni@ue and "ive advanta"es and disadvanta"es of each. 7. Detail ho) you arrived at your conclusion re"ardin" the identity of your un3no)n and ho) you arrived at that conclusion. %. sin" your results )ith 3no)n compounds )hat )as the effect of the diluent on your analysis by the different techni@uesG

,is/osal Chlorinated solvent and other or"anic solvent )aste must be placed in the appropriate container. 1ther )aste may be disposed of do)n the drain )ith copious amounts of )ater.

;uestions 'ssumin" a linear calibration curve0 calculate the M purity of each un3no)n. Ao) accurate )ill this result be in the presence of the diluentG Fi"ure a )ay to compensate for the presence of the diluent and "et an accurate result. 17

9ist the !2:DR : )eb site0 ))).s)"dru".or" and loo3 up the section on methods of analysis. <=plain )hy there are three distinct cata"ories of analytical methods and describe the difference bet)een them. #. If the dru" you )ere "iven did not match up )ith any of the spectra or color tests you used0 )hat could you do to identify the dru"G 4. 2hat determines the )avelen"th of absorption ma=ima for 9 spectraG
1.

7. !uppose you )ere usin" IR to analy5e these dru" mi=tures. Describe ho) you )ould @uantitate the concentration of cocaine in a mi=ture )ith su"ar usin" this techni@ueG Be specific. %. Codeine0 heroin0 and morphine have very similar structures. Dra) these structures and e=plain ho) you could tell them apart by IR spectroscopy. ,. 9isit the !2:dru" site and revie) the comments on validation. Describe )hat you consider to be the most important tests you )ould perform to validate a spot test for cocaine. 7. Find an article describin" a thin layer chromato"raphy *&LC. or other chromato"raphic test for the determination of marihuana. 2rite a brief *1-pa"e. revie) of the article )hich needs to be a summary and criti@ue of the article in your o)n )ords. &he article must be from a peerrevie)ed Kournal0 *1pp..

1%

3. Trace analysis: inks, fibers, and glass Scenario 2 ' ransom letter )as found in a plastic ba" inside the home of a 3idnapped child. ' suspect )as arrested0 and several pens found in their home )ere collected and labelled. sin" thin layer chromato"raphy0 determine if the in3 from any of the pens "athered match that used in the ransom note. Furthermore0 several small fibers and "lass particles )ere discovered in the plastic ba". sin" chemical tests0 polari5in" li"ht microscopy and F&IR0 identify the composition of the fibers and compare them to materials collected from the suspects home. 'naly5e "lass particles usin" refractive inde= measurements and atomic spectroscopy. +recautions $Aoid loo9in7 directly at -: li7ht 3a5 &LC .ntroduction &he analysis of in3s and dyes is an important techni@ue in the arsenal of the document e=aminer. In3 analysis can be used to detect for"ery of documents0 to chec3 for bac3datin" of documents or falsification of si"natures. In its more sophisticated format0 in3 analysis has even been used to provide estimate of the date at )hich a particular document )as si"ned. !ome reasons for forensic in3 analysis include tests for the adulteration of numbers to increase the amount of money bein" char"ed0 tests to determine if the in3 used in t)o parts of a document came from the same pen0 or tests to compare the )ritin" used in a threatenin" letter )ith pens ta3en from a suspectIs home. Li@uid in3s used in ballpoint pens contain a mi=ture of substances0 )hich include dyes0 stabili5ers0 solvents0 and resinous binders. Certain manufacturers also add fluorescent mar3ers or heavy metals to aid in product identification. &hin layer chromato"raphy *&LC. is the preferred techni@ue for in3 analysis. In &LC0 a uniform layer of silica0 alumna or some other adsorbant is placed on a "lass plate. &he in3 samples are punched out of the paper0 e=tracted in pyridine0 and spotted onto the &LC plate usin" a "lass capillary as a transfer pipette. &he plate is then placed in a "lass tan3 containin" a layer of solvent at the bottom. &his solvent mi=ture is the chromato"raphic eluent used to separate the mi=ture of dyes present in the sample. &he distance a particular dye moves relative to the solvent front is 3no)n as the Rf value. Su//lies and )6ui/ment0 ' &LC tan3s0 silica &LC plates0 bea3ers0 scissors or syrin"e punch *a )ide bore syrin"e needle cut flat for punch holes in paper0 /lastic or )ood sheet for use )ith punch. :lass capillary tubes for spottin" plates0 1.;ml tubes )ith v bottoms. :lass transfer pipettes and bulbs0 9 li"ht bo=es. B. Rea"ents6 pyridine0 ethyl acetate0 butanol0 ethanol0 and methanol. +rocedure0 )</erimental Setu/6 1. /repare # solvent mi=tures for the &LC e=periments6 Mi=ture 1 contains 17 ml of ethyl acetate0 7 ml of ethanol and , ml of )ater. Mi=ture # contains 1$ ml of butanol0 #ml of ethanol and 4 ml of )ater. /lace each mi=ture in a separate &LC tan3 in the hood and cover )ith a "lass lid. #. Cut or punch out 1$ small paper dots from the in3ed line in the ransom letter. &hese dots must be e<tremely small and contain only the in9ed /art o3 the /a/er. se a syrin"e needle that has been s@uared off as a punch. 1n another separate piece of paper ma3e test )ritin"s )ith the pens obtained from the suspects apartment. Cut or punch dots from each letter. /lace each set of dots in 1,

to a small conical vial. In the hood0 add 1- # drops of pyridine to each tube. BCarnin7 /yridine has a stenchD *ee/ it in the hoodD ,is/ose all solAent in the hood as =ellE Be sure to use a minimal amount of pyridine or the sample )ill be too dilute. Let sit for 1$-1% minutes. If necessary "ently )arm or vibrate each sample to aid the e=traction. 'dd additional pyridine if necessary. 4. /repare t)o &LC plates by li"htly dra)in" a strai"ht line )ith a pencil across each plate 1 inch from the bottom. 7. !pot the plates usin" the capillary tubes by pacin" them into the e=tracted samples and dra)in" out a small amount of solution. 'pply a small spot0 let dry and repeat until a very small0 clearly visible spot appears on the plate. Be careful to not let the spot become too bi" or your e=periment )ill be ruined. %. <=amine the level of solvent in the &LC tan3. &he solvent can not be above sample spots )hen the plate is placed in the tan30 it should be appro=imately N inch up in the tan3. ,. /lace the plates in each tan3P be sure each sample is properly labelled in pencil at the top of the plate. 2atch as the solvent front moves slo)ly up the plate. 2hen the front is Q or so up the plate0 remove the plate0 mar3 the solvent from )ith a pencil0 and place in the hood to dry. 7. &a3e the dry plate and e=amine usin" both visible and 9 li"ht. Calculate Rf for each band. Rf O distance from ori"in of sample band8distance from ori"in of sample front. 2hich in3s have common ori"inG Do any sho) fluorescent bandsG 3b5 Fiber $nalysis 1. sin" the chemical tests listed belo)0 attempt to determine the composition of the fibers in your ba""ie. * If the amount of fiber is limited0 perform the non-distructive tests listed belo) first. . #. <=amine your un3no)n fiber and the 3no)n fibers provided by your instructor under the polari5ed li"ht microscope. *ta3e pictures if possible. For each fiber e=amine usin" the follo)in" conditions6 a. no polars R note morpholo"y b. one polar R rotate sta"e and note any effects on color due to orientation c. crossed polars R rotate sta"e0 note positions of e=tinction0 note color effects. Chec3 for birefrin"ence. 4. &a3e your fibers to the infrared microscope and obtain spectra of each fiber. Identify maKor pea3s and correlate )ith functional "roups present in the polymeric matri= of the fiber.

17

Sa3erstein8 R5 Criminalistics Lab 1anual8 +rentice ?all8 1""!8 /5 162

3c5 Glass $nalysis &his e=periment involves a series of demonstrations on the analysis of "lass. /lease revie) your handouts on "lass analysis and report to Dr. 'lmirals lab for further instructions.

,is/osal /lace or"anic )aste in proper containers. 'cids and bases may be disposed of do)n the drain )ith copious amounts of )ater. ;uestions0 1. Can &LC be used to determine conclusively )hether any t)o pens are the sameG #. 2hat advanta"es does &LC have over other chromato"raphic techni@ues for chemical analysisG 4. 2hat )as the difference bet)een the t)o solvent systems and )hy did they produce different resultsG 7. Describe the procedure you )ould use to determine the difference bet)een a ban3 note and a counterfeit note produced by a color photocopier. 1;

%. &LC can be used to date in3s. <=plain ho) this is done. !ee the article by Cantu et al. in your handouts or perform a literature search ,. Dra) the polymeric structure of each fiber you identified. 7. Compare the information obtained by IR to that from polari5ed li"ht microscopy. ;. Fiber analysis has been a 3ey factor in the solution of a number of important criminal cases. <=plain ho) e=perts can ans)er the @uestion - )hat is the possibility these sets of fibers appeared in the suspectLs house purely by chanceG *1pa"e.. Dou may use a scientific article to bac3 up your claims. 'ttach the article to your report.

1+

4. !plosi"es analysis: #pot Tests, FT$%, &apillary electrop'oresis Scenario 2 ' bomb )as placed in the department chairs mailbo= and all the "rade forms )ere destroyed. ' search )as performed of the li3ely culprit and some commercial fire)or3s )ere recovered. Dour Kob )ill be to analy5e a sample of the fire)or3 recovered from the scene and determine its components. Dou )ill then burn the material to determine if it is an e=plosive and if the residue pattern matches that recovered from an e=tract of the "rade forms. .ntroduction !pot tests0 IR spectroscopy0 and capillary electrophoresis are valuable techni@ues in the analysis of inor"anic e=plosives. &ypically an inor"anic e=plosive consists of inor"anic o=idi5ers such as potassium nitrate0 potassium perchlorate0 potassium chlorate0 and fuels such as 'l0 Fe0 ! and C. ' variety of spot tests and microcrystalline tests can be used to detect the o=idi5ers and fuels0 includin" the "reiss rea"ent and diphenyl amine for nitrates0 !ilver nitrate and methylene blue for chlorates and the formation of /russian blue for the detection of iron. For F&IR there are specific bands indicative of nitrate *14;4 cm-1. and chlorate *ca +$$cm-1.. !ulphates0 perchlorates and other components )ill also produce results. Lastly the inor"anic o=idi5ers )ill produce specific ions )hich can be determined by capillary electrophoresis or ion chromato"raphy. &he "oal for this e=periment is to use your laboratory s3ills to determine if the material is e=plosive0 and if so the nature of the o=idi5er and fuels. +recautions @ Sul/huric acid is corrosiAe5 Sodium hydro<ide is corrosiAe5 1i< in o/en containers $5 Flame test. /lace a small amount of the material on the tip of a spatula. /lace under a flame and observe reaction. %5 1icrosco/y B3or both burned and unburned materialE 1. 2earin" "loves0 e=amine the evidence by carefully cuttin" it open )ith a 3nife or scalpel. *Rinse blade )ith DI )ater prior to use. #. Recover the e=plosive material and e=amine it under the microscope. ?ote morpholo"y and loo3 for crystalline material. <=amine under crossed polars and note any specific features. 3E +re/are or obtain the 3ollo=in7 rea7ents aE Greiss rea7ent 3or nitrites0 /repare $.#% " napthol in ;$ mL of 4$M acetic acid and $.% " sulfanilic acid in %$ mL of 4$M acetic acid. 'dd one drop of each to test plate. Loo3 fro 1ran"e color. bE ,i/henlyamine test 3or nitrates /repare %m" diphenyl amine in %$ml of concentrated ACL0 loo3 for blue color as indicator of nitrate. cE +erchlorate and chlorate 'dd 1 drop conc. A#!17 *loo3 for bro)n "as indicative of chlorate.0 add a small amount of En to un3no)n to reduce the CL17-. 'dd '" ?14 and loo3 for )hite precipitate *Cl.. 'lso place # drops of solution of un3. in spot plate. 'dd # drops of saturated 5inc sulphate0 add # drops of 1$" potassium nitrate in 7$mL )ater *be)are contamination. and add 1 drop $.$4" methylene blue in 1$$mL of )ater dE $luminium #$

/lace 3no)n or un3no)n in 4 )ells of a spot plate. &est samples )ith conc. ACL0 1M ?a1A0 and conc. A?14. se microscope to test for effervescence. 'l )ill not effervesce )ith A?14. eE iron /lace concentrated H7Fe*C?., in a "lass slide add drop of un3no)n dissolved in ACl . Move t)o drops to"ether under microscope and loo3 for blue color. 3E Sul3ur /lace material on a capped slide. Aeat "ently )ith burner and loo3 for sublimation of sulphur8odours. .R +rocedure 1btain an IR spectrum for each of the follo)in" compounds potassium perchlorate0 potassium chlorate0 potassium sulfate potassium nitrite and potassium nitrate bet)een the )ave numbers 7$$ and 7$$$ cm-1. Collect at least ,7 one second scans *7$+, data points8scan.. Compare the spectra of your burned and unburned un3no)n )ith the above results. Loo3 for specific spectral bands. Aint6 separation of components by recrystalli5in" out of methanol may improve results. Be sure Me1A is completely "one prior to analysis. CF) +rocedure Dissolve a very small amount of your 3no)n and un3no)n samples in DI )ater. Filter and dilute 18%$. /lace in each in a "lass vial and ta3e upstairs to room 471 for analysis by capillary electrophoresis. Determine individual ions via comparison )ith standards Method for 'nions6 %$ cm fused silica capillary 7%um ID 1.; mM /otassium dichromate visuali5in" a"ent *for indirect detection. Diethylene triamine osmotic flo) modifier *to reverse osmotic flo). Borate buffer #mM borate 7$mM boric acid *pA to 7.; )ith diethylene triamine. 2avelen"th #$%0#;$ nm for detection of ions by indirect *#;$. and direct *#$%. methods. 'naly5e at #$39 )ith a # second pressure inKection. ,iscussion 1. 2hich set of techni@ues provided the most definitive information in your e=perimentsG #. :ive the specific bands for the individual components in the F&IR analysis. 4. 2hat differences did you see bet)een pre and post blast analysis and )hyG 7. 2as the fuse material different from that of the bul3 e=plosiveG Ao) could you tellG 2hat other differences did you seeG %. Ao) definitive )ere the spot tests compared to the instrumental techni@uesG

;uestions0 1. !ulfur is a material added to many lo) e=plosives. 2hat role mi"ht it play in the composition of the e=plosiveG #. It is also important to loo3 for different cations in lo) e=plosives. 'mmonium nitrate is a common e=plosive in minin". /otassium salts are used in blac3 po)der. Describe t)o different methods you could use to tell the difference bet)een ammonium and potassium. 4. Contamination from nitrates used in foods and other commercial products is a real problem in e=plosives residue detection. If you used these e=periments to determine )hether someone had #1

handled e=plosives0 )hat controls )ould you use in your e=periments to avoid contamination from these and other sourcesG 7. For an e=plosive composed of potassium perchlorate0 sulphur and su"ar0 )hat ions )ould you e=pect to find in the post blast residueG %. 'nother type of e=plosive commonly used in the ! is smo3eless po)der. Find a paper describin" a method of analysis for smo3eless po)ders and "ive the paper a critical evaluations. *1 pa"e.

,is/osal Chlorinated solvent and other or"anic solvent )aste must be placed in the appropriate container. 1ther )aste may be disposed of do)n the drain )ith copious amounts of )ater.

##

(. Drug e!traction in urine: )&*+# Scenario @ ' urine sample )ill be provided for you from the pilot of the plane you )ill ta3e to Florida ne=t )ee3. ' blan3 urine sample )ill also be provided. /erform a dru" screen by performin" a li@uid8li@uid e=traction of basic dru"s from urine follo)ed by separation and identification usin" :C8M!. If dru"s are present0 report the concentration found in the urine. +recautions %S&F$ is 3lammable and harm3ul by inhalation5 .ntroduction :as Chromato"raphy *:C. is a )idely used techni@ue in forensic chemistry. Compounds that are not hi"hly volatile or li3ely to decompose at the hi"her temperature used in :C are usually H3C Si CH3 derivatised prior to analysis. &he most common type of derivative is a methyl silane. &his functional "roup increases the volatility of CH3 O the compound. &here are a number of different silyatin" rea"ents available. &he choice of rea"ent depends upon the functional CF3 C N Si CH3 "roups to be derivatised. &he rea"ent used here *?-1bis*trimethylsilyl. trifluororacetamide0 B!&F'. is a multi-purpose CH3 rea"ent )hich is capable of derivati5in" a )ide ran"e of functional "roups. &he mass spectral detector is probably the most important detector for :C in forensic chemistry. &his is partly due to the lo) limits of detection that the detector has0 but mainly because of the structural information provided by the fra"mentation ions produced. CH3 +rocedure Remove % ml of urine and adKust to a pA of 1$-11 usin" ?A7 1A *appro=imately $.;ml. /lace in a small separatory funnel. 'dd %ml 1-chlorobutane. !ha3e0 remove 7 ml of the or"anic layer into a test tube0 and evaporate to dryness by blo)in" a steady stream of nitro"en over the tube. Ma3e sure your sample is completely dry. 'dd #%ul B!F&' to the dry tube. &he reaction )ill not )or3 if any moisture is present. Cap and let stand 1$ minutes. 2arm if necessary. InKect $.% SL of this onto the :C8M!. Repeat # more times. Determine the precision of your analysis by calculatin" the standard deviation of the pea3 areas. Determine the identity of your dru" usin" the M! library and throu"h the inKection of the samples provided by the &' /repare an e=ternal standard calibration "raph. sin" the stoc3 solutions provided by the &' dilute this solution so that your calibration "raph covers the concentration ran"e 1 T"8mL to 1$ T"8mL. !tart your calibration standard preparation at the derivati5ation sta"e. Dour calibration "raph should have at least % points in it. Discussion 1. 2as your derivati5ation successfulG 2hy or )hy notG #. /rovide a table of the specific pea3s and "ive fra"mentation mechanisms for three of these pea3s in your mass spectrum )hich convinced you that you had correctly identified your dru" of interest 4. For this particular dru" )hat mi"ht a typical concentration be in blood or urineG 7. <=plain )hy it )as necessary to adKust the pA of the urine to 1$-11G %. :ive the slope0 the correlation coefficient and standard deviation of the y estimate for your results and use this data to provide an estimate of the reliability of your result.

#4

,is/osal Chlorinated solvent and other or"anic solvent )aste must be placed in the appropriate container. 1ther )aste may be disposed of do)n the drain )ith copious amounts of )ater.

;uestions 1. sin" the paper in your supplementary notes describe 4 different methods for the derivati5ation of dru"s for :C analysis. 2hich procedures are recommended for cocaine0 cannabis0 morphine and amphetamine and )hyG #. 2hat factors influence the recovery of a li@uid8li@uid e=tractionG 4. Derivati5ation is especially important for the analysis of dru" metabolites. <=plain the reason metabolites are more polar than their parent dru" and )hy this ma3es them easier to derivati5e. 7. :ive the structure of B!&F'. !ho) the reaction of B!&F' )ith ben5oylec"onine. %. Derivati5ation alters the molecular structure. !ometimes this is an advanta"e in M! analysis0 )hyG List this and other advanta"es of the use of derivati5ation rea"ents. ,. &)o mass spectrometric techni@ues sometimes used in to=icolo"ical laboratories to aid in dru" analysis are selective ion monitorin" and M!8M!. <=plain )hat these are and ho) they can be useful in to=icolo"ical analysis. 7. :C8M! is used as a confirmatory test in dru" analysis. Immunoassay is commonly used as an initial0 screenin" tool. Describe ho) immunoassay functions and e=plain ho) some dru" users can adulterate their urine to fool this test. ;. Find and criti@ue an article on the use of li@uid-chromato"raphy8mass spectrometry for forensic dru" analysis.

#7

,. -%#./ -/-01#$#: )&*F$D Scenario 2 ' restaurant )as burned to the "round and the fire inspector suspects arson. !amples of the restaurant carpet )ere believed to be treated )ith an accelerant. Dou have been provided )ith a sample from the restaurant0 as )ell as blan3 and spi3ed burnt carpet samples to determine if accelerant )as used0 )hat type0 and the amount. .ntroduction &here are a number of mechanisms that can be postulated to result in a fire of un3no)n ori"in. 1ne of these is arson. It is the role of the fire investi"ator to see3 the source of the fire. If arson is suspected the investi"ator )ill collect debris from suspect locations and place it in a metal paint can or specially formulated sealed plastic ba". pon arrival at the laboratory0 a sample of the headspace of the paint )ill be ta3en and a charcoal strip )ill be suspended above the debris. &he can )ill then be "ently heated or simply left sit overni"ht. &he charcoal strip is then e=tracted usin" a small amount of C!# and a small amount of the e=tract is inKected into the "as chromato"raph. In order to interpret the results of an arson analysis it is important to understand the ho) crude oil is refined and manufactured. &he follo)in" table illustrates the nomenclature used in the petroleum distillation process6 Class G 1 Li"ht /etroleum Distillates *L/D. # :asoline 4 Medium /D 7Herosene % Aeavy /D $ nclassified H/ea9 s/readI based on n@al9ane carbon Gs C7-C; C7-C1# C;-C1# C+-C1, C1$-C#4 9ariable )<am/les /etroleum ethers0 poc3et li"hter fuels0 rubber cement solvents0 lac@uer thinners 'utomotive "asoline0 some lantern fuels Charcoal starters0 paint thinners0 mineral spirits0 dry cleanin" fluids0 torch fuels U 1 fuel oil0 Ket-' *aviation. fuel0 insect sprays0 charcoal starters U # fuel oil0 diesel fuel !in"le compounds such as alcohols0 acetone0 or toluene0 =ylenes0 isoparaffinic mi=tures some lamp oils0 campin" fuels0 lac@uer thinners0 duplicatin"

Su//lies and e6ui/ment +rocedure Carpet *#B C #B. samples )ere all cut from one lar"e piece. !amples consist of a /yroly5ed carpet0 and carpets spi3ed )ith the follo)in" accelerants6 :asoline *#%$ Tl.0 Li"hter Fluid *#%$ Tl.0 &urpentine *#%$ l. and Diesel Fuel *#%$ Tl.. &he carpet sample from the house )as ta3en from a 1$C#$ ft. room. 'll carpet samples )ere ba""ed in nylon )ith activated charcoal strips *'lbrayco Laboratories0 %$$ Corporate Ro)0 Crom)ell0 C& $,71,P /h6 ;,$-,4%-44,+.. &he /yroly5ed sample )as heated by propane torch until the carpet started to burn. 'ccelerant samples had listed amounts applied to carpet and )ere i"nited by match. !amples )ere burned to consume half of the accelerant. Burn time )as estimated by burnin" #%$ Tl of the accelerant only0 in a 4B diameter open dish. 'll pyroly5ed and accelerant samples )ere ba""ed directly after burnin". 'll samples )ere heated V 1$$ C for +$ min. 'nalysis Conditions #%

column temp <CD "as

DB-%M! 1% m = $.#% mm = $.#% Tm 7$ C *#min. 1% C8min to #,% C

?# carrier detector temp InKector temp

4% cm8sec

split flo) ma3e up "as

1$$ ml8min

Chec9 to ma3e sure these conditions are )hat your system is usin" to avoid delays . Before startin" chec3 to ma3e sure the :C column is properly functional and clean. Ba3e column and run blan3s if necessary. /repare a sample of "asoline0 ,$M evaporated "asoline0 pristane and phytane0 and diesel fuel (O&)0 For this laboratory you )ill be inKectin" standards and samples that are diluted *.%$ Tl of sample in 1$ ml methylene Chloride.. &he C%-C#$ n-al3ane standard is diluted in pentane. )</erimental0 1. 'naly5e 1.$ Tl of the C%-C#$ standard )ith the above temperature pro"ram. #. &a3e the charcoal strips from nylon ba"s and place in 1.; ml vials )ith appropriate label. 4. In the hood usin" syrin"es provided add %$$ Tl methylene chloride to a vial containin" a charcoal strip. 7. 'naly5e all carpet samples by :C. Due to lac3 of time do not run blan3 :C analyses bet)een samples unless the column has been overloaded. InKect 1 Tl of sample into the :C. %. 'naly5e 1.$ Tl sample of a "asoline sample )hich has been evaporated to ,$M of its ori"inal volume and then diluted. Compare this result to a fresh sample. ,. Run a diesel fuel standard as )ell. ,iscussion 1. 2hat )ere the complete conditions for sample preparation and instrument operationG #. 2hat is the ran"e of pea3s that are present0 li"ht medium or heavyG Is there a broad or narro) distribution of pea3sG 4. 're characteristic class features for all of the compounds presentG 7. Do you see pea3s in the un3no)n that correspond )ith pea3s in the C%-C#$ standardG %. se these features to limit your possibilities and then run standards to test and confirm your hypothesis on the identity of your un3no)n. ,. 2hat )as the effect of the )eathered *evaporated. "asolineG 2ould you have difficulty in distin"uishin" evaporated "asoline from diesel fuelG 2hat could help you in reco"nition of differencesG 7. 2hat )as the effect of the burned carpet on the "asoline analysisG Did it complicate the resultsG

;uestions 1. &here are several methods for the detection of arson. &hese include passive headspace *this lab. dynamic headspace0 solid phase microe=traction and distillation. Describe each method and "ive advanta"es8disadvanta"es of each. #. In this e=periment you used a burned carpet as a control. From your results0 is it necessary is it to obtain a control0 or )ould a neat sample of the accelerant doG 4. !uppose your suspect claimed that she had spilled an li"hter fluid last )ee3 on the carpet. Ao) )ould you verify her claimG 7. /ristane W /hytane are indicators of a heavy petroleum distillate. In )hich accelerant should you easily find themG 2hat particular n-al3anes do they elute nearG /ristane *#0,01$017-tetramethylpentadecane. Mol. 2t. #,;.%4 C'!. ?1. 1+#1-7$-, /hytane *#0,01$017-tetramethylhe=adecane. Mol. 2t. #;#.%% C'!. ?1.,4;-4,-;

#,

%. Find an article that discusses the use of :C8M! in arson analysis and the advanta"es of sin"le ion monitorin". 2rite a brief *1-pa"e. revie) of the article )hich needs to be a summary and criti@ue of the article in your o)n )ords. &he article must be from a peer-revie)ed Kournal0 internet and company articles are not acceptable. /lease see a &' if you have trouble findin" an article. 'ttach the article to your lab report.

#7

-22 /D$3 -: &.0.4% T #T % -) /T# Cobalt isothiocyanate &est +recautions (o s/eci3ic /recautions5 Rea"ent /repare a #M )8v solution of cobalt isothiocyanate in )ater. Method 1. 'dd rea"ent to the test substance in a test tube. Indications Development of a blue-"reen colour indicates that cocaine *or surro"ates. may be present. ,u6uenois@LeAine &est +recautions $cetaldehyde boils at 21 C8 is hi7hly 3lammable and harm3ul5 $Aoid inhalin7 it and handle it in the hood5 Chloro3orm is harm3ul8 use it in the hood5 ,is/ose o3 chloro3orm =aste in mar9ed containers /roAided5 Rea"ent Dissolve vanillin *1 ". in ethanol *+%M0 %$ mL. in a "lass-stoppered bottle. 'dd fresh acetaldehyde *$.1% mL.. !tore the rea"ent in a cool0 dar3 place. Discard the rea"ent )hen it ac@uires a deep yello) colour. 'cetaldehyde is shipped in a sealed "lass bottle. Before you brea3 the seal0 cool the bottle in ice )ater to reduce the vapour pressure inside the container. !core the "lass tubin" on top of the bottle )ith a trian"ular file and brea3 the tubin" carefully. /our the contents into a bro)n "lass bottle and store in a cool0 dar3 place. Method 1. /lace a small amount of plant material in a small test tube. 'dd 1# drops of the rea"ent and stir. Decant the li@uid into another test tube and record the li@uid colour. #. 'dd an e@ual volume of concentrated ACl to the above li@uid and mi=. Record the mi=ture colour. 4. 'dd a fe) drops of chloroform to the above mi=ture and a"itate. Identify the layers and record the colour of each layer. Indications ' colour chan"e from "rey to "reen throu"h blue to violet blue su""ests the presence of cannabis. Distinction from roasted coffee and patchouli oil is re@uired. 1nly )ith cannabis is the violet colour e=tracted into the chloroform layer. Compound Initial Colour Colour e=tracted by CACl4 layer 9iolet ?one

Cannabis 9iolet-blue Coffee *roasted. 9iolet-bro)n #;

/atchouli 1il &ea *leaves.

9iolet :reen-Blue

?one ?one

1ther natural products such as basil0 bay leaf0 marKoram0 nutme"0 rosemary0 sa"e0 thyme or tobacco "ive no colour. 1andelin &est +recautions Sul/huric acid is corrosiAe5 1a9e all dilutions in an o/en bea9erDDD Rea"ent Dissolve ammonium vanadate *$.% ". in )ater *1.% mL. and dilute to 1$$ mL )ith sulphuric acid. Method 'dd a drop of rea"ent to the sample on a )hite tile. Indications 9arious colours across the entire visible spectrum are "iven by a lar"e number of compounds. &he colour "iven by LiebermannLs test should also be ta3en into account )hen interpretin" the result. Aydrochlorides "ive a red colour. Red Bro)n-red 1ran"e Red-oran"e :reen-oran"e Bro)n-oran"e Dello) 1ran"e-yello) :reen-yello) :reen
'Kmaline0 a5acyclonol0 chlorprothi=ene0 diperodon *"reen.0 dofamium *bro)n.0 flupenthi=oi0 "elsemine *"reen.0 ndapamide0 me@uita5ine0 methotre=ate0 nialamide. pericya5ine0 praKmalium0 prolintane0 sodium cromo"lycate0 thiothi=ene0 =ylometa5oline ?adolol Dropropi5ine *slo).0 ethylnoradrenaline0 hydrastinine *"reen.0 lachesine *"reen.0 levamisole *"rey"reen.0 methanthelinium0 methi=ene0 methyldopa0 methyldopate0 methylpiperidyl ben5ilate *bro)n "reen.0 noradrenaline0 orphenadrine0 pipen5olate *"reen.0 poldine methylsulphate *"reenviolet.0 propantheline0 pro@uame5ine *violet.0 solanidine *violetblue.0 solanine *violetblue.0 sulindac0 thenalidine *bro)n. Cotamine *bro)n. %-Methyltryptamine Me=iletine '5aperone0 ben5tropine0 bro=aldine0 chelidonine *"reen.0 conessine0 deptropine0 desipramine *blue.0 dihydrala5ine0 diphenhydramine0 diphenidol0 diphenylpyraline0 dropropi5ine *oran"e.0 hal@uinol0 homidium0 lidofla5ine0 methacycline *oran"e-violet.0 paraphenylenediamine0 penicillamine0 proto3ylol * bro)n.0 tofenacin0 tylosin *yello)-bro)n.0 veratrine *oran"eviolet-bro)n.0 viprynium Ae=oprenaline Metho=amine 'ceproma5ine *red.0 adiphenine *blue.0 benorylate0 bephenium hydro=ynaphthoate0 biben5onium buclosamide *blue rim.0 bunamidine0 chlorproma5ine *violet.0 clefamide *bro)n.0 codeine0 coichicine0 cycla5ocine0 cyclomethycaine *bro)n.0 debriso@uine0 diaveridine0 diben5epin0 dietha5ine *violet )ith e=cess rea"ent.0 diethylthiambutene *"reen-blue.0 dimethindene0 dimetho=anate *bro)n.0 dimo=yline0 dipipanone *blue.0 dothiepin0 do=orubicin0 do=ycycline *yello).0 ethopropa5ine *violet.0 fenpiprane0 "uano=an0 harman0 hydro=yephedrine0 iso=suprine0 metanephrine0 methadone *blue.0 methdila5ine *violet.0 methocarbamol0 metho=yamphetamine0 methylenedio=yamphetamine *blue.0 -methyltryptamine *oran"e.0 metopima5ine0 monocrotaline0 niclosamide0 nitm=otine0 norharman *yello).0 norrnetanephrine0 obido=ime *blue.0 oleandomycin0 o=ymeta5oline0 peca5ine *violet.0 penta5ocine0 pera5ine *violet.0 phena5one0 phena5opyridine0 phenformin0 phenindamine0 pheno=yben5amine *violet.0 phenyltolo=amine0 pindolol0 piperaceta5ine *redviolet.0 pipo=olan *bro)n.0 prenylamine0 proflavine0 proma5ine *violet.0 pmmetha5ine *violet.0 propranolol0 reserpine0 ritodrine0 thenium0 thenyldiamine0 thiocarlide *yello).0 tranylcypromine *violet.0 trifluomepra5ine *red-violet. ?ormethadone0 opipramol Ben5octamine0 berberine *bro)n.0 edrophonium0 hydro=ystilbamidine0 3etobemidone0 metho=yphenamine0 phentolamine0 profadol *"reen.0 vilo=a5ine Ben5ydamine0 chlorphenesin 'lverine0 a5apropa5one0 ben5he=ol0 diamphenethide0 diethyltryptamine *yello).0 dihydrocodeine0 "uaiphenesin0 hordenine0 levomethadyl acetate0 norinorphine0 o=yphencyclamine0 papaverine0 terbutaline Bamethan *"reen.0 clomipramine0 deserpidine *"reen.0 desferrio=amine *violet.0 do=apram0 droperidol *"reen.0 harmine *"reen.0 imipramine *add )ater.0 maprotiline0 mebhydrolin0 metaraminol0 phena"lycodol0 phenyramidol0 pyrido=ine *"rey-"reen.0 salbutamol *blue rim bro)n rim.0 thiorida5ine *violet.0 trimipramine *add )ater.0

Dello)-"reen Blue-"reen Bro)n-"reen :rey-"reen Blue

#+

:reen-blue 9iolet

triphenyltetra5olium *slo).0 =ipamide0 =yla5ine0 yohimbine *"reen. Chlophedianol0 labetalol 'midephrine0 benperidol0 be5itmmide *oran"e.0 bisacodyl0 captodiame0 cephaloridine0 chloropyrilene *oran"e.0 clomiphene *oran"e-bro)n.0 clomocycline *bro)n.0 denatonium0 dipyridamole0 "uanoclor *oran"ebro)nyello).0 "uano=an0 he=obendine0 hydromorphone *oran"e.0 mepacrine *yello).0 mepyramine0 methisa5one *yello).0 mianserin0 morantel0 nalo=one *bro)n.0 o=yclo5anide *oran"e.0 o=yphenisatin0 o=ytetracycline *redoran"e.0 penthienate0 perphena5ine0 phenylbuta5one0 pi5otifen *"reen.0 prilocaine0 prima@uine *oran"e.0 propioma5ine0 pyrantel0 pyffobutamine0 rolitetracycline *redoran"e.0 strychnine0 tetracycline *redoran"e.0 thiethylpera5ine0 thiopropa5ate0 triacetyloleandomycin *slo).0 tridihe=ethyl0 trimepra5ine0 trimeta5idine 'nta5oline0 carphena5ine0 dimethothia5ine0 histapyffodine0 thon5ylamine 'lcuronium0 he=ocyclium0 methotrimepra5ine 'lprenolol0 bitoscanate0 butapera5ine0 napha5oline Methoserpidine0 o=prenolot0 tricyclamol Methapyrilene 'mitriptyline *"reen.0 a5apetine0 bamipine0 carbetapentane *slo).0 clidinium *"reen.0 cyclopentolate0 diphemanil0 dipyrone0 do=epin0 embutramide0 fluanisone0 fluphena5ine0 isoetharine0 isometheptene0 isopren aline0 methindi5ate0 methyl ben5o@uate0 methyser"ide0 metoclopramide0 norpipanone *blue.0 nortriptyline *"reen.0 phencl5ine0 phenylephrine0 pimo5ide0 piperidolate0 prochlorpera5ine *violet.0 propo=ycaine0 rescinnamine0 salina5id0 stano5olol0 tetrabena5ine0 thiopropera5ine *"reen violet.0 tolnaftate0 tolpropamine0 trama5oline0 tubocurarine Ben5thia5ide0 clio=anide0 cycrimine0 deco@uinate0 diclofenac0 ethomo=ane0 fluoproma5ine0 hydrastine *red.0 trifluopera5ine Metoprolol Rifampicin0 spiramycin0 thebaine Clemastine0 clofa5imine0 physosti"mine0 rifamycin !90 trimethoprim0 tripelennamine <ten5amide0 harmaline0 lyser"ic acid0 mesorida5ine0 narceine0 syrosin"opine Chlortetracycline *yello).0 cyproheptadine0 demeclocycline0 dihydroer"otamine0 er"otamine0 lymecycline * yello).0 methyler"ometrine0 nicer"oline *bro)n.0 octaphonium0 o=etha5aine0 protriptyline0 trimetho ben5amide De=tropropo=yphene0 mephenesin carbamate Dihydromorphine0 diprenorphine0 etilefrine *"reenbro)n.0 ibo"aine *violet.0 indomethacin0 lobeline0 lyser"ide0 o=ypertine0 propranolol0 tra5odone *violet. 'lphaprodine0 diamorphine0 morphine /rocyclidine

Red-violet Blue-violet Bro)n-violet :rey-violet Blac3-violet Bro)n

Red-bro)n /in3-bro)n 1ran"e-bro)n Dello)-bro)n :reen-bro)n 9iolet-bro)n :rey-bro)n :rey Blue-"rey Blac3 1ar6uiss &est +recautions

Sul/huric acid is corrosiAe5 1a9e all dilutions in an o/en bea9erDD Rea"ent Mi= 1 volume formaldehyde solution *formalin. )ith + volumes of sulphuric acid. Method 'dd a drop of rea"ent to the sample on a )hite tile. Indications 9arious colours across the entire visible spectrum are "iven by a lar"e number of compounds. Red
'lprenolol0 ben5ylmorphine *violet.0 buphenine0 dimethothia5ine0 eten5amide0 etilefrine0 fenclofenac *slo).0 fenpiprane0 fluphena5ine0 flurbiprofen0 he=oprenaline0 labetalol *bro)n-red.0 maprotiline0 mephenesin carbamate0 me@uita5ine *slo).0 mesorida5ine *violet.0 metho=yphenamine0 metopima5ine0 me=iletine0 nadolol0 penta5ocine *"reen.0 pericya5ine0 phena5opyridine0 phenoperidine0 phenylephrine0 piperaceta5ine0 prenylamine0 thebaine *oran"e.0 thiethylpera5ine *"reen.0 thiopropera5ine0 thiothi=ene0 tolpropamine0 tranylcypromine *bro)n.0 vinblastine 'lverine0 bethanidine0 diphemanil0 flupenthi=ol &hiorida5ine *blue-"reen. 'lphaprodine Fenoprofen0 metoprolol 'drenaline *violet.0 aletamine0 amphetamine *bro)n.0 anileridine *slo).0 benacty5ine *"reen blue.0 ben5ethonium0 ben5ilonium *"reen blue.0 ben5phetamine0 bunamidine *red.0 carbetapentane *slo).0

1ran"e-Red 9iolet-Red Bro)n-Red /in3 1ran"e

4$

Red-1ran"e /in3-1ran"e Dello)-1ran"e Bro)n-1ran"e Dello)

carphena5ine *red-violet.0 clidinium *blue.0 cyclandelate *slo).0 cycrimine *red.0 dehydroemetine0 dimethyltryptamine0 dipyridamole0 ethacridine *red.0 ethohepta5ine0 ethylnoradrenaline *bro)n.0 famprofa5one0 fenbufen *bro)n.0 fencamfamin0 fenethylline0 fentanyl0 harmine0 indapamide * violet.0 indomethacin0 isothipendyl0 3etobemidone0 lachesine *"reen blue.0 lymecycline0 mepen5olate *transient.0 mephentermine *bro)n.0 mescaline0 metanephrine *violet-bro)n.0 methacycline0 methanthelinium0 methindi5ate *"reen.0 methylamphetamine0 methylpiperidyl ben5ilate *"reen blue.0 %-methyltryptamine *bro)n.0 -methyltryptamine *bro)n.0 ?-methyltryptamine0 nefopam *bro)n.0 nomifensine *slo).0 normetanephrine *violet-bro)n.0 o=eladin0 o=ytetracycline0 pentapiperide0 pethidine0 phenethylamine0 phenformin *bro)n.0 phentermine0 piminodine0 pipen5olate *"reen blue.0 piperidolate0 pi5otifen *red.0 poldine methylsulphate *"reenblue.0 prima@uine0 profadol *red-bro)n.0 prolintane *bro)n.0 propantheline0 prothipendyl0 psilocybin0 rolitetracycline0 spiramycin0 tetracycline0 trimethoprim0 trimetho=yamphetamine0 tryptamine0 veratrine0 =ylometa5oline Chlorprothi=ene Diuron 1rphenadrine0 pipradrol 'mitriptyline 'criflavine *red.0 amiloride0 a5acyclonol0 ben5@uinamide0 ben5tropine0 bromodiphenhydramine0 bro=aldine0 bro=y@uinoline0 caramiphen0 chlordia5epo=ide0 chlorpheno=amine *"reen.0 chlortetracycline *"reen.0 chlorthalidone0 cinchophen0 clefamide0 clemastine *"reen rim.0 colchicine0 conessine *oran"e.0 cycli5ine0 demeclocycline *"reen.0 deptropine0 diethyltryptamine *bro)n.0 diphenhydramine0 diphenidol0 diphenylpyraline0 D1M0 do=ycycline0 etho=5olamide0 ethylmorphine *violet blac3.0 furaltadone0 hal@uinol0 hydrocodone *bro)nviolet.0 hydromorphone *red violet.0 hydro=yephedrine0 isoetharine *oran"e.0 lidofla5ine0 lora5epam0 mepacrine0 methyldopa *violet.0 methyidopate *violet.0 norcodeine *violet.0 orciprenaline0 o=ycodone *bro)n violet.0 o=yphenbuta5one0 phan@uone0 phenbutra5ate *slo).0 phentolamine0 phenyramidol0 pindolol * bro)n.0 pramo=ine *"reen.0 proflavine *oran"e.0 salbutamol0 salina5id0 sodium cromo"lycate0 solanine *violet.0 terbutaline0 tetrabena5ine0 thebacon *violet.0 tofenacin0 triamterene0 trimeta5idine *fades.0 vancomycin0 viprynium embonate0 5omepirac *1$$0 oran"e. !tano5olol Berberine0 carbaryl0 chelidonine0 harman0 norharman0 oleandomycin0 propranolol0 protriptyline0 pseudomorphine0 sulindac *slo). 'ceproma5ine *red.0 verapamil *"rey. &olnaftate Aarmaline Cyproheptadine0 deserpidine0 napha5oline0 o=ypertine0 phenindamine0 proto3ylol0 rescinnamine0 reserpine *bro)n. Clofibrate0 embutramide0 nicer"oline *"rey. Mebhydrolin0 1-naphthylacetic acid 'pomorphine *blac3.0 a5atadine0 benorylate0 bisacodyl0 buprenorphine0 butriptyline0 captodiame0 chloropyrilene0 chlorproma5ine0 clofa5imine0 codeine0 diamorphine0 diethylthiambutene0 dihydrocodeine0 dimethindene * blue.0 dimetho=anate0 do=orubicin0 do=ylamine0 ethopropa5ine0 etho=a5ene0 "uaiphenesin0 "uano=an0 he=ocyclium methylsulphate0 mepyramine0 ,-monoacetylmorphine0 morphine0 nalorphine0 normorphine0 o=prenolol0 o=yphenisatin0 peca5ine0 penthienate0 pera5ine0 perphena5ine0 pheno=yben5amine0 phenyltolo=amine0 pholcodine0 pimo5ide0 pipo=olan *"rey.0 prochlorpera5ine0 procyclidine0 proma5ine0 prometha5ine0 pro@uame5ine0 solanidine0 thenium0 thiopropa5ate0 tricyclamol0 trimepra5ine0 vilo=a5ine 'cetophena5ine0 ben5octamine0 bephenium hydro=ynaphthoate0 cephaloridine0 chlophedianol *bro)n.0 dihydromorphine0 ethomo=ane0 fluoproma5ine0 iso=suprine0 lobeline0 methdila5ine0 propioma5ine0 trama5oline0 trifluomepra5ine0 trifluopera5ine0 trimeperidine Methocarbamol0 methotrimepra5ine0 morantel0 neopine0 noscapine *fades.0 pyrantel Butapera5ine0 dopamine0 tridihe=ethyl Ben5he=ol0 diprenorphine0 o=ymorphone0 pyrrobutamine0 thenalidine De=tropropo=yphene *"reen.0 methapyrilene0 thenyldiamine Biben5onium0 carbidopa0 cycla5ocine *"reen.0 diclofenac *slo).0 dimo=yline0 dothiepin0 do=epin0 er"ometrine0 er"otamine0 erythromycin0 hordenine *"reen.0 ibuprofen *1$$0 oran"e.0 isoprenaline *violet.0 lyser"amide0 lyser"ic acid0 nalo=one *violet.0 napro=en0 narceine *"reen.0 noradrenaline0 phena5ocine0 rimiterol *blac3.0 serotonin *slo).0 syrosin"opine0 tyramine *"reen. Biperiden0 debriso@uine0 methyl ben5o@uate0 o=etha5aine0 phenprobamate0 trimeto5ine0 tripelennamine Benethamine *bro)n.0 clomocycline0 nortriptyline Ritodrine0 thymo=amine0 triacetyloleandomycin0 tylosin 'lcuronium0 bufotenine0 psilocin Clomiphene0 dietha5ine0 levomethadyl acetate *"rey-"reen.0 metho=amine *"reen. Dihydroer"otamine0 methyler"ometrine0 octaphonium Butorphanol0 diaveridinc *violet-bro)n.0 ibo"aine *oran"e.0 lyser"ide0 methoserpidine0 methyscr"ide0 pholedrine *"reen.

1ran"e-Dello) :reen Dello)-:reen Blue-:reen Bro)n-:reen :rey-:reen Blue :rey-Blue 9iolet

Red-9iolet Blue-9iolet Bro)n-9iolet :rey-9iolet Blac3-9iolet Bro)n Red-Bro)n 1ran"e-Bro)n Dello)-Bro)n :reen-Bro)n 9iolet-Bro)n :rey-Bro)n :rey

41

Blue-:rey Blac3 Blue-Blac3

'cetorphine *yello)-bro)n.0 etorphine *yello)-bro)n. Methylenedio=yamphetamine

(itric $cid &est +recautions (itric acid is hi7hly corrosiAe5 Cear 7o77les and 7loAes =hen /er3ormin7 this test Rea"ent Concetrated nitric acid. Method 'dd a drop of acid to the sample on a )hite tile. &he Mec3e rea"ent is prepared from 1" of selenious acid in 1$$ mL of concentrated sulfuric acid. 1ec9e &est +recautions Sul/huric acid is corrosiAe5 1a9e all dilutions in an o/en bea9erDD Rea"ent Mi= 1 "m of selenious acid in 1$$ mL of concentrated sulphuric acid. Method 'dd a drop of rea"ent to the sample on a )hite tile.

4#

$++)(,.J %0 S$1+L) L$% R)+OR&

<=periment 1 (ane Doe Ai"h /erformance Li@uid Chromato"raphy Forensic Chemistry (anuary ;0 1++7

44

I?&R1D C&I1? In this e=periment0 hi"h performance li@uid chromato"raphy *A/LC. )as used to separate a mi=ture of three similar al3aloids6 caffeine0 theobromine0 and theophylline. &he retention times of the three compounds )ere measured0 and a calibration model for caffeine )as constructed based on the chromato"raphic pea3 hei"hts produced by a series of si= caffeine standard solutions. &he derived calibration model )as then employed in the prediction of caffeine content in a commercial soft drin3.

/R1C<D R< Instrumentation and <@uipment. ' modular A/LC setup )as used in this e=periment0 consistin" of *1. a MiltonRRoy Model 11$$ pump0 *#. a #$ TL sample inKector0 *4. an 'lte= CR; reverse phase A/LC column0 *7. an ultraviolet detector operatin" at #%7 nm0 and *%. an output chart recorder. In solution preparation0 a Mettler '<R1,$ analytical balance )as used to )ei"h solid rea"ents. Class ' volumetric "lass)are )as used throu"hout the e=periment. !olution /reparation. For the study of the retention times of the three al3aloids0 separate 1 L solutions of caffeine0 theophylline0 and theobromine )ere prepared in deioni5ed )ater. &he )ei"hts of solute used )ere $.1$11 "0 $.1$## "0 and $.$++; for caffeine0 theophylline0 and theobromine0 respectively. In constructin" the calibration model for caffeine0 si= standard solutions )ere prepared by serial dilution from a caffeine stoc3 solution. &he stoc3 solution )as prepared by dissolvin" $.%$$$ " of caffeine in enou"h deioni5ed )ater to ma3e 1 L of solution. <mployin" %0 1$0 1%0 and #$ mL volumetric pipets0 ali@uots of %0 1$0 1%0 #$0 4$0 and 7$ mL )ere removed from the stoc3 solution and diluted to 1$$ mL )ith deioni5ed )ater. &he un3no)n for the caffeine determination )as a sample of B/epsiB0 a commercial soft drin3. Data Collection and 'nalysis. ' mi=ture of acetonitrile *#$M v8v. in deioni5ed )ater )as used as the mobile phase. &his solution )as de"assed prior to bein" used. ' flo) rate of 1.$ mL8min )as employed durin" the e@uilibration and durin" subse@uent inKections of the samples. For the study of the retention times of the three al3aloids0 three inKections of each solution )ere made. In constructin" the caffeine calibration model0 three inKections )ere made of each of the si= standard solutions. &hree inKections of the B/epsiB un3no)n )ere then made. ?o dilution of the un3no)n )as made before inKection. &he recorded chromato"rams )ere analy5ed by measurin" pea3 hei"hts )ith a ruler.

47

<C/<RIM<?&'L D'&' Retention &ime !tudy Retention &ime *sec0 J $.1 sec. Compound Caffeine &heobromine &heophylline &rial 1 1;$.1 1%7., #77.7 &rial # 1;1.4 1%%.4 #7%.; &rial 4 1;$.% 1%7.+ #7%.#

Caffeine Calibration !tudy 'li@uot 9olume *mL. %.$ 1$.$ 1%.$ #$.$ 4$.$ 7$.$ n3no)n /ea3 Aei"ht *cm0 J $.$% cm. &rial 1 #.1$ 4.;# %.$# ;.7$ 11.+$ 17.%% ,.#4 &rial # 1.#% 4.#$ %.$+ ;.4% 1$.#% 1%.47 ,.%; &rial 4 1.%$ 4.;$ %.,$ 7.7$ 11.$% 14.;$ ,.,1

C'LC L'&I1?! '?D R<! L&! Retention &ime !tudy &he retention times of caffeine0 theobromine0 and theophylline )ere analy5ed to compute the mean retention time0 standard deviation of retention time0 and M relative standard deviation. Means and standard deviations )ere computed by use of functions resident on a Ae)lettR/ac3ard Model 7#! electronic calculator. Compound Caffeine &heobromine Mean Retention &ime *s. 1;$., 1%7.+ 4% !tandard Deviation *s. $., $.7 M R!D $.4+ $.#4

&heophylline

#7%.#

$.,

$.##

Caffeine Calibration !tudy Calculation of <=act Concentrations of !tandards Cstoc3 O *$.%$$$ " caffeine81.$ L. = *1$$$ m"8". O %$$.$ m"8L O %$$.$ ppm For a "iven standard0 Cstd O *'.*Cstoc3.81$$ mL )here ' is an ali@uot volume in mL. <=ample6 *!tandard U1. Cstd O *% mL.*%$$.$ ppm.8*1$$ mL. O #%.$ ppm

&able I <=act Concentrations of !tandards !tandard U 1 # 4 7 % , Concentration *ppm. #%.$ %$.$ 7%.$ 1$$.$ 1%$.$ #$$.$

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Calculation of Mean /ea3 Aei"hts and !tandard Deviations *A/ 7#! calculator.6 &able II Mean /ea3 Aei"hts and !tandard Deviations Concentration *ppm. #%.$ %$.$ 7%.$ 1$$.$ 1%$.$ #$$.$ Mean /ea3 Aei"ht *cm. 1.,# 4.,1 %.#7 ;.1% 11.$7 17.%, !. Deviation *cm. $.77 $.4% $.4# $.4+ $.;4 $.77

Calculation of Calibration Model ' leastRs@uares calculation )as used to compute the slope and intercept of the best calibration model for the above data. &he resident leastRs@uares functions on the A/ 7#! calculator )ere used for this calculation. /ea3 Aei"ht O R$.$7;, J *$.$77##$.*Concentration. &o evaluate the @uality of the calibration model0 the correlation coefficient )as computed6 &he builtRin function of the A/ 7#! calculator )as used. r O $.++, 1n the ne=t pa"e0 the calibration curve is plotted. In the plot0 the mean pea3 hei"hts are indicated by circles0 and the leastRs@uares line is indicated by the solid line. Computation of n3no)n Caffeine Concentration &he mean pea3 hei"ht of the un3no)n )as calculated to be ,.77 cm. &his value )as used )ith the computed slope and intercept to determine the concentration of caffeine in the sample of B/epsiB. Cun3 O *,.77 J $.$7;,.8$.$77## O ;7.+ ppm

47

&alibration &ur"e for &affeine Determination

18 16 14 12 10 8 6 4 2 0 0 10 20 30 40 50 &affeine &oncentration 5ppm6 +ean 2eak Heig't 5cm6

Fi"ure I. Calibration curve for Caffeine Determination. <rror bars are based on a +%M Confidence Interval. DI!C !!I1? &he calibration model produced an e=cellent fit to the e=perimental data0 as evidenced by the hi"h value of the correlation coefficient. 2hile no formal test of accuracy )as performed0 the successful calibration model lends confidence to the determined caffeine value. &he retention time study indicated clearly that the three compounds could be separated usin" the C;Rcolumn. &his study also revealed that the retention times )ere @uite reproducible *rsd X 1 M.. ?o anomalies )ere encountered durin" the e=perimental procedure. F <!&I1?! 1. &)o e=planations for constituents not sho)in" up in the chromato"rams are *1. their possible coRelution )ith other pea3s in the chromato"ram or *#. their bein" permanently retained on the column. It is also possible that constituents may not absorb li"ht at #%7 nm0 the )avelen"th used by the detector employed here. #. <nhanced @ualitative structural information )ould best be obtained by use of an alternate detector. Mass spectrometric detection )ould perhaps

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