ReversedPhaseHPLCDeterminationofAlliininDiverseVarietiesofFreshGarlicand

CommercialGarlicProducts

Athesis

presentedto

thefacultyoftheDepartmentofChemistry

EastTennesseeStateUniversity

Inpartialfulfillment

oftherequirementsforthedegree

MasterofScienceinChemistry

by

AaronKwakuApawu

August2009

Dr.ChuNgiHo,Chair

Dr.JeffreyWardeska

Dr.PengSun

Keywords:Garlic,HPLC,Sulfoxide,Alliin,Allinase,Allicin,Thiosulfinate
 ABSTRACT

ReversedPhaseHPLCDeterminationofAlliininDiverseVarietiesofFreshGarlicand

CommercialGarlicProducts

by

AaronKwakuApawu

Alliinisapredominantflavorprecursoringarliccloves.Itinteractswiththeenzymealliinase

whengarlicclovesarecrushed,cut,orchewedtoproduceallicin,anunstablethiosulfinatethat

isthemainbiologicallyactivecomponentoffreshcrushedgarlic.Biologicalfunctionsand

healthbenefitsofgarlicinclude reductionofcancerriskinhumans,improvingimmunesystem,

andantimicrobial,antioxidant,andantihypertensiveactivities.Thequalityoffreshgarlicand

garlicproductsisusuallyrelatedtoitsalliincontentandallicinreleasepotential.Thisresearch

presentsasimple,rapid,andpreciseHPLCmethodforalliindetermination.Itinvolvestheuse

of30:70%methanol:waterand0.05%sodiumdodecylsulfatemobilephasecomposition,C 18 5

mdisccolumnofsize3.9x150m,anddetectorsetat210nm.Themethodshowedgood

reproducibilitywith0.56%4.11%relativestandarddeviations,alinearresponseofpeakareato

alliinconcentrationof0.4ng/mL80ng/mL,andaveragerecoveryof93.5%101%.

Determinationofalliinineightgarlicsamplesindicatedthehighestamountingarlictabletthat

wasexpected.Themethodpresentediseconomicalandefficientandcanbeusedinalliin

determination.Themethodgaveasatisfactorychromatogramswithmethanolhydrochloric

acidextractbutnotwithhotwaterextract.

 DEDICATION

ThisworkisdedicatedtotheApawu(Dad,mum,andsiblings)andAyaba(inlaws)

families.Yoursupportandyourunrelentingconfidenceinmehavegivenmenoexcusetofail

inthislife.

MaythegoodLordprosperyouinallyourendeavors.

 ACKNOWLEDGEMENTS

UntoGodmyeternalfatherwhohasblessedmewiththegiftoflifeandsuccessinall

mypursuitsbeglory,honor,anddominionforevermore.

IwouldliketoexpressmydeepestappreciationtomyresearchsupervisorDr.ChuNgi

Hoforhispatience,encouragement,goodjudgment,guidance,andcorrectionsthathave

contributedimmenselytothecompletionofthisproject.Iamindeedthankfulforhavingyou

asmyacademicadvisorandamentor.

IwouldalsoliketothankDr.JefferyWardeskaandDr.PenSunforbeingonmythesis

committeeandfortheirinputsthathavemadethisworkasuccess.Specialappreciationgoes

tothefacultyandstaffofETSUChemistryDepartmentfortheknowledgeandskillsIhave

acquiredinmygraduateeducation.

MysinceregratitudetomylovingwifeAlviswhoseloveandcounselhavepropelledme

untogreaterheights.

TotheBlevinsandNeddermancommunityBiblestudygroupatGraceFellowship

Church,IsayGodrichlyblessyouforbeinguntomeafamilyawayfrommyhomecountry.

IamalsothankfultoallmyfriendsandclassmatesespeciallyLaude,William,andthe

OdameAnkrahfamily.Thethingsyoudomakeyoumorethanfriends.

 CONTENTS

Page

ABSTRACT......................................................................................................................................................2

DEDICATION..................................................................................................................................................3

ACKNOWLEDGEMENTS.................................................................................................................................4

LISTOFTABLES..............................................................................................................................................8

LISTSOFFIGURES..........................................................................................................................................9

Chapter

1.INTRODUCTION.......................................................................................................................................11

BotanyofGarlic.......................................................................................................................................11

TaxonomyofGarlic.................................................................................................................................11

TheChemistryofAlliumSativumL.........................................................................................................12

BiologicalFunctionsandHealthBenefitsofGarlic.................................................................................15

2.TECHNIQUESFORALLIINDETERMINATION............................................................................................18

CapillaryElectrophoresis(CE).................................................................................................................18

SpectrophotometricMethod..................................................................................................................20

GasChromatography..............................................................................................................................21

ThinLayerChromatography...................................................................................................................25

HighPerformanceLiquidChromatography............................................................................................27

3.METHODUSEDINTHISWORK................................................................................................................30
5

 HighPerformanceLiquidChromatography(HPLC)Process...................................................................30

NormalVersusReversedPhaseHPLC.................................................................................................31

IsocraticVersusGradientElution........................................................................................................32

ColumnsUsedinHPLC........................................................................................................................33

DetectorUsedinHPLC........................................................................................................................34

ResolutionofBands............................................................................................................................36

MethodDevelopment.........................................................................................................................37

TheUseofSurfactantsinHPLC...........................................................................................................38

ResearchObjective.................................................................................................................................39

4.EXPERIMENTALPROCEDURERESULTSANDDISCUSSION......................................................................41

Reagents..............................................................................................................................................41

PlantMaterials....................................................................................................................................41

ExperimentalProcedures........................................................................................................................42

PreparationofReagents,Standard,AndStockSolutions...................................................................42

PreliminaryGasChromatographyMassSpectrometry(GCMS)Studies...........................................43

HPLCInstrumentation.........................................................................................................................45

DataAnalysis.......................................................................................................................................45

ResultsandDiscussion............................................................................................................................46

MethodDevelopmentandValidation................................................................................................46

OptimizationofWavelength...............................................................................................................46

 OptimizationofMobilePhase............................................................................................................47

OptimizationofMobilePhasepH.......................................................................................................49

OptimizationofMobilePhasewithSurfactants.................................................................................50

ReproducibityStudies.........................................................................................................................51

LinearityStudies..................................................................................................................................52

RecoveryStudies.................................................................................................................................54

ComparingMethanolHydrochloricacid(MeOHHCl)ExtractWithHotWaterExtract.....................58

ApplicationofMethod........................................................................................................................61

5.CONCLUSION..........................................................................................................................................65

REFERENCES................................................................................................................................................67

VITA.............................................................................................................................................................71

 LISTOFTABLES

Tables Page

1. Asummaryofresultsobtainedfromreproducibilitystudiesofthemethodforthemeasurementof

alliincontentingarlicsamples...........................................................................................................52

2. Resultsoflinearitystudiesshowinglinearrelationshipbetweenconcentrationandinstrumental

response...............................................................................................................................................53

3. Standardcalibrationdatausedintherecoverystudies.......................................................................55

4. Resultsforrecoverystudiescarriedoutwithhighconcentrationofstandardsolution.....................57

5. Resultsforrecoverystudiescarriedoutwithlowconcentrationofstandardsolution......................57

6. Concentrationofalliinindiversevarietiesoffreshgarlicandgarlicproduct.....................................62

 LISTSOFFIGURES

Figures Page

1. Examplesofsulfoxidesthatarepresentinalliumvegetables.Thepredominanceofeach

compoundvariesfromonevegetabletoanother.........................................................................13

2. Ezymatichydrolysisofalliinproducingallicinasmainproductandpyruvicacidasabyproduct.

Unstableallicindecomposesintoseveralsulfurcompounds........................................................14

3. SchematicDiagramofatypicalhighperformanceliquidchromatographySystem.....................31

4. ExampleofsurfactantscommonlyusedinHPLCdeterminations,................................................39

5. GCMSchromatogramofmethanolextractofgarlic....................................................................43

6. GCMSChromatogramofmethanolextractofgarlic.Temperatureprogram;linearincrease

from1700C3000Cat30C/min;then300oC3500Cat10oC/minandkeptconstantfor10

minutes..........................................................................................................................................44

7. HPLCchromatogramsofmethanolacidextractofgarlicusing(a)80%methanol:20%water,(b)

70%methanol:30%watermobilephasecompositions.(c)60:40methanol:watermobilephase

composition.Sampleswererunat1mL/minat210nmwavelength............................................48

8. HPLCchromatogramsofmethanolacidextractsofgarlicusing(a)30%methanol:70%waterand

0.05%SDS;(b)30%methanol:70%waterand0.05%CTAB;(c)30%methanol:70%waterand

0.05%OSAphasecompositions.Sampleswererunat1mL/minat210nmwavelength...........51

9. AplotofcalibrationcurveofAlliinstandardsolutionshowinglinearityofmethod.Theplot

displayserrorbarwith5%value....................................................................................................54

10. Standardcalibrationcurveusedinrecoverystudiesdisplayingerrorbar5%value....................56

 11. Chromatogramofgarlicsampleextractedwith(a)90%Methanol0.01MHydrochloricacid

comparedwith(b)chromatogramofhotwaterextract...............................................................60

12. Chromatogramsof(a)alliinstandardin(b)whitegarlicand(c)elephantgarlicobtainedusing30

%Methanol:70%waterand0.05%Sodumdodecylsulfate.Alliineluteat2.42.6minutes.......61

13. Histogramshowingthedistributionofalliinindiversevarietiesofgarlicandgarlic....................64

10

 CHAPTER1

INTRODUCTION

Garlicisgrownallovertheworldandisusedinvariousformsasfood,spices,and

medicine.GarlicisanindigenousherbofWesternAsiaandMediterraneanwhereithasbeen

cultivatedforcenturies.ThemajorgarlicgrowingcountriesincludesKorea,China,India,USA,

Spain,Argentina,andEgypt,amongwhichChinaisbyfarthelargestproducer(1).

BotanyofGarlic

Thegarlicplantismadeupoffleshyedibleclovesthatareencasedinawhiteorpink,thincoat.

Ithasleaves,stem,andflowerslocatedontheheadthatarealsoedible.Itiseasytogrowand

canbegrownallyearround.Itiscultivatedintemperateandtropicalclimates.Garlicplantgrows

wellinwelldrainedsoilandrequiresacoolandmoistperiodduringgrowthandarelativelydry

periodasitmatures.Itispropagatedusingclovesobtainedfromthebulbsandisreadyfor

harvestwhenthetopturnsyellowishorbrownish.Itisbeststoredinwellventilatedroom(2).

Freshgarlichasacharacteristicodorandisusedforflavoringandalsoasaspice.

TaxonomyofGarlic

RecenttaxonomyrevisionsplacegarlicinthefamilyAlliaceae,whichismadeupof

approximately700Species(3).ItbelongstothegenusAllium.Agreatnumberofspeciesin

thisgenusareperennialplantsthathaveundergroundstorageorgansconsistingofbulbsor

rhizome.Theyhavegreateconomicvalueaswellasenormousmedicinalimportance.The

mostcommonediblemembersincludechives,(A.SchoenoprasumL.),leek(A.porrumL.),and

11

onion(A.CepaL.)(4).Alliumspeciesarerichinsulfurcontainingcompoundsthathavebeen

identifiedtoberesponsiblefortheircharacteristicodor,flavorvariation,andbiological

activities(3).Garlic,withoutexception,isoneofthemostextensivelyinvestigatedAllium

speciesbothbychemistsandbiologistsduetothesecompounds.Itbelongstothespecies

SativumandhasthescientificnameAlliumSativumL.

TheChemistryofAlliumSativumL.

Garliccontainshighlevelsofsulfur,zinc,phosphorus,andpotassium;moderatelevelsof

selenium,vitaminA,andvitaminC;andlowlevelsofiron,manganese,calcium,magnesium,

sodium,andBcomplexvitamins.Inadditiontothese,about33sulfurcompoundsand17

aminoacidsthatincludealanine,arginine,asparticacid,asparagine,histidine,leucine,

methionine,phenylalanine,praline,serine,threonine,tryptophan,andvalinehavebeen

identifiedandisolated(5).OneuniqueconstituentgroupofalliumplantsisSAlk(en)yl

cysteinesulfoxides(ACSOs)thatareresponsiblefortheirtypicalodorandflavors(6).These

sulfoxidesincludeSMethylLcysteinesulfoxide(Methiin),SAllylLcysteinesulfoxide(Alliin),

SpropylLcysteinesulfoxides(propiin),SpropenylLcysteinesulfoxide(Isoalliin),SEthylL

cysteinesulfoxide(Ethiin)andSnButylLcysteinesulfoxide(Butiin).Thesecompoundsare

showninFigure1.

12

 O NH2
O NH2

S
S
H 2C COOH
H3C COOH

S-Allyl-L-cysteine sulfoxide S-Propyl-L-cysteine sulfoxide

Alliin Propiin

O NH2
O NH2

H3 C S
H3C S
COOH COOH
S-Ethyl-L-cysteine sulfoxide S-n-Butyl-L-cysteine sulfoxide
Ethiin Butiin

O NH2
O NH 2

S
S
H3 C COOH H3 C COOH

S-Propenyl-L-cysteine sulfoxide S-Methyl-L-cysteine sulfoxide

Isoallin Methiin
Figure1.Examplesofsulfoxidesthatarepresentinalliumvegetables.Thepredominanceof
eachcompoundvariesfromonevegetabletoanother.

Ingarlicthepredominantflavorprecursorisalliin,withlowerconcentrationofisoalliin

(sourceoflachrymatoryfactorinonion)andmithiin,andtraceamountofpropiin(7).Alliinwas

firstidentifiedin1948byStrollandSeebrook(7).Itisastablenonproteinaminoacidthat

formstheparentsulfurcompoundthatisresponsibleforthemajorityoftheodorousvolatiles

producedfromcrushedorcutgarlic(3).Alliinislocatedinthecytoplasmofthecellofagarlic

bulbandisseparatedfromallinase,anenzymelocalizedinthecellvacuole.Whengarlicis

crushed,cut,orchewed,theenzymefromthevacuoleisreleasedtoactonthealliinina

reactionthatproducesallicin(anodiferousalkylalkanethiosulfinate),withpyruvicacidand

ammoniareleasedasbyproducts(8).Thethiosulfinateformedisrelativelyunstableand

storageoverlongperiodoftimeorsteamdistilledwillcauseittoundergoanumberof

13

transformationsdependingonthetemperature(3).Atypicalenzymaticreactioningarliccell

thatproducesallicinispresentedinFigure2.

CO2

.. H
O N
H
S S
Alliinase 2 OH
+
COOH
NH2 P

Alliin Allylsulfenic acid

Aminoacrylate
-H2O

H 2O

O O
+ NH4
S
S H3C COOH

Allicin Pyruvate
(diallylthiosulfinate)

Allyl sulfides Others

-Disulfides
-Trisulfides
-Polysulfides

Ajoenes Dithines

Figure2.Ezymatichydrolysisofalliinproducingallicinasmainproductandpyruvicacidasaby
product.Unstableallicindecomposesintoseveralsulfurcompounds.
14

 Alliinasefromgarliccloveshasbeenisolatedandwellcharacterized.Lindaetal.(8)in

theirpaperpublishedin2006describedalliinaseasadimerwithtwoequalsubunits,each

having448aminoacidresidueswithatotalmolecularmassof103kDa.Itbelongstothefamily

ofglycoproteinandcontainsabout5.5%6.0%mannose.Theenzymeisknowntoformastable

complexwithamannosespecificlectin,alliumsativumagglutinin(ASA1).TheoptimumpHfor

allinaseactivityis6.5,anditsisoelectricpointwasdeterminedtobebetween6.0and7.0(8).

Allicinisthemainthiosulfinateproducedingarlic,representing70%oftheoverall

thiosulfinatepresentorformedbycrushinggarlicgloves(9).Allicinwasdiscoveredin1944by

CavalittoandBailey(9).Itisanunstablecompoundandcannotreachitstargetcellinthebody

viacirculation(10).Allicinisahighlyreactivemoleculeandreadilydegradestoformsecondary

sulfides.Allicinhasadisullfur(S(O)S)bondthatreactswithdifferentthiolcontaining

moleculesincludingSHcontainingprotein(9,11).Manyrecentstudieshaveprovidedstrong

evidencethatmostofthebiologicalfunctionsandhealthbenefitsofgarlicareattributedto

allicin.ThisismadepossiblebyitsstrongSHmodifyingandantioxidantproperties(12).In

fact,nocompoundoutsidethethiosulfinateshasbeenfoundthataccountforasignificant

portionofthepharmacologicalactivitiesofcrushedgarlicatlevelsrepresentinghuman

consumption(25g/day)(13)

BiologicalFunctionsandHealthBenefitsofGarlic

Themedicinalvalueofgarlichasbeenexploredsinceantiquity(14,15).Ithasbeenused

traditionallytotreatseveralconditionssuchascold,coughs,hypertension,highbloodpressure,

rheumatism,diarrhea,andsnakebite.(14).Itstraditionalmedicinalusevariesfromone

15

culturetoanother.Forinstance,inEastAsia,hotwatergarlicextracthasbeenusedasan

aphrodisiacandalsototreatasthma.InEngland,hotwatergarlicextractistakenorallyfor

diabetes.GarlicbulbischewedtogetherwithotherleavesinEthiopiatotreatstomachache.

InGuatemala,hotwaterextractofdriedbulbisusedexternallyforringworm,fungaldiseases

oftheskin,andskindiseasesandirritationsfromleukorrhea,vaginitis,andinfectionsofthe

skinmucosa.WaterextractgarlicisusedinJapantopromotehairgrowth.InNigeria,garlicis

driedandsoakedinjuiceofcitrusaurantifoliaandapinchofcoppersulfateandtakenorallyto

treatconvulsionsinchildren(15).

Numerouspharmacologicalactivitiesandclinicaltrialsovertheyearshaveconfirmed

thebiologicalfunctionsandthehealthbenefitsofgarlic.Forinstance,recentepidemiological

studieshadshownthatconsumptionoflargeamountofgarlicisassociatedwithreduced

cancerriskinhumans,mostlystomachandcoloncancer(16).Furthermore,studieshavealso

showntheabilityofgarlictoreducechemicalcarcinogensindifferentanimals.Otherwell

studiedbenefitsofgarlicinclude;antimicrobial,antithrombotic,antioxidant,improving

immunesystem,anticardiotoxicandantiischemiceffects,antiaging,antibacterial,

anticytotoxic,antifatigue,antifungal,antihypertensive,antihypotensive,antihypothermic,

antimutagenic,antimycobacterial,antitoxic,andantiprotozoanactivities.Inaddition,there

havebeenstudiesontheinhibitionofacetylcholinesterase,acidphosphatase,adenosine

deaminase,adherence(bacteriatohostcells),aflatoxinproduction,andalanine

aminotransferose,aswellasalamineaminotransferaseandalkalinephosphatasestimulation

(15).

16

 Extensiveinvestigationintogarlicanditsmedicinalvaluehasledtoanimprovementin

thequalityandyieldoffreshgarlicproduction.Thecountlessbenefitsofgarlicnodoubthave

givenrisetotheproductionofseveralgarlicfoodsupplementsrangingfromtabletstopowder

andarebasedoneitherallicincontentoronthepotentialtoproduceallicin(12).This

consequentlyhasinitiatedahostofinvestigationstoascertainordebunktheclaimsthatthey

havethesameessentialingredientsasrawgarlic.Oneofsuchinvestigationshasrevealedthat

thoughgarlicpowderandgranulescanserveasimportantfoodsupplement,ifstoredforalong

time,theingredientspresentinfreshgarlicareoftenlost.Inaddition,becausealliinaseis

irreversiblydeactivatedatthepHlevelinhumanstomach,ifgarlicpowderistakendirectly

therewouldonlybeaninsignificantamountofallicinthatcanbeproducedinsidethehuman

body.Basedonthisknowledge,garliccapsulescoatedwithmaterialsthatcanresisthuman

stomachconditionsinordertoprolongtheshelflifeandprotectalliinaseactivitythroughthe

stomachhavebeencommerciallyproduced.Inthisway,allicincanbereleasedonlyinthe

intestineandconsequentlydecreasethecharacteristicodorandaftertaste(17).Manyother

studieshadbeendonetofindoutthepharmacologicalbenefitsofthesefoodsupplements,but

notallofthemcanassumedequivalenceintheircompositionandintheirbiologicalresponse

(18).

17

 CHAPTER2

TECHNIQUESFORALLIINDETERMINATION

In1973,FreemanandMcBreendevelopedthefirstmethodforanalysisofvolatilesof

onionsandgarlicbasedonarapidspectrophotometricdetermination(19).Twentyyearslater,

manyothermethodswereproposed(19).Currently,anumberofanalyticalmethodshave

beenusedtodetermineSalk(en)ylcysteinesulfoxidesingarlic.Theseinclude;capillary

electrophoresis,spectrophotometricmethods,gaschromatography,micellarelectrokinetic

capillarychromatography,thinlayerchromatography,highperformanceionpair

chromatography,andhighperformanceliquidchromatography.Theycanbecategorizedinto

directmethodsthatallowdeterminationofSalk(en)ylcysteinesulfoxidescontentbeforetheir

enzymatichydrolysistoformallicinorindirectmethodsthatareemployedinthedetermination

ofdiverseproductsarisingfromtheenzymaticreaction(20).Someoftheabovenamed

techniquesinvolveasinglestepsamplepreparation,whereasothersareassociatedwith

severalstepsduringwhichtheanalyteisderivatized.

CapillaryElectrophoresis(CE)

Capillaryelectrophoresis(CE)combinesaspectsofbothgelelectrophoresisaswellas

highperformanceliquidchromatography(HPLC).Thetechniqueinvolvesseparationofspecies

withinthelumenofasmallborecapillaryfilledwithanelectrolyte.Thecapillaryisimmersedin

electrolytefilledreservoirscontainingelectrodesconnectedtoahighvoltagesupply.Whena

sampleisintroducedatoneendofthecapillary,thecomponentsofthesampleareseparated

18

astheymigratethroughthecapillarytowardstheotherend(outlet)andaredetectedbya

detectorthatsendselectronicsignaltoarecorder(21).Theseparationdependsondifferential

migrationinanelectricfield.CapillaryelectrophoresisissimilartoHPLCinmanyways,in

sampleinjectionaswellasdatapresentationandinterpretation.Itsadvantagesincludehigh

resolvingpowerthatsometimesmaybehigherthantheconventionalelectrophoresisorHPLC.

Theuseofnarrowborecapillarywithexcellentheatdissipatingpropertiesallowstheuseof

veryhighfieldstrengththatdecreasesanalysistimeandminimizesbanddiffusion.Themajor

limitationofthistechniquehasbeenreportedinapplyingittoproteinseparation.Thehigh

surfacevolumeratioofthecapillariesandthehighsurfaceactivityofthefusedsilicacapillary

giverisetothislimitation(21).

Measurementofsulfoxidesbycapillaryelectrophoresisisbelievedtohavebeenfirst

reportedbyHoriesandYamashita(6).Intheirworkpublishedin2006,theyexplainedthat

becausecapillaryelectrophoresisallowsindirectdetectionofcomponentswithnospecific

ultravioletabsorption,itseemedlikelythatitcouldseparatesulfoxideswithoutdifficult

derivation.Theadvantagesofthistechniqueasreportedintheirarticleweresimplicityand

simultaneousdetectionofpyruvateandmethiinaswellasalliinpeaks.

However,KubecandDadakora(22)mentionedthatthereportedmethodoffers

absolutelyunsatisfactoryresultsforanalyzingrealsampleextracts.Theymadethisassertion

aftertheyhadrepeatedthepreviouslyreportedprocedure.Theyattributedthistothefact

that,thecapillaryhadtoberinsedthoroughlyaftereveryrunand,inaddition,themigration

timevariationandpeakoverlaprenderedidentificationandquantificationofindividualpeaks

19

extremelyunreliable.Basedontheseaforementionedlimitations,Kubeetal.developeda

completelynewmethod.Thiswasbasedonextractionofthesulfoxideswithmethanol,

derivatizingthembyfluorenylmethylchloroformate,andsubsequentlyseparatingthemby

micellerelectrokineticcapillarychromatography.Thederivatizingagentwasknowntoreadily

formstablederivativeswithcompoundspossessingprimaryandsecondaryaminogroupsin

virtuallyquantitativeyield.Theyreportedthatthereactionproceededinaqueoussolution

withinminuteswithnotimeconsumingcleanupprocedurerequired.Inaddition,the

derivativesproducedaveryhighextinctioncoefficient,ensuringtheirspecificandsensitivity

detection(22).

SpectrophotometricMethod

Spectrophotometricmethodsemploylighttomeasurethechemicalconcentrationof

analytes.Thisabsorptionshouldbedistinguishablefromthatduetoothersubstancesinthe

sample.Theabsorptionbyasampleisproportionaltothetotalamountofmaterialthat

absorbstheincidentlightthatisreferredtoaschromophore.ThisisdefinedbytheBeer

LambertLaw;

bc[1]

where,molarabsorptivityisaconstantthatisapropertyofthematerialitselfas

wellasthewavelengthofthemeasurement;breferstothelengthofthepaththroughwhich

thelighttravelsinthesample;andc,themolarconcentrationofthematerialthatabsorbsthe

light(23).Alliincontentingarlicextractcanbeindirectlydeterminedusingthismethod.This

20

determinationisbasedontheprinciplethatalliininpresenceofallinaseproducesallicinthat

reactsrapidlywithfreethiolgroupsthroughathioldisulfideexchangereaction.Such

exchangereactioncancauseashiftintheopticalabsorptionofathiolcontainingchromohpore,

andthereforethemolarconcentrationofalliincanbecalculatedfromthedifferencein

absorbanceaccordingtotheequations:

[alliin]=[absorbancewithoutalliin][absorbancewithalliin]xdilutionx[]1[2]

where=molarabsorptivity(12).

Mironandhiscoworkers(12)usedthismethodintheirassayforallicin,alliin,and

alliinase.Intheirrecentwork,theyused4mercaptopyridine(4MP)ratherthanthepreviously

used2nitro5thiobenzoate(NTB)becausetheformerisacommerciallyavailable

chromogenricthiol.Theirdeterminationswerebasedonthereactionof4MP(whichhas

maximumabsorptionat324nm)withtheactivateddisulfidebondofthiosulnatesS(O)Sto

form4allylmercaptothiopyridine,whichhasnoabsorbanceinthisregion.Thestructureofthe

4allylmercaptothiopyridinewasconfirmedbymassspectrometry.Thedifferencein

absorbanceobtainedwasthususedtocalculatethecontentoftheanalyte.Thismethod

thoughindirect,wasreportedtobesensitive,fast,andnoncostlyandgiveshighlyefficient

throughputassayofalliin,allicin,andalliinaseingarlicextracts.

GasChromatography

Gaschromatography(GC)involvestheseparationofcomponentsofvaporizedsample

asaresultofpartitioningbetweenagaseousmobilephase(calledcarriergas)andaliquidor

21

solidstationaryphase.Thecarriergasescommonlyusedarehelium,nitrogen,orhydrogengas.

Thechoiceofcarriergasdependsonthedetectorandthedesiredseparationefficiencyand

speed(24).

Theliquidstationaryphaseisanonvolatileliquidbondedtotheinsideofthecolumnor

toafinesolidsupport.Thisisusedingasliquidpartitionchromatography.Thesolidstationary

phaseisusedingassolidadsorptionchromatographyinwhichtheanalyteisadsorbeddirectly

onsolidparticlesofstationaryphase.Theessentialelementsofgaschromatographyincludea

regulatedsupplyofcarriergas,adeviceforvaporizingthesample(injector),athermostatted

oveninwhichthecolumnishoused,adetector,andadataprocessor(25).Whenavolatile

liquidorgaseoussampleisinjectedthroughaseptumintoaheatedport,itisrapidly

evaporatedandthevaporiscarriedthroughahotcolumnbycarriergascausingseparationof

componentsofsampletotakeplace.Thecolumniskepthotenoughtoprovidesufficientvapor

pressureforanalytetobeelutedinreasonabletime.Theseparatedcomponentsflowthrough

adetectorandtheirresponsesarerecordedbyarecordingdeviceandprocessed(24).The

columnsusedaregenerallyopentubularcolumnsandpackedcolumns.Theopentubular

columnsareusedinwiderangeofanalyses.Itismadeupoffusedsilica(SiO 2 )andcoatedwith

polyimideforsupportandprotectionfromatmosphericmoisture(24).Anopentubularcolumn

hastheadvantageofbetterseparationefficienciesandgreatlyimprovedsampledetectability

foragivenanalysistimethananypackedcolumns(25).Italsogivesgreatersensitivityand

shorteranalysistime(24).Opentubularcolumnshoweverhavedisadvantagesthatinclude

22

moredemandingofinstrumentperformance,lessforgivingofpooroperatortechnique,and

possessalowersamplecapacitythanthepackedcolumns(25).

Packedcolumnsaremadeupoffineparticlesofsolidsupportcoatedwithnonvolatile

liquidstationaryphase,orthesoliditselfmaybethestationaryphase.Itoffersagreater

samplecapacitybutgivesbroaderpeaks,longerretentiontimes,andlowerresolution.

Howeveritisusefulforpreparativeseparationsorseparationofweaklyretainedsolutessuch

asgasesandlowmolecularweighthydrocarbon.Differentdetectorsareusedingas

chromatography.Theseincludeflameionizationdetector,thermalconductivitydetector,and

electroncapturedetector.Othersareflamephotometricdetector,sulfurchemiluminescence

detector,andphotoionizationdetector(24,25).

Gaschromatographygivesexcellentresolutionandhasmassidentificationcapabilities

(26).Forthisreasongaschromatographyandgaschromatographymassspectrometry(GCMS)

havebeenwidelyusedinthecharacterizationofalliumvolatiles.Gaschromatography

determination(GCFPD)ofalliiningarlicandgarlicproductswasfirstelaboratedbySaitoetal.

in1988(20).Theirmethodinvolvedderivatizingalliinwithtrifluoroaceticacidanhydride

(TFAA)followedbyGCanalysisusingashortpackedcolumn.Saitoandhiscoworkerfoundout

thatthetrifluoroaceticderivativewasunstableandconsequentlydecomposedafteritwas

exposedtosunlightfor15minutes.Thiswasthus,amajorlimitationoftheirmethod.Another

limitationthatwasalsoreportedwaspoorcolumnresolutionthatrendersthemethod

unsuitableforroutinework.

23

 Intheirworkpublishedin1992,Blocketal.(26)emphasizedthatgaschromatography

astypicallyperformedwithhighinjectorandcolumntemperaturepresentanerroneouspicture

ofthecompositionofroomtemperatureextractsofAlliinspeciesandthatHPLCprovidesa

reliablequantitativeandmeasureofwhatisactuallypresentinthespecies.

AugerandFeraryandtheirteamalsoreiteratedthatgaschromatographmass

spectrometer(GCMS)doesnotseemtobeasuitablemethodfortheanalysisoftruegarlic

flavorsasitgaveartifacts.Theyproposedhighperformanceliquidchromatographyasa

preferablealternative(27).

However,Kubecandhiscolleagues(20)reportedanewmethodallowingahighly

sensitiveandreproducibledeterminationofSalk(en)ylcysteinesulfoxides(includingminor

derivative)usinggaschromatography.Themethodwasbasedonisolationofaminoacid

fractionbyionexchangechromatographyfollowedbyderivatizationwithethylchlorofomateat

ambienttemperatureandreductionofderivatizedSalk(en)ylcysteinesulfoxidesbysodium

iodide.TheyreportedthatallpreliminaryattemptstoanalyzeSalk(en)ylcysteinesulfoxidesby

GCimmediatelyafterderivatizationfailed.Theyusedtwodifferentcapillarycolumns,various

temperatureprograms,andinjectortemperaturesandrealizedthatunderalltheconditions

therewasasubstantialdecompositionofSalk(en)ylcysteinesulfoxidessimilartowhathad

beendescribedinconnectionwithGCdeterminationofglucosinolateshavingasulfoxide

moietyinthesidechain.Theyattributedthistothepresenceofthehighlypolarizedand

extremelylabilesulfoxidegroupandsuggestedthatthebestwaytoanalyzeSalk(en)ylcysteine

sulfoxidebyGCisremovingthisgrouppriortotheinjection,hencetheuseofsodiumiodidein

24

theirmethod.Eventhoughthismethodofferedoutstandingsensitivity,excellentresolution

capacity,accuracy,andreliability,timerequirementsareaseriousdrawbackforuseinroutine

analysis.Inaddition,themethodisunabletoresolvebetweenSalk(en)ylcysteineandtheir

sulfoxides(20).

ThinLayerChromatography

Thinlayerchromatography(TLC)involvesmovementofamobilephasethroughathin

layerofsorbent(coatedonaninert,rigidbackgroundsuchasaluminium,plastic,orglass)by

capillaryaction.Theseparationofsampleisaresultofthedifferencesinmigrationofsample

componentsinthedirectionthemobilephasetravelled.Itismeasuredintermsofretention

factororretentionindex,RFgivenas;

distancetravelledbyspotfromorigin
f 3
distancetravelledbysolventfromorigin

TLC,unlikecolumnchromatography,allowssimultaneousanalysisofanumberof

samplesandstandards.Italsoallowssamplesthataredifficulttoresolvetobedevelopedin

twodifferentsolventsruninperpendiculardirections.Inaddition,becauseTLCplateisused

onlyonce,harshseparationconditionsthatcandegradeandrapidlydestroyananalytical

columncanbeusedforTLC(23).

TheconventionalTLCtechniquehasseenimprovementinthequalityofadsorbentlayer

aswellasitsmethodsofsampleapplication.Thisnewtechniquecalledhighperformancethin

layerchromatography(HPTLC)ismorerapid,efficient,andsensitivethanconventionalTLC(25).

ComparingHPTLCwithHPLC,theformerisanopenbedwhilethelatterisaclosedsystem(25).
25

Pooleetal.(25)intheirbooknotedthatthetwotechniquescomplementeachotherandhence

selectionshouldbebasedonthetypeofproblemtobesolved.EventhoughHPLCtechniques

offeragreaterseparatingpowerthanHPTLCconsideringindividualsamples,theenormous

advantagesofHPTLCovertheHPLCcannotbeoveremphasized.InHPTLCtheselectionof

mobilephasedoesnotlimitthechoiceofdetector.Thisisbecausethesolventiscompletely

evaporatedbetweendevelopmentandmeasurementsoitdoesnotinfluencethedetection

process.ThisisnotsoinHPLC,because,forexample,UVabsorbingsolventscannotbeused

withUVdetectors(25).ThedetectionprocessinHPTLCismoreflexibleandvariablethanthat

ofHPLCbecausetheformerisdependentondistanceratherthantimeeventhoughdetection

limitsunderoptimumconditionsareapproximatelythesameforbothtechniques.

HPTLCtechniquesallowsimultaneoussampleanalysiswiththepossibilityof

substantiallyreducingthetimerequiredfortheanalysisofalargegroupofsamples.Thisisnot

sointhecaseofHPLCbecauseanalysisinHPLCisbynecessityperformedinasequential

mannerduetothenatureofthemethoddevelopment(25).

Thinlayerchromatographicmethodofanalysisofgarlicconstituentshasbeen

developedinvolvingtheuseofninhydrindetectionreagenttooptimizethedifferentiation

betweencysteinesulfoxidesandotheraminoacidderivatives(19).Theshortcomingofthis

methodhasbeenreportedtobeenzymaticdegradationofalliinduetopreparationofsample

extractbyhomogenizinggarlic.Thisallowstheinteractionbetweenalliinandallinasethat

otherwisearepresentinseparationcomponentsintheintactcells(28).

26

 In2005,Niranjanandhisgroup(28)publishedaproposedHPTLCmethodforthe

analysisofgarlicanditsformulationforitsalliincontent.Thismethodinvolvesdensitometric

evaluationofalliinafterresolvingitbyHPTLConsilicagelplateswithnbutanol:aceticacid:

water(6:2:2v/v)asthemobilephase.Afterderivatizingtheresolvedbandswithninhydrin

reagent,thepeakareaswererecordedat540nmindensitometricevaluation.Theyfoundthe

relationbetweentheconcentrationofalliinandthecorrespondingpeakareatobelinear

withintherangeof250to1500ng/spot.Theyrecommendedthismethodforuseinroutine

qualitycontrolofgarlicanditsformulationduetoitsgoodprecision,specificity,sensitivity,and

accuracy(28).

HighPerformanceLiquidChromatography

Highperformanceliquidchromatographyhasbeenusedwidelyinanalysisofdiverse

varietiesofsamplessincemostcompoundsarenotsufficientlyvolatileforgaschromatography.

Itsadvantagesinclude;highspeedresolution,sensitivity(femtogramsnanograms),good

reproducibility,recovery,accuracy,precision,andeaseofautomation.Ithasproventobea

reliabletechniqueforquantitativeandqualitativedeterminationofsulfoxidesinalliumextracts

(26).ForthisreasonanumberofHPLCtechniqueshavebeendevelopedandhavebeenusedin

thedeterminationsoforganosulfurcompoundsinalliumspecies

In2003,Arnaultandhiscoworkers(20)publishedareportonarapidHPLCmethod

suitableforroutineanalysisofsulfoxides.Thismethodinvolvestheuseof3mparticle

HypurityEliteC 18 columnofdimension150x3mm,anultravioletdetectoroperatedat208

nm,andagradientelutioninvolvingtheuseofmobilephaseconsistingof(a)20mMsodium

27

dihydrogenphosphate,10mMheptanesulfonicacid,85%orthophosphoricacidand(b)

acetonitrile,20mMsodiumdihydrogenphosphate,10mMheptanesulfonicacid.Theyused

eluentscontaininganionpairingreagenttoensureasufficientseparationbetweenalliinand

themoreretaineddipeptideatverylowpH.Arnaultetal.reportedthattheirmethodthatwas

withoutderivatizationallowedsimultaneousquantificationofalliin,allicin,aswellasdipeptides

andrequiresnoparticularsamplepreparation.Themethodalsoyieldedgoodlinearityforeach

compoundandgavearuntimeof30minutes.However,thesensitivityofthemethodwas

weakercomparedtoaprecolumndervatization.

Ichikawaandhisgrouphavealsoreportedanothermethodforsimultaneous

determinationofsulfoxides(30).Themethodinvolvedonestepsamplepreparationprocedure

followedbynormalphaseandreversedphaseHPLCtechniquestodeterminethesulfoxides.

Alliin,isoalliin,methiin,cycloalliin,andLglutamylSmethylLcysteineweredeterminedby

normalphaseHPLCusinganaminopropylbondedcolumn,whereasLglutamylS(2

propenyl)LcysteineandLglutamylS(trans1propenyl)Lcysteinewereseparatedonan

octadecylsilanecolumn.Theyreportedoverallrecoveriesof97.1%102.3%andrelative

standarddeviationvaluesofintraandinterdayprecisionlowerthan2.6%and4.6%

respectively.Theadvantagesoftheirmethodincludespecificity,speed,andeaseofuse.They

confirmedthatthemethodwasusefulforchemicalandbiologicalstudiesofgarlicandits

preparations.

In2007,Diegoetal.developedandvalidatedareversedphaseHPLCassayfor

quantitativedeterminationofalliciningarlicpowderandtablets(18).Theirchromatographic

28

separationwasperformedonanRP18 e column ofdimensions124mmx4mm.Theyuseda

mobilephasemadeup50:50methanol:water,aflowrateof0.5mL/min,andultraviolet

detectionat220nm.Themethodalsoinvolvedtheuseofethylparabenasinternalstandard.

Diegoandhiscolleaguesreportedthatthemethodwaslinearforallicinconcentrationsof5.0

60.0L/mLandgaverelativestandarddeviationforprecisiontobelessthan6.14%with

accuracyabove89.11%.

Althoughthesepublicationsdemonstrategreatsuccessinthedevelopmentofhigh

performanceliquidchromatographictechniquesforthiosulfinateandsulfoxidedeterminations,

newchromatographictechniquescontinuetoevolvefromalreadyexistingoneswiththeaimof

usingenvironmentallyfriendlyreagents,improvingefficiency,andsimplicityofmethodaswell

ascuttingdownthecostforanalysis.

29

 CHAPTER3

METHODUSEDINTHISWORK

Thischapterdiscussesthemethodologythatwasusedintheproject.Itfocusesonthe

instrumentation,thechromatographicprocess,someadvantagesithasinsulfoxide

determination,andtheresearchobjective.

HighPerformanceLiquidChromatography(HPLC)Process

HPLCemployshighpressuretoforcesolventthroughaclosedcolumncontaining

chemicalgroupsboundedtoveryfineparticlesproducinghighresolutionseparation(25).The

techniqueinvolvestheuseofasolventdeliverysystem,injectionsystem,acolumn,adetector,

controller,andrecorder.Agoodchromatographicseparationrequiresunlimitedsupplyof

solvent,ahighresolutionstationaryphase,withporouspackmaterials,highpressurepump,

andadesirableflowrateofsolvent.Furthermore,thesolventshouldbevolatileandhavealow

viscosity(31).TheuseofpureHPLCgradesolventspreventsdegradationofcolumnby

impuritiesanddecreasesdetectorbackgroundsignalsfromcontaminants.Solventsare

degassedwithheliumtoeliminategasbubblesthatotherwisedegradepumpanddetector

performance(24,25).However,itisessentialtopreventdegassinginthedetectorwhere

solventpressuremaybereducedtoatmosphericpressure,inordertopreventbaselinedriftor

continuousspikes.Samplesmustbedissolvedinarelativelylargevolumeofsolventand

injectedwithvalveinjectorratherthansyringeinjection(31).Whenasampleisinjectedinto

thecolumn,itiscarriedthroughthestationarybedbythemobilephaseresultinginseparation.

30

Thesamplecomponentsflowthroughthedetectorconnectedtotheendofthecolumnthat

monitorstheseparationandthentheresultingchromatogramisrecorded(31).Figure3shows

aschematicdiagramofatypicalHPLCsystemwiththebasicunitsdescribedabove.

High Injector
Solvent Filter Pressure
Reservoir
Pump

column

Recording
Detector
Device

Figure3.SchematicDiagramofatypicalhighperformanceliquidchromatographySystem.

NormalVersusReversedPhaseHPLC

NormalphaseHPLC(NPHPLC)involvestheuseofnonpolarmobilephaseandapolar

stationaryphase.ReversedphaseHPLC(RPHPLC),asthenameimplies,referstothereversal

ofthenormalphasechromatography.Itemploystheuseofapolarmobilephaseandanon

polarstationaryphaseinthechromatographicseparation.Duetoitssimplicityandversatility,

RPHPLCcanseparateabroadspectrumofnonionic,ionizable,andioniccompounds.Because

thestationaryphasesarechemicallybonded,columnsarestableandseparationsare

reproducible.Inaddition,duetotheweaksurfaceenergiesofbondedphases,analysesare

rapidandreequilibrationtimeisshort.Itcanbeusedtodeterminephysiochemicalproperties

suchashydrophobicity,dissociationconstants,andcomplexationconstants.Thelimitationsof

31

thereversedphasetechniqueincludethefactthatsilicabasedcolumnpackingslimitsthe

usuablepHrangeto27.5.Theunreactedsilanolgroupspresentonthesilicasurfacecancause

adsorptionofthesolutetothesilicasurfacegivingrisetopoorpeakshapes.Theretention

mechanismismorecomplexthaninotherformsofchromatographyandabetter

understandingisneededtocontrolit(25).

IsocraticVersusGradientElution

Inisocraticelution,themobilephasecompositioniskeptconstantthroughoutthe

separation.Thismethodofelutionissimpleandconvenienttouse.Italsogivesgood

reproducibilityofchromatographicdata.Therearealsoalowerassaycostsandincreased

reliabilityinusingisocraticmethod.Inaddition,whensamplesareseparatedusingtheisocratic

method,thecolumnisinequilibriumwiththesamemobilephasecompositionthroughoutthe

run,sothenextsamplecanbeinjectedassoonasthelastbandfromtheprevioussamplehas

eluted.These,amongmanyothers,accountforthecommonuseofisocraticassayforroutine

work(32).However,inseparatingsamplescontainingcomponentsofwidelydifferent

polarities,theisocraticmethodisineffectiveandproducespoorresolution.Gradientelutionis

thuspreferredundersuchconditions.Atypicalgradientelutionusesaweaksolventanda

strongsolventasmobilephase.Elutionisdonebyvaryingthestrengthofthemobilephase

duringseparationbycontinuouslychangingthemobilephasecomposition(21,24).Problems

associatedwithgradientelutionincludedriftingbaselines,solventdemixing,andartifactual

peaksthatarearesultofseparatinganyUVabsorbingimpuritiesinthemobilephasecausing

32

appearanceofpeaksthatdonotcorrespondtosamplebands.Also,inusinggradientmethod

reequilibrationofthecolumnrequirealongertime(32).

ColumnsUsedinHPLC

ColumnisanintegralcomponentofHPLC.Itissensitiveandeasilydegradedbydustor

particlesinthesampleorsolventandbyirreversibleadsorptionofimpuritiesfromsampleor

solvent.Narrowcolumnareusuallyusedbecausetheyrequirelesssampleandproduceless

waste.Theyarealsousefulbecauseheatgeneratedbyfrictionofsolventflowinsidethe

columnismoreeasilydissipatedmaintainingisothermalcondition.Thecolumnispackedwith

small,rigidparticleswithnarrowparticlesizedistribution(25).Silicaisbyfarthemost

commonlyusedadsorbentinHPLC(24).ItishoweverineffectiveathighpHbecauseit

dissolvesinbase(25).Columnefficiencyisexpressedintermsoftheoreticalplate.Itisa

measureofthebroadeningofapeakduringpassagethroughthechromatographiccolumn(21).

Theefficiencyofapackedcolumnincreaseswithdecreasedparticlesize.Smallerparticlessize

leadstohigherplatenumber,highpressure,shorteroptimumruntime,andlowdetection

limit.However,smallparticlesizecancauseresistanceinsolventflowleadingtohighpressure.

TypicalparticlesizeinusetodayinHPLCcolumnsis35m(24).

ControllingcolumntemperatureiscriticalinHPLC.Heatingthecolumnusually

decreasestheviscosityofsolventandallowsfasterflow.Italsodecreasesretentiontimeand

improvesresolutionbyhasteningmasstransportofsolutes.However,increasedtemperature

candegradethestationaryphaseanddecreasecolumnlifetime(25).Exampleofanalytical

33

columnusedinreversedphaseHPLCincludeC 18 (ODS),C 8 ,phyenyl,trimetylsilyl(TMS),and

cyano(33).

DetectorUsedinHPLC

AdetectorisausefulcomponentoftheHPLCsystem.Itproducesasignalthatis

dependentontheconcentrationofsampleandrelaysitonadataprocessorfordisplayand

storage.Itsperformanceincludestheabilitytodetectacomponentoverthebackgroundof

eventandmatrixsignal,includingnoise.Thispropertyisreferredtoasselectivity(21).Another

importantparameterofthedetectoristhedetectionlimit,whichistheminimumconcentration

ofanalytethatcanbedetectedwithagivenconfidencelimit.Detectornoisemayresultfrom

instrumentelectronics,linevoltagesurgestemperaturefluctuations,flowchanges,andpulse

fromthepumpcausingachangeintheoutputsignalofthedetectorthatisnotdirectly

attributedtotheanalyte.Otherparametersaredetectordrift,whichisasteadymovementof

thebaselineeitherupordownscale,andabsolutesensitivityofthedetector(34).

Detectorsusedcanbeclassifiedintotwocategories;thebulkpropertyorgeneral

detectorsandsolutepropertyorselectivedetectors.Theformermeasuresthedifferencein

somephysicalpropertyofthesoluteinthemobilephasecomparedtothemobilephasealone.

Examplesofthiskindofdetectorincluderefractiveindex,dielectricconstant,andconductivity

detectors.Thesolutepropertyorselectivedetectorsrespondtoaphysicalorchemical

propertyofthesolutethat,ideally,isindependentofthemobilephase.Examplesofthese

detectorsarespetrophotometric,electrochemical,andfluorescencedetectors(24,34).

34

 Differentialrefractometerdetectorsmeasurethechangesinrefractiveindexofeluent

asaresultofthepresenceofsolutesastheycomeoutfromthecolumn.Itisruggedandisable

todetectconcentrationsofabout105to106g/mL.However,itcannotbeuseeffectivelywith

gradientelution(duetoachangeinbaseline)norwhenthesolventhasarefractiveindexclose

tothatofthesolute.Itisalsohighlysensitivetotemperaturechanges.

UVdetectorshavegoodsensitivityto108g/mLandisnottemperaturesensitive.Itis

alsorelativelyinexpensiveandcanbeusedwithgradientelution.Inaddition,itissensitivetoa

largenumberoforganiccompounds.However,itcannotbeusedwithsolventthathave

significantabsorptionintheUVregionorwithsamplecomponentthatdonotabsorbintheUV

region.

Indiodearraydetector,thefocusradiationsourcepassesthroughthedetectorflowcell

andisdispersedbygratingtoaphotodiodearrayfordetection.Thedetectorcanrecord

absorptionspectrainstantaneouslyandprovidesanadditionalresolvingpower.

UVVisdetectorsareselectivedetectors.Theyarethemostcommonlyuseddetectors

forHPLCandarebasedonultravioletandvisiblespectrophotometers.Theunderlyingprinciple

oftheoperationofthisdetectoristheBeerLambertlawthatrelatesabsorbanceto

concentration.Becausethepathlengthandabsorptivityforaparticularcompoundinagiven

detectorareconstant,absorbanceonlydependsontheconcentration.Examplesoflampsused

inUVdetectioninclude,cadmium,mercury,andzincdischargelamps.Theadvantagesofthe

UVVisabsorbancedetectorincludeshighselectivity,highsensitivity(10101011g),andeasy

operation.ThedetectorisnearlyuniversalandhaslowbackgroundwithmanyHPLCsolvent,
35

allowinggradientelutionwithoutexcessivebackgrounddrift.However,inusingtheUVVis

detector,analytemusthaveabsorbanceintheultravioletorvisibleregion.Anotherlimitation

isthatitcannotoperateatwavelengthbelowtheUVcutoffofthesolvent.Also,atagiven

wavelength,responsevariesbetweenmoleculesbasedontheirabsorptivity(21).

OtherdetectorsthatarealsousedinHPLCarefluorescenceandamperometric

detectors.FluorescencedetectorscanprovideselectivityovertheUVabsorptiondetectorsand

theyhavegoodsensitivity.Amperometricdetectorsareusefulindetectingelectroactive

substancesandhavewidebiologicalapplication(35).

ResolutionofBands

Theextenttowhichtwobandsaredisengagedfromeachotherdeterminesthequality

ofachromatographicseparation.Thisisreferredtoasresolution.Itisdefinedquantitatively

as;

R s =2(t 2 t 1 )/(W 2 W 1 )[4]

wheret 1 andt 2 areretentiontimesofadjacentbands,W 1 andW 2 arethepeakswidth

measuredattheirrespectivebasesinthesametimeunitsastheretentiontime.Bandsthat

overlaphavesmallvaluesofR s, whereasthosethataretotallyseparatedfromeachotherhave

bigR s values.Resolutionisdependentonretention,selectivity,andefficiencyofseparation.

Whileretentionreferstotheabilityofamoleculetointeractwiththestationaryphaseorthe

affinityofthemoleculesforthematrix,selectivityisthediscriminatingpowerofthematrixthat

36

producesdifferentialinteractionforatleasttwocompounds.Efficiencyreferstotheeaseof

movementofthesolutethroughthecolumn.Thesefactorsarerelatedas

R s =N/4(1)(k 1 /1+k 1 )[5]

where(1)istheselectivitytermthatbecomes(1)/iftheplatesandcapacityfactorsare

associatedwithsecondpeak.Nisefficiencyandk 1 referstotheretentionterm(21).

MethodDevelopment

Thissectionfocusesonestablishingthebestanalyticalconditionsforsample

determination.Newmethodsmaybenecessarybecauseofpoorexistingonesortheneedto

analyzeanewanalyte.Mostoften,newmethodsarederivedfromexistingonesorfrom

similarmethodsusedintheliterature(21).Methoddevelopmentmaybebytrialanderror.

Thisapproachisinefficientandtimeconsuming.Inordertodevelopagoodmethod,the

chromatographersindepthknowledgeofthechemistryoftheanalyteanditsmatrixis

paramount.Modelsbasedonpropertiesofanalytehavebeendevelopedinordertonarrow

downontheoptimumconditionssuitableforaparticularanalytedetermination(25).This

minimizesthetroublechromatographersgothroughduringmethoddevelopment.Developing

anHPLCmethodmaybedonebyfirstvaryingthemobilephasecompositionwiththeaimof

obtainingadequateresolution,areasonableruntime,andeasilydetectednarrowbands.Other

stepsincludevaryingthemobilephasepH,additionofsurfactants,orchangingtheanalytical

column(33).Thesuccessofthenewmethodisevaluatedbyvalidatingitsparameterssuchas

robustness,linearity,accuracy,precision,andlimitsofdetection(21).

37

TheUseofSurfactantsinHPLC

Someproteinsandpeptidesareanalyzedeffectivelyinthepresenceofsurfactants.

Thesesurfactantsenhancetheirsolubilityandpreventaggregation(21).Surfactantsaremade

upofanonpolarendthatisusuallyahydrocarbonresponsiblefortheirhydrophobic

propertiesandapolarheadthataccountsfortheirhydrophilicbehavior.Surfactantsare

groupedintoanionic,cationic,amphoteric,andnonioniccompoundsconsideringthecharge

onthehydrophilicgroup(36).Surfactantshavetheabilitytoadsorbontoaqueoussolutionat

interfaces.Whentheyadsorbontohydrophobicsurface,theynormallyorienttheir

hydrophobicendtowardsthesurfaceandexposethehydrophilicgrouptowardsthewater

makingthesurfacehydrophilic.Thesurfacetensionbetweenthesurfacesisthusreduced(37).

SurfactantsareaddedtothemobilephaseduringHPLCdeterminationstoreduceviscosityand

surfacetension.Theyincreasetheeluentstrengthanddiffusioncoefficientcausingreduction

bandwidth(21).TangandDemingstudiedtheeffectofsurfactantsinreversedphase

chromatographyandreportedthattheadditionofsurfactanttoeluentcanreducethe

interfacialtensionandthusdecreasetheretentiontimeoftheinjectedsample(38).Although,

surfactantsinchromatographyareuseful,theyshouldbeusedwithcautionastheycanbind

stronglyonthecolumnwallsandconsequentlydamageit(21).Examplesofsurfactants

commonlyusedinhighperformanceliquidchromatographyareshowninFigure4aand4b.

38

 O
O
S
O OH(a) (b)

O
O
S
O O-Na+(c)

Figure4:ExampleofsurfactantscommonlyusedinHPLCdeterminations,(a)1Octanesulfuric
acid(OSA),(b)Cetyltrimethylammoniumbromide(CTAB),(c)Sodiumdodecylsulfate(SDS)

ResearchObjective

Fromtheliteratureanddiscussionabove,theuseofHPLCasapowerfulanalytical

techniquecannotbeoveremphasized.Itsroleintheanalysisofsulfoxideshasthusbeenwell

examinedandhasbeennotedtoprovideareliablequantitativeandqualitativemeasurement

(25).

ThisworkseekstodevelopasimplebutefficientHPLCmethodforalliindetermination.

Theobjectivesofthismethodareaslistedbelow:

1. Touseasimplebuteffectiveextractionproceduretoextractalliininfreshgarlicand

garlicproducts.

2.ToestablishanoptimumreversedphaseHPLCconditionsforalliindeterminationusing:

a)SuitableHPLCinstrumentation.

b)Amobilephasecompositionthatiseconomicalandenvironmentallyfriendly.

39

3.ToestablishthefiguresofmeritsoftheproposedRPHPLCmethodbyverifyingits

lineardynamicrange,reproducibility,andrecovery.

4.Toverifytheapplicabilityofthemethodinthedeterminationofalliininfreshgarlicand

commercialgarlicproducts.

40

 CHAPTER4

EXPERIMENTALPROCEDURERESULTSANDDISCUSSION

Reagents

1. OptimumgrademethanolandHydrochloricacidusedwereACSCertifiedandproducts

ofFisherScientific(FairLawn,NJ).

2. Surfactants;SodiumdodecylSulfate(SDS),Cetyltrimethylammoniumbromide,

(CTAB)and1OctaneSulforicacidwereobtainedfromSigmaChemicalCompany

(St.Lousi,MO).

3. AlliinstandardusedwasaproductofSigmaAldrich(Steinheim,Germany).

PlantMaterials

FreshgarlicandgarlicproductswerepurchasedfromdifferentstoresinJohnsonCity

TennesseeandToledoOhio.Onefreshgarlicsamplewasobtainedfromabackyardgarden.

Varietiesoffreshgarlicandgarlicproductsusedareshownbelow:

WhiteGarlicfromOrientalMarketinJohnsonCity

GarlicTablets1fromWalMartinJohnsonCity

ElephantgarlicfromToledo,Ohio

OrganicgarlicfromToledo,Ohio

PurewhitegarlicfromToledo,Ohio

41

GarlicTablets2formKroger,JohnsonCity

Hybridgarlicfromabackyardgarden

CaliforniagarlicfromFoodLion,JohnsonCity

ExperimentalProcedures

PreparationofReagents,Standard,AndStockSolutions

Theextractionsolventusedwas90:10methanol:hydrochloricacidmixture.Thiswas

preparedandusedasextractionsolventtoinhibitenzymaticactivityonalliin.Inpreparingthe

extractionmixture,0.5mLofconcentratedhydrochloricacidwasaddedto450mLmethanolin

a500mLvolumetricflackanddilutedtothemark.

Thealliinstandardsolutionwaspreparedbydissolving10mgofpurealliinin100mLof

themethanolacidmixtureandsavedinabottle.AstockSolutionofgarlicwaspreparedby

homogenizingabout10gofgarlicclovesin25mLoftheextractionsolventusingalaboratory

blender.Thiswascarriedoutfor5minutesandthehomogenatewasquantitativelytransferred

intoa250mLbeakerusing40mLofthemethanolacidmixture.Itwasthensonicatedfor

another5minutetomixwell.Thehomogenatewasfilteredbygravityintoa100mLvolumetric

flaskandtheresiduewashedthreetimes,eachwith10mLofthesolvent.Thefiltratewas

madeuptothe100mLmarktoobtainthestocksolutionofeachsample.

Thesamestepswerefollowedusingboiledwaterasextractionsolventandthestock

solutionspreparedaswellasthatofthemethanolacidextractsweresavedinlabeledbottles

andkeptinalaboratoryrefrigeratoruntilanalysis.
42

PreliminaryGasChromatographyMassSpectrometry(GCMS)Studies

Preliminarydeterminationsofalliininthegarlicextractspreparedwereperformed

usingHewlettPackardModel5890SeriesII GaschromatographequippedwithSeries5971

massselectivedetector.TheseweredoneusingHP5MScolumnofdimensions30mx0.25mm

and0.25mfilmthicknesswithpolarstationaryphaseconsistingof5%phenyland95%

dimethylpolysiloxane.Theinstrumentwasoperatedat1700Cinjectiontemperature,1800C

interfacestemperatureandionsourcetemperatureof2000C.Thetemperatureprogramonthe

instrumentwasvariedinanattempttogettheoptimumconditionsforthealliindetermination.

Figures5and6presentssamplesofchromatogramsandmassspectraobtainedinthe

preliminarystudies.

Figure5.GCMSchromatogramofmethanolextractofgarlic.

43

Figure6.GCMSChromatogramofmethanolextractofgarlic.Temperatureprogram;linear
increasefrom1700C3000Cat30C/min;then300oC3500Cat10oC/minandkeptconstantfor
10minutes.

TheGCMSprocedureusedinthepreliminarystudiesprovedtobefutileasthe

chromatogramsobtaineddidnotgivegoodrepresentationofsulfoxidesintheextract.The

procedureyieldedinconsistentresultsandpoorresolutionofbands.Thismaybeduetothe

44

assertionthatsulfoxidesandthiosufinatesarethermallyunstableandmaydecomposeinto

severalsulfurcompoundsathightemperature(19,25,36).ThustheGCMSappearednottobe

atechniqueofchoiceforalliindeterminationsinthegarlicextractsprepared.

HPLCInstrumentation

ShimadzuLCIOASsystemwithSPD1OAUVVISdetector,aproductofShimadzu

ScientificInstrumentIncorporated,7102RiverwooddriveColumbia,MD21046wasused.Ithas

ascanrangeof190300nuandSCL1OAVPsystemcontroller.ReversedphaseHPLC

conditionsincludingC 18 5mDisccolumnofsize3.9X150mwithcolumntemperaturesetat

25oC.TheUVVISdetectorwassetat210nmafterseveraltrials.AllHPLCseparationswere

donewithisocraticelutionataflowrateof1.0mL/minandanaveragepressureof4001600

psi.

DataAnalysis

TheexperimentaldataobtainedwereanalyzedmainlywithMicrosoftOfficeExcel2007

software.Thesoftwarewasusedtocalculateaveragesoftheinstrumentalresponsefor

triplicatesrunsaswellasrelativestandarddeviations.Itwasusedinmakingcalibrationplotsof

alliinstandardsolutionsshowingerrorbarwithin5%value.Thesoftwarewasalsousedto

calculatethealliinconcentrationsinsampleswiththeaidofthecalibrationcurve.

Statisticalstudiesofdataweredonebyanalysisofvariance(onewayANOVA)using

MinitabandSPSSsoftwares.

45

 ResultsandDiscussion

Followingtheresultsobtainedinthepreliminarystudiesandtherecommendations

publishedinliteraturethatHPLCpresentsasuitableapproachindeterminingsulfoxideingarlic

extracts(26,29),anHPLCmethodwasdevelopedandusedinalliindetermination.Thissection

discussestheresultsobtainedstartingfromthemethoddevelopmentthroughtothe

applicationofthemethodinalliindetermination.

MethodDevelopmentandValidation

DevelopingthebestanalyticalconditionsforareversedphaseHPLCdeterminationwas

anintegralportionoftheentirework.Indoingso,muchemphasiswasonsimplicityofmethod

withoutcompromiseonitsefficiency.Inaddition,theuseofreagentsthatareenvironmentally

friendlyandeconomicalwasvitalatthisstage.Preliminaryexperimentsweredonetodevelop

amethodforalliindetermination.Theseincludewavelengthoptimizationandmobilephase

optimization.Aftertheseexperiments,figuresofmeritssuchasreproducibility,linearity,and

recoverystudiesweredonetoevaluatetheproposedmethod.

OptimizationofWavelength

Ultravioletvisible(UVVis)detectionofalliinhasbeenreportedatthewavelength

rangeof205nm280nm.(17,29,29).Detectionatthesewavelengthswasexploredusing

alliinstandardsolutionsandtheoptimumwavelengththatgaveahighabsorptionpeakwas

210nm.Thiswasusedinthemethoddevelopmentandsubsequentdeterminations.

46

OptimizationofMobilePhase

AsuitablesolventforHPLCanalysismustbereadilyavailableinapureformandhave

lowviscosity.Itmustalsobecompatiblewithdetectionsystemandhavelowflammabilityand

toxicity(25).Inthisstudy,themobilephasecompositionforthedeterminationofalliiningarlic

sampleswasexploredwiththeaimoffindingtheoptimummobilephasethatwillgivethebest

separation.Itisknownthatachangeinpercentorganiccompositionoftenleadstosignificant

changesinseparationfactor,,forreversedphaseHPLCandthisprovidestheeasiestwayof

optimizingbandspacing(33).Basedonthisfact,mobilephasewithdifferentsolventstrengths

beginningfrom80:20,70:30,60:40,55:45,30:70,to20:80methanol:watercompositionswere

used.MethanolwasusedbecauseithaslowviscosityandUVtransparency.Itisalsototally

misciblewithwaterwhichisreadilyavailable(33).Eachmobilephasepreparedwasdegassed

bypassingheliumgasthroughitfor15minutesandthenpurgedbeforeitwasusedinthe

optimizationstudies.Alliinstandardsolutionsandaliquotsofgarlicextractswererunwith

thesemobilephasepreparationsonthereversedphaseHPLCinstrumentalconditions

mentionedpreviouslyusinganinjectionvolumeof20Landtheirchromatogramswere

compared.

Figure7showsthechromatogramsofgarlicextractobtainedbyrunningsamplesata

flowrateof1mL/minat210nmwavelengthusing80:20,70:30,and60:40methanol:water

mobilephasecompositions.

47

 (a) (b)

Time(min.)time(min.)

(c)

Time(min.)

Figure7.HPLCchromatogramsofmethanolacidextractofgarlicusing(a)80%methanol:20%
water,(b)70%methanol:30%watermobilephasecompositions.(c)60:40methanol:water
mobilephasecomposition.Sampleswererunat1mL/minat210nmwavelength

48

 Usingthesemobilephases,allthechromatogramsobtainedhadverygoodruntimesof

about5minuteswiththealliinpeakelutingbetween2.53.0minutes.However,themajor

problemwiththesemobilephaseswaspoorresolutionofbands.Inthechromatogram(a)of

Figure7inwhichelutionwasdonewiththehighestmethanolconcentration,onlytwo

noticeablebandswereobserved.Astheconcentrationofthemethanolinthemobilephase

wasdecreasedotherpeaksshowedupinthechromatogramsbutthesepeakswerewideand

overlappedwitheachother.Alsoveryprominentwastheoccurrenceofnegativepeaks

appearingasanextensionofthesuspectedalliinpeakin(a)and(b).Varyingthesolvent

compositionfrommoremethanoltolessmethanol,representingadecreaseinsolvent

strength,improvedthebandresolutionandeliminatedthenegativepeakextension.These

comparisonsshowedthatdecreasingthesolventstrengthcouldimprovebaselineseparation

andgivebetterchromatograms.Thisobservationisinlinewithknownfactthatmobilephase

morethananyothervariablehasamajoreffectonbandresolution(33).

OptimizationofmobilephasepH

ThepHofvariousmobilephaseswasadjustedusingglacialaceticacidbuffer.Starting

fromamobilephasecompositionof80:20throughto20:80methanol:waterratios,theacid

bufferwasaddedtostudytheeffectofpHontheseparation.Itwasobservedthatadditionof

thebuffershowednosignificantimprovementintheresolutionofthepeaks.Instead,atlow

pH(lessthan3),asharpnegativepeakwasnoticedadjacenttothealliinpeakandthis

interferedwiththealliinpeakarea.

49

Optimizationofmobilephasewithsurfactants

Surfactantsareknowntoreducebandwidthsbecausetheyincreasebotheluent

strengthanddiffusioncoefficients(21).Sodiumdodecylsulfateisananionicsurfactant.It

aggregatesindiluteaqueoussolutiontoformmicellesatacharacteristicconcentrationcalled

criticalmicelleconcentrationof8.6at40oCtemperature.Inpresenceofbufferthecritical

micelleconcentrationreducesto4.6at30oC(39).Cetyltrimethylammoniumbromide(CTAB)is

acationicsurfactantandappearsaswhitecrystalpowder.Thecriticalmicelleconcentrationof

CTABincreaseswithincreasedconcentrationofmethanolunderroomtemperature(32,40,

41).

Sodiumdodecylsulfate(SDS),Cetyltrimethylammoniumbromide(CTAB),and1Octane

sulfonicacid(OSA)wereaddedtothemobilephasetoreducesolventviscosityandsurface

tension.Thebestelutionofgarliccomponentswasobservedwhensampleswererunusing

30%methanol:70%water,and0.05%sodiumdodecylsulfate.Thisgavethebestresolutionof

bandscomparedwithalltheothertrials.Theruntimewasveryshortandtherewereno

negativepeaks.Standardsolutionsofalliinwerealsorunandthealliinpeakwasdeducedto

eluteataretentiontimeof23min.Figure8showsthechromatogramsofgarlicsamplerun

usingthevariousmethanol:water:surfactantmobilephasemixture.

Theadditionofcetyltrimethylammoniumbromideand1Octanesulfonicacidgave

broadpeaksandpoorbaselineseparation.Thus,theoptimummobilephasecompositionfor

alliindeterminationwaschosentobe30%methanol:70%water,and0.05%sodiumdodecyl

sulfate.ThepHofthemobilephasewasdeterminedtobe3.5.

50

 (a) (b) (c)

Figure8.HPLCchromatogramsofmethanolacidextractsofgarlicusing(a)30%
methanol:70%waterand0.05%SDS;(b)30%methanol:70%waterand0.05%CTAB;(c)
30%methanol:70%waterand0.05%OSAphasecompositions.Sampleswererunat1
mL/minat210nmwavelength

ReproducibityStudies

Repetitionofamethodofmeasurementseveraltimesrevealshowreproduciblethe

methodis.Thisfigureofmeritisprecision.Theprecisionofananalyticalmethodisvitalin

measurement,especiallyinroutinework.Inthisstudy,theprecisionforthequantitative

methodwasdemonstratedwithtwosetsofsolutions,oneathighandanotheratlow

concentrationsofanalyte.Forthesetsofsolutionsofhighanalyteconcentration,eightaliquots

ofworkingsolutionswerepreparedbypipeting600Lofstockgarlicsolutionintoeight5mL

volumetricflasksanddilutedwithdistilledwatertothemark.Eachaliquotofthissolutionwas

runonthereversedphaseHPLCintriplicateandthealliinretentiontimesaswellasthepeak

51

areaswererecorded.Theprocedurewasrepeatedusing10Lofthestockgarlicsolution

insteadof600Landthepeakareaswerecompared.Theaveragesandrelativestandard

deviationforeachtriplicaterunwerecalculatedforalleightsamples.Theseeightaverages

werethenaveragedagainandtherelativetherelativestandarddeviationcalculated.These

resultsforboththehighandthelowanalyteconcentrationsaretabulatedinTable1.

Table1:Asummaryofresultsobtainedfromreproducibilitystudiesofthemethodforthe
measurementofalliincontentingarlicsamples.Trial1istheresultforthehigheranalyte
concentrationwhileTrial2isforloweranalyteconcentration.

Overall Trial1 Trial2

Mean 1.73E+07 2.32E+06

RelativestandardDeviation 4.11% 0.56%

Theresultsshowedthatthemethodgaveexcellentprecision.Therelativestandarddeviations

were4.11%and0.56%,forthesamplewiththehigherandloweranalyteconcentrations

respectively.Itwasquiteunexpectedthatthesetofsampleswithlowerconcentrationgave

betterprecision.Overall,theresultsindicatedtheproposedprocedurewasfeasibleforthe

determinationofalliininasample,evenatverylowconcentrationofanalyte.

LinearityStudies

Linearitymeasureshowlineartheresponseofanalyteisinrelationtoitsconcentration

(25).Thelineardynamicrangeofthemethodwasstudiedusingdifferentconcentrationsof

alliinstandardsolution.Intodifferent5mLvolumetricflasks,10L,500L,1000L,and2000

52

Lofalliinstandardsolutionsweredeliveredandtheflasksfilleduptothemarkwithdistilled

water.ThreealiquotsofeachsolutionwerepreparedandrunonthereversedphaseHPLCin

triplicateandtheirpeakareaswererecorded.Linearrelationshipbetweenconcentrationsand

correspondingpeakareaswasobtainedasshowninTable2.

Table2:Resultsoflinearitystudiesshowinglinearrelationshipbetweenconcentrationand
instrumentalresponse.Note:Threealiquotofeachconcentrationwerepreparedandrunin
triplicates.Theaveragepeakareasareshown.

Volumeofstandardin Concentration
Standard(std.)ID averagepeakarea
5mLsolution(L) (ng/mL)

1 10 0.4 1.69E+04

2 500 20.0 8.85E+05

3 1000 40.0 1.68E+06

4 2000 80.0 3.40E+06

Fromthedatatabulated,linearityofthemeasurementswasdemonstratedgraphicallyina

calibrationplotaspresentedinFigure9.Regressionequationandcorrelationcoefficientwere

calculatedfromthecalibrationcurveusingMicrosoftOfficeExcel2007.

Acommonmeasureoflinearityisthecorrelationcoefficient,R2fortheequationofthe

line.ForacalibrationcurvetobeconsideredlinearR2mustbeclosetooneandtheinterceptof

thecalibrationcurveshouldbeclosetozero(25).Basedonthesecriteriathemethodyielded

linearresponsewithvaryingconcentrations,showingagoodcorrelationcoefficient

53

of0.9998andequationoftheliney=42272x+10833.

4.0E+06
y=42272x+10833
3.5E+06 R=0.9998

3.0E+06

2.5E+06
peakarea

2.0E+06

1.5E+06

1.0E+06

5.0E+05

0.0E+00
0 20 40 60 80 100

concentration (nM)
Figure9.AplotofcalibrationcurveofAlliinstandardsolutionshowinglinearityofmethod.
Theplotdisplayserrorbarwith5%value

ThecalibrationcurvepresentedinFigure9alsoshowserrorbarsthatgiveageneral

indicationofhowaccuratemeasurementsare.Thesehavebeendisplayedwithin5%value

usingtheexcelsoftware.Byinspection,theerrorbarsshowthatthemeasurementsmadein

thelinearitystudiesweresatisfactoryandarewithinexperimentalerror.

RecoveryStudies

Intherecoverystudies,theaccuracyofthedevelopedmethodwasaccessedand

verified.Accuracydescribestheclosenessofthevalueorresultdeterminedtotheexpected

value.Itismeasuredasthepercentageofanalyterecoveredusingspikedsamples(21).

54

 Twodifferentsampleswereusedintherecoveryexperiment,thewhitegarlicandgarlic

tablet.Forthewhitegarlicsample,ninealiquotsoftheworkingsolutionswerepreparedby

pipeting10Lofstocksolutioninto5mLvolumetricflasksanddilutedtothemarkwith

distilledwater.Tothreeofthesealiquots,nostandardsolutionwasadded.Toanotherthree,

50Leachofstandardsolutionwasadded.Tothelastsetofthree,100Leachofstandard

solutionwasadded.Thesameprocedurewasrepeatedwiththegarlictablet.However,this

time0L,20Land40Lstandardsolutionswererespectivelyaddedtotheworkingsolution.

Astandardcalibrationcurvewasalsoobtainedbyrunningdifferentconcentrationsofalliin

standardsolutions.Table3showsdataobtainedfortriplicaterunsofalliinstandardsolutions

andtheresultingcalibrationcurveispresentedinFigure10.

Table3.Standardcalibrationdatausedintherecoverystudies.Note:Threealiquotofeach
concentrationwerepreparedandrunintriplicates.

StandardsolutionID VolumeofStd.in concentration

alliinpeakarea
(std.) 5mLsolution(L) (ng/mL)
Std1run1 500 20.0 1671880
Std1run2 500 20.0 1684239
Std1run3 500 20.0 1672978
Std2run1 1000 40.0 3449196
Std2run2 1000 40.0 3489779
Std2run3 1000 40.0 3469160

Std3run1 2000 80.0 5280206

Std3run2 2000 80.0 6233435
Std3run3 2000 80.0 6241045

55

8000000

y=75851x+269734
6000000 R=0.996
averagealliinpeak

4000000
area

2000000

0
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00

Alliinconcentration(ng/mL)
Figure10.Standardcalibrationcurveusedinrecoverystudiesdisplayingerrorbar5%value.

Usingthecalibrationcurvethedataobtainedfortherecoverystudiesforbothtypesof

garlicsampleswereanalyzed.Theamountofalliineachaliquotofthegarlicsampletowhich

noalliinstandardsolutionhadbeenaddedwerecalculated.Similarly,thetotalamountofalliin

allthespikedsampleswerecalculated.Fromtheseresultstheamountofalliinaddedtothe

originalaliquotsofthegarlicsampleswerecalculated.Thecalculatedamountofalliinadded

overtheactualamountaddedmultiplyby100%givethepercentofrecovery.Theresultof

thesecalculatedrecoveriesaretabulatedinTable4forthewhitegarlicsampleandTable5for

thegarlictabletsample.

56

Table4.Resultsforrecoverystudiescarriedoutwithhighconcentrationofstandardsolution.
Thesampleusedinthesestudieswaswhitegarlicandexperimentsweredoneintriplicate.

Amountadded Amount % Relativestandard

SampleId
(ng/mL) recovered(ng/mL) recovery deviation(%)

Sample+50L
1.98 2.01 102% 1.23
standardsolution

Sample+100L
3.92 3.94 101% 0.36
Standardsolution

Table5.Resultsforrecoverystudiescarriedoutwithlowconcentrationofstandardsolution.
Thesampleusedinthesestudieswasgarlictablet1andexperimentsweredoneintriplicate.

Amountadded Amountrecovered % Relativestandard

SampleId
(ng/mL) (ng/mL) recovery deviation(%)

Sample+20L
0.80 0.73 91.3% 5.76
standardsolution

Sample+40L
1.59 1.52 95.6% 2.99
standardsolution

AsshownbytheresultsobtainedintherecoverystudiestabulatedinTable4and5,the

proposedprocedureyieldedsatisfactoryrecoveries.Thatis,theaccuracyoftheproposed

methodwasgood.

Therecoveryforwhitegarlicsampleswasverygoodwithverysmallexperimentalerror

of0.36%1.23%.Theresultsfromtheexperimentsperformedonthegarlictabletwerenotas

goodbutevenstillacceptableandwithintheexperimentalerrors.Thereasonforthe

57

differencemaybeinthesmalleramountinthealliinstandardaddedtothealiquotsofthe

garlictabletsample.Thus,theerrorwaslargerandrecoverynotasgoodastheoneperformed

onthewhitegarlicsample.

Inall,therecoverystudiespresentedaninterestingresultinthatwhenhigher

concentrationsofthestandardsolutionswereaddedtobothsamples,theamountrecovered

improved.Thiswas,infact,evidentwiththesamesampleandbetweenthetwosamples.Thus

theaccuracyofthemethodwasfoundtobebetteratcomparativelyhighconcentrationsofthe

analyte.

ComparingMethanolHydrochloricacid(MeOHHCl)ExtractWithHotWaterExtract

Fromthechemistryofgarlic,welearnedthatalliinisconvertedtoallicinbytheenzyme

alliinasewhenthegarliccelliscrushed.Allicinthendecomposesrapidlyintoothersulfur

compounds(3,8).Thismeansthatinordertoidentifyandquantifyalliiningarlicsamples,

thereistheneedtoirreversiblyinhibittheenzymeactivityonalliin.Inhibitionofalliinase

activityingarlicextracthasbeendonebyeitherextractingsampleswithmethanolhydrochloric

acidmixtureortheuseofhotwaterasextractingsolvent(30).Inthemethanolhydrochloric

extraction,thehydrochloricacidprovidesanacidicmediumunfavorableenoughtohinderthe

activityoftheenzyme.Anumberofstudieshaverevealedexamplesinwhichsulfoxideshad

beenextractedwithmethanolacidicsolution.Theacidprovidesanacidicmediuminorderto

irreversiblyinhibitalliinaseactivityatapHvaluebelow3.6,whereashighmethanol

concentrationintheextractingsolventprotectstheanalyticalcolumnduetodecreasein

solubilityofcarbohydratesandproteinsingarlic(20,30).

58

 Ichikawaandhiscolleagues(30)exploredandevaluatedtheeffectofhydrochloricacid

andmethanolconcentrationsonextractionefficiencyoforganosulfurcompoundsandreported

thatincreasingmethanolconcentrationresultedinadecreaseinextractionefficiencyof

glutamylpeptidesinthepresenceof0.001MHCl.Theyattributedthistosolidificationof

preparedsamplesthatdecreasesthesolubilityofpolarcompounds.Theyalsorealizedthatat

highHClconcentrations(i.e.0.01and0.1MHCl)samplesextractedwith80%or90%methanol

showedhighercontentsofglutamylpeptidesthanthosewith95%methanol.Theyexplained

thatthismightbeduetoenhancementofsolubilityinallconcentrationsofmethanolby

suppressionofionizationofglutamylpeptidesathighHClconcentrations.Accordingto

them,thesampleextractedwith90%methanoland0.01MHClresultedinthehighestcontents

ofsulfoxidescomparedwiththatofotherextractionsolventcompositions.

Hotwaterextractionofalliiningarlicseekstoelevatethetemperatureofthegarlic

extractabovetheoptimumtemperatureof35oC37oCtoirreversiblyinhibittheenzymes

activity.Thedenaturingofenzymebeginsat42oCandtemperaturesabove60oCinactivateit

(43).Inlightoftheresearchobjectiveofgoinggreenandcuttingdownoncostwithout

compromisingtheefficiencyofthemethodofalliindetermination,thetwoextraction

procedureswerecompared.Todothis,10LofMeOHHClextractsofwhitegarlicsample

werepipettedintoa5mLvolumetricflaskanddilutedtothemark.Thiswasrunonreversed

phaseHPLCusing30:70MeOH:H 2 Oand0.05%SDSmobilephase.Waterwasheatedto

boilingandwasusedtoextractalliiningarlicusingthesameproceduredescribedformethanol

59

acidextraction.Aliquotsofhotwaterextractofthesamesamplealsowerepreparedandrun

thesamewayandtheirchromatogramswerecompared.

Methanolhydrochloricacidextractyieldedsatisfactoryresultsfromthemethod

developed.However,thehotwaterextractdidnot.Thisisevidentinthechromatograms

presentedinFigure11.

Figure11:Chromatogramofgarlicsampleextractedwith(a)90%Methanol0.01M
Hydrochloricacidcomparedwith(b)chromatogramofhotwaterextract.

AscanbeseenfromthechromatograminFigure11b,thehotwaterextractgavepoor

peaksseparationandresolution.Thus,hotwaterextract,thoughcheaperandsimple,isnot

thebestextractionprocedurefortheproposedmethod.Hence,alltheextractionsforalliin

determinationinfreshgarlicandgarlicproductsweredoneusingmethanolhydrochloricacid

mixture

60

ApplicationofMethod

Themethodforalliindeterminationinvolvestheuseofextractionsolventmadeupof

90%methanoland0.01MHClaswellasamobilephasecompositionof30%methanol:70%

water:0.05%sodiumdodecylsulfate.Separationandquantificationofalliinweredoneon5m

C 18 columninShimadzuLCsystemconnectedtoaUVVisdetectorwithoptimumwavelength

selectedtobe210nmandaflowrateof1mL/min.

Eightfreshgarlicsamplesandgarlicproductswereanalyzed.Eachgarlicsamplewas

extractedandpreparedandtriplicatealiquotswereinjectedintotheHPLC.Theresulting

chromatographicdatawereusedwiththehelpofthecalibrationcurvetocalculatetheamount

ofalliinin1gofeachgarlicsample.Figure12showschromatogramsofwhitegarlicand

elephantgarlicclovesaswellasthatofalliinstandard.

(a) (b) (c)

Figure12:Chromatogramsof(a)alliinstandardin(b)whitegarlicand(c)elephantgarlic
obtainedusing30%Methanol:70%waterand0.05%Sodumdodecylsulfate.Alliineluteat2.4
2.6minutes.

61

 Theretentiontimeofthealliinpeakwasrecordedbetween2.42.6minutes.Fromthe

chromatographicdatatheconcentrationsofalliininthevariousfreshgarlicandgarlicproducts

werecalculatedwiththehelpofacalibrationcurve.Theseresultshavebeentabulatedandare

presentedinTable6.

Table6:Concentrationofalliinindiversevarietiesoffreshgarlicandgarlicproduct.

Concentration mgofalliin/gof
Varietiesofgarlic Massofgarlic
(ng/mL) garlicclove

Purewhitegarlic 10.4030 43.2 0.207

Whitegarlic 10.2237 57.3 0.281

Elephant 10.1753 7.5 0.037

Organic 10.3695 5.2 0.025

California 10.9683 0.6 0.003

Hybrid 10.6473 30.1 0.141

Tablet1 10.5531 76.2 0.361

Tablet2 10.1147 9.9 0.049

Thedataweresubjectedtostatisticalanalysisusinganalysisofvariance(oneway

ANOVA)andtheresultsshowedsignificantdifferencesamongalliincontentinthevarietiesof

garlicat95%confidentlimit.Tablet1recordedthehighestalliincontentindicatinga

concentrationof0.361mg/gofthegarlicsample.HoweverTablet2recordedaloweralliin

concentrationof0.049mg/gofthegarlicsample.Thiswassobecausethetwoaredifferent

sampleswithdifferentbrandnamesandweremanufacturedunderdifferentconditionsand

62

specifications.Whitegarlicrecordedrelativelyhigheramountofalliinthanthepurewhite

garlic.Thesearehoweverthesamegarlicvarietybutpurchasedfromdifferentstoresin

differentcities(JohnsonCity,TNandToledo,OHrespectively)atdifferenttimes.The

expressionpurewasusedtodistinguishbetweenthem.Thepossiblereasonforthis

differencemightbetheeffectofenvironmentalfactorsonthetwosamples.Thesesamples,

thoughthesamevariety,weregrownunderdifferentenvironmentalconditions(temperature,

humidity,soilcomposition)aswellasdifferentfarmingpractices(useofsulfurcontaining

fertilizer),andwerealsokeptunderdifferentstorageconditionsbeforepurchase.The

differenceintheperiodoftimethatthesesampleshavebeenontheshelfmightalsohave

affectedthealliincontent.Thehybridtypeofgarlicthatwaslocallygrowninabackyard

gardenyielded0.141mgofalliinperagramofthegarlicclove,anamountgreaterthanthatof

Elephantgarlicthatgaveanalliincontentof0.037mg/goffreshgarlicclove.Alliincontentof

thehybridwasalsomorethanthatoftheorganicvarietyandgarlictablet2.Itwasevenso

muchmorethanthatofCaliforniagarlicthatrecordedtheleastconcentrationofalliin(0.003

mg/goffreshgarlic).Thisobservationencouragessmallscalecultivationofgarlicwhere

expensiveandsophisticatedstoragefacilitiesarenotrequiredtopreservethevegetablein

ordertoretainessentialchemicalconstituentssuchasalliin.Alltogether,thedifferences

betweenthealliinlevelscanbeattributedtogeneticandenvironmentalfactors.Agraphical

distributionofalliinlevelsinthevarioussamplesispresentedinFigure13.

63

 0.400
gramsofalliin(mg)

0.350

0.300

0.250

0.200

0.150

0.100

0.050

0.000

Figure13.Histogramshowingthedistributionofalliinindiversevarietiesofgarlicandgarlic
products

Figure12givesapictorialcomparisonofalliincontentamongvarietiesofsamples.For

example,thealliincontentinthehybridvarietyrepresentlessthanonehalfoftheamountin

tablet1,withtablet2formingaboutaquarterofthecontentinpurewhitegarlic.Alliin

concentrationintheorganicvarietycomesnextafterthatoftheCaliforniacultivarthathasthe

leastamountoftheanalyte.

Inallthemethodgaveagoodquantificationoftheanalyteinthevariousvarietiesand

canbeusefulinroutineroutinework.

64

 CHAPTER5

CONCLUSION

DevelopinganHPLCmethodthatiseconomical,efficient,andgreentobeusedinalliin

determinationisusefulduetotheenormousmedicinalvalueofthecompound.Inthisstudy,a

simplereversedphasehighperformanceliquidchromatographicmethodwasdeveloped.The

proposedmethodworksbestwithmethanolhydrochloricacidextract.Thisapproachof

extractingalliiningarlicclovesissimpleandcanbeaccomplishedinonestep.Itinvolves

homogenizinggarlicsamplewiththeextractionmixtureandfilteringittoobtaingarlicextract

containingalliinratherthanallicinthatisunstable.Theuseofhydrochloricacidinthe

extractionsolventprovidesanacidmediumtoinhibitalliinaseactivity,whereasthemethanol

protectstheanalyticalcolumn(30).Hotwaterextractofgarlicdoesnotworkwellwiththe

methoddeveloped.Thisyieldedbroadpeakswithpoorresolution.

TheHPLCmethoddevelopedutilizesthemobilephasecompositionof30%

methanol:70%waterwith0.05%sodiumdodecylsulfate.Themobilephaseiseconomicaland

moregreenbecausewaterisreadilyavailableandtheuseoflargevolumesofitreducesthe

toxicityofmethanoldrastically.ItisefficientbecausemethanolhaslowviscosityandUV

transparencyandistotallymiscibleinwater(33).Theadditionofthesurfactantsincreased

eluentstrengthanddiffusioncoefficientandconsequentlyreducedbandwith(21).However

additionofpHbuffershowednosignificanteffectorimprovementontheseparation.

65

 Thealliinpeakwasnotedataretentiontimeof2.03.0minutesandwasidentifiedin

comparisonwithalliinstandard.Reproducibilityofmethodwasgoodrangingbetweenrelative

standarddeviationof0.56%4.11%.Themethodexhibitedgoodlinearitywithcorrelation

coefficientof0.9997.Accuracyofmethodwassatisfactoryshowingaveragerecoveryof93%

101%.Theuseofthemethodinalliindeterminationinfreshgarlicandcommercialgarlic

productswassuccessful.Garlictablet1recordedthehighestlevelofalliingivinga

concentrationof7.19ng/mLin1gofthetablet.Californiagarlichad0.003mgofalliinper1g

ofthegarlicclove.Thedifferencesinalliinlevelsfoundcouldbeattributedtogeneticand

environmentalfactorssuchastemperature.Differentagriculturalpracticessuchastheuseof

sulfurcontainingfertilizersaswellastheperiodofstorageoffreshgarlichavealsobeenproven

toaffecttheiralliinconcentrations.Significantvariationswerefoundbetweenthealliin

contentofthecommerciallyproducedgarlicproductsbecausetheywerepreparedunder

differentmanufacturingconditionsandwithdifferentchemicalspecifications.Themethod

developedischeap,accurate,precise,efficient,andthuscanbeusedinroutinedetermination

ofalliininfreshgarlicandcommercialgarlicproducts.

Forfurtherwork,itwouldbehelpfultocomparethismethodwithcurrentlyaccepted

onesbydeterminationofalliinlevelsingarlicsamplesthatarecultivatedandstoredunderthe

sameconditions.Also,theeffectsoftheperiodofstorageofgarlicontheiralliincontent

shouldbestudied.

66

 REFERENCES

1. Nybe,V.E.;MiniRaj,N.;Peter,V.K.HorticulturalScience(5)Spices.NewIndia

PublishingAgency,2007,251252.

2. http://www.herbsociety.org/garlic/gdesc.php(accessedFebruary28,2009)

3. Rose,P.;WhitemanM.;MooreK.P.;ZhuZ.Y.BioactiveSalk(en)ylcysteinesulfoxide
metabolitesinthegenusAllium:thechemistryofpotentialtherapeuticagents.Journal
ofRoyalSocietyofChemistry,2005,(22),351368.

4. Kamenetsky,R.;Rabinowitch,D.H.TheGenusAllium:ADevelopmentaland
HorticulturalAnalysis.HorticulturalReview,2006,(32),329337.

5. Agarwal,C.K.TherapeuticActionsofGarlicConstituents.MedicinalResearchReviews,
1996.(16),111124.

6. Horie,H.;Yamashita,K.;Ichiro.Nonderivatizedanalysisofmethiinandalliinin
vegetablesbycapillaryeletrophoresis.J.ofChromatographyA,2006,(1132),337339.

7. Hughes,J.;Trgova,A.;Tomsett,A.B.;Jones,M.G.;Cosstick,R.;Collin,H.A.Synthesisof
theflavorprecursor,alliin,ingarlictissuecultures.ScienceDirect,2004,337339.

8. Shimon,J.W.L.;Rabinkov,A.;Shin,I.;Miron,T.;Mirelman,D.;Wilchek,M.;Frolow,F.
TwoStructuresofAlliinasefromAlliiumsativumL.:ApoFormandTernaryComplex
withAminoacrylateReactionIntermediateCovalentlyBoundtothePLPCofactor.J.of
MolecularBio,2007,366,611625.

9. Miron,T.;Rabinkov,A.;Mirelman,D.;Weiner,L.;Wilchek,M.Aspectrophotometric
AssayforAllicinandAllinase(Alliinlyase)Activity:Reactionof2Nitro5thiobenzoate
withThiosulfinates.Anal.Biochem,1998,317325.

10. MartinoDe.A.;FilomeniG.;AquilanoK.;CirioloR.M.;RotilioG.Effectsofwatergarlic
extractsoncellcycleandviabilityofHepG2hepatomacells.J.ofNutritionalBiochem,
2005,(17),742749.

11. Miron,T.;Bercovici,T.;Rabinkov,A.;Wilchek,M.;Mirelman,D.(3H)Allicin:preparation
andapplications.Anal.Biochem,2004,(331),364369.

67

 12. Miron,T.;Shin,I.;Feigenbiat,G.;Weiner,L.;Mirelman,D.;Wilchek,M.;Rabinkov,A.A
spectrphotometricassayforallicin,alliin,andalliinase(alliinlyase)withachromogenic
thiol:reactionof4mercaptopyridinewiththiosulfinates.Anal.Biochem,2002,7683.

13. LiYu;Xu;ShiYing,Sun;DaWen.Preparationofgarlicpowderwithhighallicincontent
byusingcombinedmicrowavevacuumandvacuumdryingaswellas
microencapsulation.J.offoodEng,2007,7683.

14. Boon,H.;Smith,M.ThecompleteNaturalmedicineGuidetothe50mostcommon
medicinalherbs,2004,119131.

15. Ross,A.I.MedicinalplantsoftheWorldChemicalConstituents,TraditionalandModern
Medicinaluses.Humanapress,Totowa,NewJersey,1999,2663.

16. Bergs,R.;Siess,M.H.;Arnault;JA.;Kahane,R.;Pinnert,M.F.;Vernevanut,M.F.,BonA
M.Comparisonofthechemopreventiveefficaciesofgarlicpowderswithdifferences
contentsagainstaflatoxinB1carcinogenicityinrats.Carcinogenesis,2004,25(10),1953
1959.

17. Li,Y.;Xu.Y.S.;SunD.W.Preparationofpowderwithhighallicincontentbyusing
combinedmicrowavevacuumandvacuumdryingaswellasmicroencapsulation.J.of
FoodEng.,2007,7683.

18. Diego,D.M.;Avello,M.;Mennickent.S.;FernandezM.;FernandezP.Validatedliquid
chromatographicmethodforquantitativedeterminationofalliciningarlicpowderand
tablets.J.ofsci,2007,(16),27032707.

19. LanzottiV.Theanalysisofonionandgarlic,J.ofChromatographyA,2006,(1112),322.

20. Kubec,R.;Svobodov,M.;Velis,J.GaschromatographicdeterminationofS
alk(en)ylcysteinesulfoxides.J.ofChromatographyA,1999,8594.

21. Cunico,R.L;Gooding,K.M.;Wehr,T.BasicHPLCandCEofBiomolecueles.Laboratory
RichmondCA,1998,1369.

22. Kubec,R.;Dadakova.Quantitativedeterminationofsalk(en)ylcysteineSoxides
mlebymicellareletrokineticcapillarychromatography.J.ofChromatography,2008,154
157.

23. Rubinson,K.A;Rubinson,J.F.ContemporaryInstrumentalAnalysis.PrenticeHall,Inc,
362708.

68

 24. Harris,D.C.QuantitativeChemical,seventhedition.W.HFreemanandCompany,2007,
528588.

25. Poole,F.C.;Schuett,A.S.ComtemporaryPracticeofChromatography.Elsevier,1984.
213317,353370,619640.

26. Block,E.;Naganathan,S.;Putman,D.;Zhao,S.H.HPLCAnalysisofThiosulfinatesfrom
Onion,Garlic,wildGarlic(Ramsoms),Leek,Scallion,Shallot,Elephant(GreatHeaded)
Garlic,Chive,andChineseChive.UniquelyHighAllyltoMethylRatiosinSomeGarlic
Samples.J.Agric.FoodChem.,1992,(12),24182430.

27. Lee,S.;Kim.N.;Lee,D.Analyticalandbioanalyticalchemistry.Comparativestudyof
extractiontechniquesfordeterminationofgarlicflavorcomponentsbygas
chromatographymassspectrometry.Anal.Bioanal.Chem.,2003,(377),749756.

28. Kanaki,S.N.;Rajani,M.DevelopmentandValidationofThinLayerChromatography
DensitometricMethodfortheQuantitationofAlliinfromGarlic(Alliumsativum)andIts
Formulations.J.ofAOACInternational,2005,(88),15681570.

29. Arnault;Christides,J.P.;Mandon,N.;Haffner,T.;Kahane,R.;AugerJ.Highperformance
ionpairchromatographymethodforsimultaneousanalysisofalliin,deoxyalliin,allicin
anddipeptideprecursorsingarlicproductsusingmultiplemassspectrometryandUV
detection.J.ofChromatography,2003,6975.

30. Ichikawa,M.;Ide,N.;YoshidaJ.;YamaguchiH.;Ono,K.Determinationofseven
organosulfurcompoundsingarlic.J.ofAgric.andfoodchem.,2006,54(5),15351540.

31. Hamilton,R.J.;Sewell,P.A.Introductiontohighperformancechromatography.
ChapmanandHall,1977,130,138.

32. Diaz,L.D.;Valazquez.VerificationofCriticalMicelleConcentrationwithSurfactant
Structure:Asimplemethodforcesonmicellarassociation.Art.ofchem.Edu.,2007,(12),
327330.

33. Snyder,L.R.;KirklandJ.J.Introductiontomodernliquidchromatography.Awiley
intersciencepublication,1974,137142

34. Synder;Lloyd,R;DolanW.J.HighPerformanceGradientElution.JohnWiley&sonsInc.
publication,2007,110,4251,7496,115.

35. Christian,D.G.Analyticalchemistry,Johnwiley&sonsInc.,sixthedition,604607.

69

 36. Vogt,C.;Heinig,K.Traceanalysisofsurfactantsusingchromatographicand
electrophoretictechniques.FreseniusJ.ofAnalchem.1999,(363),612618.

37. Holmberg,K.;JonssonB.;Kronberg,B.;Lindman,B.Surfactantsandpolymersin
Aqueoussolution,2ndedition,Wiley,2002,152.


38. Tang,M.;Deming,N.S.Interfacialtensioneffectsofnonionicsurfactantsinreversed
phasechromatography.Anal.Chem,1983,55(3),425428.

39. Takeda,S.;Wakida,S.I.;Yamone,K.I.;Terabe,S.Effectofpolargroupsofanionic
surfactantonmigrationbehaviorinmicellarelectrokineticchromatography.J.of
chromatographyA,1997,(781),1116.

40. Mukherjee,D.P.;Crank,J.A.;Halder,M.;Armstrong,D.A.;Petrich,J.W.Assessing the Roles
of the Constituents of Ionic Liquids in Dynamic Solvation:Comparison of an Ionic
Liquid in Micellar and Bulk Form. J.Phys.Chem.A,2006,110(37),1072510730.

41. Anderson,T.M.;Martin,E.J.;Odinek,G.J.NewComer.P.P.Effectofmethanol
concentrationonCTABMicellizationandontheFormationofSurfactantTemplates
Silica(STS).Articleofchemistryofmaterials,ACSpublication,1998,10(6),14901500.

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VITA

AARONKWAKUAPAWU

PersonaldataDateofBirth:August29th1979

PlaceofBirth:Ghana

MaritalStatus:Married

EducationBscChemistry,UniversityofCapeCoast,Ghana.2005

MSChemistry,EastTennesseeStateUniversity,Johnson

City,Tennessee.U.S.A.2009.

ProfessionalExperienceTeachingAssistant;UniversityofCapeCoast,Ghana.

20052006.

ResearchAssistant;UniversityofCapeCoast,Ghana.

20062007.

Graduate/TeachingAssistant,EastTennesseeState

University,CollegeofArtsandSciences,TN,U.S.A.

20072009.

71