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QualitativeAnalysisofCarbohydrates

QualitativeAnalysisofCarbohydrates
JulioFrancisco
FloridaInstituteofTechnology

QualitativeAnalysisofCarbohydrates

Abstract:
Thepurposeofthisexperimentwastocompareandidentifyanunknowncarbohydrate
compoundtoknowncarbohydratesamplesthroughvariousofchemicaltesting.Theseschemical
testingsincludetheBenedictstest,molischtest,inversionofsucrose,Seliwanoff'stest,Bials
test,andthinlayerchromatography(TLC).Afterperformingallthetest,theunknowncompound
wasdeterminedtobemaltose.However,furthertestingmustbeconductedtoconfirmifthe
unknowncarbohydrateistrulymaltose.

QualitativeAnalysisofCarbohydrates

Introduction:
Thefollowingexperimentwasconductedtoidentifyanunknowncompoundusingsix
differenttestingtechniques.Theunknowncompoundcanbedeterminedbycomparingthe
chemicalreactionofknowncarbohydratesbyapplyingvariouschemicaltests.Qualitativedata
werecollected.Theknowncarbohydratesusedinthisexperimentwereglucose,fructose,
galactose,arabinose,maltose,ribose,xylose,andsucrose.Thefollowingisanoverallofthe
applicationsandusageofeachtest:
BenedictsTest
TheBenedictstestisimportanttoidentifyreducingsugars.Underanalkalisolution,the
reducingsugarwouldbeheatedresultingintheenolizationofsugars.Thesesugarscan
potentiallyreduceCu2+toCu+whichresultsinprecipitationofaredcopper(I)oxide.Therefore,
theappearanceofaredbricksolutionindicatedthepresenceofareducingsugar.
TherearemanyapplicationsfortheBenedictstest.Sincethe1900samodifiedversion
ofBenedictstestwasusedtodetectglucoseintheurineinordertodiagnosesomeonewith
diabetesmellitus.However,theintroductionofClinistixandTesTapesupplantedtheuseofthe
BenedicttestsinceClinistixandTesTapeprovidedamoreaccuratereadingthantheBenedicts
test(Ackerman,Williams,Packer,Hawkes,&Abler,1958).However,duetothelimitationof
urineglucosemonitoringandtheriseofbloodglucosemonitoring,urinaryglucosetestbecome
lessfrequentlyusedfordetectionofdiabetesmellitus.Todayselfmonitoringofcapillaryblood
glucoseiscommonlyusedtodeterminethebloodglucoselevelfordiabeticpatientduetoamore
accuratereadingthanprevioustestingtechniques(Cowart&Stachura,1990).Therearemany
otherapplicationsoftheBenedictstesttodiscusshowever,suchapplicationsisbeyondthe
scopeofthispaper.

QualitativeAnalysisofCarbohydrates

MolischTest
TheMolischtestisatestforgeneralcarbohydrate.Inthistest,carbohydrateisdehydrate
toafurfuralderivativeinthepresenceofaconcentratedacid(e.g.sulfuricacid).Thefurfural
reactwithnaphtholresultingintheformationofaredorvioletcolor.Therefore,apositivetest
foracarbohydrateisdenotedbytheappearanceofaredorvioletcoloredsolution.
ResearchergenerallyusetheMolischtesttodeterminedthepresenceofcarbohydratein
varietyofsubstances,fromelastinproteintounripenedguavas(Misra&Seshadri,1968Stein&
Miller,1938).
BialsTest
TheBialstestisachemicaltestusedtodetectthepresenceofapentosemolecule.Ifa
pentosemoleculewaspresence,thentheBialsreagentwillcausethepentosetodehydrate
whichresultintheformationoffurfural.Thefurfuralfurtherreactwithorcinoltogivea
bluegreencondensationproduct.Therefore,theappearanceofabluegreencolorsolution
indicatesthepresenceofapentosemolecule.
Seliwanoff'sTestforKetose
TheSeliwanoff'stestisusedtodetectthepresenceofketose.Iftheketoseispresence,
thentheSeliwanoffsreagentwoulddehydratetheketoseandformhydroxymethylfurfural.The
hydroxymethylfurfuralwillfurtherreactwithresorcinolgivingoffaredcondensationproduct.
Therefore,thepresenceofaketosemoleculeisindicatedbytheformationofaredcolored
solution.

QualitativeAnalysisofCarbohydrates

TLCforCarbohydrate
TLCisatechniqueusedtoseparatemoleculefromacomplexmixturebasedonparticular
propertiesofthecompounds(e.g.adsorption).SomeoftheadvantageofusingTLCmethodis
thefactitissimple,expedient,inexpensive,andeffectivemethodofseparation(Tyrpie,
Bodzek,&Manka,2001).TheapplicationsofTLCtechniqueissoprofoundthatitwillgo
beyondthescopeofthepaper.
ExperimentalProcedure:
BenedictsTest
IntheBenedictstest,5mlofBenedictsreagentwasaddedto5testtubes.Then,1mlof
0.1Mofglucose,maltose,arabinose,sucrose,andxylosewereindividualtoeachtesttube.Place
inboilingwaterbathfor5to50minutes.Anycolorchangewasobserved.
MolischTest
IntheMolischtest,5mlof1:10dilutionofglucose,fructose,galactose,maltose,
arabinose,ribose,xylose,sucrose,andtwounknownswereplacedintoindividualtesttubes.
Next,2dropsofMolischreagentand3mlofconcentratedH2SO4wereaddedandobservation
wasrecorded.
BialsTest
FortheBialstest,add5mlofBialsreagentto1mlaliquotsof0.01Mglucose,
fructose,arabinose,ribose,andsucrose.Placeinboilingwaterbathforfifteenminutes.Record
appearance.

QualitativeAnalysisofCarbohydrates

Seliwanoff'sTestforKetose
FortheSeliwanoffstest,add5mlofSeliwanoffsreagentto1.0mlaliquotsof0.01M
solutionsofglucose,fructose,sucrose,galactose,andribose.Placeinboilingwaterbathfor
fifteenminutes.Recordanychangesinappearance.
InversionofSucrose
Fortheinversionofsucrose,add5mlof0.1Msolutionsofglucose,fructose,maltose,
arabinose,ribose,xylose,sucrose,andunknownsolutionsintotwodifferenttesttubes.Add5
dropofconcentratedHCltoonetesttubeandheatbothtubeinboilingwaterbathfor10
minutes.Afterward,add5mlofbenedictsreagenttotestthepresenceofreducingsugar.
ThinLayerChromatographyofCarbohydrate
IntheTLCexperiment,spot0.1Mofsugarsolutionsandtheunknownsolutionswitha
capillarytubeontoanactivatedsilicagelplate.Allowthespottodry.Afterthespothasbeen
dried,placetheplateintoaglassblockchromatocabfilledwithsolventtothe0.5cmlevel.
Allowthesolventtorisefortwentyminutes.Removetheplatefromthefromthechromatocabin
orderfortheplatestobeanalysis.

Results:
Table1.CarbohydrateIdentificationTable.Thefollowingtabledemonstratetheobservationfor
eachtestappliedonindividualcarbohydrates.Theshadedregionsrepresenttheomissionof
certaincarbohydratefromcertaintest.
Carbohydrate

Benedicts
Test

MolishTest

BialsTest

Glucose

Red
Precipitate
Formed

BrownColor Lightpeach
color

Seliwanoff's
Test

Inversionof
Sucrose

Goldcolor

Nochange

QualitativeAnalysisofCarbohydrates

Fructose

Darkbrown
color

Darkred
color

Bloodyred
color

Nochange

Galactose

Redcolor

Lightyellow

Nochange

Maltose

Ambercolor
with
precipitation
inthebottom

Redcolor

Nochange

Arabinose

Ambercolor
with
precipitation
inthebottom

DarkRed
color

Yellowcolor

Nochange

Ribose

Lightbrown
color

Lightsilver
color

Darkyellow
color

Nochange

Xylose

Browncolor

Nochange

Sucrose

Nocolor
change

Redcolor

Darkred
color

Bloodyred
color

Sucrosewasa
darkerhuein
comparisonto
other
carbohydrate
compounds

Unknown1

Afusionof
amberand
bluecolor
with
precipitation
formation

Redcolor

Peachcolor

Peach

Nochange

Unknown2

Unknown3

Unknown4

Sixcarbohydrateidentificationtestswereperformedandtheobservationwasrecorded
(Table1).First,themolischtestwasusedtodetectanycarbohydrate.Apositiveresultforthe
molischtestisindicatedbytheappearanceofaredtovioletcolor.Amongallcarbohydrate

QualitativeAnalysisofCarbohydrates

tested,fourcarbohydrateschangedtoaredcolor.Thisincludesgalactose,maltose,arabinose,
andsucrose.Ontheotherhand,glucose,fructose,ribose,andxylosechangedtoabrownish
color.
ThenexttestwastheBenedictstestwhichiscommonusetodetectreducingsugars.A
positivetestfortheBenedictstestisindicatedbytheformationofyellow,green,orred
precipitation.Glucose,maltose,arabinose,andunknownaretheonlycarbohydratesthat
indicatedapositiveresultforthebenedictstest.Furthermore,sucrosedidnotshowedanycolor
changes.
ThethirdtestwastheBialstestwhichisusedtodetectthepresenceofpentose.The
presenceofapentoseisindicatedbytheappearanceofgreenblueorolivegreencolor.The
Bialstestwasconductedonsolutionswithglucose,fructose,arabinose,ribose,sucrose,andan
unknowncompound.Noneofthecompoundsgaveanindicationofapentose.
ThenextexperimentconductedwastheSeliwanoffstest.TheSeliwanoffstestisusedto
detectthepresenceofaketose.Apositiveresultisindicatedbytheappearanceofredcolor.
Fructoseandsucrosearetheonlytwocompoundsthatgaveapositiveresultforthepresenceof
ketose.
Thelasttestwastheinverseofsucrose.Sucrosewastheonlycarbohydratethatproduced
adifferentdarkerbluethanothercarbohydratesolution.

QualitativeAnalysisofCarbohydrates

Figure1.ThinLayerchromatographyanalysisofglucose,fructose,galactose,maltose,
arabinose,ribose,xylose,sucrose,andunknown1,2,and3.Solutionsof0.1Mofsugar
compoundwerespottedontoasilicabasedgelplateandallowedtodry.Oncedried,thespotted
platesweretransferredintoaglasschromatocabcontainingofapproximately500mLsolvent.
Thesolventconsistofchloroform,glacialaceticacid,andwater(v/v30:35:5).Thesolventwas
allowedtoruntoaheightof7cmforapproximately20minutes.Next,theplatewasremoved
fromthechromatocabforfurtheranalysis.Itwasdeterminedthatunknown1hadsimilar
migrationpatternasmaltose.

Athinlayerchromatographywasconductedtoidentifytheunknowncompoundsby
comparingspotsoftheunknowncompoundstoknowncarbohydratesolution(Fig.1).Itwas
observedthatunknown1traveledthesamedistanceasmaltose.Furthermore,unknown3

QualitativeAnalysisofCarbohydrates

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traveledthesamedistanceasgalactose,andunknown4travelsimilardistanceasarabinose.
Unknown2didnotappearedintheTLC.
Discussion:
ThefirsttestwastheBenedictstest.TheBenedictstestisusedtoidentifythepresence
ofreducingsugar.Apositiveresultisdenotedbytheformationofayellow,green,orred
precipitate.IntheBenedictsexperiment,itwasrevealedthatglucose,maltose,andarabinose
gaveapositiveresultforsuchtest.Ontheotherhand,riboseandsucrosegaveanegativeresult
indicatingthatthethesetwocarbohydratesarenonreducingsugars.TheresultfortheBenedicts
experimentwasconsistentwithknownfacts.Also,theBenedictstestindicatedthatthe
unknowncompoundisareducingsugar.
ThesecondtestwastheMolischtestwhichisaneminenttestusedtodetectthepresence
ofsolublecarbohydrate.Aredtovioletcolorisapositiveindicationofacarbohydrate.Itwas
expectedthatallofthecarbohydrateusedwouldgiveapositiveresult.However,onlygalactose,
maltose,arabinose,andsucrosegaveapositiveresult.Glucose,fructose,ribose,andxylosegave
anegativeresult.Thismaybeduetotheactionofsulfuricacid(Dreywood,1946).Sincethe
carbohydratewasburnedbythesulfuricacid,furthertestingmustbeconductedtoensurethat
experimentwasperformedproperly.Furthermore,theunknowncompoundgaveapositiveresult
indicatingthattheunknowncompoundisacarbohydrate.
ThenexttestperformedwastheBialstestwhichisusedtodetectthepresenceof
pentoses.Thepresenceofpentoseisindicatedbytheappearanceofabluishgreenorolivegreen
color.NoneofthecompoundsgaveapositiveresultfortheBialstest.Thisisunexpectedsince
arabinose,ribose,xylosearepentosemoleculesand,therefore,thesemoleculesshouldindicated

QualitativeAnalysisofCarbohydrates

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apositiveresultfortheBialstest.Itcanbeassumedthatthetheremightbeanerrorthat
occurredintheexperiment.
TheSeliwanoffstestisusedtodetectthepresenceofketoses.Thecompoundsthatgave
apositiveresultwerefructoseandsucrose.Itwasunexpectedtoseethatsucrosereceiveda
positiveresultduetothefactthatsucroseisadisaccharidecomposedofbothglucoseand
fructose.ItcanbepostulatethattheSeliwanoff'sreagentreactedwiththefructosecomponentof
sucroseleadingtoapositiveresult.Furthermore,theunknowncompounddidnotgaveapositive
resultfortheSeliwanoffstest,andtherefore,theunknowncompoundcouldbepotentialbea
pentose.
Intheinversionofsucrose,theonlycompoundthatchangedappearancewassucrose.
Invertsugaristheprocessofbreakingdownsugar,suchassucrose,intoitsindividual
monosaccharidecomponents.Sincesucroseistheonlydisaccharideusedinthisexperimentthat
canbebrokendown,theresultwasconsistentwiththeexpected.Also,thistestindicatesthatthe
unknowncompoundisnotasucrosemoleculesincethecompounddoesnotundergotheprocess
ofinversion.
Thelastexperimentwasthethinlayerchromatography.Basedonthemigrationpattern,
unknown1traveledthesamedistanceasmaltose.Thisindicatedthattheunknowncompound
maybemaltose.Basedonalltheexperimentaltestsperformed,unknown1isanonsucrose,
reducingsugar.Also,theunknowncompoundcanpotentialbeapentosehowever,theBials
testmustbeperformproperlytoconfirmthattheunknowncompoundistrulyapentose
molecule.Withtheseinformationinmind,itcanbepostulatethatunknown1istrulymaltose.

QualitativeAnalysisofCarbohydrates

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Unknown2,3,and4cannotbeidentifiedduetothelackofdata.Furtherimprovement
mustbemadeforfutureexperiments.Inparticular,additionaltrialsmustbeconductedto
determineiftheexperimentisvalid.Onlyonetrialwasperformedinthisexperimentwhichdoes
notgiveagoodrepresentationofthetestperformedand,therefore,additiontrialsisessential.

QualitativeAnalysisofCarbohydrates

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