Beruflich Dokumente
Kultur Dokumente
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
10
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
11
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
12
Unknown2,3,and4cannotbeidentifiedduetothelackofdata.Furtherimprovement
mustbemadeforfutureexperiments.Inparticular,additionaltrialsmustbeconductedto
determineiftheexperimentisvalid.Onlyonetrialwasperformedinthisexperimentwhichdoes
notgiveagoodrepresentationofthetestperformedand,therefore,additiontrialsisessential.
QualitativeAnalysisofCarbohydrates
13
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