Chemistry103 Lab2:Reactionsandstereochemistryofcarbohydrates Objective:Todeterminetheidentitiesofseveralcarbohydratesthroughaseries oftests.

Introduction: TheSugars: Carbohydrates,alsoknownas sugars or saccharides,areanimportantclassof biochemical compounds,because theyfunction assources ofenergy in living organismsaswellasstructuralmaterialsforcells.Carbohydratesarecomplex biomoleculesthatcontainbothalcoholandcarbonylfunctionalgroups. Polysaccharides canbeviewedas polymers ofvarious monosaccharide units. Monosaccharides are carbohydrates that cannot be broken down into smaller carbohydrateunits.Twoexamplesofmonosaccharides,glucose(1)andfructose (2),areshownbelow.

Sinceglucoseandfructosehavebothalcoholandcarbonylfunctionalities,they areabletoform cyclic hemiacetal and hemiketal compounds.Thehemiacetal andhemiketalformsareinequilibriumwiththeopenchainforms.Sincebothof theseformsexist,onecouldpredictthatthechemistryofcarbohydrateswould besimilartothechemistryofketonesoraldehydes.Inthecaseofglucose,its open chainformwiththealdehydeshouldbeeasilyoxidized.Carbohydrates thatcanbeoxidizedareknownas reducingsugars.Carbohydratechemistryis reviewedinTimberlake. A disaccharide isacarbohydratecomposedoftwomonosaccharideunits.One

suchcompoundissucrose(3),alsoknownastablesugar.Sucroseiscomposedof afructoseunitandaglucoseunit.Thekeypointtonoteisthatthefructoseand glucoseunitsarejoinedbyan acetal linkinvolvingboththecarbonylcarbons, which means thatthe aldehyde andketoneforms are unavailable to sucrose. Therefore,sincesucroseisnotinequilibriumwithanyaldehydeforms,sucrose isconsideredanonreducingsugar.

In contrast, lactose (4) is a disaccharide composed of a galactose unit and a glucoseunit.Inlactose,thehemiacetalbondisformedwithanalcoholoxygenof glucoseandthereforethealdehydefunctionalityoftheglucoseunitisavailable. Thus,lactoseisareducingsugar.

Sucralose(Splendatm)isanartificialsweetenerthathasrecentlybecomepopular. Itsshapeissimilartosucrosecausingtobindtothesweetnesssensingreceptors in our tongues. Although this compound somewhat mimics the flavor of sucrose,thestructureisdifferentenoughthatourbodiesdonotbreakitdown thereforeitprovidesuswithnocalories.

Sucralose

Polysaccharides, on the other hand, are used for energy storage. Amylose, shownbelow,isatypeofstarch.Asyoucansee,starchesgenerallycontainlong chainsofglucoseunits.

TheTests: Inthisexperiment,youwilltestglucose,fructose,sucrose,sucraloseandstarch forreducingactionbyreactingthemwithTollensandBenedictsreagents.In theTollenstest,silverionisreducedtometallicsilver.Thesilverwillstickto thesideofthetesttubegivingitamirrorlikeappearance.Beawarethatthe silverisverydifficulttoremovefromthetesttubegenerallyruiningthetube. IntheBenedictstest,copper(II)isreducedtocopper(I);thereshouldbea definitecolorchange.TheresultsoftheTollensandBenedictstestsshouldhelp youidentifywhichunknownsarereducingsugarsandwhicharenonreducing sugars. Thestarchcanbeidentifiedbyitscolorfulinteractionwithiodine.Whena coupleofdropsofiodinesolutionareaddedtoastarchsolutionadramaticdark bluecolorresults.Whentheiodinesolutionisaddedtotheglucose,fructose, sucroseandsucralosesolutions,thereshouldbelittletonocolorchange.

A few drops of iodine in a starch solution. Thebodyusesthemonosaccharideglucoseasitssourceofenergy.Inorderfor energytobeobtainedfromsucrose,starchesandothersaccharides,theorganism mustfirstbreakthemdownintoglucose.Thisreactionentailsthehydrolysisof the glycosidic linkages catalyzed by a collection of enzymes. Enzymes are proteinsthatacceleratebiochemicalreactions. Theseenzymestendtobequite selective.Forinstance,lactaseisusedtobreakdownlactose,amylaseisusedto break down amylose and invertase (also known as sucrase) is one of the enzymesthatcanhydrolyzesucrosetoglucoseandfructose.Benedicts,Tollens and the iodine test should yield identical results for sucrose and sucralose howevertheinvertaseshouldhydrolyzesucrosewhileleavingsucraloseintact. Following action of the enzyme upon sucrose, Benedicts and Tollens tests shouldnowyieldpositiveresults.

Glucose andfructose should yield identical results forall of the above listed tests. Polarimetry can be used to determine which is which. Fructose and glucose are chiral molecules (wikipedia.org has an excellent explanation of

chirality)andthereforetheyhavetheabilitytorotate planepolarizedlight,a phenomenon that can be measured by a polarimeter. Glucose rotates plane polarized light in a quite positive (+) direction (as viewed from detector to source)whilefructosedoessoinaquitenegative()direction allowingusto differentiatebetweenthetwo. PreLabQuestionsandActivities: Prepareyourlabnotebookwithatableofthetestsyouwillberunning. 1. Listthehazardouschemicalswewillbeworkingwith. 2. Whatfunctiondoestheinvertaseenzymeperform? 3. Whichmaterialswillbereducingsugars?

Materials Glucosesolution* Fructosesolution* Sucrosesolution* Sucralosesolution* Starchsolution* Testtubes Distilledwater 37Cwaterbath InvertaseEnzyme(Sucraseenzyme) 5%Silvernitratesolution 5%Sodiumhydroxidesolution 2%Ammoniumnitratesolution BenedictsReagent Iodinesolution *ThesolutionswillbelabeledasunknownsAE. *************************************Procedure

1. Gather 5 test tubes and label them A E. Place about 1 mL of each unknownintothecorrespondingtube. 2. Add 1 or 2 drops of iodine solution to each of the tubes and record observations.Presenceofadeepblue,purpleorblackcolorisapositive testforpresenceofstarch. Withthestarchidentified,younowhave4 unknowns. 3. Properlydisposeofwaste,andwashanddrytheusedtesttubes. 4. Gather8testtubesandlabelthem. Therewillbe2testtubesforeach unknown.TollenstestwillbeperformedinonetesttubeandBenedicts intheotherforeachunknown. 5. Perform Benedicts and Tollens Tests on each of the unknowns and recordresults. Tollenstest: Toanemptytesttube,add10dropsof5%silvernitratesolution and2dropsof5%sodiumhydroxide.Addenough2%ammoniumhydroxideto justdissolvetheblackprecipitate(shakethetubetomix).Besurenottoaddan excessofammoniumhydroxide. Sometimesitisdifficulttodissolveallofthe precipitate,Add4dropsofyoursugarsolution.Formationofasilvermirroron theinteriorofthetesttubeisapositivetestforareducingsugar.Gentleheating inthe37Cwaterbathwillfacilitatethereaction. Benedictstest:Inanemptytesttube,add5mLoftheBenedictsreagentto2mL ofyoursugarsolution.Heatthesemixturesina37Cwaterbathfor10minutes. Areducingsugarwillproduceared,greenoryellowprecipitate. 6. Discardthesilveredtubesandcleantheremainingtesttubes. 7. TheresultsofTollensandBenedictstestsshouldrevealwhichunknowns aremonosaccharidesandwhicharedisaccharides.(Iseitherdisaccharide areducingsugar?)Labeltwotesttubesandplaceapproximately2mLof thedisaccharideunknownsintotheappropriatetube. 8. Add approximately2mgofinvertase enzymeto bothtubes andplace theminthe37Cbathfor2030minutes. 9. Whilethetesttubesareincubatinginthebath,polarimetrywillbeusedto identifythemonosaccharides. UsethesolutionslabeledpolarimeterA and polarimeter B for this test. Consult the operating manual and

instructorforfurtherdetails.Recordtheresults. Specificrotationsoffructoseandglucose: fructose =93 glucose =+52 10. Once the incubation time is complete, repeat Tollens test on the disaccharides. Apositivetestshouldidentifythesucrosesolution(now brokendownintomonosaccharidesbytheenzyme). Havingcompletedtheabovetests,theidentityofeachunknownshouldnowbe established. ***********PostlabQuestions 1a.Drawtheproductsoftheacidhydrolysisoflactose. b.Drawthealdehydeformoflactose. 2.WhatcanyouconcludefromtheresultsoftheBenedictsandTollenstests?In otherwords,whichsugarsarereducing,andwhicharenonreducing,according totheresultsofyourtests?Thisanswershouldbelongerthanacouplewords. 3.Describe,usingthepolarimetrydata,whatsugarsweregeneratedintesttubes 2and3.Pleasesupportyourclaimswithevidence!