experiment4 procedure

7/28/2019 Experiment4 Procedure

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Freshman Research Initiative: Synthesis and Biological Recognition streamSection 2: Amino Acids, Structure Representation & Modeling

Experiment 4: Qualitative Tests of Amino Acids

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Part1:AminoacidandProteinSolubilityThe following procedures are adapted from the experiment “QualitativeTesting for

AminoAcidsandProteins”byFrankR.MilioandWilliamM.Loffredo.

SolubilityinWater

1.Label6clean,dry4-dramvialswiththenamesofthefollowingsubstances:

glycine,glutamicacid,lysine,tyrosine,gelatin,andcasein.Place50mgofeachsubstanceinthecorrespondingtesttube.Add2mLofdistilledwatertoeachvial.

Capthevialsandshakevigorouslyfor2min.Recordyourobservationsinyourlaboratorynotebook.Savethesemixturesforthenextstep.

SolubilityasaFunctionofSolutionpH

2.Add1mLof3Msodiumhydroxidesolutiontoeachvial.Capthevialandshakevigorouslyfor1min.Recordyourobservations.

3.Add3MhydrochloricacidtotheglycinevialseveraldropsatatimeuntilthepH

isacidic.TestthesolutionpHusingapipetandpHpaper.DonotdipthepHpaper

in;usethepipetandcapillaryactiontoremoveasmallamountofthesolutionto

spotonthepaper.Capthevialandshakevigorouslyfor1min.Recordyour

observationsinyourlabnotebook.

4.Add15moredropsof3Mhydrochloricacidtotheglycinevial.Capthevialand

shakevigorouslyfor1min.Recordyourobservationsinyourlaboratorynotebook

5.RepeatSteps3and4usingtheremainingmixturespreparedinStep1.Notethatyoucansavetimebyperformingsteps3–5simultaneouslyoneachsample.

6.Dilutethemixturesinthevialswithwateranddiscarddownthedrain.Washthe

vialswithsoapordetergentsolution,andrinsethreetimeswithtapwaterand

oncewithdistilledwater.Allowthevialstodryupsidedowntoremoveasmuchwateraspossible.





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Part2:QualitativeReactionsofAminoAcidsandProteins

NinhydrinTest

7.Checkthewaterbathssetupinthehoodstoverifythattheyarebetween95–100ºC.Adjustthetemperatureifnecessary.

8.Label6clean,drained13x100-mmtesttubeswiththenamesofthefollowing

solutions:2%glycine,1%tyrosine,2%proline,2%casein,2%gelatin,and2%

albumin.Obtainthesolutionsfromyourlaboratoryinstructorandplace15drops

ofeachinthecorrespondingtesttube.Toeachofthetesttubes,add5dropsof

0.5%ninhydrin–ethanolreagentsolution.

9.Usingatesttubeholderor“hothands,”placethetesttubesintothehotwater

bathpreparedbyyourmentorsthatyoucheckedinStep7.Allowthetesttubesto

standinthebathfor5min.Usingatesttubeholderorhothands,removethetest

tubesfromthebathandplacetheminatesttuberack.Recordyourobservations

inyourlaboratorynotebook.

10.Discardthetestsolutionscontainingninhydrinintothecontainerprovidedby

yourlaboratoryinstructorandlabeled“DiscardedNinhydrinSolutions.”Rinsethetubesoncewith~2mLwaterintothewastecontainer.Washthetesttubes

withsoapordetergentsolution,andrinsethreetimeswithtapwaterandonce

withdistilledwater.Allowthetesttubestodraintoremoveasmuchwateras

possible.

BiuretTest11.Label5clean,drained13x100-mmtesttubeswiththenamesofthefollowing

solutions:2%glycine,2%arginine,2%gelatin,2%casein,and2%albumin.

Obtainthesolutionsfromyourlaboratoryinstructorandplace15dropsofeach

inthecorrespondingtesttube.Add5dropsof3Msodiumhydroxideand2drops

of0.1Mcopper(II)sulfatesolutiontoeachtesttube.UsingaseparatePasteur

pipetforeachtesttube,mixthecontents.Recordyourobservations.

12.Discardthetestsolutionscontainingthebiuretmixturesintothecontainer

providedbyyourlaboratoryinstructorandlabeled“DiscardedBiuretTest

Solutions.”Washthetesttubeswithsoapordetergentsolution,andrinsethree

timeswithtapwaterandoncewithdistilledordeionizedwater.Allowthetesttubestodraintoremoveasmuchwateraspossible.

XanthoproteicTest


solutions:1%tyrosine,2%tryptophan,2%glycine,2%albumin,and2%gelatin.





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Obtainthesolutionsfromyourlaboratoryinstructorandplace15dropsofeach

inthecorrespondingtesttube.

14.Workingunderafumehood,cautiouslyadd10dropsofconcentratednitricacid

tothe1%tyrosinetesttube.Usingatesttubeholder,placethetesttubeintotheboiling-waterbath.Warmthecontentsgentlyfor1–2min,usingtheholderto

swirlthetesttube.Removethetesttubefromtheboiling-waterbath.Placeitinto

atesttuberackandallowittocool.Recordyourobservations.

15.RepeatStep14usingtheothersolutions.

16.ToeachofthecooledsolutionsfromSteps14and15,cautiouslyadd3Msodium

hydroxideuntilthesolutiontestsslightlybasicusingpHpaper.Recordyour

observations.

17.Dilutethecontentsofeachtesttubewithwater,anddiscarddownthedrain.

Washthetesttubeswithsoapordetergentsolution,andrinsethreetimeswith

tapwaterandoncewithdistilledordeionizedwater.Allowthetesttubestodraintoremoveasmuchwateraspossible.

Millon’sTest


solutions:1%tyrosine,2%tryptophan,2%glycine,2%albumin,2%gelatinand

2%casein.Obtain2mLofeachsolution.Addeachsolutiontothecorrespondingtesttube.Add3dropsofMillon’sreagenttoeachtesttube.Usingatesttube

holderorhothands,placethetesttubesintothehotwaterbath.Heatthe

solutionsjusttotheirboilingpoints,thenuseatesttubeholdertoremovethetesttubesfromthebathandplacetheminatesttuberack.Recordyourresults.

19.DiscardthetestsolutionscontainingMillon’sreagentintothecontainer

providedbyyourlaboratoryinstructorandlabeled“DiscardedMillon’sSolutions.”Rinsethetubestwicewith~2mLofwaterintothewastecontainer.

Washthetesttubeswithdetergentsolutionandrinsethreetimeswithtapwaterandoncewithdistilledordeionizedwater.Allowthetesttubestodrainto

removeasmuchwateraspossible.

Hopkins–ColeTest


solutions:1%tyrosine,2%tryptophan,2%albuminand2%gelatin.Obtain2mL

ofeachsolution,andaddeachtothecorrespondingtesttube.Add2mLofHopkins–Colereagenttoeachtesttube.





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Note:Ifapurpleringdoesnotformattheinterfaceofthetwolayersineachtest

tube,gentlytapthesideofthetesttubeoncetoslightlymixthetwolayers.Ifa

purpleringstilldoesn’tform,consideritanegativeresult.

21.Holdingthe1%tyrosinetesttubeata45°angle,cautiouslyandslowlyadd30

dropsofconcentratedsulfuricaciddowntheinsidewallofthetesttube.Donot

mixthetwolayersthatform.Recordyourobservations.

22.RepeatStep21usingtheremainingreactionmixtures.

23.DiscardthetestsolutionscontainingHopkins–Colereagentintothecontainer

providedbyyourlaboratoryinstructorandlabeled“DiscardedHopkins–Cole

Solutions.”Washthetesttubeswithsoapordetergentsolution,andrinsethree

timeswithtapwaterandoncewithdistilledordeionizedwater.Allowthetest

tubestodraintoremoveasmuchwateraspossible.

NitroprussideTest


solutions:2%glycine,2%cysteine,2%albumin,and2%casein.Labelthefifth

testtube“blank.”Obtain2mLeachof2%glycineand2%cysteine,and4mLeach

of2%albuminand2%casein.Placeeachsolutioninthecorrespondingtesttube.

Add2mLof3Msodiumhydroxidesolutiontoeachofthe5testtubes.Add1mL

of2%sodiumnitroprussidesolutiontoeachtesttube,includingtheblank.

Recordyourobservationsinyourlaboratorynotebook.

Part3:Determiningtheidentitiyofunknownaminoacidor

proteinsolutions

Usingtheaboveprocedures,determinetheidentityoftheunknownsolutionsthatyouaregiven.Youmayuseanyofthepreviousteststodeterminetheidentityof

yourunknownsolutions.Besurethatyouuseadefinitivetesttodeterminethe

identity;anegativeresultcannotbeusedtodefinitivelyfindtheidentityofanunknown.

experiment4 procedure

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