Thinlayerchromatographyisbasedontheseparationofamixtureofcompoundsasitmigrateswiththehelpofasuitablesolventthroughathinlayerofadsorbentmaterialwhichhasbeenappliedtoanappropriatesupport.Thinlayerchromatogramcanbelookeduponasbeing"opencolumn"system.FollowingthedevelopmentofarelativelysimpleapparatusforpreparingchromatoplateswithuniformlythinlayersofadsorbentbyStahl(1964),TLCbecameverypopular.Nowadays,ready-to-useTLCplatesarenormallyused,sincetheyoffergreaterreproducibilityandhighermechanicalstrength.Differentstationaryphasesareavailablethatallowtoseparatebypartition,ion-exchange,adsorptionoracombinationofanyofthesephenomena.Adsorptionisthemostcommonmechanism.InadsorptionTLCthesampleiscontinuallyfractionatedasitmigratesthroughtheadsorbentlayer.Competitionforactiveadsorbentsitesbetweenmaterialstobeseparatedandthedevelopingsolventproducescontinuousfractionation.Aportionofthematerialtobeseparatedwillbefoundinthemobilephaseandaportionwillbeadsorbedtothesolidadsorbentparticles.Astheprocesscontinuesthevariouscomponentsmovedifferentdistances,dependingontheirrelativeaffinitiesfortheadsorbentascomparedwiththemigratingsolvent.Ingeneral,themorepolarcompoundsareheldbackbytheadsorbentwhilelesspolarmaterialadvancefurther.Polarsolventeffectthemostmovementofsamplematerialovertheadsorbent. ThemigrationofacompoundinagivenTLCsystemisdescribedbyitsRfvalue: distancetraveledbycompoundRf=distanceofthesolventfrontfromtheorigin a)becapableofretaininganyofamultitudeofmixturesoforiginalsolventsb)beairtight,toinsuremaintenanceofasolventsaturatedatmospherec)berelativelyeasytohandle a)naturalcolourb)sprayreagents;yieldscolouredderivativesonreactionwiththeseparatedspots.c)iodinevapourd)fluorescenceandUVabsorptione)autorADIography 1.Usingthetemplateprovided,marktheplateswithasharppencil,asshownhere 2.Linethechamberwithchromatographypaper.Prepare202mlofsolventsystem(Hexane:Ether:Aceticacid60:40:1)ina500mlErlenmeyerflask.Mixandpour~150mlintothechamber.Coverandletthechambersaturatewhileloadingtheplates. 3.Witha10µlcapillaryPipette,spot1-2µlofphospholipidsstandardontotheTLCplate,asshown.Makesurethespotremainssmallerthan4mmindiameter.Moveontotheotherstandards.Afterthespotshavedried,repeatloadingeachstandardsuntilyouhaveloadedapprox.10µleach.Also,load10µlofyourlipidextractononespot,andthentheremainderoftheextractasaline(i.e.,aseriesofspots). 4.Letdrythespots.Makesurethattheloadingareaisabovethesolvent.Placetheplatesinthechambertodevelop. 5.Immediatelyclosethecoverandletrunforapproximately30min,untilthesolventfronthasreachedtheupperline. 6.Removetheplateandleavetodryintherackinthefumehood.Discardthesolventinthewastecontainerprovided,removethechromatographypaperandleaveinthechamber.Leavethechamberinthefumehoodtodry. 7.Nowplacetheplateintheiodinetankinthefumehood.Youwillseethelipidsasyellowspotsafterabout5minorso. 8.Marktheedgesofthespotswithapencil.Makeatracingononionskinpaperforarecord. 10.Meanwhilepreparecolumnstoelutethelipidsfromsilicagel.Todothisinsertasmallamountofglasswoolintoapasteurpipette,labelthepipetteandleaveonthestandprovided. 11.Carefullytransferthesilicagelhavingdifferentlipidsintoappropriatecolumns.Keep7mlglassvialsbeneaththecolumns. 12.Drip1mlofchloroformintoeachcolumnexceptforthephospholipidandmonoacylglycerolcolumns.Tothelatter,add>1mlof100%methanol. 13.Shakethevialswell.Placetheminthefumehoodevaporatingset-upandevaporateoffthesolventunderastreamofN2.(AsktheinstructorbeforeusingtheN2tank). 14.StorethedrylipidsunderN2. 15.Labelandleavetheseparatedlipidinthe-20°Cfreezeruntiltransmethylation(day2). However,differencesinthesolventcompositionwillleadtoalteredmobilityofthedifferentcomponents.AcommonproblemoccurswhentheTLCtankisnotcompletelysealed,andthesolventscanslowlyevaporate.Sinceevaporationratedependsontheboilingpoint,solventslikehexanewillevaporatefaster.Asaresult,themobilephasebecomesmorepolar.Nevertheless,apositiveidentificationofyourspotsshouldalwaysbepossiblesinceyouranstandardsonthesameplateaswell.i.Adsorbent:
ThemostcommonadsorbentusedisSilicagel(silicicacidcombinedwithasmallamountofgypsumasabindingagent).Manyothermaterialhavealsoprovenusefulforspecificpurposes;forexample,aluminafortheseparationofsteroidsandwatersolublevitaminsandKieselgurforseparationofsugars.ii.Solvents:
AlthoughitisfrequentlypossIBLetoselectagoodsolventsystembytheempiricalmethods,sometimeandeffortmaybesavedbyconsiderationofsomeprinciplesofthechromatographicseparation.OfparticularimportanceinTLCaretheelutropicseriesofsolvents.Thisisaseriesofsolventsarrangedintheorderoftheirelutingpower;thatishavingincreasingABIlitytoremovecompoundsfromanadsorbent.Aknowledgeoftherelativeadsorbentcharacteristicsoftheclassofcompoundstobeseparatedisalsoimportant.Forexample,saturatedhydrocarbonsareadsorbedpoorlywhileunsaturatedhydrocarbonsareadsorbedaccordingtotheincreaseinnumberoftheirdoublebonds.iii.Development:
Theapparatususedfordevelopmentmusthavecertainfeatures:iv.Visualization:
CompoundsseparatedcanbevisualizedbyEXPERIMENTALPROTOCOL
Handletheplatescarefully.NOFINGERPRINTS.ThesewillbeshownafterexposingtoI2vapour.
9.Scrapofflipidfractionsasshown,placeinweighingpaper,foldandrolltogrindtheclumps.Results
Youshouldhaveobtainedagoodseparationofthelipidclasses,similartotheplateshownbelow.
