TLC.docx
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TLC
TLCVisualizationReagents
ThisisabriefselectionofthemanyavailableTLCvisualizationreagents.Beloweachtitleisthetypeofcompoundsorstructurewhichcanbedetectedwiththespecificreagent.Whenbeginningworkwiththesereagents,acquireanyMSDS(materialsafetydatasheets)toseeifthereareanyextraprecautionsneededinsafelyusingthem.
Beforespraying,platesshouldbewelldriedinthehoodofresidualsolventsandcomponents.Aminesandorganicacidsusedinthemobilephasesmayadverselyaffectthevisualizationreactionbeingattempted.Ifheatingtoremovethesecomponentsisdone,considerationshouldbegivensothatlossofcomponentsortheirdecompositionisavoided(byloweringthetemperatureorusingashortertimeintheoven).
Alwayssprayanyofthesereagentsontoplatesinawellventilatedhoodwhilewearingsafetyglasses.Alsoapplymoderateamountstotheplatesoitalwaysappearsdullandflat(ifitlookswet,youhavesprayedtoomuch).Youcanalwaysoverspraytoenhancethedetection.
Aluminiumchloride
Forflavonoids
Sprayplatewitha1%ethanolicsolutionofaluminumchloride.
Results:
YellowfluorescenceinlongwavelengthUVlight(360nm)
4-Aminoantipyrine/potassiumhexacyanferrate(III)(Emersonreaction)–checksomethingmissing
(Emersonreagent)forthedetectionofphenolsandarylamines
SolutionI:
1gaminoantipyrine(4-aminophenazone)in100ml80%ethanol
SolutionII:
4gpotassiumhexacyanoferrate(III)in20mlwater,fillto100mlwithethanol
Procedure:
SpraywithsolutionI
Dry5minuteswithwarmair
Placechromatograminachamberwithvaporfrom25%ammoniumsolution,makingsurethatthelayerdoesnotcontacttheliquid.
Results:
Red-orangetosalmonpinkspots
2-Aminoethyldiphenylborate,seeEthanolaminediphenylborate
Ammoniummetavanadate,ammoniummonovanadate,seeVanadium(V)sulfuricacidreagent
Ammoniummolydbate
Fordetectionofphosphoricacidderivatives
SolutionI:
1Mperchloricacidinwater/acetone(1:
1)
SolutionII:
ammoniummolybdatesoln:
5g(NH4)6Mo7O24.4H2Oin35mlsemi-conc.Nitricacidand65mlwater.
SolutionIII:
Tin(II)chloridesoln:
0.5gSnCl2.2H2Oin100ml0.5Mhydrochloricacid:
Drydevelopedchromatogramandheatto60C
Hydrolysedi-andtriphosphatesbysprayingperchloricacid(solutionI)ontothewarmplate.Afterspraying2times,dryplateslowlyat50C.Amidophosphatesmightnotbedecomposed.
Inanycase,spraythestillwarmplatewithammoniummolybdatesolution(solutionII)
Thenspraythestillwetplatewithtin(II)chloridesolution(solutionIII)
Results:
Phosphatesappearasbluetoblue-greenspots.Polyphosphatescanalsobedetectedbydippingtheplatesinasolutionofammoniummolybdate(1g)dissolvedinwater(8ml)andperchloricacid(3ml,ca.70%),
filledupto100mlwithacetone.Thenphosphatesappearasyellow-greenspotsonabluebackground.AlsoseeMolybdenumbluereactionaccordingtoDittmerandLester.
Anilinephthalate
Forthedetectionofreducingsugars
Drythedevelopedchromatogram
Spraywith0.93ganilineand1.66go-phthalicaciddissolvedin100mln-butanolsaturatedwithwater.
Brieflydrywithhotair,thenheatto105Cfor10minutes
Results:
Substancespotsshowdifferentcolorsonanalmostcolorlessbackground.Somespotsgivefluorescenceat365nm.
p-Anisaldehyde–sulfuricacid
Fordetectionofphenols,sugars,steroids,andterpenes
Spraywithasolutionoffreshlyprepared0.5mlp-anisaldehydein50mlglacialaceticacidand1ml97%sulfuricacid.
andheatto105°Cuntilmaximumvisualizationofspots.Thebackgroundmightbebrightenedbywatervapor.
Results:
Lichenconstituents,phenols,terpenes,sugars,andsteroidsturnviolet,blue,red,greyorgreen.
Fordetectionofsugars
Spraywithasolutionoffreshlyprepared1mlp-anisaldehyde,1ml97%sulfuricacidin18mlethanolandheatat110°C.
Results:
Sugarphenylhydrazonesproducegreen-yellowspotsin3min.Sugarswillproduceblue,green,violetspotsin10min.Alsodetectsdigitalisglycosides.
p-AnisidineHydrochloride
Fordetectionofcarbohydrates/sugars
Mixasolutionof3%p-anisidinehydrochlorideinn-butanol
Sprayandheatat100°Cfor2-10min.
Results:
Aldohexosesareseenasgreen-brownspots,ketohexosesasyellowspots,aldopentosesasgreenspots,anduronicacidsasredspots.
Anisidinephthalatereagent
Fordetectionofcarbohydratesandreducingsugars
Spraywithasolutionof1.23gp-anisidineand1.66gphthalicacidin100ml95%ethanol.
Results:
Hexoses,green;pentoses,red-violet–sensitivity0.5ug;methylpentoses,yellow-green;uronicacids,brown–sensitivity0.1-0.2ug.
Antimony(III)chloride
Fordetectionofflavonoids
Spraywitha10%solutionofantimony(III)chlorideinchloroform
Results:
Fluorescingspotsinlongwavelengthlight(360nm).
Antimony(III)chloride
FordetectionofvitaminsA&D,carotenoids,steroids,sapogenins,steroidglycosides,terpenes
Spraywithasolutionof25gantimony(III)chloridein75mlchloroform(generallyasaturatedsolutionofantiomony(III)chlorideinchloroformorcarbontetrachlorideisused).
Heat10minat100C,viewunderlongwavelengthlight(360nm).
Bromine/Carbontetrachloride
Fordetectionoforganothiophosphorouspesticides
Placechromatograminachamberwitha10%bromineandtetrachloridewithoutcontactwiththeliquid.
Bromocresolgreen
Fordetectionoforganicacids
Dipchromatograminasolutionof0.1gbromocresolgreenin500mlethanoland5ml0.1MNaOH
Results:
Acidsyieldyellowspotsonabluebackground.
BromthymolBlue
Fordetectionoflipidsandphospholipids
Reagent:
0.1%bromthymolbluein10%aqueousethanolmadejustalkalinewithNH4OH
Spraydriedplate.
Results:
Compoundsaboveproduceblue-greencolors;sensitivity0.1-1µg.
Chloranilreagent
Fordetectionofphenols
Spraywithasolutionof1%tetrachloro-p-benzoquinoneintoluene
Chlorine/o-tolidine
Fordetectionofofcompoundsformingchloroamines,e.g.,ureaderivatives,carbamated,antibiotics
SolutionI:
160mgo-tolidinein30mlglacialaceticacid,filledto500mlwithdistilledwater,plus1gKIsolution
SolutionII:
saturatedsolutionofo-tolidinein2%aceticacid/0.85%KIsolution(1:
1,v/v)
ProcedureA
Placechromatogram15-20mininachlorineatmosphere(e.g.,Potassiumpermanganate+10%Hydrochloricacid)
Leave5minutesatambienttemperatureuntilthechlorineisevaporatedcompletely(spraycornerofplatetoinsurenobluecolorisseen,showingcompleteabsenceofchlorine).
SpraywithsolutionI
ProcedureB
Spraywith2%potassiumhypochloritesolutioninwater
Leave1-1.5hratambienttemperature
SpraywithsolutionII
Coppersulfate/phosphoricacid
UsedasacharringreagentforpolymerboundTLCplates(thenewerhardlayerplates)
Spraywithasolutionof10%copper(II)sulfatein10%phosphoricacid
Heat5-30minat110°C
Results:
Viewfrequently(every5-10min)toseeifcoloredorfluorescentspots(at254and360nm)canbeseen.Charringcanbecontinueduntilspotsarebrown,greyorblack.
Chromosulfuricacid
SeeunderPotassiumdichromate/sulfuricacid
DDQReagent(Dichlorodicyanobenzoquinone)
Fordetectionofphenols
Spraywithasolutionof2%2,3-dichloro-5,6-dicyano-1,4-benzoquinoneintoluene
Dichlorofluoresceinreagent
Forthedetectionofsweetenerssaccharine&cyclamate
Spraywitha0.2%solutionofdichlorofluoresceinin96%ethanol
Drywithwarmair;ifnecessary,spraywithwater
Viewunder360nmUVlight
Dichlorofluorescein/fluoresceinsodiumsalt
FordetectionofN-substitutedbarbiturates
Spraywitha0.1%ethanolicsolutionofdichlorofluorescein
Thenspraywitha0.1%ethanolicsolutionoffluoresceinsodiumsalt
2,6-Dichloroquinone–4-chloroimide
Fordetectionofantioxidants,phenols,primaryandsecondaryaliphaticamines,secondaryandtertiaryaromaticamines,aromatichydrocarbons,pharmaceuticals,phenoxyaceticacidherbicides,etc
Spraywithafreshlyprepared0.5-2%solutionof2,6-dichloroquinone-4-chloroimideinethanol(reagentstablefor3weeksifrefrigerated).
Heat10minat110C;treatwithammoniavapor
p-Dimethylaminobenzaldehyde
Fordetectionofsulfonamides
Spraywithasolutionof1%p-dimethylaminobenzaldehydein5%hydrochloricacid;add5%ethanol
Detectssulfonamides
p-Dimethylaminobenzaldehyde/hydrochloricacidreagent(Ehrlich’sreagent)
Fordetectionofamines,indolederivatives
Spraywithasolutionof1%p-dimethylaminobenzaldehydeinconc.hydrochloricacid/methanol(2:
2)
Heatplatesfor20minat50C
2,4-Dinitrophenylhydrazine
Fordetectionofaldehydesandketones
Sprayplatewithsolutionof0.4g2,4-DNPHin100ml2Nhydrochloricacid,add1mlethanol
Results:
Yellow-redspotswillbeseen.
Diphenylamine
Fordetectionofglycosides,glycolipids
Reagent:
10ml10%diphenylamineinethanol,100mlHCland80mlglacialaceticacid
Spraylightly,coverplatewithanotherglassplate,heat30-40minat110°Cuntilpositiveareasappear
Results:
Glycolipidsproducebluespots.
s-Diphenylcarbazone
Fordetectionofbarbiturates
Sparywithasolutionof0.1%s-diphenylcarbazonein95%ethanol
Results:
Barbiturateswillproducepurplespots
2,2’-Diphenylpicrylhydrazyl
Fordetectionofaldehydesandketones
Reagent:
dissolve15mgof2,2’-DPPHin25mlchloroform
Spray,heat5-10minat110°C;
Results:
Yellowspotsonapurplebackgroundwillbeseen.
Dithizone
Fordetectionofheavymetalions
Dissolve20mgdithizonein100mlacetone,storeinabrownbottleinarefrigerator
Procedure:
Spraywithdithizonesolution
Spraywith25%ammoniasolution
Dittm