TLC分析分离之总结.docx

TLC分析分离之总结.docx

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TLC分析分离之总结

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