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AppendixIIIA
(Ph.Eur.method2.2.27)

Thin-layerchromatographyisaseparationtechniqueinwhichastationaryphaseconsistingofanappropriatematerialisspreadinauniformthinlayeronasupport(plate)ofglass,metalorplastic.Solutionsofanalytesaredepositedontheplatepriortodevelopment.Theseparationisbasedonadsorption,partition,ion-exchangeoroncombinationsofthesemechanismsandiscarriedoutbymigration(development)ofsolutes(solutionsofanalytes)inasolventorasuitablemixtureofsolvents(mobilephase)throughthethin-layer(stationaryphase).

Apparatus


Plates Thechromatographyiscarriedoutusingpre-coatedplatesasdescribedunderReagents(4.1.1).


Preconditioningoftheplates Itmaybenecessarytowashtheplatespriortoseparation.Thiscanbedonebymigrationofanappropriatesolvent.Theplatesmayalsobeimpregnatedbyproceduressuchasdevelopment,immersionorspraying.Atthetimeofuse,theplatesmaybeactivated,ifnecessary,byheatinginanovenat120°Cfor20min.


Chromatographictank Achromatographictankwithaflatbottomortwintrough,ofinert,transparentmaterial,ofasizesuitablefortheplatesusedandprovidedwithatightlyfittinglidisrequired.Forhorizontaldevelopmentthetankisprovidedwithatroughforthemobilephaseanditadditionallycontainsadevicefordirectingthemobilephasetothestationaryphase.


Micropipettes,microsyringes,calibrateddisposablecapillaries orotherapplicationdevicessuitablefortheproperapplicationofthesolutions.


Fluorescencedetectiondevice tomeasuredirectfluorescenceortheinhibitionoffluorescence.


Visualisationreagents Suitabledevicesareusedforderivatisationtotransfertotheplatereagentsbyspraying,immersionorexposuretovapourand,whereapplicable,tofacilitateheatingforvisualisationofseparatedcomponents.


Documentation Adevicemaybeusedtoprovidedocumentationofthevisualisedchromatogram,forexampleaphotographoracomputerfile.

Method


Sampleapplication Applytheprescribedvolumeofthesolutionsatasuitabledistancefromtheloweredgeandfromthesidesoftheplateandonalineparalleltotheloweredge;allowanintervalofatleast10mm(5mmonhigh-performanceplates)betweent