Thin Layer..Chromatography

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Thin-layerChromatographyAModernPracticalApproachRSCChromatographyMonographsSeriesEditor:R.M. Smith, LoughboroughUniversityofTechnology, UKAdvisory Panel: J.C. Berridge, Sandwich, UK, G.B. Cox, Indianapolis, USA,I.S. Lurie, Virginia, USA, P.J. Schoenmakers, Eindhoven, The Netherlands,C.F. Simpson, London, UK, G.G. Wallace, Wollongong,AustraliaOthertitlesinthisseries:ApplicationsofSolidPhaseMicroextractionEditedbyJPawliszyn, UniversityofWaterloo, Waterloo, Ontario, CanadaCapillaryElectrochromatographyEditedbyKDBartleandPMyers, UniversityofLeeds, UKChromatographicIntegrationMethods, SecondEditionNDyson, DysonInstruments, UKCyclodextrinsinChromatographyBy T Cserhati and E Forgacs, Hungarian Academy of Sciences, Budapest, HungaryElectrochemicalDetectionintheHPLCofDrugsandPoisonsR J Flanagan, Guys and St Thomas NHS Foundation Trust, London, UK, D Perrett,QueenMarysSchool of MedicineandDentistry, London, UKandRWhelpton,UniversityofLondon, London, UKHPLC:APracticalGuideTHanai, HealthResearchFoundation, Kyoto, JapanHyphenatedTechniquesinSpeciationAnalysisEditedbyJSzpunarandRLobinski, CNRS, Pau,FrancePackedColumnSFCTABerger,HewlettPackard, Wilmington, Delaware,USASeparationofFullerenesbyLiquidChromatographyEditedbyKiyokatsuJinno,ToyohashiUniversityofTechnology, JapanValidationof ChromatographyDataSystems: MeetingBusiness andRegulatoryRequirementsRDMcDowall, McDowallConsulting, Bromley, Kent, UKHowtoobtainfuturetitlesonpublication:Astandingorder plan is available for this series. Astanding order will bringdeliveryof eachnewvolume uponpublication. For further informationpleasecontact:SalesandCustomerCare, RoyalSocietyofChemistry, ThomasGrahamHouse,SciencePark, MiltonRoad, Cambridge,CB40WFTelephone: 144(0)1223420066,Fax: 144(0)1223426017,Email:sales@rsc.orgThin-layerChromatographyAModernPracticalApproachPeterE.WallVWRInternationalLtd., Poole, DorsetISBN0-85404-535-XAcataloguerecordforthisbookisavailablefromtheBritishLibrary# TheRoyalSocietyofChemistry2005AllrightsreservedApartfromfairdealingforthepurposesofresearchfornon-commercialpurposesorforprivatestudy,criticism or review, as permitted under the Copyright, Designs and Patents Act 1988 and the Copyrightand RelatedRights Regulations 2003, thispublication may notbereproduced,storedortransmitted,inany form or by any means, without the prior permission in writing of The Royal Society of Chemistry, orinthecaseofreproductioninaccordancewiththetermsoflicencesissuedbytheCopyrightLicensingAgency in the UK, or in accordance with the terms of the licences issued by the appropriateReproduction Rights Organization outside the UK. Enquiries concerning reproduction outside the termsstatedhereshouldbesenttoTheRoyalSocietyofChemistryattheaddressprintedonthispage.PublishedbyTheRoyalSocietyofChemistry,ThomasGrahamHouse,SciencePark,MiltonRoad,CambridgeCB40WF,UKRegisteredCharityNumber207890Forfurtherinformationseeourwebsiteatwww.rsc.orgTypesetbyAldenBookset,Northampton,UKPrintedbyAthenaeumPressLtd,Gateshead, TyneandWear,UKPrefaceThin-layerchromatography(TLC)iswithoutdoubt oneofthemost versatileandwidelyused separation methodsin chromatography. Commercially, manysorbentsonavarietyofbackingsarenowavailable.Moststagesofthetechniquearenowautomated (can nowbe operated instrumentally) and modern HPTLC (Highperformancethin-layerchromatography)allowsthehandlingofalargenumberofsamples in one chromatographic run. Speed of separation (development time), highsensitivity and good reproducibility all result from the higher quality ofchromatographic layers and the continual improvement in instrumentation. InadditionTLChasremainedrelativelyinexpensiveandonecaneasilyseewhyitisstill populartoday. It hasfoundauseinawiderangeofapplicationareasastheconcept of TLCis so simple and samples usually require only minimal pre-treatment.Itisoftenthoughtofonlyintermsofitsuseinpharmaceuticalanalysisandproductionandinclinical analysis, but manystandardmethodsinindustrialchemistry, environmental toxicology, food chemistry, water, inorganic andpesticideanalysis, dyepurity, cosmetics, plant materials, andherbalanalysisrelyupon TLC as the preferred approach. In its simplest form, TLC costs little, but evenincluding the more sophisticated instrumentation, it still remains less expensive persampleanalysisthan,forexampleHPLC.Thin-layerchromatographycontinuestobe an active technique in research with about 500700 publications appearing eachyearduringthe1980sand1990s.Itisthepurposeofthisbooktodescribetheadvancesmade,particularlyinthelasttwotothreedecades, whichhaverevolutionisedTLCandtransformeditintoamoderninstrumental technique. All aspects of TLChavebeenaffected, fromthe sorbent layer technology, through the spotting devices and developingequipment, tothenal detectionandquantication. Computerstoo, nowplayanimportant role inthe control of equipment andinthe computationof the vastamountofdataobtained fromscansofthedevelopedTLClayer.Thishasresultedintheabilitytostoreandretrieveimagesofchromatogramsandphysicaldataonactual separation results and conditions for future use. Instrumental planarchromatographyisnowcapableofhandlingsampleswithminimal pre-treatment,detecting components at lownanogramsensitivities and with relative standarddeviations of about 1%. It is theopinionof theauthor that thesedevelopmentsvdemonstrate that the previous image of lowsensitivity, poor resolution andreproducibility can be discarded and that TLC is now truly a modern contemporaryofHPLCandGC.ModernTLChasbecomeapowerful,reliableandcostefcientmethodforqualitativeandquantitativeanalysis.Thechapters inthis bookhavebeendesignedinsuchawaythat thereaderfollows each step of the planar chromatographic process in logical order. Hence thechoiceof sorbent is followedbypreparationof samplefor applicationandthemethods of application. The subject of chromatogram development logically comesnext withdetection, quanticationand/or videoimagingusuallybeingthenalsteps. However, sometimes further instrumental analysis is necessary, hencethenal chapter onhyphenatedtechniques. Anytheoryor necessarymathematicalequations or expressions are introducedwhenrequiredwithinthe text of eachsubject. As the objective of this book is to provide a publication or manual that canbeusedbythepractisingchromatographer, thedepthoftheoryreectsonlywhatisrequiredtoexplainwhycertainpractical stepsaretaken. It istheintentionofthe author that this bookwill be of practical value anduse tothose whoarecontemplating using TLC for the rst time, and also to those who have been planarchromatographers for some time. With these points in mind, the practical examplesof chromatographicseparations reect theeld-testedprocedures available. Thebook concentrates on the basic steps involved in TLC, providing practical guidancetoachievesuperior separationsonaTLC/HPTLCsorbent layer. For thisreasontherearesometechniquesthatarenotcovered,suchassorbentcoatedquartzrodsandthin-layerradiochromatography. However, thebasicprinciplesforoptimisedseparations described in the various chapters will still apply in these relatedtechniques. Recommendations made throughout the text to obtain acceptableandoftenhighqualityresultsaremadeonthebasis of manyyearsof practicalexperienceinplanarchromatographybytheauthor.Numerous commercial products are referred to in this book as would benecessary with any publication that discusses instrumental TLC/HPTLC. Thereferencesmadearebasedontheexperiencesof theauthor withtheseproducts,and are denitely not meant to imply that they are superior to comparable productsfromothermanufacturers.vi PrefaceContentsChapter 1 IntroductionandHistory 11 IntroductiontoThin-layerChromatography 12 HistoryofTLC 23 References 4Chapter 2 SorbentsandTLCLayers 61 SorbentSelection 61.1 Introduction 61.2 SilicaBasedSorbents 71.2.1 SilicaGel 71.2.2 SilicaGelBondedPhases 121.3 Non-silicaSorbents 351.3.1 Cellulose 351.3.2 CelluloseBondedPhases 371.3.3 AluminiumOxide 371.3.4 Kieselguhr 381.3.5 Polyamide 391.3.6 MiscellaneousStationaryPhases 391.3.7 MixedStationaryPhases 401.3.8 DualPhases 402 PreparationofTLCSheetsandPlates 402.1 HomemadeTLCPlates 402.2 Pre-coatedTLC/HPTLCSheetsandPlates 413 CuttingTLC/HPTLCSheetsandPlates 414 HumidityEffectswithTLCPlates 425 Pre-washingTLCPlates 436 UseofPhosphorescent/FluorescentIndicators 447 ChannelledTLCLayers 448 ConcentrationZoneTLC/HPTLCPlates 45vii9 HPTLCPre-coatedPlates 459.1 HPTLCSphericalSilicaGel60 4810 ImprovingResolutionwithBuffersandComplexingReagents 4910.1 ImpregnationofSorbentbeforeLayerCoating 4910.2 ImpregnationoftheReadyCoatedLayer 5011 SilverNitrateImpregnation 5012 ChargeTransferTLC 5113 PreparativeLayerChromatography(PLC) 5414 References 55Chapter 3 SamplePre-treatment 591 Introduction 592 ExtractionsfromSolidSamples 603 SolidPhaseExtractionSystems(SPE) 613.1 DiatomaceousEarth 613.2 BondedSilicaGelsandPolymers 613.3 SorbentConditioning 633.4 SampleInjection 633.5 Cleaning 633.6 RecoveryofAnalytes 634 References 64Chapter 4 SampleApplication 651 Introduction 652 SelectingtheSampleSolvent 653 TheoreticalDeterminationofSpotSize 663.1 SpotSizeandLoadingEquations 663.2 SpotCapacityEquation 684 SampleLoading 724.1 LayerDamagingEffects 744.2 HumidityConsiderations 755 SampleApplicationMethods 755.1 ManualMethods 765.2 InstrumentalMethods 775.2.1 ManualInstrumentalTechniques 775.2.2 AutomatedTechniques 785.3 LargeSampleApplication 805.3.1 ContactSpotting 805.3.2 UseofConcentrationZoneTLC/HPTLC 816 ChoiceofBandorSpotApplication 827 References 85viii ContentsChapter 5 DevelopmentTechniques 861 Introduction 862 TheTheoryofSolventMigration 863 MechanismsofTLCSeparation 903.1 Introduction 903.2 AdsorptionSeparations 903.3 PartitionSeparations 913.4 Ion-exchangeSeparations 914 SolventSelection 924.1 EluotropicSeriesandSolventOptimisation 934.1.1 SelectivityTriangles 964.1.2 PRISMAModel 995 DevelopmentChambers 1005.1 N-ChambersandChamberSaturationEffects 1005.2 Twin-troughChamber 1055.3 Sandwich,HorizontalandU-chambers 1055.4 AutomaticDevelopingChamber(ADC) 1085.5 Forced-owDevelopment(O