THERETARDATIONFACTOR(Rf)–Concept&calculationinTLC/HPTLC:
Concept Of Rf Value In TLC/HPTLC:
InTLC/HPTLC,basically,twophasesareimportanti.e.stationaryphaseandmobilephase.
Thegasphasealsoplaysanimportantroleifwearegivingsaturation.
Nowadaysstationaryphaseismostlysilicagelandmobilephaseselectiondependsonthe analyteofinterest.Usually,itcouldbeamixtureofbinary,tertiaryorquaternarysolvents.
Thecompound/Analyteofinterestisdissolvedinasuitablediluent,andappliedontothe stationaryphaseintheformofspots/bondsandtheplateisdevelopedinasuitablemobile
phase Theanalyteandthemobilephasemoveoverthestationaryphaseduetothecapillary action.
Thedifferentialmobilityofdifferentcompoundsisachievedbytherelativeaffinityof compoundstowardsthestationaryphaseandmobilephase.Thecompoundswithahigher affinitytowardsthestationaryphasemoveslowlyasthestationaryphaseretainsthe respectivecompounds
SincethestationaryphaseispolarinTLC,polarcompoundsmoveslowly.However, compoundswithahigheraffinitytowardsthemobilephasemovefasterthroughthe stationaryphase.Themobilephaseisrelativelylesspolarand,non-polarcompounds,which havelessaffinitytowardsthestationaryphase,movefasterthroughthestationaryphase.
Aftercompletionofthedevelopmentstep,separatedcompoundscanbeseenasspots/bands inshortUV(254nm),longUV(366nm)orinwhitelightdependingontheresponseofthe analyte,withthehelpofsuitableTLCphotodocumentationdevicelikeCAMAGvisualizer.
CAMAGTLCscannercanbeusedforquantification,datalikeRfvalues,areaunderthe peaketc.
Response/Migrationofanalyteisexpressedintermsofretardationfactor/ratiotofront) The retardationfactor(RF)inplanarchromatography,alsoknownastheretentionfactor,isaterm usedtodescribetherelativepositionsofzonesinthechromatogram.
Itiscalculatedbasedontheratioofthedistancestravelledbythecentreofazone“A”(b) andthedistancetravelledbythemobilephasefront(a)withrespecttotheapplication positionasshownintheabovediagram
Bydefinition, Rfvaluesarealwayslessthanone,andtheyaredisplayedwithtwodecimals AnRfvalueof1ortooclosetoitmeansthatthespotandthesolventfronttravelclose togetherandarethereforeunreliable.
IUPACdefineshRfas Rfmultipliedby100,butmostpharmacopoeiasandpublicationsuse RF.
TheRfvaluesforagivencompoundshouldstayconstantifyouarefollowingsame chromatographicconditionseverytimeyouarerepeatingtheanalysis.
WhatarethefactorswhichaffectRfvaluesinTLC/HPTLC?
TheRfvaluesdependsonthesampleapplicationpositionbutalsothelevelofdeveloping solventinthechamberwhichshouldbekeptconstant.MostsignificantlyRfvaluesmay showvariationduetohumidityintheairorthedevelopmentwhichiscarriedoutindifferent humidconditions. ThisproblemisoftenencounteredwhiledoingTLC.However,withHighPerformanceThin LayerChromatography,humiditycanbecontrolledbyusingsaturatedsolutionof magnesiumchloridehexahydratesolution.
AsHPTLCisanofficialtechniqueinUnitedStatesPharmacopoeia(USP),itsguidelines suggestdevelopmentinacontrolledenvironmentof33%humiditywhichisachievedabya saturatedsolutionofmagnesiumchloride.
CAMAGADC2(AutomaticdevelopmentChamber)isusedforisocraticdevelopment Steps likeplatedevelopment,monitoringandplatedryingiscompletelyautomaticandunder controlledhumidity
Humiditycanbecontrolledat33%byusingsaturatedsolutionofmagnesiumchloride This complieswiththeUSPguidelinesforplatedevelopmentanditensuresreproducibleRf valueseverytimeanalysisisrepeated.
WebReferences:
1. https://pediaa.com/how-to-calculate-rf-values-for-tlc/
2. https://www.camag.com/
Anchrom Enterprises Pvt Ltd is one of the leaders in HPTLC analysis Please contact us at lab@anchrom.in for HPTLC analysis of plant extracts, drugs, ingredients in cosmetics, and forensic science.