Thin layer chromatography ns

Thin Layer Chromatography

1. Thin Layer
Chromatography
Prepared By: Ms. Navdha Soni
LJ Institute of Pharmacy
Ahmedabad

2. Introduction:
•Thinlayerchromatographywasfirstdiscovered
byIzmailovandSchreiberin1938.FurtherStahl
(1958)perfectedthemethodanddeveloped
equipmentandstandardizedadsorbentsforthe
preparationofuniformlayersonglassplates.
Andthistechnique,chromatographyusingthin
layersofanadsorbentheldonaglassplateor
othersupportingmediumiscalledasthinlayer
chromatography.

3. Definition:
•Thinlayerofchromatographyisamethodif
analysisinwhichthestationaryphase(afinely
dividedsolid)isspreadasathinlayerona
rigidsupportingplateandmobilephase.
•TLCisoftennamedbyothernamessuchas
drop,strip,spreadlayer,surface
chromatographyandopencolumn
chromatography.

4. Superiority of TLC overother
chromatographictechnique:
•Simpleequipment.Lowcost.
•Shortdevelopmenttime.
•Separationofmicrogramofthesubstancescanbe
achieved.
•Anytypeofcompoundcanbeanalyzed.
•Widechoiceofstationaryphase.Maybemployed
foradsorption,partition(reversedphasealso)orion
exchangechromatography.
•Earlyrecoveryofseparatedcomponents.
•Easyvisualization.
•Sensitivityisbetter10to100timespaper
chromatography.
•Variablethicknessofthinlayers.
•Chemicallyinertstationaryphase.

5. Principle:
•The principle for separation isadsorption.
•One or more compounds are spotted on a thin layer of
adsorbent coated on a chromatographicplate.
•The m/p flows through because of capillaryaction.
•The components move according to their affinities
towards theadsorbent.
▫More affinity towards the s/p –travelsslowly
▫Lesser affinity towards the s/p –travelsfaster.
•Thus the components are separated on thin layer
chromatographic plate based on the affinity of the
components towards thes/p.

6. Steps involved inTLC:
•Selection ofadsorbent.
•Selection of glassplate.
•Coating of the adsorbent on to the glassplate.
•Activation ofadsorbent.
•Purification oflayer.
•Selection of mobilephase.
•Spotting of thesample.
•Development
•Visualization
•Quantitative and qualitativeanalysis.

7. Selection ofadsorbent:
•A large no. of coating materials areavailable.
•Adsorbents classifiedinto:

8. Adsorbent:
Acidic:
E.g.silica
For acidicanalytes
Basic:
E.g. alumina
For basicanalytes
Neutral:
E.g. keisleguhr,
Cellulose powder.
For neutralanalytes
Adsorbent:
activeHas more active sites for binding to theanalyte.
inactiveHas less active
sites for the
binding ofanalyte.

9. •Asstationaryphaseaspecialfinelygroundmatrixiscoatedontheglassplate,ametaloraplasticfilmasathinlayer(approx0.25mm).
•Adsorbentdoesn’tadheretoplateproperly.Toovercomethisproblembindersareusedlikegypsum,starch,hydratedsilicondioxideetcareaddedtotheadsorbent.E.g.SilicagelG60,whereGindicatesgypsumand60isparticlesize.
•Afluorescentindicatorlikezincsilicateareaddedtotheadsorbenttosimplifyvisualizationofspot.TheadvantageisnonfluorescentU.Vabsorbinganalytescanbedetectedonathinlayercontainingfluorescentadditive.▫E.g.silicagelGF
•Suchsubstancesshowupasdarkspotsasgreenfluorescentindicator.

10. Glassplates:
•Glass plates which are specific dimensions like 20cm
×20cm (full plate), 20cm ×10cm (half plate), 20cm ×
5cm (quarterplate).
•Microscopic slides can also be used for some
applications like monitoring the progress of
chemical reaction. The development time is much
shorter like5mins.
•They should withstand temperatures used for
drying theplates.

11. Coating of adsorbent on glassplate:
•Main aim is to get uniform thickness of thelayer.
•Many techniques areused:
•Pouring:
▫Measured amount of slurry is poured and plate is tipped back and forth to spreaduniformly.▫Disadvantage –uniform thickness cannot beensured.
•Dipping:▫Whole plate is dipped intoslurry.
▫Disadvantage –backside of plate is also coated and more amount of slurry is required even to prepare fewerplates.•Spraying:▫Suspension of adsorbent is sprayed ontoplate.▫Disadvantage –uniform thickness cannot neachieved.

12. •Spreading:
▫Widelyused.
▫The slurry after preparation poured into a TLC spreader and the thickness of layer is adjusted by adjusting a knob in thespreader.
▫Nowthespreaderisrolledontheplateortheplateis moved while applying the slurry.▫Thickness of 0 –2mm (0.25 for analyticalpurposesand2mmforpreparativepurpose).
•Pre coatedplate:
▫Ready to use thinlayersarenowavailableprecoated.
▫These areexpensive
▫Thickness varies from 0.1 to0.2mm.

14. Activation ofadsorbent:
•Coatedplatesarekeptinairfor30minandtheninhot
airovenat110°Cforanother30min.
•Thedriedplatescanbestoredinthermostatically
controlledovenorindesiccatorsandcanbeused
whenrequired.

15. Purification or washing ofplates:
•Silicagelcontainsironasimpuritywhichcausesa
considerabledistortionofchromatograph.
•Topurifytheairdriedplatesaregivenapreliminary
developmentwithmethanol-conc.Hcl(9:1,v/v).The
irongetsmigratedwithsolventfronttotheupper
edgeoftheplate.
•Theplatesareagaindriedandactivated.
•WashingcanbedoneforprecoatedTLCplatesalso.

16. Selection of mobile phase:
•Thechemicalnatureifthesamplethatistobe
separatedisknownthenitispossibletoknowa
suitablesolventsbyusingoriginalstalh’striangle
whichisinter-relatingadsorbentactivity,natureofthe
soluteandnatureofthesolvent.
•IfthetriangleisrotatedsothatatcornerMpointstothe
typeofmixturetobeseparated,thisspecifiesat
cornersSandErespectively,thenecessaryactivityof
theadsorbentandtheoptimumpolarityoftheeluent.

17. •Supposeamixturehashydrocarbonsandketones.
Thenfromstahl’striangle,itisfoundthatanactive
adsorbentisrequired,togetherwithanonpolar
solvent.
•Mixturesoftwoormoresolventsofdifferent
polarityoftengivebetterseparationthan
chemicallyhomogenoussolvents.
•Theyshouldbeaspureaspossible.

18. Application ofsample:
•Concentrationofthesampleorstandardsolutionshould
hastobeminimum.
•Thespotsshouldbeatleast2cmabovethebaseofthe
plateandspottingareashouldnotbeimmersedin
m/p.
•Aglamicrosyringeisusedforquantitativework,
howevercapillarytubescanbeusedforqualitative
work.
•Tospottheplate,simplytouchthecapillarytubeendto
thecoatedsideoftheplate.Thesolventshouldquickly
evaporateleavingmixturebehindontheplate.
•Excessivespottingleadstosmearing,smudgingand
spotoverlapwillresultmakingidentificationof
separatedcomponentsdifficult.

19. Development:
Developmenttank:
•Differentchambersofdifferentsizesareusedtohold
TLCplates
•Differentdevelopmenttanksareavailable
▫Flatbottomchambers
▫Twintroughchambers–itrequirelesssolventandtwoplatescanbedeveloped.
▫Cylindricaltanks
•Chambersaturationshouldbedone.
•Tankshouldbelinedinsidewithfilteredpaper
moistenedwithm/psoastosaturatewith
atmosphere.
•Ifchambersaturationisnotdoneedgeeffectoccurs.
•Edgeeffect:wherethesolventfrontmovesfaster
inmiddleoftheplatethanthatoftheedges.
Thereforespotsaredistortedandnotregular.

21. •Onedimensionaldevelopmentorvertical:
▫Conventionalmethod
▫Solventflowsagainstgravity,becauseofcapillary
action.(bottomtotop).
•HorizontalTLC:
▫plateiskeptinhorizontalmanner.
▫Spottingisdoneinmiddleoftheplateandm/pis
addedslowlythroughsides.
•DescendingTLC:
▫Flowofsolventisassistedbygravityandhence
developmentis
faster.
▫Solventholderisontop.

22. •MultipledevelopmentTLC:
§▫SimilartoverticalTLC.
§▫Afterdevelopingonce,theplateisdriedand
thenagainkeptinsamemobilephase(same
composition)andinsamedirection.
§▫Withoutdetectionmultipledevelopmentsaredone.
§▫Itisdonetoseparatesomecomplex
mixture(moreno.ofcompounds).

23. •Step wiseTLC:
▫Platesizeisbigger30cmplate.
▫Usuallydonewhendevelopmentdistanceislong.
▫Development:
–Allowfirstm/ptotravelupto15to18cmandthen
itisstoppedanddried.
–Nowitisagainkeptinanotherm/pand
developmentisdone.
•Two-dimensionalTLC:
▫Firsttheplatesaredevelopedinoneaxisandthe
platesafterdryingaredevelopedinotheraxis.
▫Whenlargenumberofcompoundsorcomplex
mixturesareneedtobeseparatedthismethodcanbe
followed.
▫Eithersamesolventordifferentsolventsystemcan
beused.

24. •GradientTLC:▫Isocratic–samecompositionofm/pusedthroughoutdevelopment.▫Gradient–ratioofm/pchanged.
•FractionatingTLC:
▫Spotting is done in the middle at top of the plate and
them/pisforcedthoughtheedgesoftheplate.
▫Differentfractionsarecollectedatdifferenttimes
basedtheaffinityoftheanalytetowardstheadsorbent.

25. Visualization:
•Coloredspotscanbevisuallydetected.Butfor
detectingcolorlessspots,thefollowingtechniques
areused:
•Nonspecificmethods:no.ofspotscanbedetected
butnotexactnatureortypeofcompound.
▫Iodinechambermethod:wherebrownoramber
coloredspotsareobservedwhenthepaperarekept
inatankwithfewiodinecrystalsatthebottom.
▫UVchamberforfluorescentcompounds:when
viewedunderUVchamberat254orat365nm,
fluorescentcompoundscanbedetected.

26. •Specific methods: specific spray or detecting or
visualizing agents are used to find out the nature of
compound or for identificationpurposes.
▫Eg.
–Fecl3 for phenolic and tannincompounds
–ninhydrin for aminoacids.
•The detecting techniques can also be categorizedas:
▫Destructivetechnique:whenspecificsprayagents
areusedthesamplesaredestroyedbeforedetection.
–Eg.Ninhydrinreagent
▫Nondestructivetechnique:methodslikeUV
chamber,iodinechamber,densitometrymethod
doesn’tdestroythesampleevenafterdetection.

27. Qualitativeanalysis:
•Rfvalue:
▫Rf=disttravelledbysolute/disttravelledbysolventfront▫valuerangesfrom0-1▫Idealvaluesare0.3-0.8.
▫Itischaracteristictoeachcompoundinaparticularcombinationofspandmp.
▫Theunknowncompoundcanbeidentifiedbycomparingitsrfvalueswithstandard’s.
▫ifrf=1–analytemovealongwithm/pwithoutseparating.
▫Ifrf=0.1–analyteisadsorbedtoadsorbentandnotmoving.

28. •Rxvalue:
▫Distancetravelledbysample/disttravelledby
standard.
▫Itisalwayscloserto1.
▫m/pallowedtotravelthroughouttheplate.
•Rmvalues:
▫Tofindwhethercompoundsbelongtoa
homologousseries.
▫Itisacombinedvalue.
▫DeltaRm=log(1/Rf–1).

29. Quantitativeanalysis:
•Canbecarriedoutintwoways:
•Directmethod:quantitativedeterminationisundertakendirectlyonthelayer.
•Indirectmethod:thesubstancesareremovedfromtheadsorbentandthendeterminedafterelution.•Directmethods:▫Visualassessmentofchromatogram.
▫Determinationbymeasurementofspotarea–thismethodisbasedonarelationbetweenspotandamountofthesubstancepresent.▫QuantitativeTLCincorporatingdensitometer.▫Directspectrophotometryonthinlayer-–Byevaluationofwavelengthofmaximumabsorbance.
–CharacterizationofchromatogramzonesbyreadingtheabsorptionorfluorescencecurvesdirectlyfromTLCisdonebychromatogramspectrophotometerintroducedbyzeiss,stahlandjork.

30. •Indirectmethod:
▫Doneafterelutingtheindividualspotswith
suitablesolventandthenfilteringoffthe
stationaryphase.
▫Theexactquantityofcompoundscanbe
determinedbyconventionalmethodslike
colorimetry,uvspectrophotometry,fluorimetry,
flamephotometry,electrochemicalmethodsof
analysis.
▫Thespottedareaisscoopedusingcertain
apparatus.

31. Applications:
•TLCisverycommonlyusedtechniqueinsyntheticchemistryforidentifyingcompounds,determiningtheirpurityandtheprogressofareaction.
•Separation of vitamins, antibiotics, proteins, alkaloids,glycosides.•Identification of drugs e.g. amino caproic acid, digitoxin,levodopa.•Detecting the decomposition products indrug.
•Separation of inorganicions
•Identification of organiccompounds
▫Even no. alcohols from decanol through hexacosanolcan be separated on keiselguhr G with cyclohexane as a developingsolvent

32. Thank You