Tlc

For B.Pharm, 5th Sem

1. Chromatography mainly
emphasize on TLC
PRESENTED BY
Mr. Sonam Bhutia

2. History
•Mikhail Tswett, Russian Botanist,
1872-1919
•In1903-06,Tswettusedtochromatography
toseparateplantpigments
•Hecalledthenew technique
chromatographybecausetheresultofthe
analysiswas'writtenincolor'alongthe
lengthoftheadsorbentcolumn
•Theword-Chromatographyisderivedfrom
Greek,Chromameans“color”andgraphein
means“towrite”

3. •Chromatographyisaphysicalmethodof
separationinwhichthecomponentstobe
separatedaredistributedbetweentwo
phases
•Oneofwhichisstationary(stationaryphase)
whiletheother(mobilephase)moves
throughitinadefinitedirection.
•Thechromatographicprocessoccursdueto
differencesinthedistributionconstantofthe
individualsamplecomponents.

4. Chromatography
Is a technique used to separate
and identify the components of a
mixture.
Works by allowing the molecules present in the
mixture to distribute themselves between a
stationary and a mobile medium.
Molecules that spend most of their
time in the mobile phase are carried
along faster.

5. 1.Classificationofchromatography
accordingtomobilephase:
1.Liquid chromatography: mobile phase is a
liquid.
Eg: Liquid-Liquid Chromatography(LLC),
Liquid-Solid Chromatography(LSC).
2. Gas chromatography : mobile phase is a gas.
Eg:Gas-Liquid Chromatography(GLC),
Gas-Solid Chromatography(GSC).

6. 2.Classificationaccordingtothe
packingofthestationaryphase:
1.Thinlayerchromatography(TLC):thestationary
phaseisathinlayerofadsorbent(usuallysilicagel
oralumina)coatedorsupportedonglass,plasticor
aluminiumplates.
2.Paperchromatography(PC):thestationaryphaseis
athinfilmofliquidsupportedonaninert
support/paper.
3.Columnchromatography(CC):stationaryphaseis
packedinaglasscolumn.

7. 3.Classificationaccordingtotheforce
ofseparation:
1. Adsorption chromatography.
2. Partition chromatography.
3. Ion exchange chromatography.
4. Gel filtration chromatography.
5. Affinity chromatography.

8. Thin layer chromatography (TLC)
•isamethodforidentifyingsubstancesandtesting
thepurityofcompounds.
•TLCisausefultechniquebecauseitisrelatively
quickandrequiressmallquantitiesofmaterial.
•Techniqueusedtoseparatenon-volatilemixtures.
•Performonasheetorplate(about0.25mmthick)of
glass,aluminiumfoilorplatewhichiscoatedwitha
thinlayerofsolidadsorbentmaterial.
•Theadsorbentisusuallysilicagel400-800meshsize
withbinderorwithoutabinder.

9. •Separations in TLC involve distributing a mixture of two
or more substances between a stationary phaseand a
mobile phase.
1. The stationary phase:
•is a thin layer of adsorbent (usually silica gel or alumina)
coated on a plate.
2. The mobile phase:
•is a developing liquid which travels up the stationary
phase, carrying the samples with it.
•Components of the samples will separate on the
stationary phase according to
how much they adsorb on the stationary phase versus
how much they dissolve in the mobile phase.

10. Thin Layer Chromatography (TLC)

12. TLC

13. Preparing the Chamber
•Toajarwithatight-fittinglidaddenoughof
theappropriatedevelopingliquidsothatit
is0.5to1cmdeepinthebottomofthejar.
•Closethejartightly,andletitstandfor
about30minutessothattheatmospherein
thejarbecomessaturatedwithsolvent.

14. How to prepare TLC plates.
•Putyourstationaryphase(SILICAGEL)inabowl
andaddapproximatelythesameamountof
water.OnetablespoonofSILICAGELrequires
onetablespoonofwater.Mixituntilyouget
uniformsuspension.
•Verycarefullypoursmallamountofsuspension
ontheplate.Makesureyou'renotholdingplate
bythesides–thatwillcausesuspensiontospill
overtheedgeoftheplateassoonasittouches
yourfingers.It'llneedsomepractice.

16. ACTIVATION
•Activated at 105-110°C
for 1 hr or at 60°C for
overnight.

18. Silica gel grades
•Silicagelwithabinder=SilicagelG,contain
Gypsumasabinder.
•Silicagelwithabinder&fluorescence=Silica
gelGF254.
•Silicagelwithoutbinder=SilicagelH.
•SilicagelHcontainingfluorescence
activity=SilicaGelHF254.

19. Developing the Plates
•Afterpreparingthedevelopmentchamberand
spottingthesamples,theplatesarereadyfor
development.
•Becarefultohandletheplatesonlybytheiredges,
andtrytoleavethedevelopmentchamber
uncoveredforaslittletimeaspossible.
•Whentheplatesareremovedfromthechamber,
quicklytracethesolventfront(thehighestsolvent
levelontheplate)withapencil.

21. PRE-PREPARED TLC PLATES

22. Identifying the Spots (visualization)
•If the spots can be seen, outline
them with a pencil.
•If no spotsare obvious, the most
common visualization technique
is to hold the plate under a UV
lamp.
•Many organic compounds can be
seen using this technique, and
many commercially made plates
often contain a substance which
aids in the visualization of
compounds.

23. Visualizing Agents
1.Alkaloids:Dragendorff’sreagent-orangeor
orangeyellowspots.
2.Cardiacglycosides:Antimonytrichloride-
Appearsofblueorvioletcolour
3.Sugar:Anilinephthalate-brightredcolour
4.Aminoacids:Ninhydrin-purplecolor
5.Aldehydes&Ketones:2,4-DNPHsprayin
methanolandsulphuricacidresultsinorangeor
yellowspots

24. Interpretation of Data
•TheR
f(retentionfactor)valueforeach
spotshouldbecalculated.
•Itischaracteristicforanygivencompound
onthesamestationaryphaseusingthe
samemobilephasefordevelopmentof
theplates.
•Hence,knownR
fvaluescanbecompared
tothoseofunknownsubstancestoaidin
theiridentifications.

26. Importance of TLC
•Usedfortheseparationofunknownmixture
•Todeterminethepigmentofplantcontains
•Todetectthepesticidesandinsecticidesin
food
•Toidentifythecompoundpresentinthe
mixture
•Aminoacidseparation
•Vitaminsseparation

28. References
•Analytical chromatography by Dr. G.R Chatwal
•PharmaceuticalAnalysis-II,Instrumental
MethodsbyP.C.Kamboj
•InstrumentmethodsofanalysisbyScoogand
West
•TextbookofPharmaceuticalAnalysisbyDr.
S.R.Sankar.

30. THANK YOU