Slide 1: Thin Layer chromatography By
Abhisek Bhunia Topic include : Topic include Introduction : In 1944, Consden,Gorden & Martin used filter papers for separating the Amino acids.
In 1950, Kirchner identified terpenes on filter paper.
In 1958,STAHL develop standard equipment for analysing by THIN LAYER CHROMATOGRAPHY . Introduction Principle : The Principle is based on ADSORPTION Chromatography
The component with more affinity towards the stationary phase travels slower.
The component with lesser affinity towards the stationary phase travels faster. Principle Reverse phase chromatography : In this the stationary phase is Non-polar & mobile phase is polar & it is widely used in pharmaceutical analysis.
Polar compound get eluted first
Non-polar compounds are retained for long time
Comparison of Normal phase & Reverse phase : Reverse phase chromatography Stationary phase : Stationary phase Mobile phase : Nature of the substance to be separated i e weather it is polar or non-polar.
Mode of Chromatography.
Nature of Stationary phase.
Mode Separation i e Analytical or Preparative.
Examples: 1) Petroliam ether 2) Cyclohexane
3) Acetone 4) Toluene
5) Ethyl acetate 6) Benzene
7) Alcohols 8) Water
9) Chloroform 10) Pyridine Mobile phase Glass plates : Three types : they are ,
Full plate : 20cm × 20 cm.
Half plate : 20cm × 10 cm.
Quarter plate : 20cm × 5 cm.
Microscopic slides can also be
used for Monitoring the progress of a chemical reaction. Glass plates Preparation & activation of plates : The T L C plates can be prepared by following techniques :
It is nothing but removing of water/ moisture & other adsorbed substance from the surface of any adsorbent by heating. Preparation & activation of plates Application of sample : Application of sample Development tank : The development tank
should be lined Inside
with filter paper moistened
with mobile phase to
saturate the atmosphere
& also prevent the
“ EDGE EFFECT ” . Development tank Development technique : Different development techniques are :
One dimensional development.
Two dimensional development.
Multiple development. Development technique Detecting agents : Detecting agents are two types. they are,
1) Iodine chamber method.
2) Sulphuric acid spray method.
3) UV chamber for fluorescent compounds.
4) Using fluorescent stationary phase.
(B) Specific method
1) Ferric chloride.
2) Ninhydrine in acetone.
3) Dregendroff reagent.
4) 3,5 – Dinitro benzoic acid.
5) 2,4 - Dinitro phenyl hydrazine. Detecting agents detection : The Rf value is calculated for
identification "Rf value is the
ratio of distance travelled by
The solute to the distance
travelled by the solvent front”
Distance travelled by solute
Distance travelled by solvent front detection Development of t l c : Development of t l c application : Separation of mixture of drug of chemical,biological,plant origin.
Separation of Carbohydrates, vitamin, antibiotics, proteins, etc.
Identification of drug. Ex :Amoxicillin, Levodopa
Detection of foreign substances.
To detect the decomposition products of drug. application Comparison of tlc & paper chromatography : Comparison of tlc & paper chromatography Comparison of tlc & hplc : Comparison of tlc & hplc Reference : E Stahl. Thin-layer Chromatography. A Laboratory Handbook: Springer – Verlag Berlin . Heidelberg ,New York 1969:second edition 1990.
G Chatwal,S anand.Ihstrumental Methods of Chemical analysis: Himalaya publishing house,Delhi:Edition June 1995.
Dr.S. Ravi Sankar.Text book of pharmaceutical analysis:Rx Publication,Tiruneveli:Edition 1997,1999,2001.
H.F.Waltor,J.Reyes:Modern Chemical analysis and instrumentation. International Medical Book distrubutor,Mumbai.
Thin layer chromatography is available from www.encyclopidia.org.
Thin layer chromatography is available from www.chem.vt.edu
Thin layer chromatography is available from www.orgchem.com
Thin layer chromatography is available from www.wellesly.eud Reference Slide 20: THANK YOU