Thin Layer Chromatography


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THIN LAYER CHROMATOGRAPHY A Seminar Submitted to VIJAYA INSTITUTE OF PHARMACEUTICAL SCIENCES FOR WOMEN Enikepadu,Vijayawada-521108 By Alekhya Prasanna M. Pharmacy (PA&QA)


CONTENTS Introduction Principle of TLC Types of TLC Operational techniques involved Why TLC is superior than Paper Chromatography ? Advantages of TLC Applications


INTRODUCTION Thin layer chromatography (TLC) is a technique used to separate the components of a mixture using a thin stationary phase supported by an inert backing and mobile phase.




Separation: Separations in TLC involve distributing a mixture of two or more substances between a stationary and mobile phase. The Stationary Phase : is a thin layer of absorbent (usually silica gel or alumina) coated on a plate. The Mobile Phase : is a developing liquid which travels up to stationary phase, carrying the samples with it.

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Before TLC After TLC

Types of TLC:

Types of TLC

Operational techniques involved::

Operational techniques involved: Choice of adsorbent Preparation of plate Preparation and application of sample Choice of solvent Development of chromatogram Drying of chromatogram Detection of spot Quantitative estimation


Adsorbent: Two properties mainly decide their selection Particle size Homogeneity Examples: silica gel G, silica gel H, alumina, cellulose powder etc.


CLASSIFICATION OF ADSORBENTS Weak adsorbent (Sucrose, starch) Intermediate adsorbent (silica gel, magnesia) Strong adsorbent (alumina, charcoal) Organic adsorbent (cellulose powder, Starch, Sucrose) Inorganic adsorbent (Silica, Silica Gel, Glass powder) Classification of adsorbents Based on nature Based on binding strength


Plates: Three types of plates are use. Full plate : 20*20cm Half plate : 20*10cm Quarter plate :20*5cm Microscopic slides can also be used for monitoring the progress of a chemical reaction.

Preparation and activation of plates::

Preparation and activation of plates: Preparation: Pouring Dipping Spraying Spreading Activation: Heating plates at110 o C for 30 min

Mobile phase::

Mobile phase: Selection of mobile phase depends upon Nature of substance to be separated Viscosity and polarity of stationary phase Solvent used may be single (or) double phase. Examples: n-hexane, cyclohexane, CCl 4 , benzene, etc.

Insert filter paper to saturate atmosphere with solvent :

Insert filter paper to saturate atmosphere with solvent Keep the lid on!!

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Mark a line about 1 cm from the bottom with pencil It is important to use pencil

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Place TLC plate in chamber Let things develop! Don’t let the solvent front run off The top of the plate!!

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Pull it out and mark the solvent front before it evaporates Mark spots with pencil!

Detection methods::

Detection methods: Non-Specific methods: Iodine chamber Sulphuric acid spray method UV chamber for fluorescent compounds Specific methods: Ninhydrine in acetone Dregondroff reagent

Quantitative estimation::

Quantitative estimation:

Overview of TLC::

Overview of TLC:

Advantages of TLC::

Advantages of TLC: Short analysis time All spots can be visualized Adoptable to most pharmaceuticals Low cost Uses small quantities of solvent Requires minimal training Reliable and quick Minimum amount of equipment is needed Can be performed on an analytical as well as on a preparative scale

Why TLC is superior than paper chromatography ?:

Why TLC is superior than paper chromatography ? Thin Layer Chromatography Paper Chromatography 1.It require less amount of substance. 2.Less time consuming. 3.Sharpness of separation is good. 4.Physical strength is more. 5.Corrosive reagents can be coated.   1.Requires more amount of substance. 2.More time consuming. 3.Sharpness of separation is less than TLC. 4.Physical strength is less. 5.Corrosive reagents cannot be coated.  


Applications: Purity of the sample. In chemistry: Examination of the reaction. Identification of compound. In pharmaceutical industry.


References: B. K. Sharma, Instrumental Methods of Chemical Analysis, Twenty Seventh Edition:2011, GOEL Publishing House, Meerut, India, Pg.No.241 to 246. Gurdeep R. Chatwal ,Instrumental Methods of Chemical Analysis.

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