PRANESH KUMARM.PHARM(Double Goldmedalist), Ph.D.(P)

BBAU Central Univesity, Lucknow

THIN LAYER CHROMATOGRAPHY

Table of content:

INTRODUCTION PRINCIPLE EXPERIMENTAL TECHNIQUE EVALUATION OF CHROMATOGRAM APPLICATION REFERENCES

Introduction:

Thin Layer Chromatography: “The separation of moderately volatile or nonvolatile substances based upon differential adsorption on an inert solid (stationary phase) immersed in a organic solvent or solvent mixture (mobile phase)”

Thin layer chromatography was first introduced by IZMAILOV & SHRAIBER in 1938.

Principle:

Principle of TLC

Adsorption Partition Ion Exchange Reversed Phase partition

Adsorption:

Chromatography on adsorbents such as silica gel and alumina has widely been employed for the fractionation of mixture of non polar material into class of compounds that migrate at different rate by virtue of their respective polarities.

Eg- Hydrocarbons are easily separated from

acids,alcohols & aldehydes.

Partition:

Separation of mixture is effected by virtue of difference in the solubilities of its components in developing solvent and the stationary liquid phase .

Ion Exchange: In ion exchange system the rate of migration

of a compound can be found by the total charge of ionised group per molicule.

Reversed Phase Partition:

The stationary phase is non polar and the mobile phase is polar .

R P partition has widely use for fractionation of hydrocorbons and other non polar petroleum products.

Experimental Techniques:

Coating materials

Preparation of thin layer in plates

Activation of adsorbents

Sample Application

Development Tank

Solvent system

Plate Development

Detection of Component

Evaluation of chromatogram

Coating materials:

A large no. of coating materials are used which are commercially produced as a thin adsorbents.

Adsorbents Acidic or Basic Components to be separated

Silica gel Acidic Acidic and neutral substances

Alumina Basic Basic and neutral

Cellulose powder Neutral Water soluble compounds

Preparation of thin layer in plates:

Methods:

Pouring

Dipping

Spraying

Spreading

Precoated

plates

Activation of adsorbents:

After making thin layer in plates to remove as completely as possible the liquid associated with the thin layer.

This is done by drying the thin layer plate for 30 min in air and then place in oven at 110 C for 30 min.

Sample application:

Agla microsyring is generally used for transferring the sample solution on thin layer .

Development tank:

In TLC the plate is placed in a development chamber at an angle of 45 .

The bottom of chamber is covered up to 1 mm by the solvent.

Solvent system:

Trial & Error: If one does not know about the nature of

component of mix to be separated the best elute is found by trial & error method.

Methods

Trial & error

Stahl’s triangle

Stahl’s triangle:

If one knows the chemical nature of solute to be separated it is possible to know a solvent by using Stahl’s triangle.

hydrophilic M non polar E Lipophilic

polar Inactive active S

Plate development:

The plate is placed in the closed container saturated with developing solvent.

Detection of component:

Most of methods are used for detection of separated salute on TLC.

One can see coloured compound usually. One can detect colourless compound by UV light

or with treatment of visualising agent.Eg. Iodine vapour Bromocresol green Ninhydrine Dragondroff reagent

Evaluation of chromatogram:

Methods

Qualitative Quantitative

Direct Indirect

Qualitative:

This is done by the help of measurement of migration parameters.

Migration paramet

ers

Rf Rx RM

Rf values:

solvent front

component B

component A

origin

dSdB

dA

Rf of component A =

dA

dS

Rf of component B =

dB

dS

The Rf value is a decimal fraction, generally only reported to two decimal places

Rx & RM Values:

Distance travel by substance from origin line

Rx = Distance travel by std sub x from the origin line

RM = log(1/Rf -1)

Quantitative :

Direct method:Visual

assesment of chromatogram

Measurment of spot area

Spectrophotometry

Dencitometry

Indirect method:

Methods

Gravimetric

UV spectroscopy

Fluorimetry

Colourimetry

Calorimetry

Flame photometry

Applications:

For checking the purity of sample. Examination of reactions. Identification of organic compounds. Separation of amino acids. Separation of inorganic ions. Progress of chemical reaction . As a purification process.

References:

1. R.A.Day, Jr. A.L.Undewood(1987). Analisis Kualititatif. Edisi ke Empat, ms: 474-529.

2. David G. Watson(2005). Pharmaceutical analysis. Edisi ke-2, ms 315-331

3. http//orgchem.colorado.edu/hndbooksupport/TLC/TLCprocedure.html

4. Chatewal R.G.Instrumentatal methods of chemical analysis,fifth edition 2002 Himalaya publishing house pg no. 2.599