Thin Layer Chromatography - Instrumental Methods of Analysis B. Pharma 7th Semester

Thin Layer Chromatography

Contents

•       Thin layer chromatography

•       History

•       Principle involved

•       Applications of TLC

Practical requirements of TLC

•       Stationary phase

•       Glass plates

•       Preparation and activation of TLC plates

•       Application of sample

Objectives

By the end of this session, students will be able to:

Ø  Explain the principle involved in Thin layer Chromatography

Ø  Outline the components of TLC

Ø  Discuss the preparation and activation of TLC plates

Thin layer chromatography

History

•       In 1938, Izmailov and Shraiber separated plant extracts using 2 mm thick and firm layer of alumina on glass plate

•       In 1944, Consden, Gorden and Martin used filter papers for separating amino acids

•       In 1950, Kirchner identified terpenes on filter paper and

•       Later glass fiber paper coated with alumina

•       In 1958, Stahl developed standard equipment for analyzing by thin layer chromatography

Principle

•       Principle of separation is adsorption

•       One or more compounds are spotted on a thin layer of adsorbent coated on a chromatographic plate

•       Mobile phase solvent flows through because of capillary action (against gravitational force)

•       Components move according to their affinities towards adsorbent

•       Component with more affinity towards stationary phase moves slower

•       Lesser affinity moves faster

•       Components are separated on a thin layer chromatographic plate based on affinity towards stationary phase

Advantages of TLC

•       Simple method and cost of equipment is low

•       Rapid technique and not time consuming like column chromatography

•       Separation of µg of the substance can be achieved

•       Any type of compound can be analysed

•       Efficiency of separation- very small particle size can be used to increase the efficiency of separation

•       Detection is easy and not tedious

•       Capacity of thin layer can be altered

•       Analytical and preparative separations can be made

•       Corrosive spray reagents can be used without damaging the plates

•       Needs less solvent, stationary phase and time for every separation when compared to column chromatography

Practical Requirements

•       Stationary phase

•       Glass plates

•       Preparation and activation of TLC plates

•       Application of sample

•       Development tank

•       Mobile phase

•       Development technique

•       Detecting or visualizing agents

Stationary Phase

•       Several adsorbents can be used as stationary phase

•       Composition and ratio in which they have to be mixed with water or the solvents to form a slurry

Name	Composition	Adsorbent : water ratio
Silica gel H	Silica gel without binder	1 : 1.5
Silica gel G	Silica gel + calcium sulphate	1 : 2
Silica gel GF	Silica gel + binder + fluorescent indicator	1 : 2
Alumina Neutral Basic Acidic	Al2O3 without binder	1: 1.1
Cellulose powder	Cellulose with binder Cellulose without binder	1 : 6 1 : 5

Glass Plates

•       Specific dimensions like 20 cm x 20 cm (full plate)

•       20 cm x 10 cm (half-plate)

•       20 cm x 5 cm (quarter plate)

•       Width of commercially available TLC spreader is 20 cm

•       Can prepare plates of different dimensions without using TLC spreader

•       Microscopic slides can also be used for some applications like monitoring the progress of a chemical reaction

•       Development time is much shorter than 5 minutes

•       Glass plates should be of good quality and should withstand temperatures used for drying the plates 

Preparation and Activation of TLC Plates

•       Slurry which is a mixture of stationary phase and water is prepared by using the ratio mentioned earlier

•       After preparing the slurry, TLC plates can be prepared by any of the technique

Pouring

•       Slurry is prepared and poured on the glass plate which is maintained on a levelled surface

•       Slurry is spread uniformly on the surface of glass plate

•       Plates are dried in an oven

•       Drawback is uniformity in thickness cannot be ensured

Dipping

•       Two plates are dipped in to the slurry and are separated after removing from slurring and are dried

•       Drawback is large quantity of slurry is required even for preparing fewer plates

Spraying

•       Resembles that of using perfume spray on a cloth

•       Suspension of adsorbent or slurry is sprayed on a glass plate using a sprayer

•       Drawback is layer thickness cannot be maintained uniformly all over plate

Spreading

•       Best technique where a TLC spreader is used

•       Glass plates of specific dimensions (20 cm x 20 cm/ 10 cm / 5 cm) are stacked on a base plate

•       Slurry after preparation is poured inside the reservoir of TLC spreader

•       Thickness of adsorbent layer is adjusted by using a knob in the spreader

•       Normally a thickness of 0.25 mm is used for analytical purpose

•       2 mm thickness for preparative purpose

•       Spreader is rolled only once on the plates

Activation of Plates

•       Plates are allowed for setting (air drying)

•       Done to avoid cracks on the surface of adsorbent

•       Plates are activated by keeping in an oven at 100 ⁰C to 120 ⁰C for 1 hour

Activation of TLC plates

•       Nothing but removing water/ moisture and other adsorbed substances

•       By heating at high temperatures

•       Activated plates can be stored in thermostatically controlled oven or in desiccator

•       Used whenever required

Application of sample

•       To get good spots, concentration of sample or standard solution has to be minimum

•       2-5 µl of a 1% solution is spotted using a capillary tube or micropipette

•       Spots can be placed at random or equidistant from each other by using a template with markings

•       Spots should be kept atleast 2 cm above the base of the plate

•       Spotting are shouldn’t be immersed in the mobile phase in the development tank

•       Atleast 4 spots can be spotted conveniently on a quarter plate (20 cm x 5 cm)

Summary

•       In 1958, Stahl developed standard equipment for analyzing by thin layer chromatography

•       Mobile phase solvent flows through because of capillary action (against gravitational force)

•       Components move according to their affinities towards adsorbent

•       Very small particle size can be used to increase the efficiency of separation

•       Needs less solvent, stationary phase and time for every separation when compared to column chromatography

•       Glass plates should be of good quality and should withstand temperatures used for drying the plates 

•       Plates are activated by keeping in an oven at 100 ⁰C to 120 ⁰C for 1 hour

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