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Thin Layer Chromatography Instrumental Methods of Analysis

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 of Thin layer chromatography

• 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 in Thin layer chromatography

• 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)

Advantages and Limitations of Thin Layer Chromatography

Advantages of TLC:

Simplicity: TLC is easy to set up and execute, making it accessible for both beginners and experienced analysts.
Cost-Effective: It’s a cost-effective technique that requires minimal investment in equipment and materials.
Rapid Results: TLC provides quick results, allowing for efficient decision-making in research and analysis.

Limitations of TLC:

Limited Resolution: TLC may have limited resolving power compared to more advanced chromatographic techniques like High-Performance Liquid Chromatography (HPLC).
Sensitivity: It may not be as sensitive as other methods, making it less suitable for trace analysis.
Quantitative Analysis: While TLC is excellent for qualitative analysis, it may not be the best choice for precise quantitative measurements.

Summary