Thin Layer Chromatography Instrumental Methods of Analysis

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

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

•       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

Frequently Asked Questions (FAQs)

1. What are the key components of a TLC setup?
   • The key components include TLC plates, sample applicators, developing chambers, and visualization tools.
2. How does TLC differ from other chromatographic techniques?
   • TLC relies on a thin layer of adsorbent material for separation and is typically simpler and more cost-effective than techniques like HPLC or GC.
3. What is the significance of choosing the right solvent system in TLC?
   • The solvent system’s polarity affects the separation of compounds on the TLC plate, making it a crucial factor in the analysis.
4. What are the main applications of Thin Layer Chromatography?
   • TLC is widely used in pharmaceuticals, natural product analysis, food analysis, and environmental monitoring, among other fields.
5. What are the limitations of TLC as an analytical technique?
   • TLC may have limitations in terms of its separation power and sensitivity when compared to more advanced chromatographic methods.

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