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
Name | Composition | Adsorbent : water ratio |
Silica gel H | Silica gel without binder | 1 : 1.5 |
Silica gel G | Silica gel + calcium sulphate | 1 : 2Â Â |
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 platesÂ
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 platesÂ
•      Plates are activated by keeping in an oven at 100 0C to 120 0C for 1 hour
Frequently Asked Questions (FAQs)
- What are the key components of a TLC setup?
- The key components include TLC plates, sample applicators, developing chambers, and visualization tools.
- 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.
- 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.
- 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.
- 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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