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

Thin Layer Chromatography Instrumental Methods of Analysis

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 : 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

       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.

 For Thin layer chromatography PDF Notes Click on the Download Button

Leave a comment