Thin layer chromatography (TLC): Principle, Procedure, Applications and Advantages

Thin layer chromatography(TLC) is a type of planar chromatography which has been widely used for separation, identification and analysis of chemical compounds. It is a method of chromatography involving a thin layer of absorbent material that is coated on a glass, plastic or metal surface. It is also referred to as open column chromatography, drop strip, spread layer or surface chromatography.²

TLC technique was introduced by Izmailov and Shrabier in 1938 as an improvement to paper chromatography. Later in 1958, TLC was introduced as a procedure for analytical absorption chromatography by Stahl.² TLC is a simple, rapid, cost-effective and versatile analytical technique applicable across various diverse fields, including pharmaceutical, food science, forensic analysis, and biochemistry.

1.1 Principle

Thin layer separations are usually performed on a glass plate coated with thin and adherent layer of finely divided particles, referred to as stationary phase. The mobile phase moves through the stationary phase by capillary cation, which maybe sometimes assisted by gravity or electrical potential.¹ As the mobile phase moves across the layer from its one edge to the opposite edge, the component of a mixture separate because of the difference in affinity of the compounds towards the stationary phase and their solubility in the mobile phase. Polar compounds tend to interact strongly with stationary phase and move slowly, whereas less polar compounds move rapidly with solvent. The movement of the analyte is expressed by relative fraction.²

1.2 Procedure of TLC

A. Preparation of TLC plate

A thin-layer plate is prepared by spreading an aqueous slurry of finely ground solid on the clean surface of glass, plastic plate etc. The size of the plate is generally 20 × 20 cm.² The plate is allowed to stand still until the layer has set and adheres tightly to the surface. Conventional plates have thickness 200-250µm, having particles size of 20µm or greater.¹ Some of the common adsorbent used in TLC are Silica gel, alumina, cellulose powder, kieselgurh, etc.

B. Sample application

It is the most critical aspect of TLC, particularly for quantitative measurements. Generally, 0.01% – 0.1% solution of the sample is applied as a spot 1 – 2 cm from the edge of the plate using a micropipette, micro- syringe or Pasteur pipette, 1.5 cm apart from each other. Minimal spot diameter favors separation efficiency.

Fig: Thin layer chromatography setup. (Source: EVALUATION AND CHARACTERIZATION OF TRANSDERMAL THERAPEUTIC SYSTEMS: AN EXHAUSTIVE PICTORIAL AND FIGURATIVE REVIEW – Scientific Figure on ResearchGate.)

C. Plate development

It is the process in which a sample is carried through the stationary phase by a mobile phase. The most common way of developing a plate is to place a drop of the sample near one edge of the plate and mark its position. After the sample solvent has evaporated completely using a dryer, the plate is placed in a closed container saturated with vapors of the developing solvent. The end of the plate containing the dried sample drop is immersed into the developing solvent.¹ However, it is important to ensure that direct contact between the sample and the developer is avoided. After the developer has transversed one half or two thirds of the length of the plate, the plate is removed from the container.

D. Choice of solvent

Choosing a solvent depends upon two factors; nature of compound to be separated and material on which separation is to be carried out. The mixture of two or more solvents of different polarities often give better result than chemically homogenous solvent. Generally, polar substances such as alcohol, carboxylic acid, and amine solution should be used as polar solvent when separation is performed on cellulose or silica gel layers. Polar solvent supports greater migration and allowing better separation of compounds.

Some of the solvent system used in TLC are as follows:

E. Locating analytes on the plate

Different methods can be used to locate sample components after separation. Common methods applied to most organic mixtures include spraying with a solution of sulphuric acid or placing the plate in a chamber containing few crystals of iodine. Both the reagents react with organic compounds on the plate to yield distinct dark spots. Fluorescent material can be incorporated into the stationary phase, and the plate is examined under UV light after development. Some of the specific spray reagent used in the TLC are as follows:

F. Qualitative analysis of the spot

R_f values are calculated once the spots are visualized. The movement of the compound relative to the solvent is expressed in terms of R_f value. The ratio of the distance travelled by the substance and the distance traveled by solvent front is called R_f value. R_f values are evaluated for qualitative analysis.

G. Quantitative analysis

It can be performed by two methods:

Direct method

It includes visual comparison of the spots. Photodensitometry method and Flourimetry method are used for this purpose.

Indirect method

The distinct spots of the compounds are scraped off from chromatoplates and eluted with solvent.

1.3 Advantages of TLC

• It requires simple equipment.
• It is cost-effective and versatile technique. It can be applied to a wide variety of organic and inorganic compounds.
• Through TLC, compound separation can be performed in an hour or less.
• A wide range of choice of stationary phase is available.
• It is highly sensitive, 10 times more than paper chromatography.
• TLC provides clear distinct spot.
• Component can be recovered easily for further analysis by scraping the coating of the plate

1.4 Application of TLC

• TLC has been popularly used for qualitative and quantitative analysis over paper and column chromatography.
• Due to its high sensitivity, TLC can be used to check purity of a compound.
• It is popular as analytic tool in organic chemistry.
• Completion of a reaction can be determined by using TLC.
• Inorganic compounds, cations, anions, and organometallic compounds, etc. can be separated using TLC.
• Urine and blood in pathological laboratory can be analyzed using TLC.
• It can be used to detect presence of pesticides and insecticides in food.
• It is used in pharmaceutical industry for quality control of raw materials and finished drugs.
• It can also be used to analyze metabolic products, antibiotics and vitamins.

REFERENCE

1. D.A. Skoog, F.J. Holler, S.R. Crouch. Principles of instrumental analysis. Seventh edition. Cengage learning; 2017
2. M.R. Banjara, U.T. Shrestha. Instrumentation in Microbiology. First edition. Garuda Publication; 2021

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