First referred to in 1938 by Russian workers Izmailov and Shraiber, the method known as TLC today was used by American Chemists Meinhard and Hall in 1949 for separation of volatile oil components. Work of Kirchner and his associates and demonstration of its extensive utility by Stahl in 1958 gave impetus to the development of this technique.

TLC is one of the most popular and widely used separation techniques for all classes of natural products. Its versatility, speed, and sensitivity have established it as an analytical tool in modern pharmacopoeias.

TLC is also planar chromatography in which thin layers of sorbent (absorbing media) are coated on to a suitable support such as glass plate, plastic sheets, or aluminum foil. The mixture to be resolved is dissolved in a suitable solvent and applied as a spot, a short distance away from the edge (width side) of the plate. This side of the plate is dipped (a small angle away from the vertical) in a suitable solvent mixture without its surface reaching the applied spot. The whole set up is enclosed in an airtight chamber, such that it is saturated with the solvent vapor. The solvent rises up the plate due to capillary action and the solvent front travels up the plate to about 85% of its length. The plate is removed, the position of the solvent front marked, and the solvent allowed to evaporate. Depending on the nature of the sorbent and the solvent, mixture components get separated by either adsorption or partition (straight or reversed phase). Position of the separated components on the plate may be identified visually (coloured compounds), under UV light or by spraying with a suitable chromogenic agent.

Silica gel or silicic acid is the most popular and widely used sorbent. It is slightly acid in nature and is mixed with a binding agent such as calcium sulphate to hold it firmly to the supporting base. Silica gel of very fine particle size will adhere well even without a binder. Other sorbents used for different types of compounds are alumina (acid, basic, and neutral), celite, calcium hydroxide, kieselguhr, Magnesium silicate, Magnesium phosphate, polyamide, sephadex, PVP, cellulose, and ion exchange resins.

Fluorescent materials such as sodium flourescien, hydroxyl sulphonate, and rhodamine dyes may be included in the sorbent to facilitate detection of solutes, which quench the background fluorescence. When exposed to UV light (366 nm), these spots appear as dark spots against a greenish-yellow fluorescent background. Inorganic materials like uranyl acetate, manganese zinc silicate, zinc cadmium sulphate, zinc silicate, alkaline earth metal tungstates, and tin strontium phosphate may be included along with the sorbent to facilitate detection of fluorescent quenching compounds at UV 254 nm.

TLC plates may be pretreated with silver nitrate (argentative TLC) or cellulose for separations of isomeric compounds. In reverse phase chromatography, silica gel is treated with dichlorodimethyl silane making the sorbent layer hydrophobic. It is used for the separation of fatty acids, triglycerides, carotenoids, cholesterol esters, steroids, etc.

Like PC, TLC can also be used two dimensionally. Other modifications include electrophoretic separations, quantitative TLC (of spots by densitometric estimation based on UV/Vis absorption), autoradiography (for radioactive substances), and bioautography (biological detection of antibiotics).

Ready-mixed powders with binders are commercially available for making TLC plates of reproducible sorbent thickness. They need to be made into a slurry with water, before being laid on the plate. The films set quickly on air drying and they may be activated at about 105°C for 30 minutes prior to use. Commercially ready-to-use plates pre-coated with fine film of a range of sorbents (with or without fluorescent indicators) are now available.

The usual size of commercially available TLC plates is 20 × 20 cm. Microplates on microscope size support material are also available. Small strips of 10 × 2 cm may be conveniently cut out for use in qualitative detection. Film thickness of such pre-coated plates is characteristically of 250 µm thickness. For preparative separations, layers of thickness ranging from 0.5–10 mm may be used. Generally 1–2 mm thickness will suffice and plates are used in the sizes of 20 × 20 cm or 20 × 40 cm. Though resolving power of preparative plates may not be as good as regular analytical plates, it may still be conveniently prepared in lab for the isolation of quantities of components sufficient for complete structural characterization using IR, NMR, MS, etc.

Solvents used for TLC must be pure (free from admixed other solvents and water) and it is usual to use single or mixture of solvents in different ratios. The solvent mixture to be used for TLC development is arrived at by trial and error experimentation based on solubility characteristics of the compounds vis-a-vis the properties of the adsorbent and the mechanism effecting separation. Solvent selection is therefore one of the most important steps for the successful separation of compounds.

When silica gel, acidic alumina, magnesium silicate, or magnesium phosphate is the sorbent used; adsorption is the chromatographic mechanism effecting separation. A range of compounds such as steroids, amino acids, amine alcohols, hydrocarbons, lipids, bile acids, vitamins, alkaloids, and aflatoxins may be separated on these sorbents. Partition is the mechanism involved with sorbents such as cellulose, kieselguhr, and reversed phase silica gel. Compounds are partitioned between the water of hydration of cellulose fibers and solvents used for development. Commonly used solvents include n-hexane, cyclohexane, petroleum ether, ether, chloroform, ethyl acetate, butanol, isopropyl alcohol, ethanol, methanol, etc. Mobile phase used on silica gel plate often contain small quantities of ammonia solution, diethyl amine, acetic acid, dimethyl formamide, and pyridine.

Detection of compounds post development on TLC may be done by visual (with or without spray reagents) or UV examination. Depending on the nature of compounds, selected spray reagents are used for their detection. UV examination is usually done at 254 and 366 nms.

The developed TLC plate is either sprayed with the reagent or it is dipped in it. 50% sulphuric acid is most widely used for visualization of organic compounds (which on heating are seen as brown or black charred areas). Solutions of sulphuric acid-acetic anhydride (1:4) and Leibermann burchard reagent are used to detect steroids after heating at 140°C for 20 minutes. Vanillin-sulphuric acid reagent is used for detection of terpenes. Dragendorff’s reagent for alkaloids, p-anisaldehyde (with sulphuric acid and ethanol) is used for sugars, steroids, phenols, and terpenes, 2,6-Dichlorophenol-indophenol for organic acids, iodine for unsaturated fatty acids, phosphomolybdic acid in ethanol, or antimony trichloride in chloroform for steroids and flavonoids, 10% copper sulphate for sulphur containing glycosides and phosphotungstic acid for triterpenes.

TLC has become one of the most popular, simplest and most widely used separation technique applicable to the analysis of a vast range of compounds ranging from amino acids, sugars, fatty acids, alkaloids, lipids, vitamins, steroids, isoflavones, xanthones, and multiconstituent essential oils in addition to a range of plant pigments, nucleotides, and proteins.

High-end TLC techniques such as centrifugally accelerated TLC, over pressure layer chromatography are current innovative adaptions of conventional TLC for use in specific separations.

HPTLC is a sophisticated, advanced and an automated version of TLC, which combines the simplicity and precision of TLC with the speed and efficient quantitation, which modern instrumentation provides. It employs high-performance pre-coated silica gel plates, which give more efficient and reproducible separation than conventional grades of silica. Very small accurately measured volumes may be applied at a predetermined rate on exact locations at measured distances from neighboring spots if any. Development time is much smaller. Upon development, the spots may be evaluated by comparison with reference standards and also quantified with greater sensitivity and precision than is possible with conventional TLC.

Development of finger print profiles of plant extracts being one of the most salient quality control requirements in herbal drug standardization, HPTLC aids development of such profiles for multi constituent herbal drugs and formulations. Such profiles form important tools in the establishment of the identity of the plant as they are reproducible when performed under similar conditions of experimentation.

Merits of TLC over other chromatographic techniques

1. Economy in solvent and materials needed for development is its greatest advantage.
2. The short development time enables rapid separations compared to PC, with even time for equilibration being minimal.
3. Separated spots are more compact and better resolved from one another compared to PC.
4. Being simple, versatile, and easy to use, TLC is often used to develop solvent systems for and to monitor progress of separations for CC, GC, and HPLC.
5. There are available a wide range of ready-to-use TLC plates, pre-coated with different sorbents to choose from for any given analysis.
6. Using the right combination of sorbent and solvent, any type of separation is possible on TLC.
7. Multiple samples may be developed simultaneously, making it an extremely useful tool for routine analysis of a number of samples.
8. A highly sensitive technique, it can detect and separate even trace quantities of compounds from smaller quantities of mixture compared to PC.
9. TLC profile can act as a fingerprint record, making it suitable for monitoring identity and purity of drugs and also for detecting adulterations and substitutions.
10. TLC also gives semi-quantitative information about major phytoconstituents thus enabling an assessment of overall drug quality.
11. Reagents too drastic for PC such as concentrated sulphuric acid may be used for visualization in TLC.