Thin layer chromatography (TLC)

It stands for Thin Layer Chromatography.

It’s a simple technique used by chemists in separation of Mixtures in addition to supporting the identification of the separated compounds by comparing the “Retention Factor” of the separated compound with that of a known compound.

The TLC plate on which the Thin Layer Chromatography is performed is usually a sheet of glass, aluminum or plastic. It’s has a coat of adsorbent material ( known as stationary phase ) such as silica gel, alumina or cellulose.

First step is application of the sample to the plate, then in a capillary action- the chemist draws the solvent up the plate. Separation occurs due to the different ascendance rates of analytes on the plate.

Advantages of TLC over other types of chromatography

1. Paper Chromatography

• TLC is faster than paper chromatography.
• It’s more sensitive to many substances.
• Its preparation is usually sharper.
• It requires smaller quantity of sample.
• Potential application of different types of reagents without causing damage to the plate.

• TLC has smaller apparatus
• It has more speed
• Previous points leads to cheaper over all procedure
• It provides easier measurement of the “Retention Factor”.
• The above advantages makes it more suitable for analytical purposes rather than separation purposes.

Preparation of TLC plate

To prepare the TLC plate, first we must mix a small quantity of an inert substance such as “calcium sulfate” with adsorbent substance like “Silica gel” and water. The second step is spreading this mixture on sheet of unreactive substance such as “glass” or “plastic”. The third step is activation of the plate that results from the previous procedures by heating it in an oven at 110oC for nearly half an hour.

The adsorbent layer’s thickness depends on the purpose of TLC as in case of analytical purposes, it ranges from 0.1 to 0.25 mm. In case of preparative TLC it ranges from 0.5 to 2.0 mm.

Technique

A small amount of the sample’s solution is poured on the plate to cover about one centimeter high from the base. Then the plate is soaked in a convenient solvent like “Hexane” and put in a container that’s well sealed. Using capillary action, the solvent goes up the plate to meet the sample’s mixture which it helps to move up the plate and dissolves as well. Differences between rates of the compounds in the mixture are because each compound has different affinity to the adsorbent layer (stationary phase) from the other compounds and also each compound has different solubility in the mixture. If the solvent is changed, the compounds can be separated (using Retention Factor’s value).

Applications of TLC in the Pharmaceutical field

1. Thin-layer chromatography technique is used for separation of amino acids that are the main components of antibiotics, enzymes and hormones.
2. TLC-UV spectrometry was used to determine salidroside in 8 Rhodiola species ( Rhodiola is a herb that has been used for long time for treatment of cold and flu symptoms in addition to relieving mental and physical stresses. In China, it’s recently considered as promising anti-aging drug in addition to having causative agent for improvement of athletic performance and enhancing memory).
3. The combination of advanced chromatographic resolution of 2D-TLC with simple bioautography is used in discovering new natural products that are active against important fungal pathogens of plants.
4. Thin-layer chromatography in addition to HPLC are used for the identification of “Pogostemoni herba” which is a crude drug and a Kampo medicines-component.
5. TLC-colourimetric was used to estimate free and combined emodine, physcione and chrysophanol in Indian Rhubarb and it turned out to contain (0.07%)of free emodin of (0.08%) of C-glycosidic emodin, (0.30%) of O-glycosidic emodin, (0.40%) of free physcione, (0.18%) of O-glycosidic physcione, (0.17 %) of C-glycosidic physcione, (0.15%) of free chrysophanol, (0.06%) of O-glycosidic chrysophanol and (0.21%) of C-glycosidic chrysophanol.
6. TLC-UV Densitometric method is used in Misai Kucing Capsules for Simultaneous Quantification of Sinensetin and Tetramethoxyflavone.
7. TLC is used for determination of different compounds in analgesic tablets.
8. TLC-densitometric procedure is used for determination of the contents of Plumbagin which is a naphthoquinone that acts as antimalarial , antimalarial , anticancer, cardiotonic and antifertilityaction.
9. Quantitative Thin-Layer Chromatography is used for the determination of Propranolol in human plasma.
10. Determination of Steroidal Alkaloid Glycosides using Thin-Layer chromatography Immunostaining.
11. TLC is used to determine Phenytoin in pharmaceutical preparations and identify its Hydroxylated Urinary Metabolites.
12. Isolation of antioxidants from Perilla frutescens var. acuta fruit is carried out using TLC.
13. TLC- densitometric method is used to analyse artemisinin in the planlets of Artemisia annua L.
14. TLC is used to detect as well as identify opiates in urine in addition to reveilind adultration in heroin.
15. TLC-Densitometry is used in the Quantitative Analysis of Curcumin, Demethoxycurcumin and Bisdemethoxycurcumin in the Crude Curcuminoid Extract from Curcuma longa.