Thin Layer Chromatography

Introduction

The word chromatography is originated from the Greek word “Chroma”- “color” and “graphics”-“writing”.

Thin-layer chromatography (TLC) is a class of liquid chromatography in which the stationary phase is in the form of a thin layer on a glass, or aluminum, or plastic support. The term “planar chromatography” is frequently used for both thin layer chromatography as well as paper chromatography as each of them employs a planar stationary phase rather than a packed column. TLC or thin layer chromatography was introduced by Izmailov and Shraiber in the year 1938. TLC is usually called a drop, strip, spread layer, surface chromatography, and open column chromatography.

TLC as a technique for analytical adsorption chromatography was initially introduced by Stahl in 1958 that was mainly responsible for bringing out standard equipment for thin layers. They separated the extracts obtained from plants using alumina as a stationary phase on glass plates. Cosden, Gordon, and Martin in 1944 started using filter papers and worked on amino acid separation which met significant success. Kirchner, in 1950, was the first to isolate and identify the Terpenes.

Analyzing a sample by the use of multiple separation steps and static post chromatographic detection methods with different universal and specific visualization reagents is promising because all the sample elements are stored on the layer without any chance of loss. Commercially, a high quantity of layers are possible, and hence, thin layer chromatography is very flexible and selective.

An apparent disadvantage of TLC is that although all of the individual steps have been automated and online coupling with other chromatographic and spectrometric methods has been done, complete automation of TLC has not been realized.

Principle

Similar to other chromatographic techniques, TLC is also dependent on the principle of separation. The separation is decided by the adsorption and relative affinity of compounds towards the stationary and mobile phases. The compounds under the effect of the mobile phase (which are driven by capillary action) travel over the external surface of the stationary phase. During this movement, the compounds with higher affinity to stationary phase travel slowly while the others travel faster. Thus, the separation of constituents in the mixture is achieved. Once separation takes place individual constituents are viewed as spots at the respective level of displacement on the plate. Their nature or characters are recognized utilizing suitable detection methods.

Preparation of Thin layers in plates

While preparing the TLC plate the primary requirement is that the layer should be consistent throughout the plate. To meet these criteria, there is a large number of applicators that are available at a commercial level and are used for coating the glass plates with diverse adsorbing materials of even thickness.

Activation of Adsorbent

Activation of chromatographic plates involves the removal of liquid content from the stationary phase. For this purpose, the TLC plate is permitted to dry at room temperature followed by drying in an oven at 110 degrees Celsius for 30 minutes. The activation period of the TLC plate varies with different adsorbing materials.

Purification of Silica Gel G layers

Silica Gel G contains iron as an impurity which causes a considerable distortion of the chromatographs. Iron-free layers are obtained by giving the coated and air-dried plates a preliminary development with methanol and concentrated HCl. This leads the iron to migrate at the top level of the plate and re-activation can be performed on the TLC plate.

Sample application

Agla micro syringe is generally used for transferring the sample solution to the thin layers for quantitative work. However, the use of capillary tubes is preferred for qualitative analysis. The solvent used for the sample solution should be volatile and as non-polar as possible.

Solvent system or Mobile phase in TLC

Mobile phase is liquid in thin layer chromatography. The mobile phase is usually a combination of two to five different solvents selected empirically using trial and error, guided by prior personal experience and literature reports of similar separations. In addition, various systematic mobile phase optimization approaches involve solvent classification (selectivity) and the electronic series (strength). Solvents like diethyl ether, isopropanol, ethanol, tetrahydrofuran, acetic acid, dichloromethane, ethyl acetate, dioxane, toluene, chloroform, hexane, etc. are commonly used. TLC is usually carried out with a single mobile phase, instead of using a mobile phase gradient.

Chromatogram Development

TLC is always carried out in the elution mode. After the sample solvent has evaporated, the plate is placed in a closed container saturated with vapors of the developing solvents. After the developer has transversed one-half to two-thirds the length of the plate, the latter is removed and dried and the position of the compound is determined in several ways.

Both quantitative and qualitative analysis is possible by using TLC. In qualitative analysis, the Rf value i.e. Retention factor of every individual compound helps the analyst to distinguish between two compounds since the Rf value for every compound is unique. Quantitative analysis can be performed directly on the TLC plate itself or after removing the substance from the TLC plate and then determined after elution also called the indirect method.

Applications of TLC

• Quantitative determination of high molecular weight compounds, particularly in medical and biological research.• TLC can provide rapid, low-cost qualitative analyses and screening to obtain information such as sample stability, purity, and uniformity and to follow the path of a reaction.

• TLC has been applied virtually in all areas of analysis, including chemistry, biochemistry, biology, industrial, agricultural, environmental, food, pharmaceutical, clinical, natural products, toxicology, forensics, plant science, bacteriology, and entomology.

• Rate of organic reaction, identification of organic compounds such as alcohols, alkaloids, amines, amino acids, proteins, peptides, antibiotics, etc.

• Separation of an inorganic ion such as zinc and iron, separation of amino acids and vitamins.