-V. K. Gupta

Chromatography, although primarily a separation technique, is mostly employed in chemical analysis. Nevertheless, to a limited extent, it is also used for preparative purposes, particularly for the isolation of relatively small amounts of materials that have comparatively high intrinsic value. Chromatography is probably the most powerful and versatile technique available to the modern analyst. In a single step process it can separate a mixture into its individual components and simultaneously provide an quantitative estimate of each constituent. Samples may be gaseous, liquid or solid in nature and can range in complexity from a simple blend of two entantiomers to a multi component mixture containing widely differing chemical species. Furthermore, the analysis can be carried out, at one extreme, on a very costly and complex instrument and at the other, on a simple, inexpensive thin layer plate, of course with varied accuracies and precisions.

Types of Chromatography:

• Paper Chromatography
• Thin Layer Chromatography
• Column Chromatography
• Gas Chromatography
• Liquid Chromatography

Paper Chromatography:  This  is an analytical chemistry technique for separating and identifying mixtures that are or can be colored, especially pigments. This can also be used in secondary or primary colors in ink experiments. This method has been largely replaced by thin layer chromatography, however it is still a powerful teaching tool. Two-way paper chromatography, also called two-dimensional chromatography, involves using two solvents and rotating the paper 90° in between. This is useful for separating complex mixtures of similar compounds, for example, amino acids.

Thin Layer Chromatography (TLC) :
This  technique is been used in separation of mixtures. TLC is performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose. This layer of adsorbent is known as the stationary phase.
After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved.

Thin Layer Chromatography finds many applications, in

• Assaying the radiochemical purity of radiopharmaceuticals
• Determination of the pigments a plant contains
• Detection of pesticides or insecticides in food
• Analysing the dye composition of fibers in forensics, or
• Identifying compounds present in a given substance
• Aflatoxins in agro products

Column Chromatography 
In chemistry this method is used to purify individual chemical compounds from mixtures of compounds. It is often used for preparative applications on scales from micrograms upto kilograms, and for cleaning the analyte of the matrix interferences, for use with more sophisticated techniques.
The classical chromatography column is a glass tube with a diameter from 50 mm and a height of 50 cm to 1 m with a tap at the bottom. The individual components are retained by the stationary phase differently and separate from each other while they are running at different speeds through the column with the eluent. At the end of the column they elute one at a time. During the entire chromatography process the eluent is collected in a series of fractions. The composition of the eluent flow can be monitored and each fraction is analyzed for dissolved compounds, e.g. by analytical chromatography, UV absorption, or fluorescence. Colored compounds (or fluorescent compounds with the aid of an UV lamp) can be seen through the glass wall as moving bands.

Gas-Liquid Chromatography (GLC) or simply Gas Chromatography (GC)
This is a common type of chromatography used in organic chemistry for separating and analyzing compounds that can be vaporized without decomposition. Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture (the relative amounts of such components can also be determined). In some situations, GC may help in identifying a compound. In gas chromatography, the moving phase (or  mobile phase) is a carrier gas, usually an inert gas such as helium or an un reactive gas such as nitrogen. The stationary phase is a microscopic layer of liquid or polymer on an inert solid support, inside a piece of glass or metal tubing called a column. The instrument used to perform gas chromatography is called a gas chromatograph.
In principle, GC is similar to column chromatography (as well as other forms of chromatography, such as HPLC, TLC), but has several notable differences. Firstly, the process of separating the compounds in a mixture is carried out between a liquid stationary phase and a gas moving phase, whereas in column chromatography the stationary phase is a solid and the moving phase is a liquid. (Hence the full name of the procedure is Gas-liquid chromatography, referring to the mobile and stationary phases, respectively.) Secondly, the column through which the gas phase passes is located in an oven where the temperature of the gas can be controlled, whereas column chromatography (typically) has no such temperature control. Thirdly, the concentration of a compound in the gas phase is solely a function of the vapor pressure of the gas.

High Performance Liquid Chromatography or High Pressure Liquid Chromatography, (HPLC)
This is a form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify and quantify compounds. HPLC utilizes a column that holds chromatographic packing material (stationary phase), a pump that moves the mobile phase(s) through the column and a detector that shows the retention times of the molecules. Retention time varies depending on the interactions between the stationary phase, the molecules being analyzed and the solvent(s) used.

Process
Chromatography involves a sample (or sample extract) being dissolved in a mobile phase (which may be a gas, a liquid or a supercritical fluid). The mobile phase is then forced through an immobile, immiscible stationary phase. The phases are chosen such that components of the sample have differing solubilities in each phase. A component, which is quite soluble in the stationary phase will take longer to travel through it than a component which is not very soluble in the stationary phase but very soluble in the mobile phase. As a result of these differences in mobilities, sample components will become separated from each other as they travel through the stationary phase.
Techniques such as HPLC and GC.  use columns - narrow tubes packed with stationary phase, through which the mobile phase is forced. The sample is transported through the column by continuous addition of mobile phase. This process is called elution. The average rate at which an analyte moves through the column is determined by the time it spends in the mobile phase.

VK Gupta
is GM-Operations & Business Development
with Shivatec-India
Email: gupta@shivatec-india.com