Download Chromatography: Principles, Techniques, and Applications and more Study notes Chemistry in PDF only on Docsity!
CHROMATOGRAPHY
- CHROMATOGRAPHY............................................................................................................................ Table of Contents
- Introduction:.......................................................................................................................................
- Principle:....................................................................................................................................
- Components:..............................................................................................................................
- Purpose:......................................................................................................................................
- Classification:.....................................................................................................................................
- Paper Chromatography:.............................................................................................................
- Column Chromatography:..........................................................................................................
- Thin-Layer Chromatography:....................................................................................................
- Partition Chromatography:.........................................................................................................
- Adsorption Chromatography:....................................................................................................
- References:.................................................................................................................................................
CHROMATOGRAPHY
Introduction:
Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture for qualitative and quantitative analysis. The Russian botanist Mikhail Tswett coined the term chromatography in 1906. It is a powerful separation tool that is used in all branches of science and is often the only means of separating components from complex mixtures.i
Principle:
Chromatography is based on the principle where molecules in mixture applied onto the surface or into the solid, and fluid stationary phase (stable phase) is separating from each other while moving with the aid of a mobile phase. The factors effective on this separation process include molecular characteristics related to adsorption (liquid-solid), partition (liquid-solid), and affinity or differences among their molecular weights. Because of these differences, some components of the mixture stay longer in the stationary phase, and they move slowly in the chromatography system, while others pass rapidly into the mobile phase, and leave the system faster.ii
Components:
Three components thus form the basis of the chromatography technique. Stationary phase: This phase is always composed of a “solid” phase or “a layer of a liquid adsorbed on the surface solid support”. Mobile phase: This phase is always composed of “liquid” or a “gaseous component.” Separated molecules. The type of interaction between the stationary phase, mobile phase, and substances contained in the mixture is the basic component effective on the separation of molecules from each other.iii
Column Chromatography:
Since proteins have difference characteristic features as size, shape, net charge, stationary phase used, and binding capacity, each one of these characteristic components can be purified using chromatographic methods. Among these methods, most frequently column chromatography is applied. This technique is used for the purification of bio-molecules. On a column (stationary phase) firstly the sample to be separated, then wash buffer (mobile phase) are applied. Their flow through inside column material placed on a fiberglass support is ensured. The samples are accumulated at the bottom of the device in a time-, and volume-dependent manner.vii
Thin-Layer Chromatography:
Thin Layer Chromatography can be defined as a method of separation or identification of a mixture of components into individual components by using finely divided adsorbent solid / (liquid) spread over a plate and liquid as a mobile phase. Thin-layer chromatography is performed on a sheet of glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide (alumina), 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.viii
Partition Chromatography:
Partition Chromatography technique is defined as the separation of components between two liquid phases viz original solvent and the film of solvent used in the column. This separation theory was introduced in the year 1940s which was published by Richard Laurence Millington Synge and Archer Martin. It is also known as
Liquid-liquid chromatography (LLC). Or if gas is the mobile phase it is called Gas-liquid chromatography (GLC).ix
Adsorption Chromatography:
Adsorption chromatography is a process of separation of components in a mixture introduced into chromatography system based on the relative differences in adsorption of components to the stationary phase present in the chromatography column. This adsorption chromatography applies to only solid- liquid or solid-gas chromatography. Because the adsorption phenomenon is inherent property of solids and hence it is used with only solid stationary phase chromatographies.x
