Paper title-Analytical techniques in chemistry Chromatography Dr M.Vishnu priya, paper: VIIIA Semester-VI Paper title-Analytical techniques in chemistry

Introduction It is a physicochemical method for separation of complex mixtures was discovered at the very beginning of the twentieth century by Russian–Italian botanist M.S. Tswett. In his paper “On the new form of adsorption phenomena and its application in biochemical Chromatography analysis”

M.S Tswett

Primarily for the separation of plant pigments Such as chlorophyll, carotinoids and xanthophylls.Since these pigments have different colours (green, orange and yellow respectively) they gave the technique its name.

Chromatographic Analysis The sample mixture is dissolved in a fluid called mobile phase, which carries it through a structure holding another material called stationary phase. The various constituents of the mixture travels at different speeds, causing them to separate. The separation is based on different partitioning between stationary and mobile phase.

Terminology Stationary phase: This phase is always composed of a solid phase or a layer of liquid adsorbed on the surface of solid support. Mobile phase/Eluent: This phase is always composed of a liquid or gaseous components. Chromatogram: A visible record or a graph showing the result of separating the components of mixture by chromatography.

PRINCIPLE Chromatographic analysis is based on the principle where molecules in mixture applied on to the surface or into the solid and liquid stationary phase is separating from each other while moving with the aid of a mobile phase.

The mobile phase is then forced through immobile, immiscible stationary phase. A component quite soluble in 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.

Rf value

Calculation of Rf value Rf value is degree of retention of a component retardation factor. It does not have units, since lower and upper cases are in distance. Here the distance is measured up to the centre of the spot. Rf value=Distance travelled by the component/Distance travelled by the mobile phase

Types of chromatography

Paper chromatography Paper chromatography is an analytical method that is used to separate coloured chemicals or chemical substances especially pigments. This is useful for separating complex mixtures of compounds having similar polarity for example amino acids. If filter paper is used, it should be a high quality paper. The mobile phase is developing solutions that can travel up to the stationary phase carrying the sample along with it.

Paper chromatography

Principle The principle involved is partition chromatography where in the substances are distributed or partitioned between to liquid phases. One phase is water which is held in pores of filter paper used and other phase is that of mobile phase which moves over the paper. The compounds in the mixture get separated due to difference in their affinity towards water(in stationary phase) and mobile phase solvents during the movement of mobile phase under the capillary action of pores in the paper.

Similer to other chromatographic techniques TLC is also based on principle of separation.

Column chromatography Column chromatography is one of the most useful methods for the separation and purification of both solids and liquids. This is a solid-liquid technique in which the stationary is solid and mobile phase is liquid. Principle Adsorption Mixture of components dissolved in the mobile phase is introduced in to the column. Components move depending upon their affinities.

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