INTRODUCTION
Chromatography is usually based on the principle of partition of solute between two phases. It usually consists of a Mobile Phase and a Stationary Phase. The Mobile Phase usually refers to the mixture of the substances to be separated dissolved in a liquid or a gas. The Stationary Phase is a porous solid matrix through which the sample contained in the mobile phase percolates. It is a physical process where the components (solutes) of a sample mixture are separated as a result of their differential distribution between stationary and mobile phases. Greek chroma meaning ‘color’ and graph meaning ‘writing’ Stationary Phase.
HISTORY
Tswet, Russian botanist (referred to as Father of chromatography) is credited for the development of chromatography.
Principle
Chromatography is usually based on the principle of partition of solute between two phases. It usually consists of a Mobile Phase and a Stationary Phase. The Mobile Phase usually refers to the mixture of the substances to be separated dissolved in a liquid or a gas. The Stationary Phase is a porous solid matrix through which the sample contained in the mobile phase percolates.
CHROMATOGRAM
A graphical presentation of detector response, concentration of analyte in the effluent, or other quantity used as a measure of effluent concentration. The retention time or volume is when a solute exits the injector and passes through the column and the detector. Data represented by the chromatogram are used to help identify and quantify the solute(s).
Because eluting solutes are displayed graphically as a series of peaks, they are frequently referred to as chromatographic peaks. These Peaks are described in terms of peak (1) width, (2)height, (3)area
CLASSIFICATION
Chromatographic methods can be classified in three different ways :-
a) Based on shape of chromatographic beds. Example - Planar and column Chromatography
b) Based on the physical state of mobile and stationary phase. Example - Gas and liquid chromatography
c) Based on mechanism of separation. Example -Ion-exchange chromatography, partition, affinity and adsorption chromatography.
Based on shape of chromatographic beds Chromatography Planar Paper Thin Layer (TLC) Column GAS (GC) Liquid (LC)
Planar Chromatography
In Planar Chromatography, stationary phase is present on a plane. The plane can be a paper impregnated by a substance acting as the stationary phase- Paper Chromatography or a Thin layer of a substance acting as a stationary phase spread on a glass, metal or plastic plate- Thin Layer Chromatography. Planar chromatography is also termed as Open Bed Chromatography.
Paper Chromatography
Paper chromatography is a liquid partition chromatography. In paper chromatography, the end of the paper is dipped in solvent mixture consisting of aqueous and organic components. The solvent soaks in paper by capillary action because of the fibrous nature of paper. The aqueous component of the solvent binds to the cellulose paper and thereby forms a stationary phase with it. The organic component of the solvent binds continues migrating, thus forming the mobile phase.
Mechanism of Separation
Mobile Phase rises up by capillary action. The testing sample is concentrated at a minute spot at the bottom of the filter paper. The sample mixture gradually rises up with the mobile phase, which is liquid. Compounds in the mixture will be separated according to their ability of solubility. More Polar substances will move slower and less polar substances will travel faster.
Procedure
A small spot of sample is applied to a strip of chromatography paper about two centimeters away from the base of the plate. This sample is absorbed onto the paper and may form interactions with it. The paper is then dipped into a solvent, such as ethanol or water, taking care that the spot is above the surface of the solvent, and placed in a sealed container.
The solvent moves up the paper by capillary action and dissolves the sample mixture, which will then travel up the paper with the solvent solute sample. Different compounds in the sample mixture travel at different rates. It takes several minutes to several hours. Analysis- Spots corresponding to different compounds may be located by their color, UV light, Ninhydrin or by treatment with iodine vapors.
Ascending paper Chromatography-
In this method, the solvent is to pool at the bottom of the vessel in which the paper is supported. It rises up the paper by capillary action against the force of gravity.
Descending paper Chromatography-
In this method, the solvent is kept in a trough at the top of the chamber and is allowed to flow down the paper. The liquid moves down by capillary action as well as by the gravitational force.
Significance of Paper Chromatography
It is very easy, simple, rapid and highly efficient method of separation. Can be applied in even in micrograms quantities of the sample. Can also be used for the separation of a wide variety of material like amino acids, oligosaccharides, glucosides, purines and pyrimidines, steroids, vitamins and alkaloids like penicillin, tetracycline and streptomycin.
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