Chromatography is essentially a process of separation of molecular mixtures. It is a very common and frequently used technique of separation of various constituents of a sample before chemical analyses. The technique is based on different rates of movement of constituents of a mixture through a porous medium under the influence of some solvent or a gas.
The porous medium is called the Stationary phase while the solvent or gas which moves through the medium is referred to as the Mobile phase. Different types of chromatographic techniques used monitoring of the environmental quality are:
1. Paper chromatography.
2. Thin layer chromatography.
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3. Column chromatography.
4. High performance (pressure) liquid chromatography.
5. Gas chromatography.
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Paper chromatography is basically a process similar to solvent extraction. It is a type of partition process in which the substances are distributed between two liquids, one in stationary phase which is held between the fibres of the paper and the other is a solvent which moves through the paper. The constituents of the mixture to be separated move at different rate on the strip of paper and appear as spots at different points on the paper.
The differential movement of the constituents of the mixture is due to the difference in their solubility in the mobile phase or the solvent. Those substances which are more soluble move faster across the paper strip as compared to those which have lower solubility. The ratio of distance traversed by the substance and the solvent from the point of origin, or the spot where the mixture is deposited is referred to as R/value.
The R/ value of a substance, which is a function of partition coefficient is characteristic. It is a constant for a given substance provided that the conditions under which chromatographic system is kept are constant with respect to temperature, type of paper used, humidity; the amount of liquid in the reservoir etc.
The process of paper chromatography is simple. Mixtured to be separated is dissolved in a solvent and is applied with the help of micropipette to form a spot at one side of the filter paper (Whatman filter paper). It is dried, applied again and dried and so on till sufficient amount of the mixture is deposited on the paper. The place where mixture is deposited is marked and a line drawn across the paper strip.
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The strip is now dipped in a suitable solvent and placed straight in a chamber to isolate it from the outside atmosphere. The desired condition of humidity and temperature is maintained in the chamber. The solvent slowly moves up by capillary action, passes over the spot where material to be separated is deposited, carrying different constituents of the mixture at different rates of flow.
At about the time when solvent has reached the tip of the strip, the chromatogram is taken out dried and suitably treated to observe the spots on it. Different constituents of the mixture are seen forming separate spots which are usually arranged in a linear series. If the constituents of the mixture are coloured they can be seen as such. If the spot are colourless they have to be developed with the help of suitable reagents to make them visible. The R/values may be calculated by measuring the distances up to which the spot travels divided by the distance up to which the solvent has moved up the strip of paper.
When the mixture to be analysed is deposited near the bottom of the strip and the solvent or the mobile phase is made to move up the paper strip, the process is referred to as Ascending paper chromatography. When the mixture to be analysed is deposited on the top of paper strip and solvent is made to run downwards from a reservoir supported on some stand near the top, the process is termed as Descending paper chromatography.
The R/values which are characteristic for each substance may be compared with known standard Revalues from which the constituents of the mixture may be identified. In order to obtain a quantitative estimate the spot on the paper strip is cut out and placed in a small amount of solvent into which the constituent of the mixture dissolve out. This solution is then analysed by means of suitable analytical techniques.