Introduction to Chromatography

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Chromatography - Key Terms

Chromatography: A technique that can be used to identify and separate substances in a mixed solution

Paper chromatography is a simple technique based on the principle that some substances dissolve better in a solvent than others.

Thin Layer Chromatography is based on the principle that some substances adhere to the molecules of the stationary phase more strongly than others.

Paper chromatography can be used to:

Distinguish between pure and impure substances

Identify substances by comparing the pattern on the chromatogram with the patterns formed by known substances

Identify substances by calculating their \(R_f \text{ value. }\)

\(\text{ Stationary/ Immobile phase: }\) The solvent and dissolved substances move along this. In the case of paper chromatography, this is the paper. In the case of Thin Layer chromatography, the stationary/ immobile phase is aluminium oxide paste or silica gel

\(\text{ Mobile phase: }\) The solvent. This could be an aqueous solvent such as water, or a non-aqueous solvent such as ethanol

\(\text{ Chromatogram: }\) The paper with the separated components on it

\(R_f \text{ value: }\) The distance the compound has risen divided by the distance the solvent has risen. Both measurements are taken from the starting positions of the samples on the paper. This value depends on the mobile phase i.e. what solvent is used and the stationary phase i.e. what type of paper is use

\( R_f \text{ value= }\frac{\text{Distance moved by the spot}}{\text{Distance moved by the solvent}}\)

This value does not change if the chromatography conditions are the same.

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How Chromatography Works

The startline, known as the baseline, should be drawn in pencil so that it doesn't run or smudge in the solvent.

The paper/TLC plate should be placed in the container so that the baseline is above the solvent. This means that the spots do not dissolve in the water.

The lid prevents the solvent from evaporating into the laboratory.

The solvent moves up the paper, carrying the spots with it.

Some spots move at different speeds according to their:

Solubility in the mobile phase

Strength of molecular adhesion to the stationary phase

The final result is known as the chromatogram.

If a substance is pure, only one spot will appear on the chromatogram.

Any colour which horizontally matches another is likely to be made up of the same molecule.

Sometimes the substances being separated are colourless. In this case, locating agents can be used to show where the spots are.

Locating agents bind to the chemicals in the spots. Sometimes, another chemical is then added, which reacts with the locating agent to produce a coloured spot, or the chromatogram is put under ultraviolet light and the locating agent glows to show where the spots are