a
Stage 1 • The solution is spotted and allowed to dry. The original spot is identified as A. • The solvent begins to move up the paper by capillary action.
b
A
Stage 2 • The solvent moves up the paper, taking different components along at different rates. A
solvent front
Stage 3 • The separation of the mixture is complete. • The different components string out along the paper like runners in a race. A
Figure 2.17 a Various stages during paper chromatography. The sample is separated as it moves up the paper. b A paper chromatogram.
S
The distance moved by a particular spot is measured and related to the position of the solvent front. The ratio of these distances is called the Rf value, or retention factor. This value is used to identify the substance: Rf =
distance moved by the substance nt front distance moved by the solven
Originally, paper chromatography was used to separate solutions of coloured substances (dyes and pigments) since they could be seen as they moved up the paper. However, the usefulness of chromatography has been greatly increased by the use of locating agents (Figure 2.18). These mean that the method can also be used for separating substances that are not coloured. The paper is treated with locating agent after the chromatography run. The agent reacts with the samples to produce coloured spots. Chromatography has proved very useful in the analysis of biologically important molecules such as sugars, amino acids and nucleotide bases. In fact, molecules such as amino acids can be ‘seen’ if the paper chromatogram is viewed under ultraviolet light. 32
Cambridge IGCSE Chemistry
The purity and identity of substances Paper chromatography is one test that can be used to check for the purity of a substance. If the sample is pure, it should only give one spot when run in several different solvents. The identity of the sample can also be checked by comparing its Rf value to that of a sample we know to be pure. Probably the most generally used tests for purity are measurements of melting point or boiling point. As we saw earlier, impurities would lower the melting point or raise the boiling point of the substance. They would also make these temperatures less precise. These temperatures have been measured for a very wide range of substances. The identity of an unknown substance can be found by checking against these measured values for known pure substances. The process of purification is of crucial importance in many areas of the chemical industry. Medicinal drugs (pharmaceuticals) must be of the highest possible purity. Any contaminating substances, even in very small amounts, may have harmful side effects. Coloured dyes (food colourings) are added to food and drinks to improve their appearance. The colourings added need to be carefully controlled. In Europe the
Original material © Cambridge University Press 2014