The fact that different amino acids carry different net charges at any particular pH permits mixtures to be separated using low or high voltage electrophoresis. The most frequently used supporting media are paper or thin-layer sheets (cellulose or silica gel) and the locating reagents already described for chromatography may be used for visualization of the spots. Separations at high voltages can be achieved more quickly than at low voltages and one of the principal advantages of the former is that salts and other substances that may be present in the sample affect the quality of electrophoretogram to a lesser extent. This permits the separation of amino acids in relatively crude extracts and untreated fluids, whereas prior to low voltage electrophoresis it is necessary to remove interfering substances such as proteins, carbohydrates and salts using the same methods as described for chromatography.
Although electrophoretic separations can be achieved using buffers over a wide range of pH values, in practice the pH values chosen are either pH 2.0 or pH 5.3. At pH 2.0 all amino acids will carry a positive charge and the basic amino acids, having the highest positive charge, will migrate furthest towards the cathode whereas at pH 5.3 migration will occur towards both electrodes depending on the charge carried. Separations at pH 5.3 are particularly useful to determine the acidic or basic nature of an unknown amino acid or dipeptide.
A two-dimensional technique involving initial separation by high voltage electrophoresis at pH 2.0 followed by chromatography is a useful means of separating similar amino acids and short peptides and does not require desalting or excessive purification of the sample (Figure 10.17).
> Electrophoresis - see Section 3.3.2.
Electrophoresis pH 2.0
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