Ampholyte in the pH-Gradient

All the isoelectric fractionations previously described took place in water. Thus they all contain the ampholyte water, which is isoelectric at pH 7. An ampholyte with pi less than 7 can thus be completely separated from another with pi larger than 7. A zone of pure water will be created between them. But if a pair of ampholytes both have a pi lying on the same side of pH 7, that is, both are less than 7, or both exceed 7, they can never be separated unless other ampholytes are present. Water as an ampholyte furthermore has the drawback of having an extremely low conductivity at its isoelectric point pH 7. 

Under the conditions prevailing during electrofocusing, water may be assumed to have its theoretical conductivity, i.e. 

Since pi is always larger than pK+, the second term in the denominator will always be larger than unity. This means that the upper limit for a is equal to Obviously a becomes larger as the difference between pK+ and the isoionic point diminishes. The conductivity of an ampholyte is directly dependent of the extent of its ionization. This means that the condition for high conductivity at the isoionic and isoelectric point is that the pair of pK values lying on either side of the pi and nearest to it are close together. 

Svensson (2) has calculated the conductivities of several ampholytes of low molecular weight. We refer the reader to his works. For example, histidine has pi = 7.81, pi — pK+ = 1.50, and conductivity 3C = 158 mhos cm 10 . At this pH, the contribution from the conductivity of the water is zero. Another example is lysine, with pi = 9.96, pi — pK+ = 0.79 and 3C(corr) = 2.162 mhos cm 10 . Thus lysine has a considerably larger conductivity at its isoelectric point than histidine. This results 

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