Remixing of Separated Zones During Elution

Remember to collect fractions of sufficiently small volume when separate zones lie close together. Sharply focused zones can lie less than 1 mm apart and still appear completely separated to the eye. This means that a volume of less than 0.5 ml separates them. Thus accurate analysis is not possible unless the fractions are considerably under 0.5 ml. 

One factor causing remixing of the zones is diffusion during the time it takes to elute the column. P. Just Svendsen (56) avoids this in his design of the column. He can elute with the voltage applied. In this way he can maintain the electrofocusing effect during the entire elution. The apparatus and technique used by Svendsen are described later. 

However, other parts of the system following after the column also cause loss of resolution during elution. In the capillary tubing the flow profile is parabolic and causes severe remixing of adjacent zones. Where 

Another way of avoiding the mixing effects of parabolic flows in capillary tubing would be to use bubbles of air in Tecnicon s Auto-Analyzer. Such an injection system would divide the flow up into short plugs of liquid about 1 cm in length, each followed by about. 1 cm of air. This ought to solve the problems. However, there is no commercially av -able apparatus adapted to column systems. 

The LKB 440 ml column is reported as giving better resolution than the 110 ml column. This observation probably confirms the above statement as to remixing in the capillary system after the column. In the 440 ml column, the volume separating two adjacent zones is larger than in the 110 ml column. The capillary system after the column and the flow photometer is the same in both cases. Remixing thus has less effect in the case of the 440 ml coliunn. 

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