Preparative isoelectric focusing in a density gradient

Density materials used are practically identical with those used on the analytical scale, e.g., sucrose, and less frequently glycerol, sorbitol, ethylene glycol, ficoll and dextran. It should be noted that for the isoelectric fractionation of proteins with p7 values beyond 8.0 other density material than sucrose should be used (sucrose tends to be dissociated at high pH) (e.g., glycerol) (Fig. 6.29). 

The sample may be introduced into the system by preparing the density gradient in an already diluted solution of the sample. In this arrangement, however, a part of the sample protein comes into contact with both electrodes and, moreover, the proteins have to traverse through pH areas that may cause denaturation. Another method is to apply the rather concentrated sample in a position that is close to the expected focusing position. In this case the density of the introduced sample should 

Most of the preparative scale isoelectric focusing separations are done at 1600 or 2000 V in 110 and 440 ml columns (commercially available). After the run has ended the column is emptied (before a certain volume of water is pumped on top of the column because hydrostatically forced emptying does not result in constant flow rate due to density differences). Recommended flow rates are 60 ml/h or 240 ml/h for the 110 ml and 440 ml columns, respectively. 

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