INFLUENCE OF ADDITIVES ON RESOLUTION AND FOCUSING EFFICIENCY IN FREE-FLOW ISOELECTRIC-FOCUSING

In theory, the resolution of isoelectric focusing (IEF) depends directly on the electric field strength. However, at higher electric field strengths with free-flow IEF, focusing of proteins is impaired by electrodispersive effects. These effects cause a widening of protein peaks sharply focused at lower voltage. This was dependent on the electric field strength and thus reduced the focusing efficiency and resolution. Using 4-nitroaniline as a visible marker substance, the influence of concentration and molecular mass of various additives on the electrodispersive effects as a function of electric field strength was investigated. The efficiency to reduce electrodispersive effects was determined by various properties of the additives. However, the molecular mass of the additives only marginally influenced electrodispersive effects. In the most favourable case, the resolution R for the separation of bovine serum albumin and haemoglobin applied as a standard mixture increased from R = 0.4 without additive to R = 3.4 with 2% saccharose and 0.3% Servalyte 3-10 as ampholyte. Improvements in the resolution for the separation for the standard mixture were independent of the ampholyte used (Bio-Lyte, Ampholine, Servalyte).