Behaviour of substituted aminomethylphenol dyes in capillary isoelectric focusing with electroosmotic zone displacement

Abstract

The behaviour of six substituted aminomethylphenol dyes, having p I values between 5.3 and 10.4, in capillary isoelectric focusing with electroosmotic zone displacement is described. Using untreated fused-silica capillaries and different neutral capillary conditioners in the catholyte, the low-molecular-mass dyes are shown to focus and elute reproducibly in the order of decreasing p I values. In the absence of proteins, the detection times of the dyes are independent of the sampled amount. Hence these substances permit the characterization of the pH gradient produced in this capillary isoelectric focusing method. With concurrent focusing of dyes and test proteins, a macromolecular impact on detection times (reduction of electroosmosis)` is revealed. The effect is shown to be dependent on the type and amount of proteins applied and has been observed with three different capillary conditioners. Nevertheless, mapping of the pH gradient with these dyes and determining the p I values of known proteins is shown to provide values in agreement with those in the literature. Hence the substituted aminomethylphenol dyes can be employed as p I markers in capillary isoelectric focusing with electroosmotic zone displacement. Further, focusing and separation of two of the six dyes by preparative recycling free fluid isoelectric focusing is described, illustrating that the substituted aminomethylphenol dyes are also applicable to other free fluid focusing methods.