BEHAVIOR OF SUBSTITUTED AMINOMETHYLPHENOL DYES IN CAPILLARY ISOELECTRIC-FOCUSING WITH ELECTROOSMOTIC ZONE DISPLACEMENT

The behaviour of six substituted aminomethylphenol dyes, having pI val ues between 5.3 and 10.4, in capillary isoelectric focusing with elect roosmotic zone displacement is described. Using untreated fused-silica capillaries and different neutral capillary conditioners in the catho lyte, the low-molecular-mass dyes are shown to focus and elute reprodu cibly in the order of decreasing pf values. In the absence of proteins , the detection times of the dyes are independent of the sampled amoun t. Hence these substances permit the characterization of the pH gradie nt produced in this capillary isoelectric focusing method. With concur rent focusing of dyes and test proteins, a macromolecular impact on de tection 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. Neverth eless, mapping of the pH gradient with these dyes and determining the pi values of known proteins is shown to provide values in agreement wi th those in the literature. Hence the substituted aminomethylphenol dy es can be employed as pf markers in capillary isoelectric focusing wit h electroosmotic zone displacement. Further, focusing and separation o f two of the six dyes by preparative recycling free fluid isoelectric focusing is described, illustrating that the substituted aminomethylph enol dyes are also applicable to other free fluid focusing methods.