CAPILLARY ZONE ELECTROPHORESIS ANALYSIS OF ACRYLAMIDO BUFFERS FOR ISOELECTRIC-FOCUSING IN IMMOBILIZED PH GRADIENTS

Studies are described using fourteen weak acrylamido acids and bases (Immobilines) as buffers and titrants for isoelectric focusing in immobilized pH gradients (IPG), plus one strongly acidic (pK 1.0) and one strongly basic (pK > 12) titrants, utilized in general to reach pH extremes in extended IPG ranges. Three fundamental properties of these buffers were evaluated: (a) resistance to alkaline hydrolysis of the amido bond; (b) hydrophilicity (so as to avoid hydrophobic interactions with the matrix during electrophoretic migration); and (c) resistance to oxidation by peroxodisulphate during the polymerization step. Capillary zone electrophoresis (CZE) was used to evaluate these properties. The acidic compounds were resolved in 100 mM acetate (pH 4) whereas the alkaline species were separated in 50 mM phosphate buffer (pH 7.7) (or pH 7.2 for the weaker compounds). All the acrylamido derivatives were detected underivatized by their absorption at 214 or 254 nm. The degradation kinetics of all compounds were monitored, after hydrolysis in 0.1 M NaOH at 70-degrees-C (up to 6 h), by CZE in 0.1 M borate buffer (pH 9). The decrease in the Immobiline peak and the appearance of its hydrolytic products (acrylic acid and a diamine in the case of the acrylamido bases) could easily be monitored and quantified in CZE by using the Gold integration system. A hydrophobicity scale was constructed by measuring the partition coefficients of the basic acrylamido derivatives in 1-octanol-water. General guidlines are given for the proper use of these chemicals and for the synthesis of additional compounds.