Synthesis and characterization of quaternary ammonium dicarboxylic acid isoelectric buffers and their use in pH-biased isoelectric trapping separations.

Two approaches are described in this paper for the synthesis of isoelectric buffers that have p/ values in the 1.5 < p/ < 4.3 range. The first synthesis relies on the alkylation of existing aminodicarboxylic acids and recovery of the ampholyte as an inner salt. The second synthesis method forms low-p/ ampholytes by reacting a secondary amine with two equivalents of an alkylester of a haloalkanecarboxylic acid, followed by hydrolysis of the intermediate in an alkaline solution and recovery of the ampholyte as an inner salt. The new ampholytes have been analytically characterized by capillary electrophoresis, high-resolution electrospray ionization-mass spectrometry, one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy, and X-ray crystallography. The isoionic solutions of the new ampholytes have high buffering capacity and conductivity, making them good pH biasers in the receiving stream in preparative-scale pH-biased isoelectric trapping separations.