Structure-stability relationship of Immobiline chemicals for isoelectric focusing as monitored by capillary zone electrophoresis

Abstract

An acrylamido buffer for isoelectric focusing in immobilized pH gradients, 1-acryloyl-4-methylpiperazine (p K = 6.85 at 25°C), was synthesized. As it is a disubstituted amide, it was thought that it would be much more resistant to alkaline hydrolysis. In reality, it degraded rapidly in 0.1 M sodium hydroxide solution at 70°C (86% in 6 h). Therefore, the stability of Immobiline buffers of p K 9.3, 8.5, 7.0 and 6.2 was investigated. Under the same hydrolysis condition, the degradation was 11%, 22%, 26% and 34%, in order of decreasing p K values. The kinetics of degradation were monitored by capillary zone electrophoresis in 0.1 M borate buffer (pH 9). The decrease of the main Immobiline peak and the appearance of its hydrolytic products, acrylic acid and a diamine, could be easily measured. The following general rules were derived: when the nitrogen engaged in the amido bond is inserted into a cyclic structure ( e.g., 1-acryloyl-4-methylpiperazine) there is very little protection against hydrolysis, and conversely, when this nitrogen carries flexible, fairly long substituents (4–5 atoms long) much stronger shielding and protection of the amido bond can be obtained. These findings helped in designing new acrylamido derivatives strongly resistant to chemical degradation.