Isoelectric buffers for capillary electrophoresis. 2. Bismorpholine derivative of a carboxylic acid as a low molecular weight isoelectric buffer

Abstract

A new compound class of synthetic isoelectric buffers is introduced, designed as a small molecule with one fully or prevailingly dissociated acidic group (such as sulfonic or carboxylic) and two partly pronated (buffering) basic amino groups attached onto a hydrophilic UV-transparent backbone. As an example, a new isoelectric compound 2,2-bis(4-morpholinylmethyl)propanoic acid (BMMPA) was synthesized by attaching two morpholine groups onto a molecule of pivalic acid. It was characterized as having an isoelectric point pI = 6.5 and exhibiting satisfactory buffering capacity at the pI. Solutions of BMMPA are transparent down to the low-UV spectral region, thus making it a potentially suitable buffer for a number of separation methods. Its use in capillary electrophoresis was demonstrated in a separation system for indirect photometric detection of anions based on an electrolyte with the anionic dye Orange G as the indirect detection probe and using BMMPA as a buffer. The use of an isoelectric buffering compound brings the advantages of a buffered electrolyte without the concomitant introduction of co-ions that would be detrimental to the indirect detection process. Submicromole per liter limits of detection for a number of inorganic and small organic ions were achieved. Optimal structural properties of the isoelectric buffer with respect to its buffering properties are discussed.