Reversed-phase capillary electrochromatography of pre-column derivatized mono- and oligosaccharides with three different ultraviolet absorbing tags

Abstract

In this research report, an in house developed octadecyl monolithic (ODM) column has been exploited in the reversed-phase capillary electrochromatography (RP-CEC) of precolumn derivatized mono- and oligosaccharides with three different tagging agents, namely 1-naphthylamine (1-NA), 2-aminoanthracene (2-AA) and 3-amino-2,7-naphthalenedisulfonic acid (ANDSA). These three derivatizing agents, which differed in their charges, nonpolar characters and optical absorption properties, led to different RP-CEC elution patterns and UV detection signals. In fact, the limit of detection of the derivatized sugars were 50 µM for the ANDSA- and 1-NA-sugar derivatives and 35 µM for the 2-AA-sugar derivatives due to the presence of three fused aromatic rings in 2-AA versus 2 fused rings in the 1-NA and ANDSA tags. Furthermore, while the longer ANDSA-oligosaccharides eluted later than the shorter ones and the ANDSA-monosaccharides, 1-NA- and 2-AA-sugar derivatives necessitated the presence of borate ions at alkaline pH in the mobile phase to form in situ charged derivatives to facilitate their separation by RP-CEC, and the elution order was the reversal of that observed with the ANDSA-sugar derivatives; that is the mono- eluted later than the larger size oligosaccharides. In addition, plots of log tR vs. number of glucose residues (nGlc) for derivatized glucose and maltooligosaccharides yielded straight lines with slopes representing log η where η is the retention time modulus (i.e., ratio of retention time of two neighboring derivatives differing in one glucosyl residue). In the case of 1-NA and 2-AA derivatives, η was smaller than unity while it was greater than unity in the case of ANDSA-sugar derivatives because the elution occurred in the order of decreasing size of the homologous sugar derivatives in the former than in the later derivatives. The prepared ODM column was stable for more than a month of continuous use, a fact that allowed a good repeatability for intraday and interday analyzes.