α,ω-Bis-quaternary ammonium alkanes as effective buffer additives for enhanced capillary electrophoretic separation of glycoproteins

R. P. Oda, B. J. Madden, T. C. Spelsberg, J. P. Landers

Research output: Contribution to journalArticle

52 Scopus citations

Abstract

The egg white glycoprotein, ovalbumin, is known to be microheterogeneous as a result of its varied glycan content. The use of 1,4-diaminobutane (DAB) as a buffer additive has been shown to be key in the high-resolution capillary electrophoretic separation of "glycoforms" of this protein [Anal. Biochem. 205 (1992) 115]. Although a separation buffer consisting of 100 mM borate and 1 mM DAB allowed for adequate separation of ovalbumin glycoforms, prolonged separation times of 35-45 min were undesirable. In the present study, the α,ω-bis-quaternary ammonium alkanes, hexamethonium bromide (C6MetBr), hexamethonium chloride (C6MetCl) and decamethonium bromide (C10MetBr) were tested as buffer additives for their effectiveness in the separation of ovalbumin glycoforms. Where 1 mM DAB gave optimal separation in ca. 45 min, 100 μM C6MetCl or C10MetBr yielded comparable resolution in less than 20 min. Results with the C10MetBr were better than those obtained with C6MetBr, indicating that there may be a correlation between effectiveness and alkyl chain length. Use of the chloride salt of C6Met afforded the same resolution as the bromide salt in slightly shorter analysis time. The rank order for their effectiveness was found to be C10MetBr > C6MetCl > C6MetBr > DAB. These results allow for speculation on the mode through which these additives exert their effect on resolution. Included in these are additive-wall coating interactions, protein-additive interactions, protein-wall interactions or any combination of these.

Original languageEnglish (US)
Pages (from-to)85-92
Number of pages8
JournalJournal of Chromatography A
Volume680
Issue number1
DOIs
StatePublished - Sep 30 1994

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

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