The influence of buffer composition on separation efficiency and resolution in capillary electrophoresis of 8-aminonaphthalene-1,3,6-trisulfonic acid labeled monosaccharides and complex carbohydrates.
The effect of buffer conditions -- varying in salt type, pH, and concentration -- on the separation of 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS)-labeled monosaccharides and complex-type carbohydrates was investigated. Different buffer systems for high and low electroosmotic flow conditions were chosen: a phosphate and a citrate background electrolyte, each at pH 2.5, a phosphate buffer, pH 9.0, and a borate buffer at pH 9.5. All buffer systems displayed differences in resolution and selectivity. Phosphate and borate buffer demonstrated the greatest selectivity changes for ANTS-labeled carbohydrates. While separation in the phosphate system relies mainly on differences in the charge-to-mass-ratio, additional selectivity can be achieved with borate complexation of glycoconjugates. The use of borate buffers improved monosaccharide separations whereas complex carbohydrates showed a loss in resolution. The citrate background electrolyte at low pH caused no significant changes in the separation performance. The pH 9.0 phosphate buffer showed a reversed migration order of the ANTS conjugates with a decreased resolution, compared to the pH 2.5 phosphate buffer, due to the strong electroosmotic flow generated under high pH conditions. An ovalbumin-derived oligosaccharide library demonstrates the significance of buffer selectivity for complex carbohydrate separations. The separation in the acidic phosphate and the alkaline borate buffer generates a different pattern and only the combination of both buffer systems allows an appropriate assessment of sample complexity.
Ciba, Corporate Analytical Research, Zurich, Switzerland.
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Klockow
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R
Amado
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HM
Widmer
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Paulus
D000143:Acids; D000468:Alkalies; D000818:Animals; D001881:Borates; D002021:Buffers; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000302:isolation & purification*; D016208:Databases, Factual; D019075:Electrophoresis, Capillary; D008969:Molecular Sequence Data; D009005:Monosaccharides / Q000302:isolation & purification*; D009281:Naphthalenes; D009844:Oligosaccharides / Q000737:chemistry; D010047:Ovalbumin / Q000737:chemistry; D010710:Phosphates
D000143:Acids; D000468:Alkalies; D001881:Borates; D002021:Buffers; D002241:Carbohydrates; D009005:Monosaccharides; D009281:Naphthalenes; D009844:Oligosaccharides; D010710:Phosphates; D010047:Ovalbumin; C046465:8-amino-1,3,6-naphthalenetrisulfonic acid