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 citriate 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.