Temperature effects in high-performance anion-exchange chromatography of oligosaccharides.

High-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection has been widely used for analysis of mono-, oligo- and polysaccharides. Many factors that affect separation of carbohydrates by HPAEC have been evaluated, however effect of temperature has not been carefully studied. In the present study, neutral and sialylated oligosaccharides from human milk and different types of N-linked oligosaccharides were analysed by HPAEC at temperatures ranging from 13 to 30 degrees C. N-Acetyl neuraminic acid, Galacturonic acid and stachyose were also analysed since they have been used as internal standards when analysing various oligosaccharides by HPAEC. All oligosaccharides showed decreased retention times with increased temperature. Even small differences (i.e. +/- 5 degrees) resulted in considerable changes in retention times. In addition, individual oligosaccharides showed relative changes in retention time with increased temperature. By changing the temperature, a switch in elution of order of individual oligosaccharides were sometimes found. These results show that retention times relative to an internal standard cannot be used for oligosaccharide identification unless temperature is carefully controlled. Regulation of temperature is also a valuable tool in achieving optimal separation of oligosaccharides by HPAEC.