Liquid ion-exchange chromatography under pressure of milk oligosaccharides using a pulsed amperometric detector

Several oligosaccharides from human milk were separated completely on a Dionex AS6 ion-exchange column under mild alkaline conditions, such that no degradation of oligosaccharides was detectable after incubation for 6 h at room temperature in the eluent buffer. In general, both the presence of fucosyl groups and branching within oligosaccharide chains tend to reduce retention times for oligosaccharides in this system. Thus, both lacto-N-fucopentaose II [beta-D-Galp-(1----3)-[alpha-L-Fucp-(1----4)]-beta-D-GlcpNAc-(1--- -3)- beta-D-Galp-(1----4)-D-Glc] and lacto-N-fucopentaose III [beta-D-Galp-(1----4)-[alpha-L-Fucp-(1----3)]-beta-D-GlcpNAc-(1--- -3)- beta-D-Galp-(1----4)-D-Glc] were eluted before lacto-N-fucopentaose I [alpha-L-Fucp-(1----2)-beta-D-Galp-(1----3)-beta-D-GlcpNAc-(1----3 )-beta-D- Galp-(1----4)-D-Glc], and all three fucopentaoses were eluted earlier than lacto-N-tetraose [beta-D-Galp-(1----3)-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-D-Glc ]. 2'-Fucosyllactose [alpha-L-Fucp-(1----2)-beta-D-Galp-(1----4)-D-Glc] and 3-fucosyllactose [alpha-D-Galp-(1----4)-[alpha-L-Fucp-(1----3)]-D-Glc] were separated completely under a wide range of conditions. Introduction of substituents at either the O-3 or O-4 of 2-acetamido-2-deoxy-D-glucose produced large differences in retention: lacto-N-tetraose and lacto-N-neotetraose [beta-D-Gal-(1----4)-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-D-Glc] could be separated by more than 8 min. The l.c. ion-exchange chromatographic system described represents a useful addition to existing methods for separating oligosaccharides derived from glycoproteins and glycolipids.