Separation of anionic oligosaccharides by high-performance liquid chromatography

Area of Science:

• Analytical Chemistry
• Carbohydrate Chemistry
• Biochemistry

Background:

• Anionic oligosaccharides containing sulfate and/or sialic acid are crucial in biological processes.
• Accurate separation and characterization of these complex molecules are essential for understanding their functions.
• Existing chromatographic methods often struggle with the complexity of these mixtures.

Purpose of the Study:

• To develop rapid and effective high-performance liquid chromatography (HPLC) methods for the fractionation of anionic oligosaccharides.
• To enable separation based on charge, sialic acid, and sulfate content, including isomeric forms.
• To provide both analytical and preparative separation capabilities for sulfated and sialylated oligosaccharides.

Main Methods:

• Ion-exchange HPLC on amine-bearing columns at pH 4.0 to separate based on net negative charge.
• Ion-exchange HPLC at pH 1.7 to separate based on sialylation and sulfation patterns.
• Tandem application of pH 4.0 and pH 1.7 ion-exchange HPLC for complete fractionation.
• Ion-suppression amine adsorption HPLC to resolve sialylated oligosaccharide isomers based on sialic acid linkage and location.

Main Results:

• Separation of anionic oligosaccharides into distinct charge groups (0 to 4 charges) at pH 4.0, independent of moiety type.
• Resolution of neutral, mono-, di-, and tetrasialylated, and various sulfated oligosaccharides at pH 1.7.
• Coelution of trisialylated and monosulfated/monosialylated oligosaccharides at pH 1.7 was resolved by sequential analysis at pH 4.0 and pH 1.7.
• Demonstrated ability of ion-suppression amine adsorption HPLC to distinguish sialylated oligosaccharide isomers differing in sialic acid linkage (α2.3 vs. α2.6) and position.

Conclusions:

• A combined approach using ion-exchange HPLC at two different pH values (4.0 and 1.7) allows for the complete fractionation of complex mixtures of sulfated and sialylated oligosaccharides.
• Ion-suppression amine adsorption HPLC effectively resolves sialylated oligosaccharide isomers.
• These tandem HPLC methods offer powerful analytical and preparative tools for characterizing oligosaccharides based on the number, type, and linkage of their anionic moieties.