Raffinose

Trisaccharides can be difficult to separate using conventional HPLC columns. The four trisaccharides panose, raffinose, isomaltotriose, and kestose have the same chemical formula, and the all have two glycosidic bonds connecting the three monosaccharides. Obelisc N was used as a stationary phase to separate trisaccharides because it is capable of multiple modes of separation. Obelisc N is a highly polar column that retains polar and charged analytes. Even though the trisaccharides differ only in regio- and stereochemistry they are resolved.

Five sugars were separated on a Primesep S2 HILIC column with LC/MS compatible conditions. Various mobile phase produce different selectivity and separation. Method can be used as alternative to aminopropyl column. Primesep S2 column is stable and does not undergo rapid hydrolysis like aminopropyl columns.

Common sugars can be separated on an Obelisc N HILIC/cation-exchange column with LC/MS compatible mobile phase. Highly polar surface of Obelisc N column provides good retention for sorbitol, sucrose, lactose and raffinose. Other sugars can be separated using this column.

In mixed-mode HILIC chromatography, selectivity of separation can be adjusted by amount of acetonitrile, amount of buffer and buffer pH. Buffer concentration and pH will affect retention of ionizable compounds to a different degree. Retention of neutral compounds can be adjusted by the amount of acetonitrile. Carboxylic acid, three amino acids and two sugars are separated by combination of HILIC and ion-exchange mechanisms. Compounds can be monitored by ELSD, Corona (CAD), LC/MS or low UV. UV-transparent mobile phase /buffer is required for UV monitoring of this mixed-mode separation. This HPLC method can be adopted as general approach for analysis of sugars, amino acids and carboxylic acids.