Two-column system for determination of glucosamine, galactosamine, and amino acids on a Beckman 121MB amino acid analyzer: separation of the anomers of glucosamine and galactosamine.

A procedure is described for the determination of glucosamine, galactosamine, and the common amino acids in glycoproteins by the use of a Beckman 121MB amino acid analyzer. The procedure employs Beckman AA10 ion exchange resin in a two-column system. Separation of the hexosamines and their anomers along with the basic amino acids on a short column and of the acidic and neutral amino acids on a long column is achieved. The use of the two-column system permits quantitation of the hexosamines and amino acids in the 100–1000 pmol range, corresponding to 2–20 μg of glycoprotein analyzed. The separation of the α and β anomers of the hexosamines is critically dependent on pH. Galactosamine yields two peaks at pH 6.20 and glucosamine one, and glucosamine yields two peaks at pH 6.74 and galactosamine one. Separation of the anomers improves at lower temperatures (25 versus 40°C) but is relatively insensitive to ionic strength (0.1 to 0.4 n in sodium).