Study of the high-performance liquid chromatographic separation of reducing sugars, applied to the determination of lactose in milk.

The loss of reducing sugars by formation of Schiff bases on amino-bonded silica or on dynamically coated amino-silica columns was investigated at two column temperatures. Depending on the type, age and temperature of the column, these losses were in the range of 0-100%. A dimethylamino-bonded silica column did not cause loss of reducing sugars, but the retention of sugars was too weak to allow separation. The analysis of reducing sugars can be carried out on diol-modified silica with diisopropylethylamine in the eluent to enhance mutarotation. It is also possible to use a cation-exchange resin (Ca2+) column, equipped with a pre-column packed with a mixed-bed ion-exchange to remove interfering salts and acids. In combination with an acetate-acetonitrile sample clean-up, this method results in coefficients of variation of less than 1% for the determination of lactose in skim-milk.