High-performance liquid chromatographic separation of some mono- and disaccharides with detection by a post-column...

The effluent from a chromatographic column was mixed with nicotinamide adenine dinucleotide coenzyme (NAD+) buffer and passed through a packed-bed reactor containing immobilized glucose dehydrogenase. Oxidation of the carbohydrates emerging from the column produced an equivalent amount of reduced coenzyme (NADH), which was detected electrochemically using an electrode modified with 7-dimethylamino-1,2-benzophenoxazine (Meldola Blue). Separation was effected in three different chromatographic systems containing a protonated ion exchanger, a calcium(II)-saturated or a lead(II)-saturated ligand exchange column. Separation, detection and k' values are reported for glucose, 2-deoxyglucose, xylose, mannose, cellobiose, lactose, ribose and glucosamine. The detection limit was 2 ng for a 20-microliter injection of glucose and the response was linear up to 6300 ng. Samples from fermentation of penicillin were analysed for lactose and glucose with the described detector. A comparison with the recordings from a refractive index detector showed that the selectivity of the enzymes and the modified electrodes are necessary for the determination of glucose and lactose.