Analysis of Carbohydrates by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAE-PAD)

Anion-Exchange Chromatography I. Mechanism of Separation Although anion-exchange chromatography has been used extensively to analyze acidic carbohydrates and glycopeptides, it has not been commonly used for analysis of neutral sugars. However, examination of the pKa values of the neutral monosaccharides listed in Table 1 shows that carbohydrates are in fact weak acids. At high pH, they are at least partially ionized, and thus can be separated by anion-exchange mechanisms. This approach cannot be used with classical silica-based columns because these matrices dissolve at high pH. Anion exchange at high pH is, however, ideally suited Introduction Methods for the liquid chromatographic analysis of carbohydrates have often employed silica-based aminobonded or polymer-based, metal-loaded, cation-exchange columns, with refractive index (RI) or low-wavelength ultraviolet (UV) detection. These analytical methods require attention to sample solubility, sample concentration and, in the case of the metalloaded cation-exchange columns, also require column heating. In addition, RI and low-wavelength UV detection methods are sensitive to eluent and sample matrix components. This usually precludes the use of gradients and often requires stringent sample cleanup prior to injection.