Abstract
This chapter describes the pre- and post-column detection-oriented derivatization techniques in the high-performance liquid chromatography (HPLC) of carbohydrates. Difficulties arise in analyzing carbohydrates by chromatography in the absence of effective chromophores to allow sensitive detection. Colorimetric analysis (post-column labeling) had long been used; however, the detection method is not suitable for derivatization purposes. Monosaccharides and oligosaccharides are detected without any derivatization by measuring their refractive indices or absorption in an ultraviolet (UV) region at 190–210 nm. Although these procedures are reasonably sensitive and suitable for chromatography, problems are often encountered with interfering peaks that arise from strongly UV-absorbing contaminating materials. Thus, their sensitivities are restricted to the nanomole order. Although a pulsed amperometric detector (PAD) combined with HPLC allows the sensitive detection of carbohydrates, PAD needs eluents with relatively high pH values, which imposes several limitations on the HPLC system and causes some modification of reducing-end sugars.
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