Mixtures of carbohydrates can be resolved into their individual components (Fig. 11-25) by many of the same techniques useful in protein and amino acid separation: differential centrifugation (see Fig. 2-24), ion-exchange chromatography (see Fig. 5-12), and gel filtration (see Fig. 6-2). Hydrolysis in strong acid yields a mixture of monosaccharides, which, after conversion to suitable volatile derivatives, may be separated, identified, and quantified by gas-liquid chromatography (see p. 264) to yield the overall composition of the polymer. For simple, linear polymers such as amylose, the position of the glycosidic bond between monosaccharides is determined by treating the intact polysaccharide with methyl iodide to convert all free hydroxyls to acid-stable methyl ethers (Fig. 11-25). When the methylated polysaccharide is hydrolyzed in acid, the only free hydroxyls present in the monosaccharides produced are those that were involved in glycosidic bonds. To determine the stereochemistry at the anomeric carbon, the intact polymer is tested for sensitivity to purified glycosidases known to hydrolyze only α- or only β-glycosides. Total structure determination for complex heteropolysaccharides is much more difficult. Stepwise degradation with highly specific glycosidases, followed by isolation and identification of the products, is often helpful. Mass spectral analysis and high-resolution nuclear magnetic resonance (NMR) spectroscopy are extremely powerful analytic tools for carbohydrates, especially when combined with selective degradation by glycosidases.
