Separation and identification of carbohydrate mixtures

Mannose, fructose and glucose may all be assayed independently using hexokinase and the appropriate additional enzymes. All the reactions can be monitored by the increase in absorbance at 340 nm as NADI is reduced to NADPH. 

D-galactose to D-galactonolactone in the presence of the coenzyme NAD+, is more specific and enzyme preparations are available for which the only other substrates are a-L-arabinose and /3-D-fucose. The generation of NADH is conveniently monitored at 340 nm and permits quantitation of the galactose  

Fermentation tests are based on the ability of yeast to oxidize the sugar to yield ethanol and carbon dioxide, although only the D-isomers are fermentable and only relatively few of these. Modem chromatographic techniques are, however, much more acceptable and paper and thin-layer techniques are useful for routine separation and semi-quantitation of carbohydrate mixtures, although GLC or HPLC techniques may be necessary for the more complex samples or for quantitative analysis. 

Both ascending and descending paper chromatographic techniques have been used and, when thin-layer supports are employed, the use of either silica gel or cellulose is applicable. As the number of carbohydrates present in the sample is often small, the careful choice of solvent will generally make it unnecessary to perform the two-dimensional separations that are often needed when large numbers of substances, such as amino acids, are present. Reference solutions of each carbohydrate can be made up in concentrations of approximately 2 g 1 1 dissolved in an isopropanol solvent (10% v/v in water) and samples of about 10 fx should give discernible spots after separation. 

Several monophasic solvent systems are useful for the separation of carbohydrate mixtures, and in all those listed in Table 9.3 the smallest solute molecules have the fastest mobility. Thus pentoses have higher RF values than hexoses, followed by disaccharides and oligosaccharides. 

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