Paper and thinlayer chromatography

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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 should give discernible spots after separation.

Solvent systems

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.

The distance moved by different oligosaccharides is a reflection of the number of monosaccharide units of which they are composed, with the smallest molecules again moving the furthest. However, in general, the distances moved by all classes of carbohydrates are small and although modification of solvent composition may result in greater overall mobility, the relative differences between the components is still low and it may be necessary to run the solvent off the end of the support to achieve a satisfactory separation. In these circumstances the distance moved by glucose is used as a reference and is given the value RF = 100 in any solvent system. The migration of another carbohydrate is reported as its Rg value:

_ distance moved by substance

B distance moved by glucose

Locating reagents

A variety of reagents can be used for visualization of the separated components and it may be useful to run duplicate chromatograms and use a different stain on each one to assist in identification of unknown spots. The most commonly used reagents make use of the chemical reactions of carbohydrates already described in the section on quantitative methods and appropriate safety precautions must be taken when using the various locating reagents. The actual reagent composition may be modified either in terms of concentration of components or by substitution of one chemical for another very similar one, although the principle of

> Thin-layer chromatography - see Section 3.2.1.

Table 9.3 Some monophasic solvents for thin-layer chromatography of carbohydrates

Solvent Proportions* Comments composition

Table 9.3 Some monophasic solvents for thin-layer chromatography of carbohydrates

Ethyl acetate

60 !

Commonly used. Gives good separation of

Pyridine

30 \

pentoses and hexoses. Will resolve glucose

Water

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