Separation of Sugar Mixtures

Ketoses may be separated from contaminating aldoses by oxidation of the latter with bromine and removal of the aldonic acids with an ion-ex-change resin (2), Ion-exchange resins are also useful in the recovery of sugar acids and of some nitrogen-containing derivatives of sugars. 

General references C. A. Browne and F. W. Zerban, Sugar Analysis. Wiley, New York, 1941 F. J. Bates and Associates, NatL Bur, Standards Circ. C440, (1942) Z. Dische, in Methods of Biochemical Analysis (D. Click, ed.), Vol. II, p. 313. Interscience, New York, 1955. 

Ion-exchange chromatography has also been used to separate components of sugar mixtures (7). The carbohydrates form complexes with borate ions (8) which behave as anions and can be separated on an anion-exchange 

By far the most widely used chromatographic technique in the carbohydrate field is paper chromatography (12y IS). This method, first used for the analysis of amino acids was used in 1946 by Partridge (16) to separate a mixture of sugars. The method enables the rapid separation of the components of some complex mixtures. Very small amounts of material can be used, and relatively simple equipment is needed. In addition the technique can be used as an aid in establishing the homogeneity of a sample and the identification of an unknown substance. 

The method involves the following steps. A small drop of the material in solution is placed at one end of a strip of filter paper. After drying, the paper is treated with a suitable solvent so that the solvent moves gradually over the sugar spot and along the paper this process is called development. After a time the paper is removed from contact with the solvent and dried the spots on the paper are identified by appropriate methods. 

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Thin-layer chromatography affords a simple, rapid and sensitive method for the qualitative and quantitative analysis of low molecular weight sugars and their derivatives. In the few years since Stahl and ELaltenbach [1] extended TLC to the separation of sugar mixtures, the method has been adopted for a wide spectrum of analytical problems. This chapter considers two classes of compounds the hydrophilic carbohydrates and their derivatives which are hydrophobic in nature. 

Fig. 1 (A) Chromatographic separation of sugars. Track 1 fructose, 2 sucrose, 3 glucose, 4 mixture of the substances in tracks 1-3, 5 mixture of substances in tracks 1-3 and 6, 6 Fructo-oligosaccharides, 7 1-kestose, 8 mixture of glucose, maltose, maltotriose and maltotetraose. (B) Absorption scan of track 5 with 200 ng each substance per chromatogram zone 1 = fructosyl-nystose, 2 = nystose, 3 = 1-kestose, 4 = fructose, 5 = sucrose, 6 = glucose.

Only model studies have so far been reported, and the method has not yet been applied to the analysis of sugar mixtures, with the exceptions noted.48,49 The recovery of glucose-14C trifluoroacetate from a column of Carbowax 20 M or SF-96 has been shown to be 5 to 14% and 3 to 5%, respectively.208 Table III (p. 112) records further details of the separation of sugars and their derivatives as trifluoroacetates. 

Many different solvent developers have been used in the separation of sugars and related compounds. Three of these, phenol-water, collidine-water, and 1-butanol-acetic acid-water,27 also commonly employed in the resolution of amino acid mixtures, are widely used. Other commonly used solvent developers are 1-butanol-ammonia-water, 1-butanol-ethanol-water,27 1-butanol-pyridine-water,61 ethyl acetate-acetic acid-water, and ethyl acetate-pyridine-water.26... 

Solvent systems in whose phase diagram there is in any danger of a miscibility gap are also critical. Even with small changes in temperature, these can lead to emulsion formation, i.e. separation of the mixture (example the DAB solvent system for the sugars fructose, glucose, lactose and sucrose). 

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