Separation and Identification of Sugars

Several methods exist for the separation and identification of sugars, though novel or unusual sugars can still present serious chemical difficulties, especially if only trace quantities are available. The methods are of two general kinds, chromatographic and electrophoretic or ion-exchange. 

GLC is not a good method for occasional use the equipment is costly, should be run frequently and needs a fair amount of skill. For regular use it is far more satisfactory and some of the newer techniques of sample injection are going a long way towards improving its effective sensitivity. 

An alternative to electrophoresis is to separate sugars by ion-exchange in borate buffer, using the same equipment as for aminoacid analysis. The method is useful for automatic analysis, but has no advantage over electrophoresis either in time, resolution or cost. 

Modern techniques of high pressure liquid chromatography and isotacho-phoresis have not yet made much impact on the biochemistry of carbohydrates, but will soon do so. Nevertheless, some problems remain, especially with regard to the detection of sugars, where procedures at least comparable in sensitivity to the use of dansyl reagents in protein chemistry are badly needed. 

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Bosch-Reig, R, Marcote, M.J., Minana, M.D., and Cabello, M.L. 1992. Separation and identification of sugars and maltodextrines by thin layer chromatography Application to biological fluids and human milk, Talanta, 39 1493-1498. 

Acyl groups have a protective role in some synthetic reactions. Gluconic acid nitrile acetates (aldonitrile acetates) are analytically suitable sugar derivatives for gas chromatographic separation and identification of sugars. An advantage... 

The paper-chromatographic properties of the common deoxy and dideoxy sugars have been treated in several reviews,2 28 a book,829 and individual publications. Common solvent-systems are 6 4 3 1-butanol-pyridine-water (Solvent A) 4 1 5 1-butanol-acetic acid-water (Solvent B) and 1-buta-nol-ethanol-water (4 1 5, upper phase, Solvent C or 3 1 1, Solvent D). The four 2-deoxy-D-hexoses and the 2,6-dideoxyhexoses may be separated as their borate complexes. 0 The use of 1-butanol-water on the one hand, and of 2-butanone-borate buffer on the other, usually provides adequate separation and, by use of a combination of solvents, these deoxy sugars may be identified. 80 The use of buffered systems has proved highly advantageous in the separation and identification of the isomeric 6-deoxy-hexoses.8 1 Other systems, such as Solvent A and 2 1 2 ethyl acetate-... 

Of particular interest to the biochemist has been the separation and identification of the phosphorylated sugars. These components have been examined with the aid of paper chromatography, and Table II shows some of the solvent systems that have been used. The general topic of the separation of sugar phosphates is adequately covered by Benson (2 ),... 

Bhushan, R., and Kaur, S. (1997). TLC separation of some common sugars on silica gel plates impregnated with transition metal ions. Biomed. Chromatogr. II 59-60. Bhushan, R., and Parshad, V. (1996a). Separation and identification of some cephalosporins on impregnated TLC plates. Biomed. Chromatogr. 10 258—260. 

In the case of the low abundance of some compounds, there are difficulties with signal overlap. To overcome these difficulties, there have been developments involving NMR hyphenation with techniques such as HPLC and mass spectrometry. In LC/NMR methods of analysis, NMR is used as the detector following LC separation and this technique is capable of detecting low concentrations in the nanogram range. This technique has been reported for the detection and identification of flavanoids in fruit juices and the characterization of sugars in wine [17]. 

Although the isolation and identification of new disaccharides, tri-saccharides and tetrasaccharides and their derivatives, either by acid hydrolysis or by controlled oxidative degradation, " would be of great help in these studies it would appear to be worth while to develop other indirect methods of approach involving the use of enzymes capable of effecting scission at specific points in the molecular complex. Better methods for the quantitative separation of sugars and their derivatives are in the process of development and it is not unlikely that in the near future it will be possible to derive formulas not only for plant gums but for the many related complex polysaccharides. 

The reaction of carbohydrates in alkaline or acidic aqueous solutions results in a myriad of products, many of which have been recognized for well over a century. The number of identified products has greatly increased in recent years, owing to the development of sophisticated techniques for separation and identification. With the exception of anhydro sugars and oligosaccharides, found as concentration-dependent, equilibrium constituents (reversion products) in acidic solutions, all of the products result from reactions of intermediates present in the Lobry de Bruyn-Alberda van Ekenstein transformation. 

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