Neutral Monosaccharides

The original publication by Sweeley and coworkers5 was concerned with the separation of a wide range of carbohydrates, from mono- to tetra-saccharides. Most of the subsequent publications have considered the quantitative analysis of mixtures of varied complexity, although two studies have demonstrated the separation of the protium from the deuterium fonns of monosaccharides.200,201 The study of mutarotational equilibria by gas-liquid chromatography has been discussed in Section IV (see p. 38). 

It should be noted that the per(trimethylsilyl) derivative of 1,6-anhydro-jS-D-glucopyranose has the same retention time as that of a-D-xylopyranose when they are chromatographed on a column of 3% 

SE-52 at 160°. Similarly, l,6-anhydro-/ -D-glucofuranose corresponds to /J-D-xylopyranose. These anhydro compounds must be formed in the acid-hydrolysis stage, and they give incorrect values for D-xylose unless their presence is detected 78 this may be done by operating the column at 135°. 

Although many analyses are performed on alditol acetates (see Section VII, p. 56), in order to avoid the formation of multiple peaks, such a reduction is not practical when the mixture contains ketoses, notably fructose. Such analyses are mainly encountered with medical samples and in the examination of sugars occurring free in Nature. Furthermore, the peak-area ratios may be used as a means of identification, to check on the completeness of trimethylsilylation,67,89 and, despite the complex chromatograms obtained from trimethyl-silyl derivatives, they have the merit of being rapidly formed.89 For all of these reasons, improvements in the separation of monosaccharides as their trimethylsilyl derivatives continue to be of considerable importance. 

Analysis of glycosaminoglycan hydrolyzates is often complicated by the presence of basic and acidic glycoses, in addition to neutral monosaccharides. Adsorption chromatography on silica gel was used to classify such a hydrolyzate, and the neutral fraction was shown to contain D-glucose, D-galactose, and D-mannose.288 Separation on ion-exchange resins may also be used.51,149 The resolution of the acidic and basic fractions is discussed in Sections IX (p. 71) and X (p. 78). 

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The content of neutral monosaccharides was determined after an acid hydrolysis, performed as follows with 72 % sulphuric acid for 1 hr at 30°C and then, after dilution to IM sulphuric acid, for another 3 hr s at 100°C. The determination was performed by GC analysis of the prepared alditol acetates (13,14)... 

The content of galacturonic acid in the fractions, obtained by gel chromatography, was determined by the carbazole method (9), and the content of neutral monosaccharides was determined by the anthron reaction (16). 

Alpenfels and coworkers studied the hydrolysis of glycoproteins and keratin fibers with 1 or 2 M hydrochloric acid for various periods of time at 100°. These investigators found that the concentration of neutral monosaccharides from hard keratin reached a maximum after hydrolysis with 2 M hydrochloric acid for 2 h at 100°, and the yield of the neutral monosaccharides was linear up to 25 mg of hair per mL of hydrochloric acid solution. The latter fact shows that a relatively large amount of protein does not interfere with the analysis of a relatively small amount of carbohydrate. 

Griggs and coworkers studied the hydrolysis of canine submaxillary mucin (CSM) by 0.5, 3, and 6 M hydrochloric acid for 1.5, 3, 4.5, 6, and 24 h at 100°. They found that use of 3 M hydrochloric acid for 3 h gives the maximal release of neutral and amino monosaccharides, with the minimal degradation of the monosaccharides liberated, although recoveries of neutral monosaccharides were only 76-88%. Guerrant and Moss also used hydrolysis with 3 M hydrochloric acid, although for 16 h at 75°, for the hydrolysis of bacterial cell-walls. 

Honda and coworkers used 4 M hydrochloric acid for 6 h at 100° for the hydrolysis of nondialyzable glycoconjugates when determining amino monosaccharides, but preferred 2 M CF3CO2H for 6 h at 100° when determining the neutral monosaccharides and uronic acids, as these compounds are subject to more-severe degradation by 4 M hydrochloric acid. They obtained complete hydrolysis (with >90% recovery of monosaccharides added prior to hydrolysis) by using these two sets of hydrolytic conditions. 

A new PET-based chemosensor for uronic and sialic acids utilizing the cooperative action of boronic acid and metal chelate was reported by Shinkai and co-workers. This group synthesized a novel fluorescent chemosensor molecule bearing both an o-aminomethylphenylboronic acid group for diol binding to a saccharide and a l,10-phenanthroline-Zn(II)chelate moiety for the carboxylate binding, which enables this sensor to discriminate between neutral monosaccharides and acidic compounds [110],... 

The ion-exchange separation usually affords individual fractions of structurally related glycosyl esters of nucleoside pyrophosphates, containing the same nucleotide residue, but differing in the structure of the glycosyl groups. Separation of the esters of N-acetylhexos-amines, uronic acids, and neutral monosaccharides from one another is also usually achieved. 

Chromatographic separation of free nucleotides from various sources usually gives the glycosyl esters of uridine 5 -pyrophosphate in three fractions, containing fhe derivatives of neutral monosaccharides, uronic acids, and 2-acetamido-2-deoxyglycoses, respectively. 

In the spermatozoa of the sea urchin Anthocidaris crassipina, nine sialoglycolipids were found, two of which were isolated and their structures established from the results of acid hydrolysis, methylation, and periodate oxidation.139 The major component of the glycolipid mixture is a disialo-glycolipid, NeuAc-(2- 8)-NeuAc-(2- 6)-Glc-Cer. The least-polar, minor component has the structure of a monosialoglycolipid, NeuAc-(2 - 6)-Glc-Cer. In these compounds, glucose is again the only neutral monosaccharide, and the sialic acid is attached to it at 0-6. 

The amount of each neutral monosaccharide in the samples can be calculated relative to the internal standard, allose, using response factors. The relative response of the detector for the individual alditol acetates can be calculated from the areas under the peaks for each alditol acetate, relative to the area under the peak for allitol acetate. There should be no peaks in the chromatogram of the water control and only one peak, corresponding to allitol hexaacetate from the internal standard allose, in the chromatogram of the TFA control. 

In this procedure, the cell walls are first treated with TFA to determine the neutral monosaccharide composition of the noncellulosic polysaccharides. The TFA-insoluble residue is then hydrolyzed using a two-stage sulfuric acid procedure to determine cellulose content. 

Plant cell wall polysaccharides are composed of varying proportions of the neutral monosaccharides rhamnose, fucose, arabinose, xylose, mannose, galactose, and glucose, and the acidic monosaccharides galacturonic acid, glucuronic acid, and 4-0-methylgIucuronic acid (see Fig. E3.2.1). Other monosaccharides have been identified but are present only in trace... 

From the carbohydrate analyses of peak II, it was determined that 780 pg of polysaccharide (i.e., polymers of neutral monosaccharides with polymer mol. wt. of 1,000-5,000) were reversibly complexed with the estimated 19.5 pg of glycopeptide contained in this peak. Hence, the calculated carbohydrate glyco-peptide weight ratio is approximately 40 1. 

Flaxseed mucilage, associated with hull of flaxseed, is a gum-like material and composed of acidic and neutral polysaccharides. The neutral fraction of flaxseed mainly contains xylose (62.8%), whereas the acidic fraction of flaxseed is comprised mainly of rhamnose (54.5%), followed by galactose (23.4%) (Cui et al., 1994a). A study by Warrand et al. (2005) found that the neutral monosaccharides were a mixture of three major families of polymers, arabinoxylans with a constant A/X ratio of 0.24, and various amount of galactose and fucose residues in the side chains. Acidic hydrolysis yields xylose, galactose, arabinose, rhamnose, galacturonic acid, fucose, and glucose (BeMiller, 1973 Erskine and Jones, 1957). 

Goes, J. I., N. Handa, S. Taguchi, T. Hama, and H. Saito. 1996. Metabolism of neutral monosaccharide constituents of storage and structural carbohydrates in natural assemblages of marine phytoplankton exposed to ultraviolet radiation. Limnology and Oceanography 41 1478-1489. 

Moers, M. E. C., M. Baas, J. W. DeLeeuw, J. J. Boon, and P. A. Schenek. 1990. Occurrence and origin of carbohydrates in peat samples from a red mangrove environment as reflected by abundances of neutral monosaccharides. Journal Cosmochimica Acta 54 2463-2472. 

Rich, J. H., H. W. Ducklow, and D. L. Kirchman. 1996. Concentrations and uptake of neutral monosaccharides along 140°W in the equatorial Pacific Contribution of glucose to heterotrophic bacterial activity and the DOM flux. Limnology and Oceanography... 

Moers, M.E.C., and Larter, S.R. (1993) Neutral monosaccharides from a hypersaline tropical environment applications to the characterization of modern and ancient ecosystems. Geochim. Cosmochim. Acta 57, 3063-3071. 

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