Anion-exchange chromatography acidic polysaccharides

Lipoteichoic acids (from gram-positive bacteria) [56411-57-5J. Extracted by hot phenol/water from disrupted cells. Nucleic acids that were also extracted were removed by treatment with nucleases. Nucleic resistant acids, proteins, polysaccharides and teichoic acids were separated from lipoteichoic acids by anion-exchange chromatography on DEAE-Sephacel or by hydrophobic interaction on octyl-Sepharose [Fischer et al. Ear J Biochem 133 523 1983]. 

De Ruiter, G. A., Schols, H. A., Voragen, A. G. J., and Rombouts, F. M., Carbohydrate analysis of water-soluble uronic acid-containing polysaccharides with high-performance anion-exchange chromatography using methanolysis combined with TFA hydrolysis is superior to four other methods, Anal. Biochem., 207, 176, 1992. 

In most cases, uronic acids are liberated from acidic polysaccharides by hydrolysis leading to irreproducible concomitant formation of lactones. Several methods to circumvent this problem have been published describing conversion of the uronic acid into methyl esters followed by reduction with borohydride or borodeuteride reagents and subsequent hydrolysis and GC-MS detection [129]. Other techniques are based on the liberation and quantification of carbon dioxide. Direct determination of uronic acid residues in hydrolyzates has frequently been performed according to colorimetric assays, which are rather insensitive and have thus mostly been replaced by high-performance anion exchange chromatography (HP-AEC) methods [130-132]. 

The elucidation of a levan polysaccharide produced by a novel halophilic isolate from soil samples taken from Camalti Saltern area in Turkey, Halomonas smyrniensis was achieved thanks to the glycosyl analysis. In particular, the microbial EPS was hydrolyzed with trifluoroacetic acid (0.5M TFA at temperature of 120°C for 1.5h) and subsequently analyzed by HPAE-PAD (High-Performance Anion-Exchange Chromatography Pulsed Amperometric Detection), a high-performance anion-exchange... 

The concentrations of monomeric carbohydrates were determined using high performance anion-exchange chromatography (HPAEC, Dionex). The content of mannose in the filtrate and in the filtrate after acid hydrolysis (using 0.5 M sulfuric acid at 120° C for 4 hours) was determined. Using this data and data from raw material analysis, the yield of mannose residues in oligo- and polysaccharides in the filtrates were determined (2, 5). 

From the known, differential complexing between boronic acids and polyhydroxy compounds, it follows that carbohydrate mixtures may be separated by column-chromatographic methods that exploit the differences. Nucleoside and nucleotide boronates have been separated on columns of anion-exchange resins,90 and sugars and alditols have been shown to be differentially retained on such resins in the sulfonated phenylboronic acid form,64 but perhaps the best uses of column chromatography in this connection have incorporated the resolving powers of insoluble polymers to which boronic acid groups have been covalently bonded. Such insoluble forms of boronates have been synthesized either by substitution of polysaccharide derivatives, or by polymerization of suitable arylboronic acids. 

This can be illustrated by our work (O Neill et al., 1986a) on the extracellular anionic polysaccharide produced by Alcaligenes (ATCC 31555) species. The polysaccharide was hydrolyzed for 1.5 h with 0.2 M trifluoroacetic acid and the acidic oligosaccharides isolated by ion-exchange chromatography. Following reduction and methylation, the methylated oligosaccharide-alditol methyl esters were separated by reverse-phase HPLC (Fig. 9). Two major and one minor components were isolated and characterized by FAB-MS, EI-MS, and H-NMR. 

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