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.
Sulfuric acid hydrolysis according to the Saeman procedure, TFA hydrolysis, and methanolysis combined with TFA hydrolysis were compared for the hydrolysis of water-soluble uronic acid-containing polysaccharides originating from fungi, plants, and animals. The constituent sugar residues released were subsequently analyzed by either conventional GLC analysis of alditol acetates or high-performance anion-exchange chromatography with pulsed-amperometric detection. It was shown that TFA hydrolysis alone is not sufficient for complete hydrolysis. Sulfuric acid hydrolysis of these polysaccharides resulted in low recoveries of 6-deoxy-sugar residues. Best results were obtained by methanolysis combined with TFA hydrolysis. Methanolysis with 2 M HCl prior to TFA hydrolysis resulted in complete liberation of monosaccharides from pectic material and from most fungal and animal polysaccharides tested. Any incomplete hydrolysis could be assessed easily by HPAEC, by the detection of characteristic oligomeric products, which is difficult using alternative methods currently in use. Methanolysis followed by TFA hydrolysis of 20 micrograms water-soluble uronic acid containing polysaccharides and subsequent analysis of the liberated sugar residues by HPAEC allowed us to determine the carbohydrate composition of these polysaccharides rapidly and accurately in one assay without the need for derivatization.
De Ruiter
G A
GA
Wageningen Agricultural University, Department of Food Science, The Netherlands.
Schols
H A
HA
Voragen
A G
AG
Rombouts
F M
FM
D000818:Animals; D000838:Anions; D002241:Carbohydrates / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D005110:Extracellular Space / Q000737:chemistry; D005658:Fungi / Q000737:chemistry; D005965:Glucuronates / Q000032:analysis; D020723:Glucuronic Acid; D006025:Glycosaminoglycans / Q000032:analysis; D006603:Hexuronic Acids / Q000032:analysis; D006868:Hydrolysis; D007067:Iduronic Acid / Q000032:analysis; D000432:Methanol; D010368:Pectins / Q000032:analysis; D010944:Plants / Q000737:chemistry; D011134:Polysaccharides / Q000032:analysis*; D012680:Sensitivity and Specificity; D012995:Solubility; D014269:Trifluoroacetic Acid; D014574:Uronic Acids / Q000032:analysis*; D014867:Water
D000838:Anions; D002241:Carbohydrates; D005965:Glucuronates; D006025:Glycosaminoglycans; D006603:Hexuronic Acids; D011134:Polysaccharides; D014574:Uronic Acids; D014867:Water; D007067:Iduronic Acid; C007819:galacturonic acid; D010368:Pectins; D020723:Glucuronic Acid; D014269:Trifluoroacetic Acid; D000432:Methanol