Uronic acids, determination

Fig. 5.—Sugars in Hydrolyzates of Total Hemicellulose from Oat Plants of Different Ages, Excluding Glucose but Including Uronic Acids Separately Determined.167 (It is assumed that, after hydrolysis, all uronic acids were present in aldobiouronic acids containing a xylose residue the proportions of xylose have been altered to include xylosyl residues equivalent to the uronic acid determined. Reproduced, by permission, from Phytochemistry.)...

Total uronic acids determined as glucuronic acid. Glucuronic and galacturonic acids were present in cell walls of CCM 859 in a molar ratio of about 2.3 1,... 

A more rapid and somewhat simpler procedure for uronic acid determination was described in 1973 (8). This method is advantageous for determining galacturonic acid in pectin, as interference by neutral sugars is reduced. This method is based on the color formation which accompanies the addition of m-hydroxybi-phenyl to heated solutions of uronic acids in sulfuric acid/boric... 

Table II. Carbohydrate compositions (weight percentage) of individual oligomer peaks purified (QAE-Sephadex or HPLC ion-exchange separation, respectively) from mixtures of citrus pectin oligomers or B fruit extracts Compositions shown are for peaks whose biological activity is described in Figure 4. Uronic acid values are based on colorimetric assay. Proportions of neutral sugars were determined by GC and adjusted so that totals equal 100%. In fact, some oligomers (G7 peaks 8, 9 and 10. B extract peak 10) produced small (less than 1 % of the total integrated area), unknown peaks in the GC chromatograms.

Blumenkrantz, N. Asboe-Hansen G. 1973. New method for quantitative determination of uronic acids. Anal. Biochem. 54 484-489. 

Uronic acid content was determined as anhydro-uronic acid (AUA) by the automated colorimetric /n-hydroxydiphenyl assay [2,3,6] using an auto-analyser (Skalar Analytical BV, Breda, The Netherlands). Corrections were made for interference by neutral sugars present in the sample. 

The determination of the sugar composition was performed with and without prehydrolysis to determine the cellulose content. Cellulose was present in soy meal and in WUS, the content was respectively 17.2 and 17.9 mol%. Both soy meal and WUS contained mainly galactose, glucose (cellulose), arabinose and uronic acids and their sugar compositions were very similar. This indicates that no sugar residues were specifically removed during the isolation procedure. 

Rha, Ara and Gal are the neutral sugar components from all the fractions. Xyl is not present in Fla and is significantly present in the hemicellulose fractions, indicating that this monosaccharide is component of hemicellulosic polymers. Chemical composition of the water fractions were determined (Table V). High protein contents and the presence of O-acetyl-groups were observed in four aqueous fractions. Neutral sugar and uronic acid composition points to inclusion of these polymers in the class of pectic polysaccharides. 

Table V. Determination of carbohydrates, proteins, uronic acids and 0-acetyl of Fractions from aqueous extractions from seeds of C. speciosa.

Neutral sugars were quantified by trifluoroacetic acid hydrolysis (3) and gas chormatography of alditol acetates (4). Uronic acids were determined by Blumenkrantz method... 

The uronic acids in polysaccharide fraction I (Table 2) were determined colorimetrically with m-hydroxybiphenyl (15). 

DF dietary fiber Cel cellulose Rha rhamnose Fuc fucose Ara arabinose Xyl xylose Man mannose Gal galactose Glu glucose U. ac. uronic acids KL Klason lignin n.d. not determined t traces Source Englyst and Cummings 1988 Englyst and others 1988 Marlett and Vollendorf 1994. 

Determination of xylan is frequently made by estimation of furfural production. Data so obtained, even when appropriately corrected for furfural that arises from uronic acid, may be high if araban is present in the polysaccharide preparation. In the absence of interfering carbohydrates, furfural estimation may lead to accurate xylan values. 

Chaplin used methanolysis for the analysis of carbohydrates in glycoproteins. His method was a variation of the foregoing procedures, with an improvement of using tert-hvAyX alcohol to remove hydrogen chloride by coevaporation, instead of prolonged trituration with silver carbonate. His method is useful for samples containing uronic acids and lipids. Mononen studied methanolysis, followed by deamination and reduction with borohydride, for determination of the monosaccharide constituents of glycoconjugates. This method was applied to a lipid-free, protein fraction of rat brain. 

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. 

The determination of the structure of a plant gum involves the establishment of its homogeneity, its equivalent weight, and rotation and its uronic acid and pentosan content by the well-known methods. The nature of the constituent sugars and the uronic acid are then determined, after hydrolysis of the gums, by ciystallization or by conversion into characteristic crystalline derivatives. As an example the hexose mannose may be identified as the free sugar or in the form of its anilide, methyl glycoside or phenylhydrazone. Autohydrolysis of the acid... 

Many authors have commented upon the problems associated with the hydrolytic step, which has been considered to be the main source of loss in carbohydrate analysis.19 The fact that, in determining uronic acids, differing results were obtained, depending on whether hydrolysis or methanolysis was employed, led others20 to conclude that... 

In model experiments,205 it was found that concentration of an aqueous solution of the aldonic acids in the presence of hydrochloric acid gives exclusively the 1,4-lactones, which, on subsequent trimethyl-silylation, give only one peak on the chromatogram. This method was successfully employed for the separation of D-galacturonic, D-glucuronic, and D-mannuronic acids. Of the 1,4-lactones examined, only the trimethylsilyl ether of D-mannono-1,4-lactone was obtained in crystalline form. However, all of the derivatives showed characteristic differences in their infrared spectra in the range of 1500 to 600 cm"1. When this method was applied to the determination of uronic acids in a variety of polysaccharides,205 it was impossible to find any hydrolytic conditions under which the uronic acids were quantitatively released and then reduced, a problem experienced by other workers.20 The method was, however, successful in affording a qualitative, microscale procedure for the identification of hexuronic acids (which otherwise are difficult to detect). 

---