Carbazole method, uronic acid

Uronic acids were traditionally measured by the carbazole reaction. In this procedure, sugars were treated with concentrated sulfuric or hydrochloric acid to yield products that reacted with carbazole. For example, Dische (1947) used carbazole for the determination of uronic acid in solutions of sulfuric acid, but the method was subject to interference from neutral sugars that were also present in the solution. The... 

Dermatan sulfate may be distinguished from chondroitin 4- and 6-sulfates in that it is not degraded by testicular hyaluronidase and, furthermore, the desulfated mucopolysaccharide is unattacked by testicular and bacterial hyaluronidases (M17). Further diflFerentiation of dermatan sulfate from hyaluronic acid and the foregoing chondroitin sulfates is readily made on the basis of color reactions given by the different uronic acid components. Dermatan sulfate shows equimolar ratios of uronic acid ihexosamine sulfate when the uronic acid content is determined by the orcinol (K7) or decarboxylation (T4) methods, whereas significantly lower values are obtained by the carbazole method (D8). 

Uronic acids in soil extracts have been determined by the carbazole method in proportions of 0.07 to 0.16 % of the total soil. In another study, the uronic acids constituted 1 to 4 % of the organic matter of the soils. The glycosiduronic acids arc not completely extracted from the soil, and... 

In all the colorimetric methods, galacturonic acid is liberated during the assay by hydrolysis of polymeric pectin. An application of the carbazole method, after extraction of pectin from various fruits and vegetables, has been described (6), as has been an automated carbazole method for monitoring uronic acid levels in pectin fractions (7). 

Although widely used, colorimetric methods for determining total ur-onic acid and hexosamine are now regarded as providing, at best, only semi-quantitative estimates of heparin. Colorimetric reactions in a strong acid, such as the carbazole reaction for the uronic acids and the Morgan -Elson reaction for the hexosamines, are not specific for hepa-... 

FIGURE 15.2 Alginate content eluted into weak alkaline solution from non-shaved kombu (NSK) and tororokombu (TK). The uronic acid content was analyzed using total components eluted from NSK and TK into phosphate-buffered saline (pH 8.0) for the determination of alginate by the carbazole method. Data are presented as the mean SE (n = 3). p < 0.005 versus NSK. 

In addition to the reactions mentioned above, there are others of this type that are used in detecting and determining specific classes of carbohydrates. The carbazole-sulfuric acid method has been much used in analysis of uronic acids and acidic polysaccharides, which give a weaker response than neutral sugars in the other methods. The presence of borate ions decreases interference by proteins and salts, increases color yield, and reduces the time required for color development. In a recommended procedure for manual analysis, the sample solution (containing 0.02-0.2 [tmol of uronic acid) is mixed (1 5, v/v) with a solution of sodium tetraborate (25mmol 1 ) in concentrated sulfuric acid and the mixture is heated at 100°C for 10 min. After rapid... 

NaOH, should be subtracted from the reading. This method is more sensitive and specific than the carbazole method. The molar absorptivity of the chromophore produced by mannuronic acid is 50% of that of the product from glucuronic acid, but the other uronic acids commonly encountered,... 

Pectin in almond kernels can be determined by extracting ground samples with water (soluble pectins) and then 0.05 M HCL at 100 °C for 90 min (insoluble pectins). Pectins can be determined as uronic acid by the carbazole method, using galacturonic acid as standard (Bitter and Muir 1962). 

Almost half of the known types of polysaccharides contain uronic acid units. Uronic acid residues may be determined by decarboxylation with 12% hydrochloric acid and the evolved carbon dioxide determined by absorption (4) or by manometric measurement (S). A micro-quantitative colorimetric method employing carbazole has been developed for uronic acids (6), (See also Part II of this chapter and Chapter XI.)... 

In the presence of strong mineral acids the uronic acids give specific colours with phenolic compounds such as naphthoresorcin, and a colorimetric method for the determination of uronic acids utilizing carbazole and sulphuric acid has been developed. A similar method which depends on the colour produced with benzidene in glacial acetic acid has also been devised . Alternatively the acids can be converted into the hydroxamic acids which give intensely coloured compounds with ferric salts. The intensity of colour can be measured and the method is sufficiently accurate to be used on the semi-micro scale . Care should be observed in the interpretation of the results as other acids such as acetic acid derived from acetyl residues, interfere. 

In heparan sulfate the amount of uronic acid estimated by the carbazole method was much higher than the one found by the decarboxylation method, an observation also made in the case of heparin. Although it has been suggested that this high value was due to the presence of sulfate groups, it is still possible that it depends on the presence of another uronic acid, besides glucuronic acid. 

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