Phenol-sulfuric acid method

In concentrated sulfuric acid, polysaccharides are hydrolyzed to their constituent monosaccharides, which are dehydrated to reactive intermediates. In the presence of phenol, these intermediates form yellow products, such as that shown in Eq. 1.6,28 with a combined maximal absorbance at 492 nm. 

The phenol-sulfuric acid method has been adapted for use with 96-well microtiter plates. Carbohydrate samples are combined with an equal volume of 5% (w/v) 

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Colorimetric Methods. Numerous colorimetric methods exist for the quantitative determination of carbohydrates as a group (8). Among the most popular of these is the phenol—sulfuric acid method of Dubois (9), which rehes on the color formed when a carbohydrate reacts with phenol in the presence of hot sulfuric acid. The test is sensitive for virtually all classes of carbohydrates. Colorimetric methods are usually employed when a very small concentration of carbohydrate is present, and are often used in clinical situations. The Somogyi method, of which there are many variations, rehes on the reduction of cupric sulfate to cuprous oxide and is appHcable to reducing sugars. 

Uronic acid was estimated by the rw-hydroxyphenyl method [4], O-Acetyl contents by the method of Hestrin [5], carbohydrate by the phenol-sulfuric acid method [6] and protein by the Hartree method [7]. 

Purity was confirmed by gel-filtration using a HPLC column packed with Asahipak GS-520HQ and elution with 100 mM sodium phosphate buffer containing 300 mM sodium chloride (pH 6.7). The content of total protein, total sugars, uronic acids, sulfates, nucleic acids, phosphate or fatty acids was assayed by the BCA [32] and Lowry method [33], the phenol-sulfuric acid method [34], the Blumenkrantz method [35], nephelometry [36], absorption at 260 nm, the Bartlett method [37] and the GLC method after methyl-esterification [38], respectively. 

To determine intracellular polysaccharide, 100 mg of powdered dry hairy roots was suspended in 10 mL of distilled water, sonicated for 1 h, and centrifuged twice at 5030y for 10 min. The collected supernatant was used to determine intracellular polysaccharide by the phenol-sulfuric acid method (19). 

Other photometric methods have been described. The phenol-sulfuric acid method (N8, R3) calls for the use of dangerous chemicals, namely, 80%... 

Another purified enzyme preparation which produces laminaripentaose from insoluble laminarin and from heat-treated pachyman is produced by a strain of Arthrobacter luteus (100,101,102) when grown on yeast cells or / -(1 —>3)-glucan. The enzyme, which was named Zymolase (also referred to as Zymolyase) appeared to be homogeneous by electrophoresis in a Tiselius apparatus and by ultracentrifugation. The molecular weight of the enzyme was estimated from ultracentrifugation to be ca. 20,500. The optimum pH for lysis of viable yeast cells was 7.5. The optimum temperature was 35°C. The optimum pH for heat-treated pachyman hydrolysis was 6.5, and the optimum temperature was 45°C. A Lineweaver-Burk plot with heat-treated pachyman yielded a Km value of 0.04% when the solubilized carbohydrate was assayed by the phenol-sulfuric acid method. Zymolase lost all its activity after incubation at 60°C for 5 min. 

Sugars in the culture medium, assayed as glucose by phenol-sulfuric acid method... 

Chemical analyses. Total carbohydrate was determined by the phenol-sulfuric acid method of Dubois (1 ), and total... 

Determined by the phenol-sulfuric acid method (13). Determined by the Folin method of Lowry et al. (jU). Quantitated by the methpd of Ames and Dubin (13) as PO H cl.O, 11.0, water. 

Enzyme Assays. Starch digestion from blends by porcine a-amylase (Sigma) was determined by measuring soluble product formation by the phenol-sulfuric acid method (12). Incubations were conducted in 20 mL of 0.05 M phosphate buffer (pH 7.0) containing 8 pieces of 1 x 2 cm starch-plastic blend and 2 /iL mL merthiolate to inhibit microbial catabolism of digestion products (13). Merthiolate did not inhibit en2yme activity or interfere with product assays. Sufficient enzyme was added to give a 100 to 200 units mL solution. Incubation temperature was 35° C. Mixtures were shaken at 50 to 70 rpm on a rotary shaker. 

The phenol-sulfuric acid method involves treatment of the solution containing the carbohydrate analyte (5—40pgml ) with an aqueous solution of phenol (5%, m/v) and then concentrated sulfuric acid a characteristic yellow color is produced, with an absorption maximum at 490 nm for hexoses, 480 mn for pentoses, deoxy sugars, and uronic acids. Only amino sugars do not react. Oligo- and polysaccharides are hydrolyzed to their constituent monosaccharides by the addition of concentrated... 

Anthrone (9,10-dihydro-9-oxo-anthracene) reacts with most carbohydrates in concentrated sulfuric acid to produce a characteristic blue color, with an absorption maximum at 625 nm. This reaction has been automated and was once widely used in analysis of dextran fractions emerging from size exclusion columns. However, as in the case of the phenol-sulfuric acid method, the presence of concentrated acid necessitates special precautions (such as the inclusion of a pulse suppressor) and for this reason, and also because of the instability of the reagent, the method is seldom used today except in manual analysis. 

Gel filtration of the reaction products was carried out on a Sephadex G-75 column (1.8 x 90 cm) using 0. 1 M NaCl as a solvent. Two mL fractions were collected and the amount of sugars in each fraction was determined the phenol-sulfuric acid method(8). 

Masuko, T., Minami, A., Iwasaki, N. et al. (2005) Carbohydrate analysis by a phenol-sulfuric acid method in microplate format. Anal. Biochem., 339, 69-72. 

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