Sugars quantitative determination

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. 

In as far as other analytical methods are concerned, many specific reactions have been elaborated for the quantitative determination of 2-deoxy aldoses. 2-Deoxy-D-ribose (2-deoxy-D-erythro-pentose), a compound which was recognized early as playing an important role in biological systems, has been of particular interest. Overend and Stacey (43) have given a critical review of the methods available until 1952 for the estimation of 2-deoxy pentoses. A recent summary of specific methods for the identification and quantitative estimation of the different classes of deoxy sugars has been prepared by Dische (13). 

Despite the above-mentioned short-comings, this approach to the estimation of those deoxy sugars which yield malonaldehyde when oxidized with periodate, seemed promising, since, as has been seen (58,59), the dye is formed quantitatively in the reaction of malonaldehyde with 2-thiobarbituric acid also, more recently, its constitution (49,57) and molar extinction coefficient (36) have been established. Thus, if conditions could be found in which malonaldehyde, while being formed quantitatively from the deoxy sugars, would be stable, an ideal method, independent of standard compounds, would be available for the quantitative determination of all of these sugars. 

Davison, P. K., Young, R. (1964). Gas chromatography of carbohydrates the quantitative determination ofthe free sugars of plants as their trimethylsilyl ethers. Journal of Chromatography A, 41, 12-21. 

Water is the only solvent in which the composition of sugars has been systematically explored. Stevens1674 has determined the composition of several aldoses in pyridine-d5 by -n.m.r, spectroscopy at 300 MHz. There are scattered data on solutions in organic solvents (mainly pyridine, dimethyl sulfoxide, and N,N-dimethylformamide), but only rarely have four (or more) components of such solutions been quantitatively determined. The data that have been encountered are collected in Table VII undoubtedly, there are others that have been missed. 

Whether eluted from columns or from thin-layer plates, the quantitative determination of sugars was traditionally based on colorimetric reactions involving the use of chemical reagents, e.g., anthrone. These detection methods have been largely replaced in modem HPLC by the refractive index detector, although ultraviolet detectors are also employed. Recently we have also seen the introduction of other types of detector (e.g., the mass detector), as will be discussed later. 

Acid chlorides are used for the quantitative determination of hydroxyl groups and for acylation of sugars. Industrial applications include the formation of the alkyl or aryl carbonates from phosgene (see Carbonic and chloroformic esters) and phosphate esters such as triethyl, triphenyl, tricresyl, and tritolyl phosphates from phosphorus oxychloride. 

When quantitative determination is necessary, the procedure is as with diffusion juice. With very small amounts of sugar, extra long polarimefcer tubes are used, or the liquid is rendered faintly alkaline with a few drops of milk of lime and evaporated, the residue being neutralised with dilute acetic add, clarified, made up to one-half or one quarter of the original volume and polarised. 

When V-acetyl-D-glucosamine is heated with dilute sodium carbonate solution a colorless chromogen is formed. The latter yields a violet dyestuff370 with p-dimethylaminobenzaldehyde in acidic solution. This reaction is the basis of the quantitative determination of A-acetylamino sugars (Morgan-Elson reaction). 

Neeser, J. R., and Schweizer, T. P. (1984). A quantitative determination by capillary gas—liquid chromatography of natural and amino-sugars (as o-mthyloxime acetates) and a study on hydrolytic conditions for glycoproteins and polysaccharides in order to increase sugar recoveries. Anal. Biochem. 142(1), 58-67. 

Figure 6 NMR spectra of HMW DOM from surface seawater, (a) C-NMR spectra can be used to quantitatively determine the functional groups, and by inference, the relative importance of different biochemical classes in HMW DOM. The spectra highlight the importance of carbohydrates (100 ppm and 70 ppm), carboxylic acids (175 ppm), and alkyl carbon (10-30 ppm), (b) H-NMR also show the importance of carbohydrates (4 ppm) and alkyl carbon (1 ppm), but additionally show that acetyl groups most likely bound to carbohydrate are an important components. N-NMR show that 80-90% of HMW DON is amide, while 10-20% is free amine. Quantitative analyses for acetate and nitrogen suggest that most amide in surface seawater is bound as A-acetyl amino sugars and protein residues. In the deep ocean HMW DOM however, most amide is nonhydrolyzable, and is of unknown molecular environment.

Reduction of Fehling s Solution.—The reaction of sugars with an alkaline tartrate solution of copper sulphate, known as, Fehling s solution, while not giving any information as to the constitution of sugars, is of importance in distinguishing certain sugars and other carbohydrates, and of still more value as the basis of analytical methods for their quantitative determination. 

One milliliter of mixed solution will react with 5 mg of glucose the empirically determined ratio is the basis for quantitative determination of the sugar. The Fehling s test is not specific to reducing sugars, because ordinary aldehydes reduce the reagent although by a different mechanism and at a different rate. 

Among the other sugars detected in the soil, only the 6-deoxyhexoses have been quantitatively determined. In Delaware soils, rhamnose and fucose, determined by quantitative paper-chromatography, amounted to 20% of the sugars. Under the conditions of furfural formation from pentoses, the 6-deoxyhexoses yield 5-methyl-2-furaldehyde this has been determined by the differential solubilities of the phloroglucides in alcohol. The proportion of 6-deoxyhexoses in some cases exceeded that of pentoses. 

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