Reducing Sugar Methods

Reducing Sugar Methods (1) a. Oxidation by Metallic Salts in Alkaline Solution 

RCHO -h Ag20 - 2 Ag -h RCOOH RCHO + 2 CuO CU2O + RCOOH 

However, the reaction does not proceed stoichiometrically. It has been shown previously (see Chapter I) that sugars with free aldehyde and ketone groups quickly undergo change even in weakly alkaline solution. Glucose, fructose, and mannose undergo a mutual interconversion until equilibrimn 

Under such circumstances it is amazing that the reaction has quantitative value. But it has been found that, although the products are many and variable, it is possible to standardize the conditions so that the amount of cuprous oxide may be used as a measure of the quantity of sugar. 

Citrates, oxalates, salicylates, glycerol, and cane sugar also stabilize alkaline solution of cupric salts. Some of these, citrates in particular, have been used in the preparation of copper solutions for sugar analysis. 

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Rates of Enolization Reactions. For a better understanding of the transformation and oxidation reactions of reducing sugars, methods have been developed to measure the primary rates of enolization (18). One of these methods depends on the rate at which tritium ions are released from aldoses-2- to the solvent. This is measured by separation of the water-, sublimation, and radiochemical assay of the water as the reaction proceeds. The rate constant is calculated from the first-order equation ... 

In 0.01 N hydrochloric acid at 95 (method not given). Reducing-sugar method. 

Davis, R.E. A combined automated procedure for the determination of reducing sugars and nicotine alkaloids in tobacco product using a new reducing sugar method ... 

Gcl-25m) equilibrated with a buffer consisting of 0.25 M acetic acid 0.05 M ammonium acetate (1 1, pH 4.0) was also used for the separation of chitosan oligosaccharides. The oligosaccharides in the effluents were determined by ninhydrin color detection at 570nm. The reducing sugar method was also used for determination of individual oligosaccharides. 

Emmerich Method. This method is for determination of trace amounts of reducing sugars in pure sucrose and white and refined sugars with reducing sugar content up to 0.15%. The test is carried out in a nitrogen atmosphere and is based on the reduction of 3,6-dinitrophthahc acid. 

Knight and Allen. This is a copper reduction method for reducing sugars in white sugar up to 0.02%. It utilizes EDTA to determine excess unreacted copper. Tests undertaken in 1994 to extend the range of this method were unsuccesshil. In spite of poor performance in ring tests, it remains an official ICUMSA method. 

Luff Schoorl. This method is for the determination of total reducing sugars in molasses and refined symps after hydrolysis. It is a copper-reducing method that forms the basis of some molasses purchasing contracts. 

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 the sugar industry, where the goal is to determine the exact amount of sucrose present, the analysis of other components is essential to determine purity. The most important of these, besides reducing sugars discussed, are moisture, ash, and color. Also relevant are methods used to determine particle-size distribution and insoluble matter. 

The principal analytical methods were developed in the mid-nineteenth century the polariscope by Ventzke in 1842, the Brix hydrometer in 1854, Fehling s method for reducing sugars, and Clerget s method for sucrose in 1846. 

Oxidation of the aldehyde group of an aldose to form a carboxyUc acid or carboxyUc acid anion is often used analytically to determine the amount of reducing sugar. The Benedict and Fehling methods measure the amount of reducing sugar present in a fluid. In these reactions, the oxidant, Cu ", is reduced to Cu". Cu" precipitates as CU2O, which can be measured in a variety of ways. In the ToUens test, Ag" is reduced to Ag. ... 

The dilute acid of HCl was used as a pretreatment agent. In the first method, bean curd refuse was pretreated without heating. An amoimt of 0.5 g bean curd refuse was dipped in 10 ml of 1 mol/1 HCl aqueous solution for 1, 3, 6 and 24 hours. In the next, 0.5 g bean curd refuse was dipped in 10 ml distilled water or HCl solution in the concentration range of 0.01-1 mol/1 and held in a steam-heated auto clave at 121 C for 1, 15 or 30 min. After neutralization with 5 mol/1 NaOH solution, the concentrations of reducing sugar, total sugar and TOC were measured for the pretreated solution. 

Figure 1. Effect of 1% (w/v) pectin (pect), 1% galactose (gal), and of the simultaneous presence of 2% glucose (glu) on the production of extracellular polygalacturonase activity. Two-stages cultures were prepared as described under methods. Polygalacturonase was assayed in the culture filtrate as reducing sugar-releasing activity using sodium polypectate as a substrate.

Figure 2. CM-cellulose chromatography of pectolytic enzymes. The activity peaks of the flow-through of a DEAE-cellulose chromatography was applied to a CM-cellulose column. The column was eluted with a NaCl (0-0.5M) continuous gradient at a flow rate of 34 ml/h. 10 ml fractions were collected and assayed for pectolytic activities Symbols (0) pectate lyase ( ) polygalacturonase (reducing sugar-releasing activity) (x) protein. Other details in Methods.

Figure 5. pH activity profile of PGE on polygalacturonate. Mcllvaine buffers were used in the pH range from 2.5 to 5,5, Reducing sugars were detected by the neocuproine method. PGE (2 mg/ml) was 80-times diluted and incubated with 500 pi of a 0.25% (w/v) polygalacturonate solution, at SO C. 

Peris-Tortajada, M., Puchades, R., and Maquieira, A. (1992). Determination of reducing sugars by the neocuproine method using flow injection analysis. Food Chem. 43,65-69. 

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