Determination of Redusing Sugar Using Dns Method

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1.0ABSTRACT
The production of reducing sugars by acid of carbohydrate cereals powder, jams (total sugar content) and jams (reducing sugar content) were performed in order to study reducing sugar production. The study involved hydrolysis carbohydrate cereals powder and jams by immersing in boiling water at environmental temperature. Acid hydrolysis was carried out through reflux boiling for 20 minutes with 10ml 0f 1.5M concentrations of diluted sulphuric acid. Reducing sugars concentrations were determined by spectrophotometry using the 3, 5 dinitrosalicylic acid (DNS) method. The first sample was operated as blank for zero. The stock solution was prepared in order to find the unknown concentration of carbohydrate cereals powder, jam (total sugar content) and jams (reducing sugar content). The absorbance of stock solution were 0.017,0.030, 0.038, and 0.099 respectively and the absorbance of carbohydrate cereals powder, jams (total sugar content) and jams (reducing sugar content) were 0.06, 0.014 and 0.138 respectively. After determining the specific absorbance of stock solution, the calibration curves was made to determine the unknown concentration of carbohydrate cereals powder, jams (total sugar content) and jams (reducing sugar content).

2.00 INTRODUCTION
Determination of total reducing is of great important in the food industries especially in industries where quality control is monitored. One method to determine the sugar concentration of reducing sugars is by heating with 3, 5 dinitrosalicylic acids (DNS) which produce a red-brown product (Miller1959) The reaction is direct, thus the method is preferred over the Benedict’s test method.

The concentration of the coloured complex can be determined with the spectrophotometer at Absorbance 540 ( http://en.wikipedia.org/wiki/3,5-Dinitrosalicylic_acid) .The sugar concentration of unknown sample can then be read off a calibration curve(standard curve) created using known sugar concentrations. The dilutions of a solution of known concentration are used to determine the concentration of unknown. Being familiar with the background information about reducing sugars and various methods used to identify them, Biotechnology students were provided with a Fructose sample solution and were required to find its concentration Maltose can be used as a standard for estimating reducing sugar in unknown samples. Constructing a standard curve / graph for maltose helps us to estimate concentration of reducing sugars present in an unknown sample and for determining the activity of amylase enzyme in forthcoming experiments. The standard curve for maltose is usually constructed using 3, 5-Dinitro salicylic acid (DNS) as the reagent. Maltose reduces the pale yellow colored alkaline 3,5-Dinitro salicylic acid (DNS) to the orange- red coloured, 3 amino,5 nitro salicylic acid. The intensity of the colour is proportional to the concentration of maltose present in the solution as per Beer Lambert's law; (http://amrita.vlab.co.in/?sub=3&brch=63&sim=156&cnt=1). This intensity change in colour is measured using a colorimeter as the absorbance at 540nm wavelength. Wave length is set to 540 nm because it is the region where orange-red colour absorbs. A series of solutions containing varying concentrations of maltose are prepared in test tubes and a known quantity of DNS is added to each. These test tubes are then heated on a water bath for few minutes and their optical densities are measured using a colorimeter. A graph is then plotted with amount of maltose on X axis and the observed optical density at Y axis. The plot thus obtained is called a standard maltose curve.

This method tests for the presence of free carbonyl group (C=O), the so-called reducing sugars. This involves the oxidation of the aldehyde functional group present in, for example, glucose and the ketone functional group in fructose. Simultaneously,...
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