INTRODUCTION Determining the sugar concentration of food samples is very important 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 acid(DNS) which produce a red-brown product Miller(1959)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. The sugar concentration of unknown sample can then be read off a calibration curve(standard curve) created using known sugar concentrations.
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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 Materials •A freshly prepared solution of DNS was provided •Sugar solution of unknown concentration •Stock sugar standard(fructose solution in saturated benzoic acid) •Working sugar standards(fructose solutions diluted 1 in 4 before use •Boiling water bath Method
A serial dilution was made by using 1ml of DNS reagent with 3ml of working sugar standards and made up to 4 tubes. One tube of unknown was made by mixing 1ml of DNS with 3ml sugar solution. A blank was prepared by adding 1ml DNS to 3ml distilled water. The tubes were covered with marble and were placed in a water bath for 5 minutes. They were cooled to room temperature and then read at 540nm against the blank. Results Below is the tabulated form of the absorbance reading obtained from the spectrophotometer at 540nm: Table 1: Absorption values of the 5 tubes TubeAbsorbance(nm)Concentration( g/ml)Dilution 10. 1522500. 20. 1501250. 25 30. 07862. 50. 125 40. 07131. 250. 0625 U(unknown)0. 145Unknown Below is the standard curve of the 4 tubes and the unknown sample: Fig 1:standard curve of the 4 tubes and the unknown fructose sample Concentration Equation of ‘line of best fit’ is y=0. 3915x + 0. 0669 By substituting our absorbance of unknown(y value) into equation, we come up with the concentration~0. 145=0. 3915x + 0. 0669 X=(0. 145- 0. 0669)/0. 3915 X=199. 8 The concentration of the unknown was found to be 199. µg/ml Discussion The experiment was done according to the lab manual instructions but the standard curve seem to be wrong. In theory the graph must be a straight line yet a bent graph was obtained in the experiment. This may be attributed to either the preparation of DNS(no longer fresh)or the handling of the cuvettes. Therefore the concentration of the unknown was found by drawing a ‘line of best fit’ through the graph. On loading the cuvettes into the spectrophotometer, an error was done by putting tube1 which has the highest concentration instead of starting with the least concentrated tube(4).
This error was quickly detected and the error was corrected . Also the tubes had cooled to room temperature when absorbance was recorded. No alternative of making the experiment faster could be found since the steps followed are very particular, for example, the tubes must be boiled in water bath for 5min . References 1. Skoog,D. A. et al (1996) Fundamentals of Analytical Chemistry,7th ed, Saunders College Publishing, New York,pg 563-573 2. Miller,G. L. (1972)Analytical Chemistry,Kalyani Publishers,New Delhi,pg 426 3. Dr Mokwena’s source.