Determination of Reducing Sugars Using Dns
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.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
Tube Absorbance(nm) Concentration( g/ml) Dilution
1 0.152 250 0.5
2 0.150 125 0.25
3 0.078 62.5 0.125
4 0.071 31.25 0.0625
U(unknown) 0.145 Unknown
Below is the
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.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
Tube Absorbance(nm) Concentration( g/ml) Dilution
1 0.152 250 0.5
2 0.150 125 0.25
3 0.078 62.5 0.125
4 0.071 31.25 0.0625
U(unknown) 0.145 Unknown
Below is the
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.