Determination of reducing sugars concentration by DNS method Aim: To determine the concentration of reducing sugars in unknown sample by DNS method. Principle: Reducing sugars have an aldehyde group which can be oxidized through redox reactions. This property of reducing sugar has been utilized in estimating their concentration in many reactions such as Benedict’s, Fehling’s and DNS method. DNS (Dinitrosalicylic acid) is one of the reagents used to estimate reducing sugars in the solution. It ’s a spectrophotometric method. 3,5-Dinitrosalicylic acid is reduced to 3 amino 5 nitro salicylic acid while oxidizing the reducing sugars. The color change can be quantified spectrophotometrically at a wavelength of 540 nm. Chemical reaction:
Reagents required: DNS reagent composition (for 1000ml): 1000ml):
Sodium hydroxide: 10g Sodium potassium tartarate: 192g Dinitrosalicylic acid: 10g Phenol: 2g Sodium sulphite: 0.5g Make up the volume to 1000ml with distilled water Standard Glucose solution: 10 mg/ml glucose solution Precaution: Add sodium potassium tartarate pinch by pinch and store the prepared DNS solution in amber colored bottle. Procedure:
1. Take 16 clean test tubes, label seven tubes as B, 1, 2 3, 4, 5, 6, U and other 8 tubes as duplicates dB, d1, d2, d3, d4, d5, d6 and dU. 2. Add 0 ml, 0.1 ml, 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml, 1 ml of standard glucose solution to tubes labeled B, 1, 2 3, 4, 5 and 6, respectively. Similarly make duplicates in duplicate test tubes. 3. Add 1 ml of unknown sugar sample in ‘U’ mark test tube. Similarly, make unknown duplicate in duplicate test tube. 4. Add 1 ml of DNS to each test tube as mentioned in the table below. 5. Incubate the tubes in boiling water bath for 5 minutes. M. Tech. I Sem (Biotechnology)
Page 1
Determination of reducing sugars concentration by DNS method 6. Cool the tubes to room temperature and add 8 ml of distilled water and mix well. 7. Adjust the spectrophotometer with a blank. 8. Take 1ml of reaction mixture in a clean cuvette, and check the absorbance at 540nm. 9. Note down the absorbance values for each reaction mixture and their duplicates. 10. Calculate standard deviation for each pair of values. 11. Plot a graph between average optical density (O.D) at 540 nm against concentration of glucose (S) and use linear regression analysis to draw the straight line with error bars based on standard deviation. 12. Estimate the slope of line, line of equation to determine the concentration of reducing sugar in an unknown sample.
Table: Experimental and observation Table
Serial number
B 1 2 3 4 5 6 Unkno wn (U)
Standard Distilled glucose water solution (ml) (ml) 0 0.1 0.2 0.4 0.6 0.8 1.0 1.0
1 0.9 0.8 0.6 0.4 0.2 -
DNS (ml)
Incubation Distilled Concentration water of glucose (ml) (µg/ml)
O.D. (540 nm) 1
1 1 1 1 1 1 1 1
s e t u n i m 5 r o f C 5 9
°
8 8 8 8 8 8 8 8
Avg O.D
Standard deviation
2
0 100 200 400 600 800 1000 ??
Observation:
The intensity of the color observed will be proportional to the concentration of glucose present in the solution.
Result: 2
The slope (∆ O.D / ∆ S), equation of line (Y = mX + C) and goodness of fit (R ) from standard curve drawn between O.D. vs. concentration of glucose are _________, _________ and_________, respectively. The reducing sugar concentration in the unknown sample is _________ µg/ml.
M. Tech. I Sem (Biotechnology)
Page 2
