DETERMINATION OF REDUCING SUGAR USING THE DINITROSALICYLIC ACID (DNS)

COLOURIMETRIC METHOD

INTRODUCTION

OBJECTIVES

APPARATUS AND REAGENTS

Double-beam spectrophotometer
Analytical balance
Plastic-Glass cuvettes
Beakers
Vortex mixer
Pipettes
Volumetric flasks
Test-tubes and racks
Water-bath (boiling)
Spatula
Wash bottle
Stop-watch
Ice water
Glucose standard
3,5-dinitrosalicylic acid (DNS)
2 Molar NaOH
Sodium potassium tartrate tetrahydrate
Distilled water

PROCEDURES
1. Preparation of DNS Reagent
Solution A: 10g of DNS were dissolved in 200mL NaOH with warming and
vigorous stirring.
Solution B: 300g sodium potassium tartrate tetrahydrate were dissolved in 500mL
distilled water.
 Both solution A and B were mixed and made up to 1L with distilled water.

2. Preparation of Sample
For solid sample:
(a) Approximately 3g of the solid sample was weighed accurately into a beaker and
then 50mL of distilled water were added. The mixture was warmed and stirred for
5 minutes (or until thoroughly dissolved).
(b) 100mL of the mixture were filtered into a 100mL volumetric flask. The residue
was washed into the volumetric flask with a small amount of distilled water and
then make up to a volume (100mL). The sample solution was mixed well with
repeated inversion of the flask.
(c) 10mL of sample solution were diluted to 250mL with distilled water in a volumetric
flask and mixed well by inversion of the volumetric flask (if necessary, further
dilution may be conducted).

For liquid sample:

(a) 5mL of the liquid sample were pipetted and filtered into a 100mL volumetric flask.
The residue was washed into the volumetric flask with a small amount of distilled
water and then made up to volume (100mL). The sample solution was then mixed
well with repeated inversion of the flask.
(b) 10mL of sample solution were diluted to 100mL with distilled water in a volumetric
flask and mixed well by inversion of the flask (if necessary, further dilution may be
conducted).

3. The line of best fit was drawn by using only the points in the linear region. The linear
regression (R2) for the standard curve was calculated (If the R2 is less than 0.98,
repeat the steps, starting from Step 3 of the Preparation of Glucose Standard
Solutions).
4. The concentration of the sample solution was calculated from the graphs obtained.

RESULTS

1. Record of absorbance data obtained:

Absorbance data for DNS colorimetric method

Standard/Sample Absorbance Reading at 496nm
Absorbance 1 Absorbance 2 Average
Concentration
 (mg/mL) Absorbance±SD

Blank 0 0 0
Solid sample 0.596 0.628 0.612±0.023
(strawberry jam)
Liquid sample 0.395 0.417 0.406±0.016
(apple juice)
1 mg/mL 0.561 0.557 0.559±0.003
2 mg/mL 1.255 1.259 1.257±0.700
3 mg/mL 1.888 1.811 1.850±0.054
4 mg/mL 2.256 2.231 2.244±0.018
6 mg/mL 2.471 2.469 2.470±0.001
8 mg/mL 2.530 2.528 2.529±0.001

Standard curve graph

*graph and calculation

(a) State the glucose concentration range for the linear curve:
(b) Write the equation derived from the linear curve:
(c) Write the linear regression obtained:

Calculation
(a) Show one example of calculation for preparing a desired concentration of
glucose solution from the stock solution given.
(b) Show the calculation for determining sample concentration from the equation
derived from the linear standard curve.

DISCUSSIONS

CONCLUSION