EXPERIMENT 2

DETERMINATION OF REDUCING SUGAR USING THE

DINITROSALICYCLIC (DNS) COLOURIMETRIC METHOD
OBJECTIVE:
1. To determine the concentration of reducing sugar by using the
Dinitrosalicyclic (DNS) colourimetric method
2. To studies the application of UV-Visible Spectrophotometry
3. To use the standard curved to determine the concentration of
samples
INTRODUCTION
According

to

Dubois

et.

al

(2000),

Dinitrosalicyclic

(DNS)

colourimetric method is a tests for the presence of free carbonyl group

(C=O), which is reducing sugars. This involves the oxidation of the
aldehyde functional group present. 3, 5-dinitrosalicylic acid (DNS) is
reduced to 3-amino, 5-nitrosalicylic acid under alkaline conditions.
3, 5 Dinitrosalicyclic acid + Glucose (Reducing sugar)

3-amino-5-

nitrosalicyclic acid
+ Gluconic acid
The above reaction scheme shows that one mole of sugar will react with
one mole of 3, 5-dinitrosalicylic acid and reduced to 3-amino-5nitrosalicyclic acid
On

the

other

hand,

the

application

of

spectrophotometric

(colorimetric) method is by using light absorption to determine the

concentration of a molecule which absorbs light where this device known
as uv-vis spectrophotometer (double beam spectrophotometer) measures
the transmittance by calculating the ratio of the two intensities. These
obey the Beer-Lambert Law where it relates the absorbance of light by a
sample to the concentration of the absorbing species. In the Beer-Lambert
Law, the absorbance is directly proportional to the concentration of the
absorbance and also to the path length. In this experiment, the reducing

sugar concentration of two samples which are apple juice and strawberry
jam will be determined by using colourimetric (spectrophotometric)
method

MATERIALS
1. Double-beam spectrophotometer

10. Spatulas

2. Analytical balance

7. Wash bottle

3. Plastic/Glass cuvettes

8. Stop-watch

4. Beakers

9. Ice water

5. Vortex mixer

10. Glucose standard

6. Pipettes

11. 3, 5-dinitrosalicylic acid (DNS)

7. Volumetric flasks
8. Test-tubes and racks
tetrahydrate
9. Water-bath (boiling)

12. 2 Molar NaOH

13.

Sodium

potassium

tartrate

14. Distilled water

METHODS
A. Preparation of DNS reagent
1. Solution A was prepared first by dissolving 10 g of DNS in 200 mL 2
M NaOH with warming and vigorous stirring
2. Solution B was then prepared by dissolving 300 g sodium potassium
tartrate tetrahydrate in 500 mL distilled water
3. Both solution A and B was then mixed together before make up to 1
L distilled water
B. Preparation of the Sample
a) Solid sample

1. 3 g of solid sample was weighed accurately into a beaker and 50 mL

of distilled water was added. The solution was stirred until
thoroughly dissolved
2. The solution was then filtered into a 100 mL volumetric flask. The
residue was then washed into the volumetric flask with a small
amount of distilled water and the volume was making up to 100 mL.
The solution was then mixed well by repeated inversion of the
volumetric flask
b) Liquid sample
1. 5 mL of the liquid sample was pipetted
2. The liquid sample was filtered into a 100 mL volumetric flask. The
residue was washed into the volumetric flask with a small amount of
distilled water and make up to 100 mL volume. The liquid sample
was then mixed well with repeated inversion of the flask
3. 10 mL of sample solution was diluted to 100 mL distilled water in a
volumetric flask. The solution was then mixed well by inversion of
the volumetric flask
C. Preparation of Glucose Standard Solutions
1. 1.0 mL of distilled water was pipetted into a test tube and labelled
as blank
2. 1.0 mL of each concentration of glucose standard solution was
pipetted in order into appropriately labelled test tubes
3. 1.0 mL of sample solution prepared was pipetted into a test tube
and labelled as sample (The sample, standard and blank was
prepared in triplicate before testing was conducted)
1. 1.0 mL of DNS reagent and 3 mL of distilled water was then added
into prepared test tubes. The solution was then mixed well with a
vortex mixer
2. The test tubes was arranged in a test tubes rack before it was
placed in the boiling water bath for exactly 5 minutes to allow the
reaction between the DNS reagent and glucose to occur
3. The test tubes was then transferred immediately into a container of
iced water after 5 minutes in water bath
4. 10 mL of distilled water was then added to each test tube and mixed
well by using vortex mixer

5. The maximum wavelength was then determined by measuring the

absorbance spectrum of 4 mg/mL of glucose standard solution by
using a double-beam spectrophotometer.
6. The spectrum was measured within the wavelength range of 400 to
600 nm. The spectrophotometer wavelength was set by using the
highest absorbance obtained
7. The absorbance for each standard was measured for each standard
solution and the sample solutions
8. RESULT
Absorbance data for DNS colorimetric method
Standard/

Absorbance reading at

: 477 nm

Sample

Absorban

Absorbanc

Absorbance

Absorbance

Concentration

ce 1

e2

Average

(mg/mL)
0 (Blank)
2
4
6
8
10
15
Liquid Sample

0
0.456
0.826
1.099
1.312
1.315
1.060
0.068

0
0.443
0.825
1.097
1.223
1.319
1.067
0.039

0
0.434
0.831
1.096
1.310
1.373
1.152
0.038

SD
0
0.444
0.827
1.097
1.282
1.336
1.093
0.048

(Apple Juice)
Solid Sample

0.072

0.067

0.061

0.067

(Strawberry Jam)

2. Standard curve graph

a) State the glucose concentration range for linear curve
mg/mL 6 mg/mL
b) Write the equation derived from the linear curve
0.1925 x
c) Write the linear regression obtained

: y =
: 0.9852

CALCULATIONS
1. Show

one

example

of

calculation

for

preparing

desired

concentration of glucose solution from the stock solution given

To prepare 4 mg/mL glucose standard solution:C1V1 = C2V2
100 mg/mL x V1 = 4 mg/mL x 100 mL
V1 = 4 mL
2. Show the calculation for determining sample concentration from the
equation derived from the linear standard curve
1. Liquid sample (apple juice)
Y = mx
0.048 = 0.1925 x
X = 0.2494
Concentration:= X x dilution factor (Final dilution / Initial dilution)
= 0.2494 x (100/10)
= 2.494 mg/mL
2. Solid sample (Strawberry jam)
Y = mx
0.067 = 0.1925 x
X = 0.3481

Concentration:
= X x dilution factor (Final dilution / Initial dilution)
= 0.3481 x (250/10)
= 8.7025 mg/mL
DISCUSSION
Through the experiment, the concentration of reducing sugar which
are in apple juice (non-added sugar) and strawberry jam was determined
by using the Dinitrosalicyclic (DNS) colourimetric method. Both samples
which are solid sample and liquid sample was first prepared by weighing
approximately 3 g of solid sample and pipetting 5 mL of the liquid sample
respectively. The samples were then undergoing several dilutions after
being filtered properly into 100 mL of volumetric flask. The solid sample
solution was then diluted again by using ratio 1: 25 while the liquid
sample was diluted to 1: 10 dilution ratio. This is to ensure the
concentration in the sample is not too high and causing the sample
absorbance values is above highest point in the standard curve.
After that, a series of glucose standard solutions of 2 mg/mL, 4
mg/mL, 6 mg/mL, 8 mg/mL, 10 mg/mL and 15 mg/mL was prepared by
diluting 100 mg/mL glucose stock solution by using distilled water in 100
mL volumetric flask. Volume of glucose stock solution required to make
dilution was determined by using this formula:C1V1 = C2V2
Meanwhile, the measurement of absorbance was prepared by
pipetting each 1.0 mL of glucose standard solution and samples solution,
1.0 mL of DNS reagent and 3.0 mL of distilled water into the test tubes.
DNS reagent is a reagent used to determine sugar content especially
glucose. Meanwhile, the sample was prepared triplicate to minimize
human error. All the solution was mixed properly by using vortex before it
was placed in boiling water bath for 5 minutes to allow the reaction to

occur. In hot solution, the DNS reagent will reacts with the reducing sugars
producing the reddish-brown product (3-amino-5-nitosalicil acid).
As the concentration of the glucose standard solution increases, the
colour of the solution will be darker. As the more sugar there is, the more
carbonyl groups there are hence resulting in darker solution. After 5
minutes the test tubes was immediately transferred to container of ice
water to stop the reaction from occurring.

The concentration of the

coloured solution was then measured with the uv-vis spectrometer at 477
nm.
From the results, the absorbances average was calculated and
reducing sugar standard curve (linear and non-linear) was constructed to
calculate linear regression (R2) and obtained equation derived from
standard curve. From the equation, the concentration of samples solution
(liquid and solid sample) can be calculated where it was obeying the BeerLambert Law. The calculations show that the concentration of liquid
sample (apple juice) is 2.494 mg/mL meanwhile the concentration of solid
sample (strawberry jam) is 8.7025 mg/mL.
The results show that the concentration of solid sample (strawberry
jam) is higher than liquid sample (apple juice) as the strawberry jam
contains a lot of glucose. This is because; the strawberry jam was made
with higher amount of sugar where it can inhibit the growth of bacteria
hence extending the shelf life of the jam. Meanwhile, apple juice that was
used is non-sugar added typed where there is no sugar added in their
juices preparation. The sugars were only obtained from the fruits itself
which is from apple. According to Malaysian Food Law and Regulation
(1985), the soluble solids for apple juice shall not less than 11.5 g.
Meanwhile, for jam the soluble solids should be at the 65 per cent of
soluble solids.
During the experiment, they are some sources of error that occur
which may affect the accuracy of the result. For example, there are
parallax error that happen when the eyes does not directly proportional to

the reading of the pipette. Poor pipetting technique may also contribute to
the error besides there might be the present of bubbles during pipetting.
In addition, enzyme mixture (DNS) might not be prepared correctly or the
stability limit had exceeded. To minimize these errors various precautions
should be taken such as, make sure our eyes are directly proportional
during reading the pipette measurement. The fresh enzyme mixture was
prepared and re-assay. Lastly, three consecutive of measuring need to be
carried out to eliminate the errors of the handlers.
CONCLUSION
In conclusion, the concentration of reducing sugar in apple juices
and strawberry jam was determined by using the Dinitrosalicyclic (DNS)
colourimetric method which are 2.494 mg/mL and 8.7025 mg/mL
respectively.
REFERENCE
C. Michael, (2003), How does Dinitrosalicyclic Acid stops the enzymesubstrate reaction in

enzyme assay, Retrieved October 6, 2003 from

http://www.researchgate.net/post/How_does_Dinitrosalicylic_Acid_stops_th
e_enzy

me-substrate_reaction_in_enzyme_assay

M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers and Fred Smith, (2000),
Determination of Reducing Sugar by Colourimetric Method and Other
Substances, Anal.
April

Chem., 1956, 28 (3),

26,

pp

350356

2000

Retrieved

from

http://pubs.acs.org/doi/pdf/10.1021/ac60111a017
Malaysia. (1985). Food Act 1983 and Food Regulations 1985: Revised 7th
October, 1985: Act 281. Kuala Lumpur: MDC