FST606

Instrumental Analysis of Food

NAME

MATRIC NO.

GROUP AS246 5A

TITLE OF EXPERIMENT Experiment 1: Determination of reducing sugars

using the dinitrosalicylic (DNS) colourimetric method

LECTURER’S NAME DR. RASEETHA VANI SIVA MANIKAM

 Experiment 1: Determination of reducing sugar using the Dinitrosalicylic (DNS)
Colourimetric method

INTRODUCTION:
Spectroscopy is an analysis technique of how light and other radiation are
absorbed and interacted by matter, in which the processes are influenced by the
wavelength of the radiation. In other words, it is used to estimate the quantity of light
that passes through a sample, and to assess the quantity of absorbed light, it needs
to be compared to the initial intensity of light entering that sample. In this experiment,
the UV-vis double beam spectrophotometer is used to quantitatively measure the
absorber concentrations in a solution as well as to calculate the ratio of two levels of
light intensities which both follow Beer Lambert’s Law.
When reducing sugars, such as aldehyde or keto groups, are present,
reducing sugars are formed, and it becomes a sugar with a reducing action.
Glucose, maltose, and lactose are some examples of reducing sugars. When other
molecules react with reducing sugars, the oxidation-reduction chemistry process
takes place. Some frequently used methods in quantifying reducing sugar are the
Somogyi-Nelson Method and the DNS method. In this experiment, the DNS
colourimetric method is specifically used to detect the presence of the carbonyl
group (C=O) in reducing sugar. When the DNS combines with reducing sugar and
other reducing molecules, it produces 3-amino-5-nitrosalicylic acid, an aromatic
compound that absorbs light significantly at 540 nm, allowing for a quantitative
spectrophotometric assessment of the quantity of reducing sugar present.

OBJECTIVES:
The objectives of this experiment are to determine the concentration of
reducing sugar using the Dinitrosalicylic (DNS) colourimetric method and to find out
the samples concentration by utilizing the standard curve.
MATERIALS AND APPARATUS:
- Double-beam spectrophotometer
- Analytical balance
- Plastic Glass cuvettes
- Beakers
- Vortex mixer
- Pipettes
- Volumetric flasks
- Test-tubes and racks
- Water-bath (boiling)
- Spatulas
- Wash bottle
- Stop-watch
- Ice water
- Glucose standard
- 3,5-dinitrosalicylic acid(DNS)
- 2 Molar NaOH
- Sodium potassium tartrate tetrahydrate
- Distilled water

METHODS:
RESULTS:
1. Table 1: Absorbance reading for standard and sample concentration 
Glucose Standard Solution/ Sample Absorbance Reading at 496 nm
Concentration (mg/mL)
Rep 1 Rep 2 Average absorbance

Blank 0 0 0
Solid Sample (Strawberry Jam) 0.596 0.628 0.612

Liquid Sample (Apple Juice) 0.395 0.417 0.406

Glucose standard solution Level 1 0.561 0.557 0.559

(1 mg/mL)

Glucose standard solution Level 2 1.255 1.259 1.257

(2 mg/mL)

Glucose standard solution Level 3 1.888 1.811 1.850

(3 mg/mL)

Glucose standard solution Level 4 2.256 2.231 2.244

(4 mg/mL)

Glucose standard solution Level 5 2.471 2.469 2.470

(6 mg/mL)

Glucose standard solution Level 6 2.530 2.528 2.529

(8 mg/mL)

Non-linear region
 Graph of absorbance against concentration of glucose

3
2.47 2.53
2.5 2.24
Absorbance reading (nm)

2 1.85

1.5 1.26

1
0.56
0.5
0
0
0 1 2 3 4 6 8
Concentration of glucose (mg/mL)

Figure 1: Non-linear graph of average absorbance against the

concentration of glucose (mg/mL)

Graph of absorbance against concentration of glucose

2.5 2.24
f(x) = 0.58 x + 0.03
Absorbance reading (nm)

2 R² = 0.99 1.85

1.5 1.26

1
0.56
0.5
0
0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
Concentration of glucose (mg/mL)

Linear region

Figure 2: Linear graph of average absorbance against the concentration

of glucose (mg/mL)

2. Standard curve graph

 a) State the glucose concentration range for the linear curve.
The glucose concentration range for the linear curve is from 1mg/mL to 4
mg/mL.
b) Write the equation derived from the linear curve.
The equation derived from the linear curve is y = 0.5779x + 0.0262.
c) Write the linear regression obtained.
The linear regression obtained is R2 =0.9925.

CALCULATION:
a) Show one example of calculation for preparing a desired
concentration of glucose solution from the stock solution given.
C1V1 = C2V2
C1 = 100 mg/ml V1 =?
C2 = 4 mg/ml V2 = 100 ml

(100mg/ml) x V1 = (4mg/ml) x (100ml)

V1 = 4ml
Therefore, 4ml of 100mg/ml of glucose solution is needed to prepare glucose
standard solution of 4 mg/ml concentration.

b) Show the calculation for determining sample concentration from the

equation derived from the linear standard curve.
Equation: y=0.5779x + 0.0262
Solid sample (Strawberry Jam) Liquid sample (Apple Juice)
Average absorbance, y= 0.612 Average absorbance, y=0.406

0.612=0.5779 x +0.0262 0.406=0.5779 x+ 0.0262

0.5779 x=0.612−0.0262 0.5779 x=0.406−0.0262

(0.612−0.0262) (0.406−0.0262)
x= x=
0.5779 0.5779

x=1.0137 mg/mL x=0.6572 mg/mL

 ≈ 1.01 mg/mL ≈ 0.66 mg/mL

Concentration of = 1.01 mg/ml Concentration of = 0.66 mg/ml

strawberry jam apple juice

DISCUSSION:
In simpler terms, reducing sugar is defined as sugar that can act as
reducing agent and owns a free carbonyl group. Some few examples of reducing
sugars include glucose, fructose, and maltose. When other compounds react with
reducing sugars, the oxidation-reduction chemical process will take place. From the
experiment that has been carried out, the concentration of reducing sugars in
samples of strawberry jam and apple juice were analysed by using the DNS
colourimetric method. The method was used to test for the presence of free carbonyl
group (C=O) in reducing sugars as many reagents can be reduced by reducing
sugars. In this process, the aldehyde functional group in glucose and the ketone
functional group in fructose are both oxidised.
Based on the obtained results, the average absorbance value for glucose
standard solution concentration of 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 6mg/mL and
8mg/mL are 0.559, 1.257, 1.850, 2.244, 2.470 and 2.529 respectively. From the
data, we could observe that, the higher the concentration of glucose standard
solution, the higher the average absorbance. Thus, we could conclude that the
glucose standard solution concentration is directly proportional to the average
absorbance. On the other hand, in order to determine the concentration of both solid
and liquid samples of strawberry jam and apple juice, the linear standard curve was
used, where an equation which is y = 0.5779x + 0.0262, was derived from it. The
average absorbance values for strawberry jam and apple juice are 0.612 and 0.406,
respectively. By substituting the average absorbance of both samples into the
equation derived from the curve, the concentration of strawberry jam and apple juice
were calculated. The calculated concentration for strawberry jam is 1.01 mg/ml while
0.66 mg/ml for apple juice. It is found that strawberry jam has a higher absorption
value than apple juice. This is due to the fact that a high absorbance in the sample
indicates a high concentration. It is somehow connected to Beer’s Law, which states
that concentration and absorbance are related to each other. Thus, when more light
is blocked when passing through a solution with higher concentration, it is because
of the amount of large molecules contained in it. On top of that, the value for the
linear regression obtained from the curve, is R2 =0.9925.

Furthermore, a few errors might occur while handling this experiment which
can contribute to the inaccuracy of the results. One of them is when the eye level is
not perpendicular to the reading scale of the apparatus. This will lead to a parallax
error. Other than that, while preparing the solid sample, a glass rod must be used
instead of the spatula when stirring the solid samples. This is to avoid the filter paper
from being torn. Moreover, another error that could affect the result is when
transferring the test tubes from the boiling water bath to the iced bath. This is
because the transfer must be done immediately to stop the reaction between the
glucose and the DNS reagent.

CONCLUSION:
In conclusion, we were able to determine the concentration of reducing sugar
using the Dinitrosalicylic (DNS) colourimetric method in both solid and liquid samples
of strawberry jam and apple juice. Strawberry jam has a concentration of 1.01 mg/ml,
which is higher than apple juice, with a concentration of 0.66 mg/ml. Besides, the
average absorbance value for strawberry jam and apple juice are 0.612 and 0.406,
respectively.

REFERENCES:
Miller, G. L. (1959). Use of Dinitrosalicylic Acid Reagent for Determination of Reducing

Sugar. Analytical Chemistry, 31(3), 426–428. https://doi.org/10.1021/ac60147a030

Penner, M. H. (2017). Basic Principles of Spectroscopy. Food Science Text Series, 79–88.

https://doi.org/10.1007/978-3-319-45776-5_6

Wolstenholme, R. (2020). Ultraviolet–Visible and Fluorescence Spectroscopy. Analytical

Techniques in Forensic Science, 115–143.

https://doi.org/10.1002/9781119373421.ch6