LABORATORY REPORT OF

INSTRUMENTAL ANALYSIS OF FOOD (FST 606)

NAME : FATIN NADIAH BINTI SAYPOL ANWAR

MATRIC NUMBER : 2019704597

GROUP : AS246 5A1

LECTURER’S NAME : DR. MARINA BIINTI ZULKIFLI

EXPERIMENT 1 : DETERMINATION OF REDUCING SUGARS USING THE

DINITROSALICYLIC (DNS) COLOURIMETRIC METHOD
TITLE:
Determination Of Reducing Sugars Using The Dinitrosalicylic (Dns) Colourimetric Method

INTRODUCTION

It was 1921 when Sumner established the dinitrosalicylic acid (DNS) method to determine
reducing sugars in normal urine and diabetic urine. Measurement of amount of reducing sugars
in quantitative spectrophotometric contained in a component was discovered by the reaction of
dinitrosalicylic acid (DNS) with reducing sugars and other reducing molecules by formation of
3-amino-5-nitrosalicylic acid, an aromatic compound that strongly absorbs light at 540nm.
(Goncalves et al. (2010)

Determination of sugar concentration of food samples is vital in industries where quality

control is monitored. Example in food industry is the content of reducing sugars in milk. Milk’s
major constituents are lactose, fats and proteins. So, these method is based on assumption made
that the only reducing sugars in milk is from lactose. Another example is in medical system
where it is widely used to quantify carbohydrates level in blood. Index of reducing sugars is its
intensity in colour.

However, it has been discerned that besides the usage of high volume of samples and DNS
reagent needed, this process is time-consuming especially when large samples are used.
Therefore, the adaptation of using Uv-vis spectrophotometer (double beam spectropohotometer)
as means to determine the concentration of reducing sugar has been used by performing reaction
and absorbance measurement in the same vessel.
OBJECTIVE

1. Determination of the concentration of reducing sugar by using the Dinitrosalicyclic

(DNS) colourimetric method
2. To observe the application of UV-Visible Spectrophotometry
3. To utilize the standard curved to determine the concentration of samples

APPARATUS AND REAGENTS

1. Double beam spectrophotometer 2. Test-tubes and racks

3. Analytical balance 4. Water-bath (boiling)
5. Plastic/glass cuvettes 6. Spatulas wash bottle
7. Beakers 8. Stop-watch
9. Vortex mixer 10. Ice water
11. Pipettes 12. Glucose standard
13. Volumetric flasks 14. 3,5-dinitrosalicycle acid (DNS)
15. 2 Molar NaOH 16. 2 Molar NaOH
17. Distilled water

PROCEDURE

1. Preparation of DNS reagent

Solution A: 10g of DNS was dissolved in 200ml 2M NaOH with warming and vigorous
stirring.
Solution B: 300g sodium potassium tartrate tetrahydrate was dissolved in 500ml distilled
water. Both solution A and B was mixed and filled up to 1L with distilled water.
 2. Preparation of sample

For solid sample:

a) Approximately 3g of solid sample (strawberry jam) was weighed accurately into a beaker
and then 50ml was added. Warmed and stirred for 5 minutes
b) Strawberry jam was 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 volume
(100ml)
c) 10ml strawberry jam was diluted to 250ml with distilled water in a volumetric flask and
mixed well by inversion of the volumetric flask.

For liquid sample:

a) 5ml of sample (apple juice) was pipetted
b) 100ml of apple juice was filter into volumetric flask. The residue was washed into
volumetric flask with small amount of distilled water and then filled up to volume
(100ml). It is then mixed well with repeated inversion of flask.
c) 10ml of apple juice solution was diluted to 100ml with distilled water in a volumetric
flask and mixed well by inversion of the volumetric flask.

3. Preparation of Glucose Standard Solutions

a) Glucose stock solution of 100 mg/mL concentration was prepared. The glucose solution was
transfered into a 100ml volumetric flask. The residue was washed into the volumetric flask with
small amount of distilled water and then filled up to 100ml volume. Mixed well with repeated
inversion of the flask.
b) 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 by dilution of glucose stock solution using distilled water in 100mL volumetric
flasks.
 Use equation where : C1V1 = C2V2 (preparation of standard solution)

C1 = concentration of glucose stock C2 = concentration of glucose to be

solution (100 mg/mL) prepared
V1 = volume of glucose stock solution V2 = volume of glucose solution to be
required to make dilution prepared

c) It was mixed well by repetition of inversion of the flask to ensure proper mixing.

4. Absorbance Measurements

a) 1.0mL of distilled water was pipetted into a test tube and labelled as ‘Blank’
b) 1.0mL of each concentration of glucose standard solution was pipietted in other appropriate
labelled test tubes.
c) 1.0mL of sample solution was pipetted into a test tube and labelled as ‘sample’.
[Blank, standard and sample prepared in triplicate]
d) In each test tube prepared above, 1.0ml DNS reagent and 3.0ml of distilled water were
added. Mixed well using a vortex mixer.
e) All test tubes were arranged in test tube racks and placed in boiling water bath for exactly 5
minutes to allow reaction between DNS reagent and glucose standard to occur.
f) After 5 minutes ended, the reaction was immediately stopped by transferring the test tubes
into a container of iced water.
g) 10ml of distilled water was added to each test tube and mixed well using vortex mixer.
h) The maximum wavelength was determined by using double-beam spectrophotometer by
measuring the absorbance spectrum of 4mg/ml glucose standard solution. Wavelength range
that was used is 400 to 600nm. Then, the spectrophotometer’s wavelength was set using the
highest absorbance obtained.
i) The absorbance for each standard solution and the sample solutions was measured.
RESULTS
Table 1 : Absorbance reading for standard and sample concentration

Table 2 : Graph of sample concentration vs average absorbance

Sample concentrati on vs average absorbance graph

3

f(x) = 0.4 x
2.5 R² = 0.98 2.53
2.47
2.24
average absorbance

2
1.85

1.5
1.26
1

0.5 0.56

0 0
Blank 1 mg/mL 2 mg/mL 3 mg/mL 4 mg/mL 6 mg/mL 8 mg/mL

sample concentration
Standard curve graph

a) State the glucose concentration range for linear curve : 0mg/mL - 4mg/mL
b) Write the equation derived from the linear curve : y = 0.4002x
c) Write the linear regression obtained : R2 = 0.9279

CALCULATIONS

Preparation for desired concentration of glucose solution from the stock solution
given

C1V1 = C2V2

mg mg
100 mL x V = 1 mL x 100mL

V = 1mL

C1V1 = C2V2

mg mg
100 mL x V = 2 mL x 100mL

V = 2mL

Determination of glucose in apple juice:

1. Equation, y = 0.4002x
Strawberry average absorbance, y = 0.406

y = 0.4002x
0.406 = 0.4002x
0.406
x = 0.4002
x = 1.0145 mg/mL
2. Concentration of glucose in original jam

1.0145 100 100

 
= 1ml 5ml 20ml

= 101.45 mg/mL

 Therefore, 0.1014 glucose in 1 mL apple juice

 10.14g glucose in 100g apple juice
 10.12% glucose in apple juice
Determination of glucose in strawberry jam:
1. Equation, y = 0.4002x
Strawberry average absorbance, y = 0.612

y = 0.4002x
0.612 = 0.4002x
0.612
x = 0.4002
x = 1.5292 mg/mL

2. Concentration of glucose in original jam

1.5292 1000 100

 
= 1ml 5ml 20ml

= 152.92 mg/mL

 Therefore, 0.15292g glucose in 1 mL strawberry jam

 15.292g glucose in 100g strawberry jam
 15.29% glucose in strawberry jam
DISCUSSION

According to Vaclavik and Christian (2014), reducing sugars are sugars that has a free
carbonyl group. Example of reducing sugars are glucose, galactose, lactose and maltose.
Reducing sugars give brown colours to baked goods which is known as Maillard reaction. A free
carbonyl group of a reducing sugar reacts with a free amino group on a protein when heated and
the result is a brown colour. The dinitrosalicylic (DNS) method is used in this to estimate the
concentration of reducing sugars in two samples, strawberry jam and apple juice. It tests the
presence of free carbonyl group (C=O), the so-called reducing sugars. Each sugar must be
calibrated as different reducing sugars yield different intensity of colour. Below is an example of
glucose in reducing sugars when tested.

In this experiment, there were two samples that were used to identify amount of glucose
which were strawberry jam for solid and apple juice for liquid. These samples were prepared in
series of standard/sample concentration accordingly in blank, 1 mg/mL, 2 mg/mL, 3 mg/mL, 4
mg/mL, 6 mg/mL and 8 mg/mL. It is done by diluting 100 mg/mL glucose stock solution by
using distilled water in 100 mL volumetric flask. For the glucose stock solution it is determined
by using the formula of C1V1 = C2V2.. Meanwhile, the absorbance is done by addition of 1.0ml
DNS reagent to know sugar content and 3.0ml of distilled water. Vortex mixer is used for the
solution to mix evenly.
 After calculating the absorbance’s average and reducing sugar standard drawn, the linear
regression (R2 = 0.9279) is obtained along with the equation derived (y = 0.4002x). The
absorbance of each sample concentration were 0.000, 0.559, 1.257, 1.850, 2.244, 2.470 and
2.529. The higher the sample concentration, the higher the absorbance (nm) recorded.
Wavelength is set to 540nm as it is the region where orange-red colour absorbs. Then, they were
heated in water bath because at a higher temperature, the alkaline mixture of reducing sugar
along with DNS, will develop into intense red colour. In addition, the more sugar there is, the
more carbonyl group, hence resulting in darker solution.

Uv-vis spectrophotometer (double-beam spectrophotometer) is used to determine the

concentration of a molecule by using light absorption. According to Haven, Tetrault, Schenken
and Wiley (1995), Uv-vis spectrophotometer has two light paths, both from same light source.
Alternation between sample and reference cells of light causes the readout device to convert
electrical signal from each detector to absorbance units. In the Beer-Lambert Law, the
absorbance is directly proportional to the concentration of the sample used and light path which
is width of cuvette. This law states that there must be a linear relationship between concentration
and absorbance as seen in the graph to enable calculation of its absorbance.

The result showed that the concentration for strawberry jam is 0.612 mg/mL and for apple
juice is 0.406 mg/mL. Strawberry jam has a higher concentration meaning the reducing sugar
content is higher. According to Malaysian Food Law and Regulation (1985), fruit jam shall be
the product prepared by boiling one or more types of sound fruits, whether raw, processed or
semi-processed, with permitted sweetening substance, with or without added pectin. This could
be that the sweetness from the fruit itself and addition of sugar cause it to have a higher sugar
content.

During the experiment, there might be a few errors that has been done causing the regression
to be (R2 = 0.9279) when the range is supposed to be above 0.9800. It could be failing of
properly aligning meniscus with volume mark or using a dirty pipette which effects the solution
to be contaminated. Besides, it could also be that the (DNS) mixture be contaminated duirng
mixing process. To avoid this errors from happenings, eyes should always be in level with the
pipette and (DNS) mixture should be prepared carefully.
CONCLUSION

As a conclusion, determination of reducing sugar by using dinitrosalicylic (DNS)

colourimetric method has been successfully accomplished. Therefore, based on the calculation
done, it can be seen that the concentration of reducing sugar in strawberry jam is 152.92 mg/mL
while for apple juice is 101.45 mg/mL.
REFERENCES
Construction of Maltose Standard Curve by DNS Method .(n.d.). Retrieved from
http://vlab.amrita.edu/?sub=3&brch=64&sim=163&cnt=2

Gonçalves, Cristiana & Gomes, Nelma & Rodriguez-Jasso, Rosa & Teixeira, José & Belo,
Isabel. (2010). Adaptation of dinitrosalicylic acid method to microtiter plates. Analytical
methods. 2. 2046-2048. 10.1039/C0AY00525H.

Malaysia. (1985). Food Act 1983 and Food Regulations 1985: Revised 7th October, 1985: Act
281. Kuala Lumpur: MDC

Vaclavik, V. A., & Christian, E. W. (2014). Essentials of Food Science. New York, NY:
Springer New York