FACULTY OF ENGINEERING TECHNOLOGY

DEPART MENT OF CHEMICAL ENGINEERING TECHNOLOGY

PRINCIPAL OF BIOCHEMISTRY LABORATORY

LABORATORY REPORT

COURSE CODE BNN 30104

EXPERIMENT NO. 2

GLUCOSE ANALYSIS USING

EXPERIMENT TITLE
COLORIMETRIC ASSAY WITH DNS
REAGENT

DATE 10 OCTOBER 2018 (WEDNESDAY)

GROUP NO. 3
LECTURER/INSTRUCTOR/TUTOR 1) TS. DR. SITY AISHAH BINTI MANSUR
2) N. MASAYU BINTI MASLAN
DATE OF REPORT SUBMISSION 17 OCTOBER 2018

ATTENDANCE/PARTICIPATION/DISIPLINE: /5%

INTRODUCTION: /5%

PROCEDURE: /5%

RESULTS & CALCULATIONS /15%

ANALYSIS /15%

DISTRIBUTION OF MARKS FOR DISCUSSIONS: /20%

LABORATORY REPORT: ADDITIONAL QUESTIONS /15%

CONCLUSION /10%

SUGGESTIONS & RECOMENDATIONS /5%

REFERENCES: /5%

TOTAL: /100%

EXAMINER COMMENTS: RECEIVED DATE AND STAMP:

 STUDENT CODE OF ET HICS

DEPARTMENT OF CHEMICAL ENGINEERING TECHNOLOGY

FACULTY OF ENGINEERING TECHNOLOGY

I hereby declare that I have prepared this report with my own efforts. I also admit to not accept

or provide any assistance in preparing this report and anything that is in it is true.

1) Group Leader
Name : SYUHAIMI BIN YUSOF
Matrix No. : AN160144

2) Group Member 1
Name : NURUL AFIQAH BINTI ABDUL GHAFAR
Matrix No. : DN160324

3) Group Member 2
Name : DEONG JING MEI
Matrix No. : DN160284

4) Group Member 3
Name : HO CON NIE
Matrix No. : AN160197
OBJECTIVES

The objective of this experiment is to understand the principle of DNS reaction on reducing sugar.

LEARNING OUTCOMES

a. At the end of the study, student will be able: a. To conduct a colorimetric assay independently.

b. To understand the principles of DNS reaction on reducing sugar, particularly glucose, by

colorimetric assay.

c. To analyse faults and carry out problem-solving techniques in colorimetric assay

INTRODUCTION

This technique tests for the presence of free radical (C=O), of the questionable reducing
sugars. This involves the reaction of the organic compound practical cluster gift in, for instance,
aldohexose and therefore the organic compound practical cluster in fruit sugar. at the same time,
3,5-dinitrosalicylic acid (DNS) is reduced to 3-amino,5-nitrosalicylic acid below alkali
conditions:

Oxidation

Aldehyde group Carboxyl group

Reduction

3,5-dinitrosalicylic acid 3-amino,5-nitrosalicylic acid

The higher than reaction theme shows that one mole of sugar can react with one mole of three,5-
dinitrosalicylic acid. However, it's suspected that there are several side reactions, and also the actual
reaction ratio is additional sophisticated than previous mentioned. The sort of side reaction depends on the
precise nature of the reducing sugars. Different reducing sugars usually yield different color intensities;
therefore, it is necessary to calibrate for every sugar. Additionally to the oxidization of the carbonyl teams
within the sugar, alternative aspect reactions like the decomposition of sugar additionally competes for the
supply of three,5-dinitrosalicylic acid. Consequently, cellulose will have an effect on the standardization
curve by enhancing the intensity of the developed color.
 Although this is often a convenient and comparatively cheap methodology, thanks to the
comparatively low specificity, one should run blanks diligently if the colorimetrical results are to
be taken properly and accurately. One will confirm the background absorption on the first polyose
substrate answer by adding cellulase, straightaway stopping the reaction, and measurement the
absorbance, i.e. following precisely the same procedures for the particular samples. Once the
results of unnecessary compounds be situated glorious, one will effectively embrace an alleged
internal common place by first absolutely developing the colour for the unknown sample; then, a
known quantity of sugar is extra to the present sample. The rise within the absorbance upon the
second color development is equal the progressive quantity of sugar added.
PROCEDURE

1. 6 test tubes of set A were prepared and labelled with 1A, 2A, 3A, 4A, 5A and 6A. 6 test tubes
of set B were also labelled as 1B, 2B, 3B, 4B, 5B and 6B. 2 test tube of set A with two
unknown concentration of glucose is labelled as SA1 and SA2. 2 more test tube of set B with
two unknown concentration of glucose is labelled as SB1 and SB2.
2. 1mg/mL of glucose stock solution was pipetted out in the range of 0.2 mL, 0.4 mL, 0.6 mL, 0.8
mL and 1.0 mL into 10 separate test tubes starting from test tube 2. Test tube 1 was not being
filled and left it blank. Unknown concentration test tube is filled with 1.0 Ml of unknown
concentration solution each.
3. The volume of each test tube was filled up with ultra pure water to 1.0 mL.
4. 2.0 mL of DNS reagent was added to each test tube.
5. The mixtures of all test tubes were mixed well using vortex mixer.
6. The test tubes were covered by using parafilm and were heated in a 90°C water bath.
7. The test tubes were taken out from water bath after 5 minutes and they were cooled down to
room temperature.
8. 7.0 mL of distilled water was added into the test tube except for the unknown concentration
testubes.
9. The mixtures in test tubes were mixed well again using vortex mixer.
10. The cuvettes were labelled with 1 to 6 and about 1.0 mL of the solution in test tube 1 to 6 for
set A were poured into the labelled cuvettes accordingly. Same goes to the unknown sample
also being labelled.
11. The cuvettes were placed into a uv-vis spectroscopy and the readings of absorbance at the
wavelength of 540nm were recorded in the table.
12. Steps 9 to 10 were repeated for solution in set B.
13. The average reading of the absorbance was calculated and the standard curve was plotted.
RESULTS AND CALCULATIONS
a) The concentration of standard glucose solution: 1.0 mg/mL
0.1𝑔 1000 𝑚𝑔 𝑚𝑔
× = 1.0
100 𝑚𝐿 1𝑔 𝑚𝐿

Table 1: Absorbance Value of Solution Prepared

Tube Sample Volume of Concentration Absorbance (540 nm)
No. Sample (mL) (mg/mL) 1 2 Average

Standard
1 0.0 0.0 0.000 0.000 0.000
Glucose

Standard
2 0.2 0.02 0.153 0.157 0.155
Glucose

Standard
3 0.4 0.04 0.327 0.323 0.325
Glucose

Standard
4 0.6 0.06 0.518 0.501 0.5095
Glucose

Standard
5 0.8 0.08 0.705 0.685 0.695
Glucose

Standard
6 1.0 0.10 0.901 0.858 0.8795
Glucose

7 A 1.0 0.0163 0.145 0.137 0.141

8 B 1.0 4.626 × 10−4 0.003 0.005 0.004

b) Calculation for Standard Glucose Concentration:
 Tube 1: 0.00 mL
C1 V1 = C2 V2
(1.0 mg/mL) (0.00 mL) = C2 (10 mL)
C2 = 0.0 mg/mL

 Tube 2: 0.20 mL
C1 V1 = C2 V2
(1.0 mg/mL) (0.20 mL) = C2 (10 mL)
C2 = 0.02 mg/mL

 Tube 3: 0.40 mL
C1 V1 = C2 V2
(1.0 mg/mL) (0.40 mL) = C2 (10 mL)
C2 = 0.04 mg/mL

 Tube 4: 0.60 mL
C1 V1 = C2 V2
(1.0 mg/mL) (0.60 mL) = C2 (10 mL)
C2 = 0.06 mg/mL

 Tube 1: 0.80 mL
C1 V1 = C2 V2
(1.0 mg/mL) (0.80 mL) = C2 (10 mL)
C2 = 0.08 mg/mL

 Tube 1: 1.00 mL
C1 V1 = C2 V2
(1.0 mg/mL) (1.00 mL) = C2 (10 mL)
C2 = 0.10 mg/mL
c) Standard Curve graph

Absorbance Vs Concentration (mg/mL)

1

y = 8.6464x
0.9

0.8

0.7

0.6
Absorbance

0.5

0.4

0.3

0.2

0.1

0
0 0.02 0.04 0.06 0.08 0.1 0.12
Concentration (mg/mL)

Figure 1: Graph of Absorbance Vs Concentration (mg/mL)

d) Estimation the concentration of glucose in:
From the Standard Curve graph, the concentration (x – axis intercept) of sample A and B
solution can be obtain by substituting the average absorbance value in y – axis value.

 Sample A
Given the linear equation is:
y = 8.6464x
0.141 = 8.6464x
x = 0.0163 mg/Ml

Concentration of Sample A prepared solution,

C1 V1 = C2 V2
C1 (1.00 mL) = (0.0163 mg/mL) (10 mL)
C1 = 0.163 mg/mL

In 200 mL of sample A, has ratio of glucose to water (1 : 199),

0.163 𝑚𝑔 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 199 𝑚𝐿 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
× = 32.437 𝑚𝑔/𝑚𝐿
1 𝑚𝐿 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 1 𝑚𝐿 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒

Hence, the concentration of glucose in Sample A is 32.437 mg/mL

 Sample B
Given the linear equation is:
y = 8.6464x
0.004 = 8.6464x
x = 4.626 × 10−4 mg/Ml

Concentration of Sample A prepared solution,

C1 V1 = C2 V2
C1 (1.00 mL) = (4.626 × 10−4 mg/mL) (10 mL)
C1 = 4.626 × 10−3 mg/mL

In 100 mL of sample B, has ratio of glucose to water (1 : 99),

4.626 × 10−3 𝑚𝑔 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 99 𝑚𝐿 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
× = 0.4579 𝑚𝑔/𝑚𝐿
1 𝑚𝐿 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 1 𝑚𝐿 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒

Hence, the concentration of glucose in Sample B is 0.4579 mg/mL

e) Amount of Glucose in 250 mL of sample
 Glucose Sample A
Concentration of Sample A in 250 mL of solute,
C1 V1 = C2 V2
(32.437 mg/mL)(200 mL) = C2 (250 mL)
C2 = 25.9496 mg/mL

25.9496 𝑚𝑔 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
× 250 𝑚𝐿 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 = 6487.4 𝑚𝑔
1 𝑚𝐿 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒

Hence, the amount of glucose in 250 mL sample is 6487.4 mg

 Glucose Sample B
C1 V1 = C2 V2
(0.4579 mg/mL)(100 mL) = C2 (250 mL)
C2 = 0.18316 mg/mL

0.18316 𝑚𝑔 𝑜𝑓 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
× 250 𝑚𝐿 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 = 45.79 𝑚𝑔
1 𝑚𝐿 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒

Hence, the amount of glucose in 250 mL sample is 45.79 mg

ANALYSIS
Referring to Table 1, the solution with standard glucose were prepared such as the
concentration keep increasing. This is by manipulated the volume of standard glucose with 1.0
mg/mL concentration. The volume varies from 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL. Absorbance is the
value obtain from quantitative analysis using Ultraviolet – visible spectroscopy (UV – VIS) at 540
nm wavelength. The desired concentration of each solution were duplicate to obtain an average
and results that are more accurate. The duplication of sample help to identify if the transferring
technique of solution into each test tube was correct. If the solution were transfer equally accurate,
the result will show less variability and the result can be accepted.
From Figure 1, graph of absorbance vs concentration, the graph shows that the relationship
was directly proportional. As the concentration of the glucose increasing the absorbance value will
increasing. Sample A and B were prepared solution with an unknown concentration. By taking 1
mL of each sample, and using the same procedure as the preparation of standard glucose solution,
the absorbance of the samples can be obtain. By observing the absorbance value only, sample A
has absorbance nearest to the absorbance of 0.02 mg/Ml standard glucose. While sample B has
absorbance closest to 0.00 mg/Ml standard glucose concentration. Using standard curve graph of
absorbance vs glucose concentration as reference, gives the value of unknown glucose
concentration.
DISCUSSION
In this experiment, students are required to perform the glucose analysis using colorimetric
assay with DNS reagent. Extensively use of 3, 5-Dinitrosalicylic acid (DNS) in estimation of
reducing sugars as it can detect the presence of free carbonyl group (C=O) in the reducing sugars.
A reducing sugar forms an aldehyde or ketone when in basic condition. The aldehyde functional
group of glucose and the ketone functional group of fructose will oxidise and reacts the 3,5-
dinitrosalicylic acid (DNS) to 3-amino-5-nitrosalicylic acid, an aromatic compound with
maximum absorption at 540 nm, allowing a quantitative spectrophotometer measurement of the
amount of reducing sugar present. (Anamaria. N. et, al., 2012)

Figure 1: Chemical Reaction for DNS method

During this reaction, water is used up as a reactant and oxygen gas is released. The
formation of 3-amino-5-nitrosalicylic acid which under alkaline conditions is converted to a
reddish-brown coloured complex which results in a change in the amount of light absorbed at
wavelength 540 nm.
We had prepared two sets of solution with the same composition to ensure the accuracy of
the calibration curves. The calibration curve obtained from the relation between glucose
concentration and the absorbance values, measured at 540 nm, were all linear. The linearity of the
calibration curves was measured by duplicate of standards at six different concentration levels.
(Anamaria. N. et, al., 2012) From the result obtained, it shows that as the concentration of stock
glucose solution increase, the absorbance also increases. The graph shows an equation which are
y = 8.6464x, where y is the absorbance of the mixture and x is the concentration of the stock
glucose solution. This means as x concentration of glucose is in the solution, there will be 8.6464
times of absorbance for that solution.
 Figure 2: Test tube for sample A (left) and sample B (right)

Figure 3: Test tube for calibration curve with known glucose concentration
In this experiment, there are 2 sample of unknown concentration of glucose need to be
determined which labelled as sample A and sample B. The sample A gives an average absorbance
of 0.141 while sample B gives an average absorbance of 0.004 at 540 nm wavelength. After
calculation, the sample A have a glucose concentration of 32.437 mg/ml whereas sample B has a
glucose concentration of 0.4579 mg/ml. Thus, sample A is having more glucose in solution than
sample B.
ADDITIONAL QUESTIONS
1. Define ‘reducing sugar’? Name and sketch structure of a reducing sugar. (2 marks)
Reducing sugars possess a free carbonyl group (either an aldehyde or keto group
Types of reducing sugars include glucose, fructose, glyceraldehyde, lactose, arabinose and
maltose. Sucrose is an example of reducing sugar.

2. DNS is an important reagent in this type of reaction. Identify the starting materials and
explain how to prepare DNS reagent. (3 marks)
The starting materials for DNS reagent are 3,5-Dinitrosalicylic Acid (DNSA) with sodium
potassium tartrate solution. To prepare DNS reagent, first we dissolve 1g of 3,5-
Dinitrosalicylic Acid in 20mL 2M sodium hydroxide, NaOH. Then add slowly 30g sodium
potassium tartrate (MW=282.2 g/mol) and dilute to a final volume of 100mL using distilled
water.

3. What is the control in this experiment and the significance of control? (2 marks)
The control of this experiment is the volume of the DNS reagent and the total volume of the
solution. Time reaction also needs to keep constant for the entire sample as it will affect the
concentration of the glucose.

4. Explain why specific wavelength is employed for this experiment. (3 marks)

Wavelength in 540nm is the region where orange-red colour absorbs. The DNS reagent used
contains sodium potassium tartrate which gives the solution a milky yellow colour, however it
turn colourless or transparent orange yellow colour with the present of sodium hydroxide. The
colour of the reagent changes from yellow to orange or red depend the concentration of
reducing sugar present in the sample. Therefore, other wavelength is not suitable to be
employed as it will absorb different colour.
5. High intensity of colour could lead to error in readings of the OD. Analyse this situation
and propose the best solution for the problem. (3 marks)
The DNS test can detect concentrations of glucose between 0.5 mM (0.09% glucose w/v) and
40 mM (0.72% glucose w/v). When the reading lead to error is indicating that the sample
contains large concentration of glucose, and had excedd the maximum concentration of
glucose that can be detected. The best solution for the problem is by doing dilution for the
sample. Dilute the sample will reduce the concentration of glucose in the sample. However, the
original concentration of glucose in original sample still can by calculated.

6. Identify TWO (2) other methods available for testing sugar and discuss the advantages
and disadvantages of these methods? (5 marks)
Methods Benedict's Test Fehling's Test
 The test only used a single  The test has ability of
Advantages solution, and has no caustic aldehyde containing sugars
properties, thus it easier to to reduce blue Cu2+ ions to
handle and more stable Cu+ ions
 It forms a visible positive
result of formation of brick
red precipitate
 It takes too long time for  Only detect the presence of
Disadvantages testing. aldehydes but not ketones
 The results could be
inaccurate if there were any
substances or contaminants
left in the test tubes.

7. Recommend TWO (2) preventive measures during experimental work for the best
outcomes. (2 marks)
One of the preventive measures for a better outcomes is improved the technique of dilution and
using micropipette. The technique of the dilution and using micropipette are very important
especially to prepare the standard curve. The little error in dilution can lead to a significant
effect to the reading of the concentration of the glucose in the sample. The other preventive
measure is make sure the cuvette is free from any dust and clear before put in the uv-vis
spectroscopy. Clean with non-abrasive cuvette tissue is recommended. The present of dirt on
the cuvette will block the light to pass through the sample and effect the reading as well.
CONCLUSION
The objective of the experiment is achieved as we understand the principle of DNS reaction on
reducing sugar. In the experiment, we use UV-VIS Spectroscopy for Dinitrosalicylic (DNS)
method to detect the concentration of glucose in two different samples, which labelled as sample
A and sample B. The experiment is duplicated to get the more accuracy of results by calculating
the average value. For the reading, the sample A gives an average absorbance of 0.141 while
sample B gives an average absorbance of 0.004 at 540 nm wavelength. After calculation, the
sample A has a glucose concentration of 32.437 mg/ml whereas sample B has a glucose
concentration of 0.4579 mg/ml. Thus, we can conclude that sample A contains more concentration
of glucose in solution than sample B. Our hypothesis by observing the colour intensity of the
sample is also accepted, the higher the intensity of colour, the greater the concentration of glucose
in solution. Sample A have higher intensity of colour compared to sample B, thus we assume
sample A will contains more glucose compared to sample B. The final result had support our
hypothesis where sample A has more glucose in solution than sample B.

SUGGESTIONS AND RECOMMENDATIONS

During the experiment, there area unit some suggestions and suggestions that may be
improved to urge additional correct result and higher reading. One among the suggestions is
improve the techniques victimisation-measuring device. Eye should be perpendicular to the scale
to avoid parallax error because the very little modification within the volume can have an effect on
the concentration of glucose content. Next, we tend to should ensure the mixture is mixed well
before heated. Combine the mixture using vortex mixer for couple of minutes is usually
recommended to make sure that everyone mixtures have mix along.
Furthermore, the test tube should lined with parafilm well once heated in water bath
therefore once the evaporation occur; the mixture will not evaporate everywhere. Time for all test
tubes being heated additionally should be same because of it would have an effect on the reaction
occur within the mixture. Thus, once place in or taken out at a similar time for all the test tubes.
Once taken out from the water bath, quiet down the solutions to temperature fully to let the
reaction between glucose and DNS chemical agent fully done.
Moreover, before out the mixture in the cuvette into uv-vis spectroscopic analysis, ensure
the surface of the cuvette is free from any dirt and clear. Non-abrasive cuvette tissue is usually
recommended to use for cleansing method. Thus, once the white uv-vis pass through the mixture,
the ray will not being block by any dirt and that we can get a far better reading. Lastly, for ready
of the trials, use a similar uv-vis spectroscopic analysis as a result spectroscopic analysis may need
different customary.
REFERENCES
1. HiMedia Laboratories Pvt Limited. The principle of DNS method. Retrieved from
http://himedialabs.com/TD/HTBC003.pdf

2. Miller, G.L., Use of dinitrosalicylic acid reagent for determination of reducing

sugar, Anal. Chem., 31, 426, 1959.
3. Anamaria. N., Vioroca. P., Manuela. M. M., Cosmin. I., Beatrice. A. V. and Vasile. O. .
(2012). Adapting the Reducing Sugars Method with Dinitrosalicylic Acid to Microtiter Plates
and Microwave Heating. J. Braz. Chem. Soc., Vol. 23, No. 12, 2176-2182, 2012.
4. DNSA reagent base. Retrieved on 15 October 2018 from
http://www.ncbe.reading.ac.uk/materials/enzymes/dnsareagent.html
5. DNS Method. Retrieved on 15 October 2018 from
http://vlab.amrita.edu/?sub=3&brch=64&sim=163&cnt=2
6. National Centre for Biotechnology Education. (2016). DNSA reagent-Instructions for
preparation and use. University of Reading.
7. Test for Reducing Sugars. Retrieved on 15 October 2018 from https://sciencing.com/test-
reducing-sugars-5529759.html
APPENDIX