UNIVERSITI KUALA LUMPUR

MALAYSIAN INSTITUTE OF CHEMICAL & BIOENGINEERING

TECHNOLOGY
LABORATORY REPORT
SUBMISSION FORM

To: Sir Muhammad Sharir Bin Abdul Rahman Code Subject: CBD30003
From: Student ID. No.:
1. George Remang Anak Gambang
2. Nurul Izzah Bt. Zamri
3. Afiqah Bt. Md Zulkifli
4. Nur Sabriena
Group Number: 1 Date of Experiment: 21 August 2018
Title of Experiment: Colorimetric Analysis for Reducing Sugar

Received by: Date of Submission: 31 August 2018

Note: Submission is after 1 week. Late submission will be penalized.

*To be filled by the marker*
VERY POOR GOOD VERY EXCELLENT
CRITERIA POOR GOOD
1 2 3 4 5
1.0 INTRODUCTION & OBJECTIVES (1 PAGE
ONLY)
1. State the background to the experiment 3 6 9 12 15
conducted.
2. State the objectives of the experiment
2.0 PROCEDURE (1 PAGE ONLY)
1. Summarize step by step used in performing this 3 6 9 12 15
experiment.
3.0 RESULTS & DISCUSSIONS (MINIMUM 2
PAGES)
1. Data are presented as deemed suitable with
complete label and units in tables and/or graphs.
8 16 24 32 40
2. Explanations of the referred tables and/or graphs
are presented after it.
3. Discuss on the findings and relations to the
theory and objective of experiment.
4.0 CONCLUSIONS (1 PAGE ONLY)
1. Summary of the results to relate the findings or 3 6 9 12 15
results with the theory applicable to the experiment.
5.0 REFERENCES 1 2 3 4 5
6.0 FORMAT & LANGUAGE 2 4 6 8 10
TOTAL
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Content Page Number

Introduction and Objective (Saberina) 3 and 4

Procedure (Afiqah) 5

Result and Discussion (George) 6,7 and 8

Conclusion and References (Izzah) 9 and 10

Apendix 11

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Introduction

A sugar is a carbohydrate that is abundantly present in the sugarcane and sugar beet
plants. Sugar is composed of hydrogen, oxygen and carbon (Laura Foist, 2018). These natural
sugars are otherwise known as carbohydrates, an essential macronutrient (Natalie Rizzo,
2017). One of these groups is called simple sugar, which are monosaccharides and
disaccharides (Laura Foist, 2018). Monosaccharides include glucose, galactose and fructose,
which are all reducing agent. A reducing sugar is a chemical term for a sugar that acts as a
reducing agent and can donate electrons to another molecule. Specifically, a reducing sugar is a
type of carbohydrate or natural sugar that contains a free aldehyde or ketone group. Reducing
agent can react with other parts of food, like amino acids, to change the colour or taste of the
food (Natalie Rizzo, 2017).

There are two methods that commonly used in determination of reducing sugar which
are Somogyi method and DNS method. The Somogyi Nelson’s method is one of the classical
and widely used methods for quantitative determination of reducing sugar. The reducing sugars
when heated with alkaline copper turtrate reduce the copper from the cupric to cuprous state
and cuprous oxide is formed. When cuprous oxide is treated with arsenomolybdic acid, the
reduction of molybdic acid to molybdenum blue takes place. The blue color developed is
compared with a set of standards in colorimeter at 620 nm (Biocyclopedia, 2018). Compare to
Somogyi method, the DNS (3, 5 dinitrosalicyclic acid) method is more suitable and easier. 3,5
dinitrosalicyclic acid (DNSA) is used to detects the presence of free carbonyl group of reducing
sugar. This involves the oxidation of the aldehyde and ketone functional group. During this
reaction, DNSA is reduced to 3-amino-5-nitrosalicyclic acid (ANSA) which under alkaline
conditions is converted to reddish brown coloured complex which has an absorbance maximum
of 540 nm (HiMedia).

Reactions between reducing sugars and free amino groups in foods, without any
catalytic involvement of enzymes, lead to nonenzymatic browning (Maillard reaction) causing a
reduction in nutritive value and certain physiological or toxicology effects. The browning reaction
develops during both home cooking and industrial processing of foods. Since Maillard reaction
occurs so frequently ans Maillard reaction products are present in practically all meals, an

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understanding of its biological implications is of importance (P.A. Finot, 2003). Besides that,
oxidation occurs when a molecule loses one or more electrons, and reduction is when the
molecule gains one or more electrons. Oxidation-reduction reaction is important in maintaining
human life by creating an energy source for the body. This process requires a catalyst, called a
reducing agent (Marci Sothern, 2017).

Other than that, the important of reducing sugar in medicine is for diabetes test.
Fehling's solution was used for many years as a diagnostic test for diabetes, a disease in which
blood glucose levels are dangerously elevated by a failure to produce enough insulin (type 1
diabetes) or by an inability to respond to insulin (type 2 diabetes). By measuring the amount of
oxidizing agent reduced by glucose, determining the concentration of glucose in the blood or in
the urine is possible to enables the right amount of insulin to be injected to bring blood glucose
levels back into the normal range. In addition, reducing sugar also contribute in food industry
which determine the food quality. The level of reducing sugars in wine, juice, and sugarcane are
indicative of the quality of these food products, and monitoring the levels of reducing sugars
during food production has improved market quality. The conventional method for doing so is
the Lane-Eynon method, which involves titrating the reducing sugar with copper(II) in Fehling's
solution in the presence of methylene blue, a common redox indicator. (Wikipedia, 2018)

Objective

The objective of this experiment is to learn the method used in the determination of th ereducing
sugar based on the colorimetric analysis. Two method will be used namely the Somogyi Nelson
method and DNS method.

4
Procedure flowchart

5
Result

Concentration (mg/L) Absorbance at 540nm

0 (Blank) 0.0000
200 0.2457
400 0.5295
600 0.7540
800 1.0308
1000 1.3313
X (Unknown) 0.6514
Table 1

Table 1 above shows the concentration of glucose solution diluted from 1000 mg/l into
concentration of 0(blank),200,400,600,800 and 1000 mg/l by using dilution formula. Unknown
glucose concentration is already prepared and examined same as other different solution. After
obtaining result from Uv-vis spectroscopy concentration of glucose versus absorbance standard
curve constructed. Unknown concentration obtain is 0.6514.

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Dilution: M1V1=M2V2

Concentration: 0 mg/l

M1V1=M2V2
1000(V1)=0(5ml)
V1= 0ml of glucose + 5ml of distilled water

Concentration: 200 mg/l

M1V1=M2V2
1000(V1)=200(5ml)
V1= 4ml of glucose + 1ml of distilled water

Concentration: 400 mg/l

M1V1=M2V2
1000(V1)=400(5ml)
V1= 3ml of glucose + 2ml of distilled water

Concentration: 600 mg/l

M1V1=M2V2
1000(V1)=600(5ml)
V1= 2ml of glucose + 3ml of distilled water

Concentration: 1000 mg/l

M1V1=M2V2
1000(V1)=1000(5ml)
V1= 5ml of glucose + 0ml of distilled water

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Discussion

This experiment is to learn the method used in the determination of the colorimetric
analysis by using DNS (3,5 dinitrosalicylic acid) method.

Given stock with concentration of 1000 mg/l is diluted into 0(Blank), 200, 400, 600, 800
and 1000 mg/l using dilution formula which is M1V1=M2V2. There are seven test tube prepared
including unknown glucose concentration each of the test tube added with 1.5 ml of DNS (3,5
dinitrosalicylic acid). The test tube is then heated using water bath for 5 minutes and cooled
under running tape water. 10 ml of distilled water added into the solution and using Uv-vis
spectroscopy the absorbance is read under wavelength of 540nm.

The result obtain shows as the concentration of the glucose increase the absorbance
also increase. Which mean according to the theory of DNS (3,5 dinitrosalicylic acid) method, the
experiment was successful. Based on graph have constructed, the unknown concentration can
be determined which the absorbance of unknown at wave length of 540nm is 0.6514. It can be
assumed that the concentration is between 400 mg/l with absorbance of 0.5295 and 600 mg/l
with absorbance of 0.7540 which specifically the unknown concentration of glucose is 500 mg/l.

During the experimental the purpose of boiling the solution for 5 minutes is when the
reaction take place nitro group of the third carbon is reduce into amines group by reducing
compound. Also the purpose of test tube is cooled under running tape water is to make sure
there are no more further reaction occur. Miller, G. L. (1972) This is because according to the
theory of DNS (3,5 dinitrosalicylic acid) method, 3,5-Dinitrosalicylic acid (DNS or DNSA, IUPAC
name 2-hydroxy-3,5-dinitrobenzoic acid) is an aromatic compound that reacts with reducing
sugars and other reducing molecules to form 3-amino-5-nitrosalicylic acid

Furthermore the observation during the experiment shows different gradient of orange
colour in different concentration of glucose. When alkaline solution of 3,5-dinitrosalicylic acid
reacts with reducing sugars for example Glucose and lactose it is converted into 3-amino-5-
nitrosalicylic acid with orange color. Intensity of the colour is an index of reducing sugar contain
in the solution. Miller, G. L. (1972)

8
 DNS assay can be used as an accurate analytical method for the evaluation of reducing
sugar both in pure solution as well as in supernatant from enzymatic saccharification of purified
cellulosics if the glucose is the sole product. Rivers, D. B. (1984)

Conclusion

In conclusion, the objectives of this experiment were to learn the method used in the
determination of the reducing sugar based on the colorimetric analysis. Two method will be
used namely the Somogyi Nelson method and DNS method. Based on the result obtained, it
can deduced that the concentration of the glucose increase and the absorbance also increase.
This is because according to the theory of DNS (3,5 dinitrosalicylic acid) method, 3,5-
Dinitrosalicylic acid (DNS or DNSA, IUPAC name 2-hydroxy-3,5-dinitrobenzoic acid) is an
aromatic compound that reacts with reducing sugars and other reducing molecules to form 3-
amino-5-nitrosalicylic acid, which strongly absorbs light at 540 nm. The objective was achieved.

There were a few possible errors that can occur during the experiment. Firstly, using a
wet or dirty test tube will cause the lack of concentration. Contamination of glassware is the
most common cause of poor results when making qualitative and quantitative observations.
Second, did not clean the cuvette properly cause the absorptions reading cannot provide a
consistent results. Finally, to avoid the error, we should use a small amount of solution to rinse
down the walls of the glassware, then dispose of that small amount of contaminated solution
properly.

9
References

“Greenan, N. S., R.L. Mulvaney, and G.K. Sims. 1995. A microscale method for
colorimetric determination of urea in soil extracts. Commun. Soil Sci. Plant Anal. 26:2519-
2529”.

M. DUBOIS, K. A. GILLES, J. K. HAMILTON, P. A. REBERS, and F.SMITH. 1956.

Colorimetric Method for Determination of Sugars and Related Substances

Miller, G. L. (1972). Estimation of reducing sugar by dinitrosalicylic acid method. Analytical

Chemistry,
31, 426-428.
Rivers, D. B., Gracheck, S. J., Woodford, L. C., & Emert, G. H. (1984). Limitations of the DNS
assay for reducing sugars from saccharified lignocellulosics. Biotechnology and
bioengineering, 26(7), 800-802.

Reducing vs. Non-Reducing Sugars: Definition & Comparison, Laura Foist, 2018, study.com,
[Online]. [Accessed 22nd August 2018]. Available from World Wide Web:
https://study.com/academy/lesson/reducing-vs-non-reducing-sugars-definition-
comparison.html#lesson
The Definition of Reducing Sugars, Natalie Rizzo, 2017, livestrong.com, [Online]. [Accessed
22nd August 2018]. Available from World Wide Web:
https://www.livestrong.com/article/386795-the-definition-of-reducing-sugars/
Methodology for Carbohydrates, 2018, Biocyclopedia, [Online]. [Accessed 22nd August 2018].
Available from World Wide Web:
https://biocyclopedia.com/index/plant_protocols/carbohydrates/reducing_sugars_by_Nel
son-Somogyi_method.php
Carbohydrates Estimation Teaching Kit (Quantitative), HiMedia, [Online]. [Accessed 22nd
August 2018]. Available from World Wide Web: http://himedialabs.com/TD/HTBC003.pdf
Browning Toxicology of Nonenzymatic Browning, Encyclopedia of Food Sciences And
Nutrition (Second Edition), P.A. Finot, 2003, [Online]. [Accessed 22nd August 2018].
Available from World Wide Web:
https://www.sciencedirect.com/science/article/pii/B012227055X001309
Common Reducing Agent, Marci Southern, 2017, [Online]. [Accessed 22nd August 2018].
Available from World Wide Web: https://sciencing.com/common-reducing-sugars-
6193580.html
Reducing sugar, 2018, Wikipedia, [Online]. [Accessed 22nd August 2018]. Available from World
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 Wide Web: https://en.wikipedia.org/wiki/Reducing_sugar
Appendix

Serial dilution after being boiled for 5 min

From blank, 200,400, 600, 800 and 1000 mg/l concentration also unknown concentration

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