FACULTY OF BIOTECHNOLOGY AND BIOMOLECULAR SCIENCES

Department of Biochemistry

BCH 3005 BIOLOGICAL CHEMISTRY

Experiment 8 : Mucic Acid Test

Programme : BACHELOR OF SCIENCE (HONORS) IN BIOCHEMISTRY

Experiment Date : 27 / 12 / 2022

Lecturer : Assoc. Prof. Dr. Syahida Ahmad

Laboratory Officer : En. Raimi Maskan

En. Mohd Cyrill Kamal

Group : GROUP 3

Name Matric No.

KINISHAA A/P TAMIL SELVEN 215393

AMIRAH NATASYA ALIA BINTI MOHD SANI 214896

WONG WEI HAO 217340

AZIB BIN HASIM 214987

ARIESSA BALQIS BINTI MOHAMAD AZILEE 214040

 INTRODUCTION
Mucic acid test is a test that is very specific and highly used to detect the presence of
galactose. Positive result of mucic acid test where colourless crystals formed,
indicates that the particular sample contains galactose. The absence of such crystals
shows that the sample does not contain galactose or any of its derivatives.
Monosaccharides will yield dicarboxylic acid upon treating with concentrated nitric
acid (HNO3) in this experiment. Lactose can form mucic acid as it will undergo
hydrolysis of the glycosidic bond between the glucose and galactose.

OBJECTIVE
To produce dicarboxylic acid from monosaccharides

MATERIALS AND APPARATUS

1. Concentrated nitric acid (HNO3)
2. Galactose
3. Glucose
4. Lactose
5. Unknown sample
6. Test tubes
7. Water bath (boiling point)
8. Glass rod
9. Glass slides
10. Microscope

PROCEDURE

50mg galactose 50mg glucose 50mg lactose 50mg unknown sample

 1mL distilled water added to each test tube.

1mL concentrated HNO3 added to each test tube.

Test tubes heated boiling water bath for1 hour.

5mL distilled water added to each test tube.

Test tubes let to stand and cool slowly.

Crystals formed were examined with a microscope.

RESULTS
Sample Crystal formed
Galactose

Glucose
Lactose

Unknown sample

DISCUSSION
Based on the observation above, it can be seen that galactose and the unknown
sample formed colourless crystals at the end of the experiment. Initially, in glucose,
the formation of crystals were absent. This happens because glucose does not have
any galactose since glucose is the simplest sugar. Glucose is a monosaccharide and
cannot be broken down into any smaller substances anymore. Thus, glucose showed
negative test result in the mucic test. Besides that, in galactose, colourless crystals
were present. Mucic acid test is a very specific for the presence of galactose, where it
will show a positive test result by forming colourless crystals. The reagent,
concentrated nitric acid used in this experiment is a strong oxidizing agent. It can
convert galactose into galactaric acid. This galactaric acid is the acid that also known
as mucic acid which is a dicarboxylic acid of galactose. The insoluble crystals formed
in galactose indicates the formation of mucic acid (SAPKOTA,2020). So, the
galactose sample showed a positive results where crystals can be examined under a
microscope. Meanwhile, the formation of colourless crystals in unknown sample same
as in the galactose. Thus, this shows that the sample contains galactose in it. Lastly, in
lactose, no crystals could be observed under the microscope. Lactose is a disaccharide
of glucose and galactose monomers. So, lactose should have given a positive test
result by forming crystals. However, unfortunately it did not. In order to crystals form
of n lactose, lactose should hydrolyse its glycosidic linkage between glucose and
galactose. Then, the concentrated nitric acid will oxidize those both monomers into
tetrahydroxy acids to produce the mucic acid. But due to some errors made during the
experiment, the crystals are not formed. The errors are may be because of some
contaminated materials or reagents like the cotton wool used to close the test tubes. It
also could be due to the duration of water bath. It must be prelonged for sometime.
Thus, lactose showed a negative test result.
 The principle of the mucic test is the most of the monosaccharides’ oxidation by
nitric acid produce dicarboxylic acids that can be soluble. However, galactose
oxidation produce mucic acid that are insoluble.Other than galactose, lactose also
produce a mucic acid because of the hydrolysis of glycosidic linkage between glucose
and galactose monomers. The insoluble, galactosaccharide acid or mucic acid crystals
separate out from the main component. Nitric acid has the ability to oxidize aldehyde
and primary alcohols groups at carbon 1 and carbon 6 of galactose to form crystal
precipitation (SAPKOTA,2020).The reaction of the mucic test is as following :

CH2OH-(CHOH)4-CHO + HNO3 → CH2OH-(CHOH)4-CHO-NO3 + H+

Application of mucic test in daily life is in galactosemia test. As we know that

mucic acid test is a test that used to detect the presence of the galactose,
galactosemia test is a blood or urine test that verify for the enzymes that needed to
convert galactose to glucose,which was a sugar that used as a source of energy for
human body. People with galactosemia do not have one of the enzymes, thus a high
levels of galactose accumulate in the urine or blood. The accumulation of galactose
in the baby's blood can cause liver damage, eating disorders, and intellectual disability.
These damages can develop within a few weeks once the baby starts to drink milk.
Babies with galactosemia need low galactose foods to gain weight and prevent brain
damage, liver problems, infections and cataracts. Galactosemia is a hereditary disease
that is passed down from parents to children. Galactosemia tests are usually done to
find out if a baby has any symptoms (MICHIGAN,2021).

CONCLUSION
Mucic acid test will produce the dicarboxylic acids from the monosaccharides of
galactose. Mucic test widely used to detect the presence of the galactose and so in
galactose sample and unknown sample in this experiment. From the experiment
conducted, the crystal formations were observed under a microscope in galactose and
unknown sample which indicated the presence of the galactaric acid.

REFERENCES

1. Sapkota, A. (2020, November 19). Mucic Acid Test- Definition, Principle,

Procedure, Result, Uses. Microbe Notes. Retrieved January 18, 2022, from
https://microbenotes.com/mucic-acid-test/#:%7E:text=The%20most%20important%20
use%20of,of%20lactose%20or%20agar%2Dagar
2. Wikipedia contributors. (2020, October 18). Mucic acid. Wikipedia. Retrieved
January 18, 2022, from
https://en.wikipedia.org/wiki/Mucic_acid

3. What is mucic acid test and its principle, Biology. (n.d.). Expert’s Mind.
Retrieved January 24, 2022, from
http://www.expertsmind.com/questions/what-is-mucic-acid-test-and-its-principle-3018
0071.aspx