Nursing Biochemistry Laboratory (NurBio Lab) 1

Laboratory Manual

Group No.___ Date Performed :07/10/19

Subject/Section: NurBio –C 1st year Instructor’s Initials :___________
Members:
1. Sangkula, jainab
2. Sakirani ,Ziarich

Data Sheet
Carbohydrates Part II: Specific Reactions Activity
4

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University, Zamboanga City, Philippines.
Chemistry Laboratory 204: Organic Chemistry and Biochemistry 2
Laboratory Manual

Rationale:
Carbohydrates are a class of natural compounds that contain either an aldehyde or a ketone group
and many hydroxyl groups , they are often called polyhydroxy aldehydes or ketones. A
monosaccharide consists of a single carbohydrate molecule, containing between 3 and 7 carbons.
Examples of monosaccharides are glucose and fructose. A disaccharide consists of two
monosaccharides that are linked together. Sucrose and lactose are disaccharides. A polysaccharide
consists of many monosaccharides linked together. Starch, pectin, glycogen, and cellulose are
examples of polysaccharides.

Objectives:
I. To study the properties of the carbohydrates
II. To determine the identification of the unknown carbohydrates by
the test that have been used

Mucic Acids Test:

Is another name of galactaric acids. Is a name for the product of the reaction. Is a type
of sugar that is less sweet that the glucose. It is considerd a nutritive sweetener
because it has food energy. It is a C-4 of glucose. G Oxidation of most monosaccharides
by nitric acid yields soluble dicarboxylic acids. However, oxidation of galactose
yields insoluble mucic acid. Lactose will also yield a mucic acid due to
hydrolysis of glycosidic linkage between its glucose and galactose subunits.Being
insoluble, galactosaccharide acid crystals separate out.

Figure.1

Seliwanoff’s Test:
Seliwanoff’s Test distinguishes between aldose and ketose sugars.
Ketoses are distinguished from aldoses via their ketone/aldehyde functionality. If the
sugar contains a ketone group, it is a ketose and if it contains an aldehyde group, it is
an aldose. This test is based on the fact that, when heated, ketoses are more rapidly
dehydrated than aldoses. Perform this test with glucose, fructose, maltose and sucrose.
The specific chemical equation for this specific event shown the below:

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Figure.2
This is the effect or the specific result for the seliwanoff test, the test is for the
distinguished of the group of aldehyde or ketones.

Bial’s Orcinol Test:

Bial’s Test is to determine the presence of pentoses (5C
sugars). The components of this reagent are resorcinol, HCl, and ferric chloride.
In this test, the pentose is dehydrated to form furfural and the solution turns
bluish and a precipitate may form. Perform this test with ribose and glucose. The
experiment will distinguish between the hexose and pentose. They react with the
bials reagent and are converted to the furfural presence of the substance.
Components. Nitric acids and dicarboxylic acids. The nitric is a oxidation that
terminals group of aldose, but leaves the secondary hydroxyl group unchanged.
While in the dicarboxylic formed from galactose is called mucic acids and is
insoluable in cold aquesous solution.

This is the specific chemical composition and the form of the chemical test of the Bial’s:

Data

Name of Test Test Added Reagents Results

Solutions
A. Mucic Acid galactose (s) + Nitric Acid Presence of crystals
Test

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(Search for glucose (s) None

Theoretical
Results Only) None
lactose (s)

Unknown

B. Seliwanoff’s 1% fructose Appearance of cheery red at

Test 1:54
1% glucose 1% glucose: No changes
even after 15
1% sucrose munutes 1% sucrose:
+ Seliwanoff’s appearance of cherry red in
Reagent 10 minutes and 3 seconds
1% maltose 1% maltose: it became
orange in 15 minutes
Unknown Unknown: apperance of
cherry red in 1 minute and
40 seconds
C. Bial’s 1% xylose 1% xylose: it became blue
Orcinol Test in 6 minutes and 57
seconds. In 8 minutes it
became green
1% glucose 1% glucose: it became light
green in 29 minutes and 10
seconds
1% lactose + Bial’s Reagent 1% lactose: it became dark
green in 29 minutes and 10
seconds
Unknown Unknown: it became blue in
5 minutes and 25 seconds.
In 7 minutes and 42
seconds it became muddy
brown.

Osazone Formation:
Filter the solution in another test tube and keep the filtrate in a
boiling water bath for 20 minutes. Allow the test tube to cool down ina minutes.
Glucose, fructose and mannose produce the same osazone because of the similarities in
their molecules structure. Involves the reaction of the significant concept of the reducing
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Laboratory Manual

of the sugar with the excess of phenylhydrazines when kept at the boiling point. Sucrose
as an example, it does not form the osazone crystalline because it is not a reducing
sugar and it does not have the characteristic of the free carbonyl group.
It is a normal routine test in order to identify the sugars. In this test, as the name
indicates, osazones are involved, we will like to discuss. Carbohydrates have many
classes and one of the class of them is Osazone and being not an original carbohydrate,
it is rather a derived form which we get when sugar do a reaction with a lot of
phenylhydrazine.

Figure.4

D. Osazone Formation (Search for Theoretical Results Only)

Test Time of Color Shape Sketch

Solutions Formation
(hour:minute
:second)
(00:00:00)
Needle
30 mins shape or
Broom
1% glucose Stick

57 mins Like tight

balls of
1% fructose needles

25 mins Like
sunflowers
stroke
1% galactose

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Chemistry Laboratory 204: Organic Chemistry and Biochemistry 6
Laboratory Manual

powder
28 mins puff-shaped
crystals.
1% lactose

Needle
59 mins shape or
Broom
1% maltose Stick

47 mins Like tight

balls of
needles
1% sucrose

N/A
1%
arabinose

N/A
Unknown

Identity of the Unknown: _________________________________________

Iodine Test:
The test is used for the detection of the starch in the solution. The blue
black color is due to the formation of the starch iodine complex. Starch contains
polymers of α-amylose and amylopectin which form a complex with iodine to give the
color blue black color.

Figure

Iodine test is an indicator for the

presence of starch. Iodine solution (iodine
dissolved in an aqueous solution of

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Chemistry Laboratory 204: Organic Chemistry and Biochemistry 7
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potassium iodide) reacts with starch producing a blue-black color. Apply this test to all
the polysaccharides provided. The substance is a detection of the starch composition
from the sugar solution.

Figure (Iodine)

Alcohol Test:
Adding sweeteners to ethanol solutions is a common method of inducing
rats to consume ethanol. However, it has usually been assumed that it is the sweet taste
and/or the calories contained in the sweet solution that controls consumption. The
present experiment examined the role of ethanol in controlling responding reinforced by
ethanol or an ethanol/sucrose mixture compared with sucrose solutions of various
concentrations.

Name of Test Test Added Results

Solutions Reagents
E. Iodine Test It became intense blue color
1% starch

+ Iodine It became pale brown as the

1% glycogen color of iodine
Solution
It became pale brown as the
1% glucose color of iodine
F. Alcohol Test N/A
(Search for 1% starch
Theoretical
Results Only) + 10% Ethyl N/A
1% glycogen
Alcohol
N/A
1% glucose

Fermentation Test: Many carbohydrates can undergo fermentation in the presence of

yeast. The carbohydrate is the food source for the yeast, and the products of the
fermentation reaction are ethanol and carbon dioxide gas. C6H12O6 à 2 CH3CH2OH + 2
CO2 (g) Glucose Ethanol Fermentation is used in the processes of making beer and
wine, where the alcohol produced by the yeast is the desired product. Not all sugars,
however, can be used by yeast as a food source. You will test which sugars ferment in
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University, Zamboanga City, Philippines.
Chemistry Laboratory 204: Organic Chemistry and Biochemistry 8
Laboratory Manual

the presence of yeast and which ones do not. The evidence of fermentation will be the
evolution of carbon dioxide gas. In the test, a quantity of a solution (containing yeast
and the sugar to be tested) will be trapped in an upside-down small test tube. After a
few days, you will check to see if a gas bubble has formed in the test tube. If there is a
gas bubble, it means fermentation did occur.
 Positive: The development of a yellow color in the medium is
indicative of a positive carbohydrate fermentation reaction.
 Negative: Lack of yellow color development is indicative of a
negative carbohydrate fermentation reaction.

Illustration of the fermentation of sugar solution.

G. Fermentation (Search for Theoretical Results)

Test Solutions Added Reagents Height of Empty Space (mm)

1% glucose N/A

1% fructose N/A

1% maltose N/A

1% sucrose N/A

1% starch N/A

ANSWERS TO QUESTIONS:
Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
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1. Write the chemical equation involved in the reaction between concentrated nitric acid
(HNO3) and glucose?I would use the equivalent of glucose, what I do want you to know
is that the reactive site on the glucose molecules is the CHO at one end of the molecules
in fact, it is the double bonded oxygen atom which is react with the nitric acid. The
−¿ (CHOH )4
nitric acid will dissociate as CH 2 -CHO + ----> HNO 3❑ CH 2 OH-
OH ¿
¿+¿¿
(CHOH )4 -CHO- NO3 + H
2. Cite at least 3 other sugars which will give a positive result in the music acid test?
Why will these give positive results? Mucic acid test is highly specific for galactose and
the formation of colorlesscrystals is its positive result. The reagent used which is
concentrated HNO3i s a strong oxidizing agent and it converts galactose to
galactaric acid which is the dicarboxylic acid of galactose and it is the one known
as mucic acid. The muci cacid produced is the one that takes the f o r m o f
crystals since it is relatively insoluble.
3. What sugars form the characteristic osazone crystals? How do the crystals differ?
Sugar known as disaccharides include maltose, lactose, and sucrose. The first present
osazone crystals that are shaped like sunflowers, while lactose osazone crystals are
more akin to tight balls of needle.
4. What is responsible for the intense blue color formed in the iodine test for starch?
According to (Al de man, n.d) a polymerisation reaction occurs between the starch and
iodine. Further iodine congregates within the structure leading to the deep blue colour.
5. What is the purpose of grinding with stand and TCA in preparing glycogen? According
to (Vamsee, n.d) Glycogen is extracted from animal tissue. This tissue is actually pretty
tough and to liberate the glycogen from it, the tissue has to be ruptured, broken down
nicely, so that all the glycogen is available for extraction. This grinding process is aided
by the presence of sand and TCA (Tri-chloro acetic acid). TCA is a highly corrosive agent
and helps in rupturing the tissue.

CONCLUSION: carbonhydrates are the most abundant classes of a bioorganic molecules

on the planet earth. Although their abundant in the human body is relatively low,
carbohydrates constitute about 75% by mass of dry plant materials. Green (chlorophyll-
containing) plant produces carbohydrates via photosynthesis. In this process, carbon
dioxides from air and water from the soil are the reactant, and sunlight absorbed by
chlorophyll in the energy source. There are several test to use in order to determine a
simple whether it is carbohydrates or not, if it is a monosaccharidesor disaccharides.

Reference:
 Laplante,J.P., & Nolin, C. (2014) https://www.ncbi.nlm.nih.gov/pubmed/8561301
 Toren,Z.,&Iliyan,S.(2008).
http://dept.harpercollege.edu/chemistry/chm/100/dgodambe/thedisk/carbo/bial/
bials.htm
 Christensen, L.(2001). https://www.micro.iastate.edu/video/microbiology-007-
carbohydrate-fermentation-test
 Freud, S. (1961). http://allmedtests.com/osazone-test-principle-procedure/

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Chemistry Laboratory 204: Organic Chemistry and Biochemistry 10
Laboratory Manual

 Mester, L.; El Khadem, H.; Horton, D. (1970). "Structure of saccharide

osazones". Journal of the Chemical Society C: Organic (18):
2567. doi:10.1039/J39700002567

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.