Nursing Biochemistry Laboratory (NurBio Lab) 1

Laboratory Manual

Laboratory Procedure Activity

Carbohydrates II: Specific Reactions 4

INTRODUCTION

This activity is the second part on carbohydrates. The parts on fermentation and mucic
acid test require more time, so it is advised to perform these immediately.

Specific Reactions
Carbohydrates are commonly isomers, but certain reactions due to particular
arrangement of atoms in the molecules distinguish them from each other.

In Seliwanoff’s test, resorcinol is used as the complex-forming agent to provide the color
reaction, although the exact nature of the colored product is not known. Since resorcinol
favors electrophilic aromatic substitution reaction, the reaction may be similar to the
Molisch’s test. Ketoses give a cherry red color within a few minutes (approximately 2
minutes) while aldoses require a longer time. This occurs because ketoses existing in the
furanose or pyranose form can readily attain, under acidic conditions, the 2-
ketofuranose structure which favors the formation of furfural by dehydration.

In the Bial’s Orcinol test, furfural from the dehydration of pentoses with orcinol form a
blue or green color, with the color varying with the concentration of FeCl3; while the
hydroxymethylfurfural from hexoses gives a muddy brown color.

Concentrated nitric acid, HNO3, oxidizes both C ends of the sugar, at the aldehyde and
at the primary alcohol groups, forming saccharic acids. The galactose isomer of this acid
is called mucic acid. Thus, with the use of this test, formation of a fine white precipitate
identifies galactose.

All reducing sugars condense with reagents such as hydroxylamine or phenylhydrazine.

With phenylhydrazine in the cold, a phenylhydrazone is first formed.

H O H N
NHC 6H5
H OH + H2N.NHC 6H5 H OH + H2O
(CHOH) 3 (CHOH) 3
CH 2 OH CH 2 OH

At 100C, in the presence of an excess of phenylhydrazine, further reaction takes place

involving the second carbon atom in aldoses and the first carbon atom in ketoses
forming an osazone.

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Nursing Biochemistry Laboratory (NurBio Lab) 2
Laboratory Manual

NHC 6H5
H N H N
NHC 6H5
H OH
+ H2N.NHC 6H5 N NHC 6H5 + C6H5NH 2 + NH 3
(CHOH) 3 (HOHC) 3
CH 2 OH CH 2 OH

Note that three molecules of phenylhydrazine are required, two of them incorporated
into the osazone structure. Additional products are aniline and ammonia.

The sugar osazones are well-crystallized compounds, each with a characteristic form.
Since the reaction involves the first two C atoms whose asymmetry is destroyed,
fructose, glucose and mannose all form the same osazone. The limitation of the reaction
to these two carbons is due to intramolecular bonding.

NHC 6H5
H N
H
NC 6H5
H N

The crystalline forms of sugar osazones are sufficiently characteristic to enable them to
be used for identification although such identification should be supported by
determination of the melting point of a recrystallized specimen.

Fermentation is the process of converting hexose sugars to alcohol and CO2, through the
action of mixture of enzymes in yeast called zymase. Hexoses are easily fermented,
pentoses are not. For disaccharides to be fermented they must first be hydrolyzed to
their monosaccharide components by other enzymes in yeast like invertase.

The chemical steps on the fermentation of glucose by yeast are very similar to the
anaerobic breakdown of glycogen by muscle extracts.

Polysaccharides such as amylase and amylopectin of starch, glycogen and dextrin form
characteristic colored complexes in iodine. Starch gives an intense blue color with iodine
due to the linear chain of glucose units of amylase which undergo helical formation. The
helix contains six glucose units per turn, so the inside of the helix is enough to
accommodate large molecules like iodine. Branched polysaccharides with interrupted
helices like amylopectin, yield less intense colored complexes. Highly branched
polysaccharides with shorter segments and hindered helix formation such as glycogen
yield only pale brown complexes with iodine.

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Nursing Biochemistry Laboratory (NurBio Lab) 3
Laboratory Manual

APPARATUS/MATERIALS CHEMICALS/REAGENTS

Bunsen burner 30 mL 1% glucose

Iron ring 25 mL 1% starch
Iron stand 20 mL 1% acetic acid
100C thermometer 18 mL 1% fructose
1 glass slide 18 mL 1% sucrose
1 pc fermentation tube 18 mL 1% maltose
1 pc 12-holed spot plate 15 mL 1% sugar solution E
test tubes 12 mL Bial’s reagent
droppers 10 mL Seliwanoff’s reagent
test tube rack 4 mL concentrated nitric acid
2 mL 1% lactose
2 mL iodine
2 mL 10% alcohol
1 mL 1% sugar solution B
1 mL 1% galactose
1 mL 1% arabinose
1 mL 1% sugar solution C
1 mL 1% sugar solution D
1 mL 1% glycogen
1 mL 1% xylose
1 mL 1% sugar solution A
1 g phenylhydrazine
1 g yeast
1 g galactose
1 g glucose
1 g lactose

IMPORTANT: One group member may begin will the mucic acid test
 (procedure C) and another with fermentation since these will entail more
time.

PROCEDURE

A. Mucic Acid Test (DO NOT PERFORM. Just search for theoretical results.)

1. Label four test tubes labeled 1, 2, 3 and 4. Place the following:

test tube 1 – a pinch of galactose
test tube 2 – a pinch of glucose
test tube 3 – a pinch of lactose
test tube 4 – 1 mL of the unknown sugar solution
2. To each tube, add 1 mL of water and 1 mL concentrated nitric acid, HNO3.

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Nursing Biochemistry Laboratory (NurBio Lab) 4
Laboratory Manual

3. Heat the tubes in a boiling water bath, for one hour, then allow them to cool to
room temperature. Induce crystal formation by scratching the inner side of the
test tube with a clean stirring rod.
4. If no crystals appear, let stand until the next laboratory period. Examine the
crystals under a low power microscope and draw/sketch as seen.
5. Confirm the solubility of the crystals by adding 2 mL distilled water to the tubes
where the crystals have formed. Note carefully which tube shows water insoluble
crystals.

B. Seliwanoff’s Test

1. Prepare 5 test tubes. Label them with numbers 1, 2, 3, 4 and 5. Place in each
test tube 2 mL Seliwanoff’s reagent. Then add 2 drops of the following solution
to the corresponding test tubes:
test tube 1 – 1% fructose solution
test tube 2 – 1% glucose solution
test tube 3 – 1% sucrose solution
test tube 4 – 1% maltose solution
test tube 5 – the unknown sugar solution
2. Immerse the tubes in boiling hot water bath. Observe the changes in each test
tube during the first 15 minutes. Record the color and time of its formation in
each solution tested.

C. Bial’s Orcinol Test

1. Prepare four test tubes labeled 1, 2, 3, and 4. Into each, place 3 mL of Bial’s
reagent, Then add 3 drops of the following sugar solutions:
test tube 1 – 1% xylose solution
test tube 2 – 1% glucose solution
test tube 3 – 1% lactose solution
test tube 4 – the unknown sugar solution
2. Heat the solutions in a boiling water bath until a color develops. Note the color
formed and the time needed for each color to develop.

D. Osazone Formation (DO NOT PERFORM. Just search for theoretical results.)

1. Into each eight test tubes, place 1 mL of the following:

test tube 1 – 1% glucose solution
test tube 2 – 1% fructose solution
test tube 3 – 1% galactose solution
test tube 4 – 1% lactose solution
test tube 5 – 1% maltose solution
test tube 6 – 1% sucrose solution
test tube 7 – 1% arabinose solution
Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Nursing Biochemistry Laboratory (NurBio Lab) 5
Laboratory Manual

test tube 8 – the unknown sugar solution

2. Add a pinch of phenylhydrazine to each test tube. Mix thoroughly and stopper
the tubes with cotton. Place them in a boiling water bath for thirty (30) minutes.
Allow cooling slowly at room temperature. Rapid cooling may distort the crystals.
Record time of formation of crystals.
3. To examine the crystals formed, use a wide tip dropper to transfer a drop of the
solution containing suspended osazone crystals to a microscope slide. Examine
the crystals under low power microscope. Compare the crystals of the unknown
solution with the osazones if known sugars or consult photomicrographs of the
osazones. Describe and draw the crystals in the corresponding spaces provided
in the data sheet.

E. Iodine Test

1. On a spot plate or in small tubes, separately place 10 drops of 1% solutions of

the starch, glycogen, and glucose. To each spot, add a drop of iodine solution.
Note the color formed.

F. Alcohol Test (DO NOT PERFORM. Just search for theoretical results.)

1. Using the same sugar solutions in different tubes, drop 10% ethyl alcohol until a
change is noted in the solution. Record the results.

G. Fermentation (DO NOT PERFORM. Just search for theoretical results.)

1. Prepare a smooth suspension by grinding 0.5 g yeast in a mortar with 15 mL of

the sugar solution. Pour 5 mL of the suspension into a regular size test tube.
2. Fill a fermentation tube (2-3 inches long) to overflowing with the same
suspension.
3. Cover the mouth of the fermentation tube with the thumb and taking care that
none of the contents will spill out, drop it upside down into the big test tube.
4. Place the fermentation set up in a beaker of warm water (maintained 40C) so
that the level of the liquid in the tube is the same as the beaker. Keep for 90
minutes. Agitate the test tube in the water bath from time to time. Gas should
collect in some test tubes before the end of the period. Measure the height of
empty space in the small test tube.

PROPER DISPOSAL: Dispose of solutions in the proper waste bottles (as

acid or basic wastes, and organic or inorganic wastes). Dispose of dilute
sugar samples under running tap water.

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Nursing Biochemistry Laboratory (NurBio Lab) 6
Laboratory Manual

QUESTIONS

1. Write the chemical equation involved in the reaction between concentrated nitric
acid (HNO3) and glucose?
2. Cite at least 3 other sugars which will give a positive result in the music acid
test? Why will these give positive results?
3. What sugars form the characteristic osazone crystals? How do the crystals differ?
4. What is responsible for the intense blue color formed in the iodine test for
starch?
5. What is the purpose of grinding with sand and TCA in preparing glycogen?

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Nursing Biochemistry Laboratory (NurBio Lab) 7
Laboratory Manual

Group No.___ Date Performed :___________

Subject/Section:__________ Instructor’s Initials :___________
Members:
1. _____________________
2. _____________________
3. _____________________

Data Sheet Activity

Carbohydrates Part II: Specific Reactions 4

Data

Name of Test Test Added Reagents Results

Solutions
A. Mucic Acid galactose (s)
Test
(Search for
Theoretical glucose (s)
Results Only) + Nitric Acid
lactose (s)

Unknown

B. Seliwanoff’s 1% fructose
Test
1% glucose

1% sucrose + Seliwanoff’s
Reagent
1% maltose

Unknown

C. Bial’s 1% xylose
Orcinol Test
1% glucose
+ Bial’s Reagent
1% lactose

Unknown

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Chemistry Laboratory 204: Organic Chemistry and Biochemistry 8
Laboratory Manual

D. Osazone Formation (Search for Theoretical Results Only)

Test Time of Color Shape Sketch

Solutions Formation
(hour:minute
:second)
(00:00:00)

1% glucose

1% fructose

1% galactose

1% lactose

1% maltose

1% sucrose

1%
arabinose

Unknown

Identity of the Unknown: _________________________________________

Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University, Zamboanga City, Philippines.
Chemistry Laboratory 204: Organic Chemistry and Biochemistry 9
Laboratory Manual

Name of Test Test Added Results

Solutions Reagents
E. Iodine Test
1% starch

+ Iodine
1% glycogen
Solution

1% glucose

F. Alcohol Test
(Search for 1% starch
Theoretical
Results Only) + 10% Ethyl
1% glycogen
Alcohol

1% glucose

G. Fermentation (Search for Theoretical Results)

Test Solutions Added Reagents Height of Empty Space (mm)

1% glucose

1% fructose

1% maltose

1% sucrose

1% starch

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