DOÑA REMEDIOS TRINIDAD ROMUALDEZ MEDICAL FOUNDATION, INC.

Calanipawan Road Tacloban City

COLLEGE OF BIOMEDICAL SCIENCE
DEPARTMENT OF CHEMISTRY

BIOCHEMISTRY
EXPERIMENT NO. 5

Qualitative Test for Carbohydrates

Name: Date: ___________________
Course and Year: ______________________ Rating: _________________
DISCUSSION:
A carbohydrate is a polyhydroxy aldehyde, a polyhydroxy ketone or a compound that yields these
compounds hydrolysis. They are the most abundant bioorganic molecules in Earth, comprising about 60-
90% of the total weight of plants in the form of cellulose and starch. Cellulose serves the structural
support and starch provides source of energy for plants.
Carbohydrates are classified according to its molecular size; these are monosaccharides,
disaccharides, oligosaccharides and polysaccharides. Monosaccharides are the most basic unit of
carbohydrates and they cannot be broken down into simpler forms upon hydrolysis. It exists as single
polyhydroxy aldehyde or a polyhydroxy ketone with 3-7 carbon atoms. Most common form of
monosaccharide exist at 6-carbon atom. Physical characteristics of a monosaccharide are; it exists as
white, crystalline solids that are water-soluble. Glucose is the most important monosaccharide; it is a
normal constituent of blood and tissue fluids and is the primary substrate for ATP production.
Oligosaccharide is composed of 2-10 monosaccharide unit that are bonded to each other. Disaccharides
are a combination of 2 monosaccharide unit that are covalently bonded to each other via glycosidic
linkage formation following a removal of water. Like monosaccharide; they exist as white crystalline
solids that are water-soluble. Polysaccharide is a polymeric form of carbohydrates that is composed of
many covalently bonded monosaccharides. They exist in linear form or in branch type with homogenous
or heterogeneous monomers. They are known to perform different types of biological function but most
of the time they are broken down for energy production.
Carbohydrates are detected qualitatively using different types of tests that react with
carbohydrates according to its properties:
1. Molisch Test is a general test for carbohydrates. All carbohydrates regardless of its molecular size
give a positive purple ring at the junction.
2. Reduction Test is to test the reducing properties of carbohydrates. All carbohydrates that has free
aldehyde group reacts with Cu2+ complex ion to form a red-orange precipitate of copper(I) oxide
(Cu2O). There are 3 tests that are commonly performed under this reduction test. Fehlings test is
the reaction of carbohydrates with alkaline copper-tartrate complex producing brick-red
precipitate. Benedict test uses alkaline copper-citrate complex as oxidizing agent producing a red-
orange or occasionally yellow precipitate. Lastly is the Barfoeds Test, this is a special type of
reducing test because it distinguish reducing monosaccharide and reducing disaccharide in an
acidic medium using copper acetate.
 3. Seliwanoff tests separates ketohexoses and aldohexoses. Ketohexoses will give a cherry red color
within few minutes while aldoses require larger time to react producing red-orange solution.
Monosaccharide with 3-5 C atoms will produce greenish color solution. Disaccharides and
polysaccharides will eventually hydrolyze that will react to the test.
4. Iodine Test detects the presence of polysaccharide specifically starch. Iodine is adsorbed onto the
surface of the polysaccharide, forming a colored complex producing a blue or purple solution
indicating a positive result. Linear form of starch; amylose will produce purple color solution
while a more complex branched starch; amylopectin and glycogen will produce yellow or brown
solution.

OBJECTIVES:
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MATERIALS:
Molisch reagent, Benedict’s reagent, Fehling’s reagent, Barfoed’s reagent, test tubes
PROCEDURE
Molisch Test:
Label six test tubes with number 1-6. Place 1 mL of each of the following solutions of the five the
test tubes. Add distilled water to the sixth test tubes to serve as the control.
1. 1% xylose solution
2. 1% glucose solution
3. 1% fructose solution
4. 1% sucrose solution
5. 1% starch solution
6. Distilled water (control)
To each test tube, add 3 drops of Molisch reagent. Shake each test tube to ensure mixing. With the test
tube held at 45 ⁰ angle, carefully and slowly run 10 drops of concentrated H 2SO4 solution down the wall
of the test tube so that the two layers form. Note the color at the point where the two layers meet,
Results:
Sample Observation
1% xylose solution
1% glucose solution
1% fructose solution
1% sucrose solution
1% starch solution
Distilled water (control)

What is the expected positive result for Molisch test? ________________________________________

Which carbohydrate gave positive result? _________________________________________________
Did any carbohydrate(s) require an extended time to give positive results? _______________________
Benedict’s Test
Place 1 mL of each of the following into seven (7) test tubes: (% xylose solution, 1% glucose solution,
1% fructose solution, 1% sucrose solution, 1% lactose solution, 1% starch solution, Distilled water
(control). Add 5 mL of the Benedict’s reagent to each solution. Shake the solution and place all 7 test
tubes in a boiling water bath at the same time. Remove the test tubes after 5 minutes and allow them to
cool. After 15 minutes, observe any changes that occurred in each test tube.
Fehling’s Test
Dilute 2 mL Fehling’s solution (1 mL Fehling’s A and 1 mL Fehling’s B) with 8 mL water. Put 1 mL of
the diluted solution in the seven test tubes. Take one of the test tubes, while heating on a water bath; add
1% glucose solution drop by drop. Note the result.
Repeat the test on the other remaining test tubes, using different 1% sugar solution of xylose, fructose,
sucrose, lactose, starch and the t-control. Record your observations in the table provided.
Sugar Observation
Benedict’s Test Fehling’s Test
Xylose
Glucose
Fructose
Sucrose
Starch
Water

What is a reducing sugar? _________________________________________________________

What is the positive result Benedict’s and Fehling’s test? What is the working principle for both of these
tests? Explain.
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Which carbohydrate are reducing sugars? ______________________________________________
Which are non-reducing disaccharides? Non-reducing monosaccharide? _______________________
Barfoed’s Test
Prepare six (6) test tubes, label them from 1 -6. Place 1 mL of each of the following sugars in the test
tubes (xylose, fructose, lactose, maltose, sucrose, glucose, starch). To each solution, add 5 mL of the
Barfoed’s reagent. Shake each sample, Place the test tubes in boiling water bath at the same time.
Remove them after 5 minutes and let cool after 15 minutes., make your observation and record them on
the table provided.

Sample Observation
xylose solution
glucose solution
fructose solution
lactose solution
maltose solution
sucrose solution
starch solution
Which carbohydrates gave positive result for Barfoed’s test?
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What is the expected positive result for this test? What is its working principle?
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Picric Acid Test
Label seven test tubes from 1-7. Place 1 mL each of the following sugars, xylose, fructose, galactose,
maltose, sucrose, lactose, and starch solution. Add 1 mL of saturated picric acid solutions to each test tube
and add 2 drops of NaHCO3. Mix thoroughly. Boil each test tube for 1 minute and observe the changes in
color.

Sample Observation
xylose
fructose
galactose
maltose
sucrose
glucose
starch

What principle describes the color changes of some sugars? ________________________________

Which of the tests performed is commonly used in clinical laboratory for the analysis of urine sugars?
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