Republic of the Philippines

CEBU TECHNOLOGICAL UNIVERSITY

BIOCHEMISTRY LABORATORY
Experiment No. 2
TESTS FOR SPECIFIC SACCHARIDES

NAME: Pila, Mary Ella Mae C. DATE SUBMITTED: March 30, 2021
YEAR & SEC.: BSN I-B

Introduction
Carbohydrates are one of the three principal classes of foods, and is a major source of energy in our
diets. Carbohydrates can be characterized in a number of different ways based on the number of
carbons (pentose, hexose), the functional group (aldose, ketose), formation of rings (furanose,
pyranose), and isomers (D or L) and the number of monosaccharide units in the molecule (mono, di, and
polysaccharides). The following are the test for determination of the properties and for the
differentiation of carbohydrates

A. Barfoed’s Test
It distinguishes test from reducing monosaccharides to reducing disaccharides by controlling the pH
and time of heating.

I. Objective:
To be able to distinguish between the monosaccharides and the disaccharides using the barfoed's
test.

II. Materials Required

Test tubes
Test tube stand
Pipettes
Water bath
Vortex
Barfoed’s reagent: 0.33M solution of copper acetate is added to 1% acetic acid.
Sample (Carbohydrates sol.)

III. Procedure: https://www.youtube.com/watch?v=yQfMqvOxPrc

1. Take 1ml of test sample in dry test tube.

2. Take 1ml of distilled water in another tube as control.
3. Add 2ml of Barfoed’s reagent to all the tubes.
4. Keep in boiling water bath.
5. Look for the development of brick red precipitate.
6. Note the time taken to develop the color

IV. Observation:
The Barfoed’s reagent is mixed in two tubes having maltose solution and glucose solution,
respectively. The positive reaction of the test is when a brick red precipitate appears on the mixture
 after heating the test tubes and determines the monosaccharide while the negative reaction of the
test is when there is no reaction happening on the test tube that determines the disaccharide. Upon
heating both of the test tubes in a boiling water for three minutes, a brick red precipitation is seen on
the test tube with the glucose solution on it as the conversion of cupric ions in the Barfoed's reagent
to cuprous ions with the formation of copper (I) oxide has resulted the color, so the glucose solution
is a monosaccharide. The maltose solution on the other hand has no reaction therefore it is a
disaccharide.

• Limitation of Barfoed’s Test

This test cannot be used to detect sugar in urine as urine contains Cl– ions, which might interfere
with the reaction.
If a higher concentration of disaccharides is present in a sample, it might give a positive result.

B. Seliwanoff’s test
Seliwanoff’s test is used to distinguish aldoses from ketoses. On treatment with conc. Acid,
ketoses are dehydrated more rapidly to give furfural derivatives and on condensation with
resorcinol give cherry red complex. The test will be answered by fructose, sucrose and other keto
containing carbohydrates
I. Objective
To distinguish ketoses from aldoses. To detect the presence of ketohexoses in a given sample.

II. Materials
o Test solution: 5 % Glucose, 5 % Sucrose, 5 % Fructose.
o Seliwanoff’s reagent (0.5% resorcinol in 3N HCl)
o Water bath
o Pipettes
o Dry test tube

III. Procedures https://www.youtube.com/watch?v=0sf14uvPgK4

1. Take 1ml of sample in test tube and take 1ml of distilled water in another tube as control.
2. Add 3ml of Seliwanoff’s reagents in both test tube
3. Keep the test tubes in water bath for 1-2 minutes.
4. Look for the development of red color.

IV. Observation and Result

According to the video provided, a formation of the cherry red-colored complex appears after
heating the prepared solution with the Seliwanoff’s reagent in another test tube for 2 minutes. This
test tube contains sucrose solution, HCI, and Sodium Hydroxide Solution, which was added with 2ml
of Seliwanoff’s reagent. The other prepared test tube with only a sucrose solution, HCI, and Sodium
Hydroxide Solution did not change its color after heating the test tube. The formation of the cherry
red-colored complex indicates a positive result which means that the given sample contains ketoses.
The absence of such color or the appearance of the color after a prolonged period suggests a negative
result which means that the test sample does not have ketoses.
C. Bial’s Test
Bial’s test is useful in distinguishing pentoses sugar from hexoses sugars. Pentoses ( such as ribose
sugar) form furfural in acidic medium which condense with orcinol in presence of ferric ion to give
blue green colored complex which is soluble in butyl alcohol.

I. Objective
To detect the presence of carbohydrates and distinguish pentoses from hexoses sugars.

II. Materials
test reagent: 5 % Glucose, 5 % Ribose, 5 % Fructose
Bial’s reagent
Water bath
Dry test tubes
Pipettes

III. Procedures https://www.youtube.com/watch?v=JboA8Ghyz5A&t=40s

a. Take 2ml of Bial’s reagent in test tube.

b. Add 4-5 drops of test solution to this reagent.
c. Keep in water bath for 30 seconds.
d. Look for the development of bluish green color.

IV. Observation and result

A formation of a blue-green/bluish complex is the indicator of a positive result from the Bial’s test.
Other colors that may show are considered negative results. The positive result determines the
pentoses sugar while the negative result is identified as hexoses sugar. Three test reagents were used
in the experiment, namely, Glucose, Ribose, and Fructose. Fructose showed a greenish-brownish
complex while glucose showed a yellowish complex. The Ribose, on the other hand, showed a blue
complex. This is due to the glucose and fructose showing different colors and they are identified as
hexoses and the ribose that showed the blue complex is the pentose.

D. Osazone test (Phenyl hydrazine test)

https://www.youtube.com/watch?v=a2wyx6nZ-s8
Osazone test is a chemical test used to detect reducing sugars. This test even allows the
differentiation of different reducing sugars on the basis of the time of appearance of the complex.
This test is also termed Phenyl hydrazine test based on the reagent used for this test.

I. Objective
To detect reducing sugars. To differentiate reducing sugars from non-reducing sugars. To distinguish
different reducing sugars between each other.

II. Materials
• Osazone reagent: 0.5 g of phenylhydrazine hydrochloride and 0.1 g of sodium acetate.
• Glacial acetic acid
• Test sample
• Test tubes
• Test tube rack
 • Pipettes
• Vortex
• Water bath
• Microscope

III. Procedure

1. Take 5 ml of test solution in a clean, dry test tube.

2. Add 0.3 g of osazone mixture and five drops of glacial acetic acid to the test tube.
3. Mix it well and warm the test tube gently in the water bath if required to dissolve all the
elements.
4. Keep the test tube in boiling water and observe the formation of crystals at various time points.
5. Observe the shape of the crystal under low magnification under a microscope

V. Observation and result

Osazone crystals formed when viewed under the microscope have different shapes depending on
the type of carbohydrate present. Needle shaped crystals that are formed in 2 minutes indicate
fructose. Needle shaped crystals that are formed in 5 minutes indicate glucose. Balls with thorny edge
shaped crystals that are formed in 20 minutes indicate galactose. Sunflower shaped crystals that are
formed in 30-45 minutes indicate maltose. Cotton ball-shaped crystals that are formed in 30-45
minutes indicate lactose.

Tabulate the result

Carbohydrate Time of formation (min) Crystalline structure

Fructose 2 minutes Needle shaped crystals

Glucose 5 minutes Needle shaped crystals

Balls with thorny edge

Galactose 20 minutes
shaped crystals

Maltose 30-45 minutes Sunflower shaped crystals

Cotton ball/powder puff

Lactose 30-45 minutes shape/similar to a
hedgehog shape

Uses of Osazone Test

• This test is the only test that can be used to distinguish lactose from maltose during the
identification of unknown sugars.
• This is a simple, cheap, and relatively less time-consuming test for the identification and
differentiation of different sugars encountered in clinical practice.
 • This test can also be used for locating sugars in plant tissues.
Limitations of Osazone Test
• This test gives a positive result for sucrose when if boiled for 30 minutes or more even though
sucrose is a non-reducing sugar.
• This test is not effective if the sample contains a mixture of different sugars.
• Similarly, large quantities of sugars are required for a positive result.

SUMMARY/DATA ANALYSIS/ RESULT

Carbohydrate test Observation Result
Barfoed’s Test A brick red precipitation was The mixture of glucose solution and Barfoed’s
observed in the glucose solution reagent resulted to a positive reaction through
mixed with Barfoed’s reagent. the presence of a brick red precipitation after
On the other hand, no changes heating, which indicates that glucose is a
in the color of the maltose monosaccharide. While the mixture of maltose
solution were observed. solution and Barfoed’s reagent, which resulted
to a negative/no reaction, indicates that
maltose is a disaccharide.
Seliwanoff’s Test After heating the first test tube The cherry-red colored complex observed in
with the mixture of sucrose the second test tube, indicates that the
solution, HCI, and Sodium mixture of Sucrose solution, HCI, and Sodium
Hydroxide solution, no change Hydroxide solution, added with Seliwanoff’s
of color was observed. On the reagent will have a positive result, which
other hand, when the first test means that the test sample contains ketoses,
tube was added with specifically ketohexose. While the first test
Seliwanoff’s reagent, after it was sample, which gave no change of color,
heated, a cherry-red color indicates that it contains aldose.
appeared.
Bial’s Test The test sample with the Both Fructose and Glucose test samples
mixture of fructose solution and showed different colors other than blue or
Bial’s reagent had a greenish bluish and greenish, which means that the two
and brownish color. On the had a negative result of the test, indicating that
other hand, a yellowish complex fructose and glucose are hexoses sugar. While
color was observed in the the ribose is the only test sample that resulted
glucose test sample. Lastly, the to a bluish complex color, which means that it
test sample with ribose showed has a positive result with the indication that
a blue color. ribose is the pentose.
Osazone Test The different carbohydrates Each of the carbohydrates has different
(Phenyl Hydrazine (fructose, glucose, galactose, formation time and different crystalline
Test) maltose, and lactose) have structures:
different crystalline structures • Fructose – 2 minutes, needle shape crystals
and formation time. Some • Glucose – 5 minutes, needle shape crystals
carbohydrates appear to have • Galactose – 20 minutes, balls with thorny
spiky appearance, other formed shaped crystals
sunflower structure and cotton • Maltose – 30-45 minutes, sunflower shaped
ball structure. crystals
• Lactose – 30-45 minutes, cotton ball/powder
puff shape/similar to hedgehog shape
CONCLUSION

In conclusion, carbohydrates can be classified by its different properties and characteristics with
the help of (1) Barfoed’s Test that determines the presence of monosaccharides and disaccharides with
the help of each carbohydrate solution and the Barfoed’s reagent, (2) Seliwanoff’s Test that
distinguishes aldoses from ketoses through the presence of red and blue color or the absence of these
colors, (3) Bial’s Test that distinguishes pentoses sugars from hexoses sugars through the presence of
any bluish-green or yellowish color, and (4) Osazone Test that detects reducing sugars through
formation of crystalline structures. The tests mentioned above have been effective and could be very
helpful in determining sugars in food nutrition.

References:

Manual Compiled by Asuncion Monsanto ChE, Hedeliza A. Pineda, PhD. Mast- ChE
https://slideplayer.com/slide/2752134/
https://www.youtube.com/watch?v=a2wyx6nZ-s8
http://www.khalidshadid.com/uploads/3/9/2/0/3920808/carbohydrates_main.pdf
https://microbenotes.com/osazone-test/#objectives-of-osazone-test