Experiment # 7 26/30

TESTS FOR SPECIFIC CARBOHYDRATES

Group Number: Group 6 Year/Section: BN 1-A

Date Performed: September 28, 2022 Date Submitted: October 5, 2022
Members: Florendo, Franz Axel G.
Tingson, Abigail M.

Objective: To study and perform certain test which indicate the presence of specific
carbohydrates.

Materials/Apparatus: Bunsen burner, tripod, water bath or 500-mL beaker, spot plate, test
tubes, test tube holder, glass slide and microscope, 5% solutions of ribose,
glucose, fructose, galactose, maltose, lactose, and sucrose, Seliwanoof’s
reagent, Bial’s reagent, conc. HNO3, solid galactose, solid glucose, 1% solution of
starch, dextrin, and gum Arabic, iodine solution.

Procedure:

A. MUCIC ACID TEST

This test is based on the formation of a crystalline dicarboxylic acid that is

insoluble in dil. HNO3, a reaction that is unique to galactose and galactose compounds.
1. Place 0.5 g each of solid lactose, galactose, and glucose separately into 3 test
tubes.
2. To each test tube, add 1 mL of water and 1 mL of conc. HNO3. Mix until the solid
dissolves.
3. Place the tubes in a boiling water bath for 1.5 to 2 hours, and allow to stand
overnight.
4. Carefully mount the crystals on a glass slide and observe under the microscope.
5. Check the solubility of the crystals remaining in the test tubes by adding 2 mL of
water to each test tube. Micic acid (galactaric acid) will not dissolve under these
conditions.
MUCIC TEST : Test for Galactose

Samples Observed Results Analyses

0.5 g of Lactose +

● Crystals formed at the

bottom of the test tube.

0.5 g of Galactose
+
● Crystals formed at the
bottom of the test tube.

0.5 g of Glucose
_

● No crystals formed at the

bottom of the test tube.

ANALYSIS: The Mucic acid test is a test that is highly specific and is used for the detection of
the presence of galactose and lactose. In this experiment, only lactose and galactose have
crystals formed at the bottom of the test tube indicating a positive result. On the other hand, no
crystals formed in the glucose indicating a negative result. In addition, the formed crystals are
insoluble in water.
 B. SELIWANOFF’S TEST

This is a time color reaction that is specific for ketones. Seliwanoff’s reagent is a
solution of resorcinol in HCl solution, ketoses undergo dehydration to yield furfural
derivatives more rapidly than do aldoses. Further, most furfural derivatives will form
complexes with resorcinol which are colored. Consequently, the relative rates of color
development in a solution containing a sugar, HCl, and resorcinol provide evidence of
the aldose or ketose nature of the sugar in question.

1. Place 1 mL each of the 7 sugar solutions separately into 7 test tubes.

2. Add 2 mL of Seliwanoff’s reagent. Mix well.
3. Place in a boiling water bath.
4. Record the time required for any change in color to occur. Leave the tubes in the
water bath for 10 minutes before recording negative results.

SELIWANOFF’S TEST : Test for Ketoses

Samples Observed Results Analyses

● The first solution

1 mL of 5% Fructose that changed
Solution color into dark
red. +
● The color
changed around
0.45 seconds

● The second
1 mL of 5% Sucrose solution that
Solution changed color
into dark red. +
● The color
changed around
1.5 mins.

● The third
1 mL of 5% Ribose solution that
Solution changed color
into green. _
● The color
changed around
4 mins.
 ● The fourth
1 mL of 5% Galactose solution that
Solution changed color
into dark red. +
● The color
changed
around 4.05
mins.

● The fifth
1 mL of 5% Glucose solution that
Solution changed color
into dark red. +
● The color
changed
around 4.15
mins.

● The sixth
1 mL of 5% Lactose solution that
Solution changed color
into dark _
orange.
● The color
changed
around 4.25
mins.

● The last
1 mL of 5% Maltose solution that
Solution changed color +
into dark red.
● The color
changed
around 4.35
mins.

ANALYSIS: Seliwanoff’s test is a test for ketoses and is used to differentiate between sugars that have a
ketone group (ketoses) and an aldehyde group (aldoses). This is a timed color reaction specific to
ketohexoses. In the experiment, fructose, sucrose, galactose, glucose, and maltose indicated a positive
result in which they yielded a cherry red color. Moreover, it is expected that fructose and sucrose will have
a positive result because both of them belong to the ketone group (ketoses) and only both of them turned
dark red in less than 3 minutes. On the other hand, ribose and lactose indicated a negative result due to
the reason that both of them belong to the aldehyde group (aldoses). Thus, when heated, ketoses are
dehydrated more rapidly compared to aldoses.

Then what about glucose and maltose? What are they?

 C. BIAL’S TEST

This test is a color reaction that is specific for pentoses. It involves the
decomposition of these sugars when heated with conc. HCl to furfural, which condenses
with orcinol (3,5-dihydroxytoluene) to form blue to green colored compounds.

1. Place 3 mL of Bial’s reagent into each of 7 test tubes and heat to boiling.
2. Add 5 drops of the sugar solution to be tested separately into the tubes.
3. Mix and note the results.

BIALS’S TEST : Test for Pentoses

Samples Observed Results Analyses

3 mL of 5% Fructose ● Turned into

cloudy green _
Solution
color.

3 mL of 5% Sucrose ● Turned into

cloudy green _
Solution
color.

3 mL of 5% Ribose ● Turned into a

blue green
Solution
color.
+
 3 mL of 5% Maltose ● Turned into a
dark green _
Solution
color.

● No change in
3 mL of 5% Glucose color
observed. _
Solution
● Remains to be
yellow brown
color.

3 mL of 5%Galactose ● Turned into a

Solution yellow brown _
color.

● Turned into a
3 mL of 5% Lactose light green
color. _
Solution

ANALYSIS: Bial’s test is useful in distinguishing pentose sugar from hexose sugar. In the
experiment, only ribose indicates a positive result which is classified as pentose sugar.
Pentoses (such as ribose) form furfural in an acidic medium that condenses with orcinol in
presence of ferric ions to give a blue-green colored complex that is soluble in butyl alcohol. On
the other hand, fructose, sucrose, galactose, glucose, lactose, and maltose indicate a negative
result which they are classified as hexose sugars.
 D. IODINE TEST

Starch, a nutrient or storage polysaccharide found in seeds, tubers, and roots of

plants, consists of two components: amylase, which is not branched but helical with a
hollow core, and amylopectin, which is highly branched and helical.

Iodine solution is used to differentiate helical polysaccharides. Amylose can

accommodate iodine atoms. Thus, iodine complexes with amylose to produce a deep
blue-black color. As the amylase is hydrolysed to amylodextrin, the color with iodine
changes from blue to purple or red violet to colorless. Amylopectin, which is branched
and helical, gives a purple or red violet color with iodine. Different samples of glycogen
give the iodine solution colors varying from wine red, red,reddish brown to brown. The
color differences are apparently related to the extent of coiling in the different samples of
glycogen. Non Helical polysaccharides give no color with iodine.

1. Place 3 drops each of 1% solutions of starch, dextrin, gum arabic, and agar-agar
separately on a spot plate.
2. Add 1 drop of iodine solution to each sample. Note the change in the color of the
iodine solution by comparing the results with the original color of iodine.

IODINE TEST : Test for detection of Starch

Samples Observed Results Analyses

ideally bluish-black
3 drops of 1% Starch ● Produced
Solution a _
yellowish
color.
(+)

3 drops of 1% Dextrin ● Produced

Solution a dark
purple or
blue-black +
color.

should be reddish or
red-violet
 3 drops of 1% Gum ● Produced
Arabic Solution a yellowish _
color.

3 drops of 1% Agar-agar ● Produced

Solution a bright _
yellow
color.

ANALYSIS: The iodine test is a test for detecting the presence of starch. In the experiment, only
dextrin solution indicates a positive result which detects the presence of starch, the sample
turns purple or blue-black in color when a few drops of potassium iodide solution are placed on
the solution. On the other hand, starch, gum arabic, and agar-agar indicate a negative result
which detects the absence of starch, the solutions produced a yellow-colored complex.
Specifically, the scratch that turned into yellow color was due to be broken down or hydrolyzed
into smaller carbohydrate units, thus, the blue-black color is not produced in this solution.
Therefore, this test can also indicate the completion of hydrolysis when a color change does not
occur. NO HYDROLYSIS took place.

Questions:

1. Why would glucose and maltose give positive results upon prolonged boiling with
Seliwanoff’s reagent?

ANSWER: Glucose and maltose would give positive results upon prolonged boiling with
Seliwanoff’s reagent due to the high concentration of glucose or other sugar that
interferes in changing its color. Prolonged boiling will transform glucose to fructose by
the catalytic action of acid which will result in cherry red complex giving a false-positive
result. Also, maltose is a sugar made out of two glucose molecules bound together to
make a positive result in Seliwanoff’s test.

2. Why is boiling necessary in Bial’s test?

ANSWER: Boiling is necessary for Bial’s test because it enhances the dehydration of
pentoses to yield furfural which in turn condenses with orcinol to form a blue-green
precipitate. The intensity of the color produced depends on the concentration of HCl,
ferric chloride, orcinol, and the duration of boiling.
 3. Each polysaccharides tested gives a different color result with the Iodine test. Explain
the reason briefly.

ANSWER: Each polysaccharide tested gives a different color result from the Iodine test
because of the formation of structures of the polysaccharides. The basis for the Iodine
test is that starch is a coiled polymer of glucose. Iodine interacts with these coiled
molecules and becomes bluish-black.
*the intensity of th color depends on the degree of coiling; the more helical, the darker

References:

Sapkota, A. (2020). Mucic Acid Test- Definition, Principle, Procedure, Result, Uses. Retrieved from
https://microbenotes.com/mucic-acid-test/#:~:text=Mucic%20acid%20test%20is%20a

‌ ondal, S. (2021). Tests for specific carbohydrates : Anthrone test ; Mucic acid test ; Osazone test ; Test
M
for non-reducing sugars ; Bial ’ s test . Anthrone Test Principle Uses of Anthrone Test Limitations of
Anthrone Test Mucic acid test Principle Mucic acid Test Procedu. November.

Karki, G. (2018). Tests for specific carbohydrates: Seliwanoff’s test, Bial’s test and Iodine test - Online
Biology Notes. Retrieved from
https://www.onlinebiologynotes.com/tests-for-specific-carbohydrates-seliwanoffs-test-bials-test-and-iodin
e-test/

(n.a). (2019). Iodine test Definition and Examples - Biology Online Dictionary. Retrieved from
https://www.biologyonline.com/dictionary/iodine-test