Experiment #4

TESTS FOR SPECIFIC CARBOHYDRATES

Group #: Year/Section:
Date Performed: Date Submitted:
Members: Instructor:

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

Materials: 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,
Seliwanoff's reagent, Bial's reagent, conc. HNO3, solid lactose, solid galactose, solid glucose, 1% solution of
starch, dextrin, and gum Arabic, agar-agar, and 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 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 at least an hour. Allow it to stand overnight.
4. Carefully mount the crystals on a glass slide and observe under the microscope (optional).
*Or mount the crystals on a watch glass and take photos of these crystals. Compare the crystal
structures.
5. Check the solubility of the crystals remaining in the test tubes by adding 2 mL of water to each tube.
Mucic acid (galactaric acid) will not dissolve under these conditions.

(a) Describe your observation and the results. ____________________________________________

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B. SELIWANOFF'S TEST

This is a timed color reaction that is specific for ketoses. Seliwanoff's reagent is a solution of resorcinol
in HCl. In conc. 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 to each. Mix well.
3. Place in a boiling water bath using a 250 mL beaker.

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 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.

(a) Describe your observation and the results. __________________________________________

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(b) What does a change in color indicate in Seliwanoff's Test?

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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. HCI to furfural, which condenses with orcinol (3,5-dihydroxytoluene) to
form blue to green colored compounds.

1. Place 2 mL of Bial's reagent into each of 7 test tubes.

2. Add 5 drops of the sugar solution to be tested separately into the tubes.
3. Heat in a water bath for 3-5 minutes or until a color changed is observed.

(a) Describe your observation and the results. __________________________________________

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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.
Nonhelical polysaccharides give no color with iodine.

1. Place 5 drops each of 1% solutions of starch, dextrin, gum Arabic, and agar-agar separately in a test
tube.
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.

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 (b) Describe your observation and the results. __________________________________________
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Questions:
1. What does Mucic acid test specifically detect?
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2. What is the significance of leaving the tubes in the water bath for 10 minutes before recording negative
results?
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3. What type of sugars does Bial’s test detect?

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4. How do the colors observed in the iodine solution correlate with the nature of the polysaccharides being
tested?
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5. Complete the table below by specifying the results if positive or negative.

Mucic Acid Seliwanoff’s Bial’s Iodine
Lactose
Galactose
Glucose
Ribose
Fructose
Maltose
Sucrose
Starch
Dextrin
Gum Arabic

Conclusion:
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