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Chem 152: Biochemistry laboratory
Department of Chemistry
School of Science and Engineering
Loyola Schools, Ateneo de Manila University
Carbohydrates
Gloves are not needed for this experiment. Please make sure you bring one “sugar-free” sample for the “hidden sugars”
part of the experiment. (One unique sample per student)
Introduction
Carbohydrates are polyhydroxyaldehydes or polyhydroxyketones, or polymers of these. The functional groups present
provide a handle for analyzing these compounds. The carbohydrates are also chiral molecules and are optically active. The
optical activity is useful for analyzing and identifying sugars using polarimetry.
The simplest carbohydrates are known as monosaccharides. Examples include glucose and fructose. When two simple
sugars are joined, the resulting product is a disaccharide. Examples of disaccharides include sucrose, maltose, and lactose.
A large number of monosaccharides joined together is called a polysaccharide. Starch and cellulose are examples of
polysaccharides. Both are made of glucose molecules joined together, but differ in how these glucose molecules are joined
together. Oligosaccharides are shorter chains of 2-6 monosaccharide units
Pre-lab:
 No need for a reagent/hazard list for this experiment. Please just be wary of the harmful effects of the corrosive
acids and bases to be used for this experiment.
 Plan out which samples are important as positive and as negative controls for the tests. (You may do this in table
form).
 Plan the order of your work! Some of the experiments have waiting times that may be used to do the other parts. The
order the experiments are written below need not be the order that you do them!
Data Sheet
 On your lab notebook, create a table containing the substances tested, tests conducted and the tests results.
 Explain briefly (one paragraph) why the results are observed.
Procedure
I. Standard Tests (Individual)
1. Molisch’s test:
a. Place 2 mL of carbohydrate solution in a test tube and add a few drops of Molisch’s reagent and mix well.
b. Add 1-2 mL of concentrated H2SO4 carefully down the side of the tube so as to form a layer beneath the
aqueous solution. A purple ring at the interface indicates a positive reaction.
2. Iodine test:
a. Place 2 mL of each carbohydrate solution in a test tube and add 3 drops of dilute iodine solution. Unbranched
macromolecules give a blue color, branched macromolecules a reddish-black color.
3. Benedict’s test:
a. To 5 mL of Benedict’s solution (cupric sulfate in a mild alkaline medium) add about 1 mL of each
carbohydrate solution. Boil in a water bath for 2 min. The production of a green, yellow, or red precipitate
indicates a positive reaction for reducing sugars.
4. Barfoed’s test:
a. To 5 mL of Barfoed’s reagent (cupric sulfate in a weak acidic medium) add 1 mL of carbohydrate solution.
Boil in a boiling-water bath for 3-4 minutes. A red precipitate or definite red cloudiness is a positive reaction.
5. Seliwanoff’s test:
a. To 5 mL of Seliwanoff’s reagent add 5-6 drops of carbohydrate solution. Boil in a boiling-water bath for 30 s.
A red coloration or red precipitate is positive for keto-sugars and for disaccharides containing a keto-sugar.
II. Hidden Sugars (by pair – 2 samples)
Perform the above tests on products that have been labeled as “sugar-free.” Try to dissolve solids in deionized water first.
Colored liquids must be diluted to avoid interference with observations. Each student must perform the test on his/her
product. The class will then share the data. (ie. 10 students= at least 10 samples)
III.Hydrolysis of Carbohydrates (by pair)
6. Take enough (about 5 mL) of the di-, oligo-, and polysaccharide samples and to each, add about 0.5-1 mL
concentrated H2SO4. Heat in a water bath for 10 minutes.
 Run the above tests on the hydrolysate.
IV.TLC on Carbohydrates (In groups of five)
1. Prepare the TLC plates, activating them at 120C for 30 minutes if necessary.
2. With a pencil, mark about 1 cm from the bottom of the TLC plate and spots about 1 cm apart.
3. Using clean capillaries, spot standard solutions of sucrose, glucose, fructose, (option: xylose, ribose, and
rhamnose), as well as 2 of the samples that you hydrolyzed.
4. Place the TLC plate in a developing chamber containing n-butanol:acetic acid:ethyl ether:water in the ratio
9:6:3:1 by volume.
5. After running the chromatogram, detect the spots by spraying anisaldehyde reagent, then set the TLC plate on a
hot plate (about 100oC) until well-developed.
Sugar
Expected Rf
Color with anisaldehyde
Sucrose
0.09
Violet
Glucose
0.22
Blue
Fructose
0.27
Violet
Xylose
0.40
Gray
Ribose
0.47
Blue
Rhamnose
0.55
Green