Lab 2

Identification of Biomolecules
Our physical bodies are essentially a collection of both common and some exotic chemicals. Many of
these chemicals are simple inorganic combinations such as sodium chloride, hydrochloric acid,
molecular oxygen, and carbon dioxide. Most of the different kinds of chemicals comprising our bodies
are larger more complex organic molecules. The biochemical reactions that are occurring constantly
within our cells synthesize new, larger molecules or decompose larger molecules into smaller pieces.

Anabolism is a term used for all the synthesis reactions occurring at any time; Catabolism is a term that
refers to all the decomposition reactions occurring at any time. Metabolism is a term that refers to ALL
of these reactions together. While our bodies can metabolize a wide variety of organic molecules, the
vast majority belong to three major groups: carbohydrates, lipids and proteins.

Carbohydrates are composed of carbon, hydrogen and oxygen atoms in a ration of (CH2O)n where n can
be any number depending on the complexity of the carbohydrate. Simple sugars such as glucose and
fructose are called monosaccharides. More complex carbohydrates such as starches are polymers of
these monosaccharide units and are called polysaccharides. Simple carbohydrates are broken down or
catabolized in a process called glycolysis which provides the cells with most of its energy.

Lipids, including fats and steroids are composed of carbon, hydrogen and oxygen atoms. They are
important components of cell membranes and are used as hormones and for energy storage. Excess
food is usually stored as fat in adipose tissue cells.

Proteins are constructed from long chains of amino acids and contain carbon, hydrogen, oxygen,
nitrogen and sulfur atoms. Proteins provide the major structural components of our cells and therefore
our bodies. Other proteins serve as enzymes which are the major catalysts that facilitate complex
biochemical reactions in our cells

We can perform simple tests to identify the presence of some of these kinds of molecules by adding
indicators to a solution to be tested. A change in color or other physical characteristic indicates the
presence or absence of a particular kind of organic molecule.

A. Simple carbohydrates (sugars).

Benedicts solution causes some sugars to turn green, yellow, orange or red when heated to boiling. The
color of a positive reaction depends on how much sugar is present (green indicates low levels; red high
sugar levels)

B. Complex carbohydrates (polysaccharides or starches).

Lugol’s iodine causes a solution containing starch to turn dark blue to black. The more starch there is the
darker the color.

C. Lipids (fats and oils).

Large amounts of concentrated lipids leave a translucent spot on absorbent paper after drying.
D. Proteins (and Polypeptides)

Biuret solution causes a protein solution to turn pink or violet.

The first step in learning to detect these chemicals is to perform control tests with substances known to
contain or not to contain specific chemicals. You will perform each of the above tests on a “positive” and
a “negative” solution (the “negative” is usually water). After completing the tests, you will see both the
positive and negative results for each of the different kinds of molecule above. Then you can compare
your experimental tests to these control results to see if any of the different kinds organic molecules are
present in each test (unknown) solution.

Experimental protocol
MATERIALS

Test tube rack, 2% Glucose solution, Benedict’s solution

Beakers for water bath, 2% Starch solution, Biuret solution

Homogenized plant & animal tissue, 2% Protein solution, Sudan III or IV

Hot plate Fat (oil), Lugol’s Iodine solution

A. INDICATOR TESTS FOR THE PRESENCES OF MACROMOLECULES

1. Benedict’s solution: small sugars + Benedict’s + HEAT => green/yellow/orange/red

2. Iodine: starch + iodine => dark blue / black color

3. Sudan: oil + Sudan => colored oil

4. Biuret: protein + Biuret => purple/lavender

5. amino acid + Biuret => pink

Procedure:

1. Fill a 600 ml beaker 1/3 full with deionized water, place it on a hot plate and heat until it is boiling.
Keep this water bath at a boil as you will need it later.

2. Keep an eye of the boiling water so that it does not completely evaporate.

3. The boiling water will be needed for several tests.

4. Label four test tubes and fill them as listed below:

Test tube #1 = 1 ml of glucose solution

Test tube #2 = 1 ml of starch solution

Test tube #3 = 1 ml of lipid solution

Test tube #4 = 1 ml of protein solution

1. Add 1 ml of Benedict’s solution to each of the four test tubes.

2. Note the initial color of each mixture for each tube.

3. Place all four test tubes in the heated water bath and note the color change when it is heated
sufficiently (5 minutes or so).

4. Use the test tube holder when removing the HOT test tubes.

5. In the presence of small sugars, Benedict’s will change to either: green, yellow, orange or red color.

6. Record your observations on the worksheet.

Test for the presence of starch:

1. Clean the four test tubes thoroughly and prepare them as listed above.

2. Add 3 drops of iodine.

3. In the presence of starch, iodine changes to a navy blue/black color.

4. Record your observations on the worksheet.

Test for the presence of fats:

1. Clean the four test tubes thoroughly and prepare them as listed above.

2. Add 3 drops of Sudan.

3. In the presence of fats, you will notice red colored oil throughout.

4. Record your observations on the worksheet.

Test for the presence of protein:

1. Clean the four test tubes thoroughly and prepare them as listed above.

2. Add 1 ml of Biuret.

3. In the presence of proteins, Biuret will change to a purple/lavender color.

In the presence of amino acids, Biuret will change to a pink color.

4. Record your observations on the worksheet.

5. Thoroughly clean each test tube.

In the above tests you determined simple identifying tests for the presence of macromolecules. You will
now use these tests on various food items to see if they contain all or some of the four classes of
macromolecules.

Procedure:

1. Develop a hypothesis or hypotheses regarding the macromolecules that may be present in each food
item to be tested.

2. Setup four test tubes for the first food item to be tested and label #1 – #4.

3. Add 1 ml of the first food item to each test tube. Use only the liquid portion.

4. To test tube #1, add 1 ml of Benedict’s solution, mix and heat for 5 minutes.

NOTE: Allow the solution to cool and observe the precipitate for any color changes.

5. To test tube #2, add 4 – 5 drops of iodine solution and mix.

6. To test tube #3, add 3 – 4 drops of Sudan solution and mix.

7. To test tube #4, add 1 ml of Biuret and mix.

NOTE: Allow at least 2 – 5 minutes for a reaction to occur.

8. Record your observations on the worksheet.

9. Thoroughly clean and dry each test tube.

10. Repeat steps 3 – 9 for the remainder of the food items to be tested.

A. INDICATOR TEST RESULTS

Positive and negative tests for indicator solutions:

Fill in the table below by placing a mark in the appropriate box for a positive (+) and a negative (-) result
for each test.

Glucose Starch Fat Protein

1. What are the building blocks (monomers) for each of the following complex molecules?

a) Carbohydrates:

b) Proteins:
c) Fats:

d) Nucleic acids:

2. If Biuret was added to a test tube and resulted in a lavender color, what does that mean?

3. What is the indicator for starch?

4. A student added Benedict’s solution to a lemonade solution and waited 5 minutes. Did he get an
accurate reading? Briefly explain.

5. Benedict’s, iodine, Sudan and Biuret are chemical indicators. What is a chemical indicator?

6. What is a dehydration synthesis?

B. MACROMOLECULE ANALYSIS IN VARIOUS FOOD ITEMS

7. Develop a hypothesis regarding the possible macromolecules in food items.

8. Did your experimentation confirm your hypothesis?

The presence of macromolecules in food items:

Fill in the table below by placing a mark in the box that corresponds to the presence of a particular
macromolecule in each substance indicating a positive test reaction.

Benedict’s (small Iodine (starch) Sudan (fats) Biuret (protein &

9. Were there any results that did not agree with the composition of foods?

10. Did you have any results that were unexpected? If so, briefly list and explain them.

11. From your experience with the taste of the food items above, did any substance test positive with
the Benedict’s solution that normally does not taste sweet?

12. Let’s say you tested an unknown sample with Biuret and iodine solutions that resulted in a
purple/lavender and black color respectively. What can you conclude about this sample?