Practical 4 — Testing for Biological

Molecules
This practical is aligned to:

Learning outcome
• Demonstrate basic laboratory skills.

Assessment criteria
• Recognise basic chemical groups and describe their role in the behaviour of biologically
important molecules (carbohydrates, fats, proteins and nucleic acids).
• Write and present a report on experimental methodologies and findings.
• Solve well-defined but unfamiliar problems using correct procedures and appropriate
evidence.

4.1 Introduction
The nutrients in the food you eat supply your body with energy for growth and repair. These
principal substances include carbohydrates, proteins and fats. Carbohydrates make up a
group of organic compounds that include sugars and starches, which are important in
supplying your body with energy. Some starches provide your body with indigestible fibre or
roughage, which aids digestion. Proteins are organic compounds important for growth and
repair. Lipids (e.g. fats) are organic compounds that can supply as much as four times the
amount of energy as carbohydrates or proteins.

Using chemical indicators

We can test for the presence of these important compounds in food by using chemical
reagents that react in predictable ways in the presence of these nutrients.
Work in an area appropriate for handling chemicals that may stain furniture or the floor, if
spilt. Wear proper safety equipment including a laboratory coat.

4.2 Outcome
At the end of this practical, students should:
• Demonstrate an understanding of the basic chemical reactions common to food groups,
e.g. lipids, simple sugars, complex sugars and proteins.
• Integrate this understanding with functional groups in complex compounds.
4.3 Risks and Hazards
NOTE!
• This experiment poses a biohazard risk due to the reagents used for chemical
reactions.
• The experiment also poses minimal risks of burns due
to the use of hot water baths/hot plates and cuts due to broken or chipped
glassware.
• All reagents used pose a health hazard if these chemicals come into contact with the
eyes or respiratory system if swallowed.
• Avoid breaking test tubes!

4.4 Risk Control Measures

• Acid-resistant laboratory coats, latex gloves and safety goggles should be worn for
the entire duration of the experiment.
• Follow the rules of GLP, which include the removal of PPE when you step outside the
laboratory for lunch and toilet breaks.
• Ensure that the experiment is performed in a well-ventilated area to prevent
respiratory distress.

4.5 Equipment and Materials

• 10× 125mm test tubes; 1per test sample.
• Test tube rack
• 3ml plastic Pasteur pipette
• Small beakers
• Glass marking pen
• Hot water bath (or hotplates)
• Laboratory thermometer
• Bunsen burner
• Safety goggles
• Different foods to be tested for sugar, starch, protein, and fat content
• Solutions/reagents to be used to test the different food groups

4.6 Experimental Methodology

PART 1: Testing known samples
The purpose of the following is for students to observe colour changes in cases where the
food sample being tested for is present. Hence, showing what the "positive" results will look
like. Your set-up should look like Figure 1. Label your test tubes as per Table 1.
 Figure 1: Test tube arrangement for part 1

Table 1: Labels and reagents to be added per test tube for Part 1

Test 1 :Simple sugar test with Benedict's reagent

Benedict's solution is used to test for simple sugars, such as glucose. It is a clear blue
solution of sodium and copper salts. In the presence of simple sugars, the blue
solution changes colour to green, yellow and brick-red, depending on the amount of sugar.

Method
1. Make sure all the test tubes used are clean. There should be no indication of residue.
2. To test tube 1, add 5ml of liquidised food sample to be tested. Remember to label the
tubes clearly as per Table 4.1.
3. Add 5ml of distilled water (negative control) to test tube 2
4. Add 10 drops of Benedict's solution to each test tube. Carefully heat the test tubes by
Suspending them in a hot water bath at about 40-50°C for five minutes.
5. Note any colour change. If sugar is present solution will turn green, yellow or brick-red,
depending on sugar concentration. You will know that a colour change has taken place by
comparing it with your negative control.

Test 2: Starch test using Lugol's iodine

Lugol's iodine is used to identify the presence of starch. The solution is yellow-brown, but
when it reacts chemically with starch, a blue-black substance called "iodide starch" is
produced. In this test, we will place drops of Lugol's lodine into test tubes with
different kinds of food. In the end, the test tube sample with the darkest colouring will
contain more starch than the other.

Method
1. Make sure all the test tubes used are clean with no indication of residue.
2. Add 5ml of liquid to be tested to test tube 3. Remember to label the tubes clearly.
3.Add 5ml of distilled water (negative control) to test tube 4.
4.Add three drops of Lugol's iodine solution to each test tube. Mix the sample thoroughly.
5.Observe any colour changes. If starch is present, a blue-black precipitate will form.
Compare the colour of each sample. Darker blue-black represents more starch present. You
will know that a colour change has taken place by comparing it with your negative control.
6.To test tubes that show a positive colour change, flame boil directly until the colour
disappears. Record this change as well. What does this mean?

Test 3: Protein test with Biurtet's reagent

Biuret solution is used to identify the presence of protein. Biuret reagent is a blue solution
that, when it reacts with protein, will change colour to pink-purple.

Method
1. Make sure all the test tubes used are clean without an indication of residue.
2. Add 5ml of liquid to be tested to test tube 5. Remember to label the tubes clearly.
3. Add 5 m lof distilled water (negative control) to test tube 6.
4. Add three drops of Biuret reagent solution to each test tube. Shake gently to mix
5. Note any colour change. Proteins will turn the solution pink or purple. You will know that
a colour change has taken place by comparing it with your negative control. What does this
mean?

Test 4: Lipids with Sudan Red solution

Sudan ||| solution is used to identify the presence of lipids. Sudan reagent is a red solution
that, when it reacts with lipids, will stain the lipid molecules red.

Method
1. Make sure all the test tubes used are clean without any indication of residue.
2. Add 5ml of liquid to be tested to test tube 7. Remember to label thetubes clearly.
3. Add 5ml of distilled water to test tube 8 (negative control).
4 . Add 5ml of Sudan reagent solution to each test tube. Shake vigorously to mix. Allow
a minute to stand and observe
5.Note any colour change. Lipids will stain red. You will know that a colour change has
taken place by comparing it with your negative control.
Test 5: Lipids with ethanol emulsion for fats and oils
The ethanol emulsion can be used to determine the presence of a broad range of naturally
occurring lipids. This test need only be done on samples that have shown positive with
Sudan ||| Solution.

NOTE
The laboratory technician must optimise the test by testing various concentrations of
ethanol starting at 70% and increasing at 5% intervals till 90%. The laboratory technician
must only provide students with the optimal alcohol concentration for this test. Students are
to research online on how the reagents are prepared.

Method
1.Make sure all the test tubes used are clean without any indication of residue.
2.Add 2ml of liquid to be tested to test tube 9. Remember to label the tubes clearly.
3.Add 2ml of distilled water or a food sample that was previously negative in the Sudan Red
test (negative control) to test tube 10.
4.Add 3ml of the prepared ethanol concentration reagent to each test tube. Shake
vigorously to mix.
5.Note the layer of cloudy white suspension that forms at the top of the solution. This is an
emulsion. Note the quantity of emulsions that form for the various foods. What does this
mean?

PART 2 :Testing unknown samples

You will now be given an unknown food sample. Your task is to use all tests done in Part 1 to
determine which biomolecules are present in the unknown sample. Your set-up should now
look like this:

Figure 2: Test tube arrangement for Part 2

Table 2: Labels and reagents to be added per test tube for Part 2

Method
1.Add 3ml of liquidised unknown food sample to five test tubes.
2.In each test tube add the respective reagents, according to Table 4.2.
3.Determine the contents of the unknown food sample, based on your results.