Biology

Key unit competence
Test for biological molecules in a variety of contexts, such as identifying the contentsof mixtures of molecules and to follow the activity of digestive enzymes

Learning objectives
By the end of this unit, I should be able to:
–– Write out procedures in the identification of biological molecules
–– Explain the importance of the reagents used in the identification of biological molecules.
–– Carry out tests for the identification of biological molecules
–– Compare reducing and non-reducing sugars
–– Appreciate the importance of identification of food values in the food industry and in processing and packaging.
–– Show resilience making observations on color changes during food tests

Introductory activity
You are given solutions containing different food stuffs including maize flour, vegetable cooking oil, and egg white sugar cane liquid and passion fruit. Using prior knowledge of biological molecules to suggest the type of biological molecule in each one of them. Suggest the chemical tests used to identify each
of the molecules.

6.1 Test for carbohydrates
Activity 6.1
Materials required:
Starch powder, Irish potatoes juice, prepared porridge, Iodine solution, beakers, droppers, source of heat and test tubes

a. Test for starch
Procedure
–– Mix 1g of starch powder with 100ml of water
–– Boil the mixture while stirring; then cool the solution
–– Boil the mixture while stirring; then cool the solution
–– Put 2ml of starch solution in a test tube labeled 1, 2ml of Irish potato juice in a test tube labeled 2 and 2ml of prepared porridge in a test tube labeled 3
–– In each test tube put 2 drops of Iodine solution and shake
–– Record your observation and draw a conclusion

b. Test for reducing sugar
Requirements
Glucose powder, beaker and test tube, Benedict solution, Bunsen burner, droppers Procedures
–– In the beaker mix 1cm3 of water and 1g of glucose powder.
–– Pour the prepared solution of glucose in a test tube and
–– Add 2ml of benedict’s solution and heat
–– Record your observation.
Biological molecules are grouped into organic molecules including carbohydrates, proteins, lipids, nucleic acids and vitamins. They also contain inorganic molecules such as minerals and water. The first four organic molecules are called macromolecules because they are required in organism in large quantity. Carbohydrates including starch, reducing and non-reducing sugars appear in this category and are the main energy producers in the organisms. Others, including lipids and proteins are needed for building organisms while vitamins protect the organisms against diseases. We need to ensure that what we take from diet have all required biological molecules.

a. Test for starch.
Carbohydrates such as starch are tested by mixing a sample with 2-4 drops of iodine or Lugol’s solution. If the sample contains starch the solution will turn from a yellowbrown color to a dark purple/dark blue (Figure 6.1). The color change is due to a chemical reaction between the large carbohydrate molecule and the iodine ions. If the sample does not contain starch the solution remains yellow-brown.

b. Testing for reducing and non-reducing sugar
The presence of reducing sugar can be tested by using benedict reagent. Benedict solution has copper ions that have a light blue color. When this solution is heated in the presence of simple reducing sugars such as glucose, the blue color of copper ions changes from a light green color to rusty orange-brown color (Figure 6.2).

If the color of Benedict reagent persists, the sugar tested is not a reducing sugar. Note that there is no special reagent to test for non-reducing sugar, but by the addition of HCl, non-reducing sugars can be hydrolyzed to reducing sugars. To test the presence of reducing sugars, a solution of sodium hydroxide is needed to neutralize the acidity because Benedict reagent works better in neutral solution

Self-assessment 6.1
A student prepared carbohydrate solution labeled C. Perform the following experiment to confirm whether C1 is starch, reducing sugar, or non-reducing sugar.

a. Is this solution a carbohydrate?
b. Specify the role of Hydrochloric acid and sodium hydroxide used at stage 3 in the table above.

6.2 Test for proteins
Activity 6.2
Materials required
Milk, eggs, soybeans, test tubes, beakers, mortar for crushing beans, 1% NaOH or
1% KOH solution, 0.1M of CuSO4 solution and Millon’s reagent.

Procedure
–– Extract the white fluid from an egg

–– Prepare an extra of soya bean and 10ml of fresh milk
–– Put 2ml of albumen solution in a test tube labelled A1 and 2ml in A2
–– Put 2ml of milk solution in a test tube labelled B1 and 2ml in B2
–– Put 2ml of soya bean solution in a test tube labelled C1 and 2ml in C2
–– Put 1ml of KOH or NaOH solution in each of the test tubes A1, B1, and C1. Shake the mixture and add 1ml of CuSO4 solution in each (A1, B1, and C1)
test tube
–– Put 1ml of Millon’s reagent in each of test tubes (A2, B2, and C2). Shake the
mixture and thereafter boil the three test tubes (A2, B2, and C2).
The Biuret reagent is used to test for the presence of proteins. It contains copper ions with blue characteristic color. During the copper ions react with protein molecules and causes the biuret solution to turn from a light blue color to purple if proteins are present.

The test can also be done by using Millon’s reagent, which in the presence of proteins, the Millon reagent changes from colorless to pink.

Self-assessment 6.2
1. You are provided with the sample of the substance M and A. Carry out the following experiments and complete the table below.

2. Carry out the same experiment using the substance A and compare your findings with M.
3. Which of the substance A and M contain proteins?

6.3 Test for lipids
Activity 6.3
Materials required - Olive oil, test tubes, ethanol, water, sudan III solution

Procedure:
Use olive oil to carry out the following experiments Add 2 cm3of olive oil in the test tube:
–– Add 5 cm3 of ethanol followed by 5 drops of water.
–– Shake the mixture and record your observation.
–– To another test tube containing 2 cm3 of olive oil:
–– add 5 drops of Sudan III solution
–– Shake thoroughly and examine the mixture in the test tube after few minute and record your observations The presence of lipids can be determined by using Sudan III indicators, which are fat-loving
molecules that are colored. During the test for a solution containing lipids, two results are likely to be found: there is either the separation of layers indicating the levels of water and lipid, or the dye migrates toward one of the layers. If the mixtureis composed of water, the conclusion is that the lipids are not present. In this case, the Sudan III indicator will form small micelles/droplets and disperse throughout the solution. A positive result indicates the lipid layers sitting on top of the water layer with a red-orange color. When using ethanol for testing lipids the presence of the color changes from colorless to milky (emulsion test).

Self-assessment 6.3
You are provided with a solution X. Use Sudan III indicator to test the presence of lipids in the solution X.

6.4 Test for vitamin C (Ascorbic Acid).
Activity 6.4
Squeeze the orange fruits to extract the juice and carry out the following test.

Which of the two solutions give a positive solution for DCPIP?

Vitamin C is tested by using DCPIP (Dichlophenol Indophenol). Its positive (presence of vitamin C) test decolorizes DCPIP, while the negative (absence of vitamin C) test is indicated by the persistence blue color of DCPIP.

Self-assessment 6.4
In this experiment you are to press a tomato fruit (s) to get juice out of it. Use the juice to carry out the test for vitamin C Draw a table of results that includes the procedure, observation and conclusion.

End of unit assessment 6
1. Biological molecules are divided into:
a. Organic molecules and inorganic molecules
b. Carbohydrates and starch
c. Lipids, carbohydrates and water
d. Carbohydrates, food and potatoes
2. Name the reagents that are used to test for the following food substances
a. Lipids
b. Starch
c. Reducing sugar
3. You are provided with the following specimen:
Specimen A: Sorghum
Specimen B: Irish potatoes
Specimen C: Oranges
Specimen D: Sunflower seeds
a. Carry out chemical tests to determine the composition of the above seed to
tell whether they are composed of proteins, fats, starch or vitamin C.
b. Draw the table of used reagent, procedure and observation in (a)
4. Some drops of fresh pineapple juice are added drop by drop to DCPIP solution.
The deep blue color of the DCPIP quickly fades.
a. Explain why the blue colour disappeared?
b. What is the importance of this food substance to the human body?
5. The result of food tests on unknown sample are shown below. Copy and complete the table to show the conclusions which could be drawn from these tests.

6. This is a practical question to be conducted using provided materials and reagents to determine the food nutrients in each solution: You are provided with the following solutions, A (sucrose 0.5%), B (1%starch), C (dilute hydrochloric acid) and D (sodium hydroxide) and 6 test tubes labeled 1 to 6. Use the reagents provided to determine the chemical nature of the substance present in the solutions. Indicate your observations and conclusions in the table below:

a. Why was it necessary to boil solutions A and B with solution C in test (3) and (6)?
b. Why was solution D added to test tubes 3 and 6?