Introduction:

All living things are made up of one or more cells, and the cells in turn contain many kinds of molecules.  In this lab we will be looking at several different macromolecules (large molecules): proteins, carbohydrates, and lipids (specifically fats).  Various chemicals will be used in this laboratory to test for the presence of these molecules.  Most often, you will be looking for a particular color change.  If the change is observed, the test is said to be positive because it indicates that a particular molecule is present.  If the color change is not observed, the test is said to be negative because it indicates that a particular molecule is not present.

You will be using these tests to determine which of the macromolecules are present in various samples of food.

In all of the procedures, you will need to include a distilled water sample as a control.  Usually, a control goes through all the steps of the experiment but lacks one essential factor (the experimental variable).  This missing factor allows you to observe the difference between a positive result and a negative result.  If the control sample tests positive, you know your test is invalid.  Some tests may also contain other controls to make sure certain additives are not contaminated with the substance for which you are testing.
 

Proteins:

Protein molecules are long chains of amino acids joined by peptide bonds.

Biuret reagent, which is a blue color, contains a strong solution of sodium or potassium hydroxide (NaOH or KOH) and a very small amount of very dilute copper sulfate (CuSO₄) solution.  The reagent changes color in the presence of proteins or peptides because the amino group (H₂N-) of the protein or peptide chemically combines with the copper ions in biuret reagent.
 

Carbohydrates:

Carbohydrates include sugars and molecules that are chains of sugars.  Glucose is a simple sugar, also known as a monosaccharide.  Sucrose, or table sugar is a disaccharide, two sugar units bonded together.  Starch is a polysaccharide, a long chain of glucose units.

Benedict’s reagent reacts with many sugars (both mono- and disaccharides) after being heated in a boiling water bath.  Increasing concentrations of sugar give a continuum of colored products ranging from green through yellow and orange to orange red.

Iodine solution reacts with starch to produce colors ranging from a brownish to blue black.
 

Lipids:

Lipids are hydrophobic molecules which are insoluble in water and soluble in solvents such as alcohol and ether.  Lipids include fats, oils, and cholesterol.

Lipids do not evaporate from brown paper, instead leaving an oily spot.  Lipids also do not mix with water, forming a separate layer, usually on top of the water.  However, some molecules mix with both water and lipids, and can be used to mix the two.  These molecules are known as emulsifiers.  The liver produces bile salts which act as emulsifiers in the digestive tract.  Soaps and detergents also act as emulsifiers.
 

Summary of tests:

 

Biuret Reagent	Benedict’s Reagent	Iodine Solution	Brown Paper
Reacts with proteins or peptides, turns purple (protein) or pink (peptides)	Reacts with sugars, turns green through yellow to orange (green, less sugar, orange, more sugar)	Reacts with starch, turns dark brown to black	Lipids leave oily spot

Procedure:

Take some time to plan with your lab partner what tests you will do, and in what order before beginning the procedures.

There are available in the lab a variety of different types of common food.  Choose at least 3 foods and test each for the presence of protein, carbohydrate (both starch and simple sugars), and fats.  Be sure to plan your experiments before starting.

Form a hypothesis for each sample you have chosen to test.

Samples will need to be suspended in water for most tests.  Using a mortar and pestle if necessary, break each sample to be tested into small pieces and suspend the pieces in a small amount of distilled water.

Also availble are samples of each of the types of molecules for which you will be testing.  Use these samples to try out the tests so that you will know what a positive result looks like.

Be sure to include a blank control (distilled water) with each test so you know what a negative result looks like.  You may also include a positive control, a sample which you know contains the substance for which you are testing.

The procedures for testing for each type of molecule are given below.

Proteins and Peptides

Proteins:

1. Use a separate test tube for each sample to be tested, as well as one for a control.
2. Label each test tube.
3. Place about 1 mL of each sample (and control) in its test tube.
4. Add 5 drops of copper sulfate solution to each tube.
5. Add 10 drops of potassium hydroxide solution to each tube and mix.
6. Record the tube contents and final color in a data table.
7. Conclusions: which tubes contained protein?

Carbohydrates: Sugars and starch

Starch

1. Use a separate test tube for each sample to be tested, as well as one (or two) for a control.
2. Label each test tube.
3. Place about 1 mL of each sample (and control) in its test tube.
4. Add 5 drops of iodine solution to each tube and mix.
5. Record the tube contents and final color in a data table.
6. Conclusions: which tubes contained starch?

 Sugar

1. Use a separate test tube for each sample to be tested, as well as one (or two) for a control.
2. Label each test tube.
3. Place about 1 mL of each sample (and control) in its test tube.
4. Add about 2 mL of Benedict's reagent to each tube and mix.
5. Heat the tubes in a boiling water bath for 5-10 minutes.
6. Record the tube contents and final color in a data table.
7. Conclusions: which tubes contained sugar?

Lipids

1. Place a small sample of the material to be tested on a square of brown paper.
2. Place a small drop of water on the square of brown paper.
3. Compare the drop of water to the sample.
4. Wait at least 5 minutes.  Evaluate which substance impregnates the paper and which is subject to evaporation.  Record your results.
5. Conclusions: which sample contained lipids?

Conclusion Questions:

1. Why do experimental procedures include control samples?
2. How would you test an unknown solution for each of the following:
1. Sugars
2. Fat
3. Starch
4. Protein
3. Assume that you have tested an unknown sample with both biuret solution and Benedict’s solution and that both tests result in a blue color.  What have you learned?
4. What purpose is served when a test is done using water instead of a sample substance?
5. Compare your results.

Lab report:

Lab reports must include the following:

1. A Title to the lab.  A Purpose: What was studied in this lab, and why did we study it?
2. Procedure: a brief description of each type of test, what constitutes a positive test and what constitutes a negative test.
3. All data tables.
4. For each food sample, state your hypothesis and your conclusions.  Did your results confirm or refute your hypothesis?
5. Answers to questions.
6. A brief analysis of what worked in this lab and what didn't work, and why.