AP Biology Biochemistry Lab: Organic Molecules Mystery Lab
A cell is a living chemistry laboratory in which most functions take the form of interactions between organic (carbon-containing) molecules. Most organic molecules found in living systems can be classified as carbohydrates, fats, proteins, or nucleotides. Each of these classes’ molecules has specific properties that can be identified by simple chemical tests.
In this lab you will learn to identify three of the four major types or organic molecules: carbohydrates, fats and proteins.
Exercise 1: Carbohydrates
The basic structural unit of the carbohydrates is the monosaccharide (or single sugar). Monosaccharides are classified by the number of carbons they contain: for example, trioses have three carbons, pentoses have five carbons, and hexoses have six carbons They may contain as many as 10 carbons.
Monosaccharides are also characterized by the presence of a terminal aldehyde group or an internal ketone group. Both of these groups contain a double bonded oxygen atom that reacts with Benedict’s reagent to form a colored precipitate.
When two monosaccharides are joined together, they form a disaccharide. If the reactive aldehyde or ketone groups are involved in the bond between the monosaccharide units, the disaccharide will not react with Benedict’s reagent. If only one group is involved in the bond (as in maltose), the other is free to react with the reagent. Sugars with free aldehyde or ketone groups, whether monosaccharides or disaccharides, are called reducing sugars. In this exercise, you will use Benedict’s reagent to test for the presence of reducing sugars.
Monosaccharides may join together to form long chains (polysaccharides) that may be either straight or branched. Starch is an example of a polysaccharide formed entirely of glucose units. Starch does not show a reaction with Benedict’s reagent because the number of free aldehyde groups (found only at the end of each chain) is small in proportion to the rest of the molecule. Therefore, you will test for the presence of starch with Lugol’s reagent (iodine/potassium iodide).
Part A: Benedicts
When Benedict’s reagent is heated with a reactive sugar, such as glucose or maltose, the color of the reagent changes from blue to green to yellow to reddish-orange, depending on the amount of reactive sugar present. Orange and red indicate the highest proportion of these sugars. (Benedict’s test will show a positive reaction for starch only if starch has been broken down into maltose or glucose units by excessive heating.)
Formulate a hypothesis and predict what you might expect to find for each of the sugars to be tested with Benedict’s reagent.
1. Water 2. Table sugar
3. Glucose 4. Potato mixture
5. Onion mixture 6. Banana mixture
1. Set up a row of six test tubes. Use a piece of paper to mark test tubes 1-6.
2. To the test tubes, add 1 ml of the solutions listed above, matching each number to the number on the tube.
3. Add ½ dropper full (approx. 1 ml) of Benedict’s reagent to each tube.
4. Mix the reagent and the sample by agitating the solution in each tube from side to side. Record the original color of each tube’s contents.
5. Heat the test tubes in a boiling water bath for 3 minutes. Record any color changes in your data.
A5a. Why did you test water with Benedict’s reagent?
A5b. Which sugars reacted with Benedict’s reagent? Why?
A5c. Which sugars did not react with Benedict’s reagent? Why?
A5d. Explain your results for each
A5e. Did the results for each test support your hypothesis?
Part B: Lugol’s
Lugol’s reagent changes from a brownish or yellowish color to blue-black when starch is present, but there is no color change in the presence of monosaccharides or disaccharides.
Formulate a hypothesis and predict what you might expect to find for each of the items you tested above.
1. Prepare another six tubes as indicated in step 1 above.
2. To the test tubes, add 1 ml of the solution listed above, matching each number to the number listed on the tube.
3. Record the original color of each tube’s contents.
4. Add several drops of Lugol’s reagent to each tube, mix and immediately record results. Do not heat the test tubes in the Lugol’s test.
B4a. Which sugars reacted with Lugol’s reagent? Why?
B4b. Which sugars did not react with Lugol’s? Why not?
B4c. Explain your results for each.
B4d. Did the results for each test support your hypothesis?
B4e. From the results of the Benedict’s and Lugol’s tests, would you conclude that a potato and an onion stores its carbohydrates as sugars or starch? How do you know?
Exercise 2: Lipids
The word lipid refers to any of the members of a rather heterogeneous group of organic molecules that are soluble in nonpolar solvents such as chloroform, but insoluble in water. Although lipids include fats, steroids, and phospholipids, the exercise will focus primarily on fats.
Triglycerides, a popular topic in discussions of diet and nutrition, are the most common form of fat. They consist of three fatty acids attached to a glycerol molecule. Triglycerides are found predominantly in adipose tissue and store more energy per gram than other types of compounds. At room temperature, some lipids are solid (generally those found in animals) and are referred to as fats, while other are liquid (generally those found in plants) and are referred to as oils. Vegetable oil, a liquid fat, is a mixture of triglycerides.
Since both solid and liquid fats are nonpolar, you will test for their presence by using Sudan IV, a nonpolar dye that dissolves in nonpolar substances such as fats and oils but not in polar substances such as water.
1. The familiar grease spot is the basis of a very simple test for fats. On a piece of unglazed paper, such as brown wrapping paper or a paper towel, place one drop of oil and one drop of water. Allow the drops to dry.
Describe the difference between the oil spot and the water spot after a period of drying.
The Sudan IV test is a more useful laboratory test for fats. Formulate a hypothesis and predict what you might expect to find for each of the substances listed below:
1. butter 2. vegetable oil
3. crisco grease 4. water
5. milk
2. Label five tubes in sequence, 1 through 5. Add approx. 1 ml of each substance listed above to the appropriate tube. Add 3 drops of Sudan IV to each tube. Mix and then add 2 ml of water to each tube. If fats or oils are present, these will appear as floating red droplets or as a floating red layer colored by Sudan IV. Record the reaction that occurs in each of the test tubes.
2A1. Why do the droplets float, rather than mix with water?
2A2. Which substances reacted with Sudan IV? Why?
2A3. Which substances did not react with Sudan IV? Why not?
Exercise 3: Proteins
Proteins are made up of one or more polypeptides, which are linear polymers of smaller molecules called amino acids. Amino acids derive their name from the amino group and the carboxyl group (acidic) that each possesses. Polypeptides are formed when amino acids are jointed together by peptide bonds between the amino group of one amino acid and the carboxyl group of a second amino acid.
The biuret reagent reacts with peptide bonds and therefore reacts with proteins, such as egg albumin, but not with free amino acids, such as glycine and alanine.
The biuret reagent is light blue, but in the presence of proteins it turns violet. Other types of molecules may cause other color changes, but only the violet color indicates the presence of polypeptides.
Formulate a hypothesis and predict what you might expect to find for each of the substances to be tested with biuret reagent
1. distilled water 2. egg albumin
3. vanilla shake mix 4. Potato mixture
1. Obtain four clean test tubes and number them from 1 through 4.
2. To the test tubes, add 2 ml of the solutions listed above, matching each number to the number on the tube.
3. Add approx. 1 ml of biuret reagent to each tube.
4. After an incubation period of 2 minutes, record your results and determine whether the solution treated contains protein. Base your conclusions only on the presence or absence of the violet color.
Which substances reacted with biuret reagent? Why?
Which substances did not react with biuret reagent? Why not?
Exercise 4: Unknown Samples
A number of commercial products and food samples containing a variety of compounds are available for testing. Using reagents and methods from the previous exercises, identify the kinds of molecules contained in your unknown solutions. Pick 4 solutions to test.
Conduct all tests according to the directions in exercises 1-3. Record the unknown number and results.
Laboratory questions
1. Explain the limitations of Benedict’s test in determining whether or not sugar is present in a certain food product. Why do all monosaccharides, but only some disaccharides, react with Benedict’s reagent?
2. What did you learn about the specificity of the biuret reagent?
3. The leaves of many plants are coated with a waxy substance that cause them to shed water. How would you expect this substance to react in the Sudan IV test?
4. What are some good sources of carbohydrates, proteins, and fats in a typical breakfast, lunch and dinner?
5. What nutrients would you obtain from red meat? Do nutritionists recommend that some fats be present in your diet? What would you use these for?
6. Some vitamins should not be taken in excess. Which ones? Why?
7. Some amino acids are called essential amino acids. What does this mean? Fatty acids with more than one double bond are considered, as essential fatty acids. Animals cannot make fatty acids with more than one double bond. What are the sources of essential fatty acids?
8. In winter, plants exchange the saturate lipids in their membranes for unsaturated lipids. Unsaturated lipids are “bent” and keep the membranes more fluid because they cannot be stacked closely together. Of what advantage would this be for herbaceous plants that live through the winter? (Hint: what happens to bacon grease or the grease on top of soup when you put it in the refrigerator?)
9. Utilizing your unknowns, explain which unknown coordinates with each nutrition label and explain your reasoning.
BONUS: We took qualitative measurements to obtain the presence of each macromolecule. Investigate ways that quantitative measurements could determine the presence of: glucose, starch, lipids and proteins.