5.2: Exercise

Exercise A: Testing for Carbohydrates

The basic structural unit of carbohydrates is the monosaccharide (single sugar), which contains either a terminal aldehyde group or an internal ketone group. Both of these functional groups contain a double-bonded oxygen atom that reacts with Benedict’s reagent. When two monosaccharides are joined together, they form a disaccharide. Some disaccharides retain their reactive functional group; others involve these functional groups to form the new bond between the monosaccharides and no longer react with Benedict’s reagent. Monosaccharides and disaccharides with free aldehyde or ketone groups are called reducing sugars since they are able to reduce the alkaline solution of cupric ions (Cu²⁺) found in Benedict’s reagent to cuprous ions (Cu⁺), forming an orange-colored precipitate composed of cuprous oxide (Cu₂O). In this exercise, we will use Benedict’s reagent to test for the presence of reducing sugars.

Polysaccharides are formed when many monosaccharides join together to form long chains of carbohydrates. Starch is a polysaccharide composed entirely of glucose molecules. Starch does not react with Benedict’s reagent because the number of free functional groups is too low (found only at the ends of the polysaccharide). Thus, we test for the presence of starch with Lugol’s reagent (iodine/potassium iodide, I₂KI) instead.

Part 1: Benedict’s Test for Reducing Sugars

When we heat a mixture of Benedict’s reagent with reducing sugars, the color of the reagent will change from blue to green/yellow/reddish-orange, depending on the amount of reducing sugar present in the sample (red = most). Benedict’s reagent might show a false-positive reaction for starch if the starch has broken down due to excessive heating.

Data Table for Carbohydrates

Data Table for Carbohydrates	Benedict’s Test (reducing sugars)		Lugol’s Test (starch)
	Color before boiling	Color after boiling	Color before adding I2KI	Color after adding I2KI
1) Distilled Water
2) Starch
3) Glucose
4) Maltose
5) Sucrose
6) Onion juice
7) Potato slice
8) Milk

• Examine the carbohydrates data table. Create a hypothesis and a prediction for at least one of the samples you will be testing using Benedict’s test:

Hypothesis: ___________________________________________________________________

Prediction: ____________________________________________________________________

What is the independent variable? ________________________________________________

What is the dependent variable? __________________________________________________

Procedure

1. Set up a row of 8 test tubes using a test tube rack. Label them 1 through 8.
2. Add 2 mL of each solution (from the data table) to each tube, matching each number to the number on the tube.
3. Add 2 mL of Benedict’s reagent to each tube and mix gently. Record the initial color of each.
4. Heat the tubes in boiling water bath for 5 minutes. Record your color changes in the data table.

• Which samples tested positive for reducing sugars? ____________________________________
• Why did we test water with Benedict’s reagent? _______________________________________
• Did the results from your test support your hypothesis? Yes / No Explain: _________________ ______________________________________________________________________________

Part 2: Lugol’s Test for Starch

Lugol’s reagent changes from a brown-yellow color to a blue-black color when starch is present. There is no reaction (no color change) with monosaccharides or disaccharides.

• Examine the previous data table. Create a hypothesis and a prediction for at least one of the samples you will be testing using Lugol’s test:

Hypothesis: ___________________________________________________________________

Prediction: ____________________________________________________________________

What is the independent variable? ________________________________________________

What is the dependent variable? __________________________________________________

Procedure

1. Set up another row of 8 test tubes in your test tube rack. Label them 1 through 8.
2. Add 1 mL of each solution (from the data table) to each tube, matching each number to the number on the tube. Record the initial color of each sample in your data table.
3. Add 2-3 drops of Lugol’s reagent to each tube and mix well. Immediately record the final color change in the data table. (We do NOT heat these tubes.)

• Which samples tested positive for starch? ____________________________________
• From the results of your tests, do you think that a potato stores its carbohydrates as sugars or as starch? ________________ Explain: ____________________________________________________________________________________________________
• How does an onion store its carbohydrates? ________________ How can you tell? ________________________________________________________________________________
• Did the results from your test support your hypothesis? Yes / No Explain: _______________________________________________________________________________________________

Exercise B: Testing for Lipids

Lipids are not soluble in water, but are soluble in nonpolar solvents. Although fats, steroids, and phospholipids are all types of lipids, this portion of the lab focuses primarily on fats. Triglycerides are the most common form of fat, and are composed of one glycerol molecule attached to three fatty acids. Triglycerides are found predominantly in adipose tissue and store more energy per gram than carbohydrates and proteins. Depending on their fatty acid composition, lipids may be solid or liquid at room temperature. Since both solid and liquid fats are nonpolar, we will test for their presence using Sudan IV, a nonpolar dye that dissolves nonpolar substances such as fat.

Part 1: Grease Spot Test for Lipids

Another very simple test for fats is the familiar “grease spot” test. On a piece of unglazed paper (i.e. brown paper grocery bags), place one drop of vegetable oil and a separate drop of water. Allow the drops to dry. Describe the difference between the oil and water drop after drying: ___________ __________________________________________________________________________________

Data Table for Lipids

Data Table for Lipids

Substance	Sudan IV Solubility Reaction (note the color and if + or – for lipids)
1) Distilled Water
2) Vegetable oil
3) Onion juice
4) Cola (regular)
5) Hamburger juice

Part 2: Sudan IV Test for Lipids

The Sudan IV test is more useful laboratory test for fats. Note: Sudan IV is toxic. Use gloves and caution when handling this reagent.

• Examine the data table for lipids. Create a hypothesis and a prediction for at least one of the samples you will be testing using the Sudan IV test:

Hypothesis: ___________________________________________________________________

Prediction: ____________________________________________________________________

What is the independent variable? ________________________________________________

What is the dependent variable? __________________________________________________

Procedure

1. Set up a row of 5 test tubes using your test tube rack. Label them 1 through 5.
2. Add 1 mL of each solution (from the data table) to each tube, matching each number to the number on the tube.
3. Add 3 drops of Sudan IV to each tube. Mix and then add 2 mL of DI water to each tube.
4. 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 reactions in the lipid data table.

• Which samples tested positive for lipids? ____________________________________________
• Why do the red droplets float? ____________________________________________________
• Did the results from your test support your hypothesis? Yes / No Explain: _________________ ______________________________________________________________________________

Exercise C: Testing for Proteins and Amino Acids

Proteins are composed of one or more polypeptides, which are polymers of smaller subunits known as amino acids. Each amino acid contains an amino group and a carboxyl group. Polypeptides form when amino acids are joined together by peptide bonds between the amino group of one amino acid and the carboxyl group of another.

Biuret reagent reacts with the peptide bonds found within proteins, such as egg albumin, but not with free amino acids like glycine or alanine. A separate reagent, ninhydrin reagent, reacts with the amino group of free amino acids, but not with polypeptides.

Part 1: Biuret Reagent Test for Proteins

Biuret reagent is light blue, but in the presence of proteins it turns violet/purple. Other types of molecules may cause other color changes, but only the violet color indicates presence of polypeptides.

• Examine the data table for proteins. Create a hypothesis and a prediction for at least one of the samples you will be testing using biuret reagent:

Hypothesis: ___________________________________________________________________

Prediction: ____________________________________________________________________

What is the independent variable? ________________________________________________

What is the dependent variable? __________________________________________________

Procedure

1. Set up a row of 5 test tubes in your test tube rack. Label them 1 through 5.
2. Add 2 mL of each solution (from the data table) to each tube, matching each number to the number on the tube.
3. Add 2 mL of biuret reagent to each tube. Mix well and incubate at your desk for 2 minutes.
4. After the 2 minutes, record your results in the data table below and determine if protein is present or absent in each solution. Base your results only on the presence or absence of a violet color.

Data Table for Proteins

Data Table for Proteins

Substance	Color with Biuret Reagent after 2 Minutes	Protein Present (+) or Absent (-)
1) Distilled water
2) Egg albumin
3) Potato starch
4) Glucose
5) Amino acid

• Which samples tested positive for proteins? __________________________________________
• What does this test tell you about the biochemical composition of starch or glucose? ________ ______________________________________________________________________________
• Did the results from your test support your hypothesis? Yes / No Explain: _______________________________________________________________________________________________

Part 2: Ninhydrin Test for Amino Acids

Ninhydrin reagent turns purple or violet when free amino groups found in amino acids are present in the solution. When ninhydrin reagent reacts with the amino acid proline, however, it turns yellow. This is because proline’s amino group is not free. Rather, its amino group is part of the ring structure of the molecule. Note: Ninhydrin is poisonous. Use gloves and caution when handling this reagent.

• Examine the data table for amino acids. Create a hypothesis and a prediction for at least one of the samples you will be testing using the ninhydrin test:

Hypothesis: ___________________________________________________________________

Prediction: ____________________________________________________________________

What is the independent variable? ________________________________________________

What is the dependent variable? __________________________________________________

Procedure

1. Divide a piece of filter paper into 4 quadrants with a pencil. Label the quadrants A, B, C, and D.
2. Place 1 drop of each solution (from the data table) to each quadrant with the corresponding letter. Allow the spots to dry.
3. Apply 1 drop of ninhydrin reagent to each spot. Caution: avoid skin contact – wear gloves.
4. Allow the paper to dry at room temperature for 20-30 minutes. (You can speed up the reaction by passing the filter paper lightly over a warm hotplate.) Record the final color in your data table for amino acids.
5. One of the solutions distilled water, two contain amino acids other than proline (methionine or alanine), and one contains proline. Complete the table, indicating the content of each solution.

Data Table for Amino Acids

Data Table for Amino Acids

Solution	Final Color with Ninhydrin	Type of Molecule in Solution
A
B
C
D

Exercise D: Testing Unknowns (assigned unknowns: #_________ and #__________)

Use knowledge gained in the previous exercises to identify the organic molecules present in unknown solutions. Your instructor will assign unknowns to each group. Conduct all tests according to the directions found in Exercises A-C. Record your results in the table below. Meet with other groups to exchange data results until your table is complete.

Hints/Tips: Avoid cross-contamination with pipettes between solutions. Wash glassware thoroughly between experiments if needed.

Data Table for Unknowns

Data Table for Unknowns

Unknown Tested	Positive (+) or Negative (-) Results
	Benedict’s Test	Lugol’s Test	Biuret Test	Ninhydrin Test	Sudan IV Test
1:
2:
3:
4:
5:
6:
7:
8:

• Your instructor will provide you with a list of the possible unknown contents. Once you have completed the data table, match the names of the unknowns to the numbers in your table by writing the name of the unknown next to each respective number.