Independent variable in benedict's test

Macromolecules: Carbs, Proteins, and Lipids – Good Eats Badge
(Adapted from Biology Laboratory Manual 10th Edition, by Darrell Vodopich and Randy Moore)

Most organic compounds in living organisms are carbohydrates, proteins, lipids, or nucleic acids. Each of these macromolecules is a polymer made of smaller subunits, or monomers. These monomers are linked together in a reaction known as dehydration synthesis, which is an energy-requiring process in which a molecule of water is removed and the two subunits are bonded covalently. We can break the bonds between the subunits to release energy and separate the monomers gain by adding a water molecule in a process known as hydrolysis.

Scientists have devised several biochemical tests to identify the major types of organic compounds in living organisms. Each of these tests involves two or more treatments: (1) an unknown solution to be identified, and (2) controls to provide standards for comparison. As its name implies, an unknown solution may or may not contain the substance that the investigator is trying to detect. Only a carefully conducted experiment will reveal its contents. In contrast, controls are known solutions. We use controls to validate that our procedure is detecting what we expect it to detect and nothing more. During the experiment, we compare the unknown solutions’ response to the experimental procedure with the control’s response to that same procedure.

In our scientific method lab, we introduced the concept of a control group: a group that is subjected to the same conditions as the experimental group EXCEPT for the independent variable. This was to show that any differences observed were due to the independent variable alone and not to other factors within the procedure. But what if there were no differences detected between the groups? Could you be sure that it was due to the independent variable and not that there was a problem with the experimental procedure? A way to be sure is to introduce a new kind of control group: the positive control. A positive control contains the variable for which you are testing and should react in a predictable way in the procedure. For example, if you are testing a solution to see if it contains protein, an appropriate positive control is a solution that is known to contain protein and there should result in a positive reaction in your test. A positive reaction indicates that your test is working as expected and shows you what a positive result should look like. You can then compare the unknown and other samples with this known positive to check for the presence of the molecule.

Even with a positive control we still need to show that any positive reaction is due only to the variable that we are testing. Therefore, we still need the negative control. The negative control does not contain the variable for which you are searching. It contains only the solvent (often distilled water with no solute) and should not react in the test. A negative control shows you what a negative result looks like.

In this lab, we are going to use chemical tests to detect macromolecules in known and unknown solutions. We will employ both negative and positive controls to be sure that our tests are working properly and that we can trust our results. You will be working in groups of 4 students (your table) to be “food detectives” and solve the mystery of your unknown solution.

Experimental Procedures
Log-in to the Hayden-McNeil lab simulation website (http://courses.haydenmcneil.com) and click on the “Biological Molecules” simulation. Read through the background material provided and then click on the gray arrow at the bottom of the page. Open up the simulation by clicking on the green button. The simulation directions are available on the website and below. Use the simulation for Procedures 1-5.

Carbohydrates:

Carbohydrates are made up of monosaccharides. Many monosaccharides such as glucose and fructose are reducing sugars, meaning that they possess chemical groups that can reduce weak oxidizing agents like the copper in Benedict’s reagent. Benedict’s test identifies reducing sugars based on their ability to reduce the cupric ions (blue) to cuprous oxide (green to reddish orange) at high pH. A green solution indicates a small amount of reducing sugars, and reddish orange indicates an abundance of reducing sugars. Non-reducing sugars like disaccharides (sucrose) are not able produce a color change.

Starch is a form of carbohydrates produced by plants. It is a highly coiled polymer of individual glucose units. Iodine interacts with only coiled polymer molecules of glucose (like starch) and becomes bluish black. Iodine does not interact with uncoiled sugars such as disaccharides (sucrose) and monosaccharides (glucose) and therefore remains yellowish brown in the presence of these sugars.

In summary – what color changes are we looking for with each test?

Color Changes
Positive Result
Negative Result
Benedict’s Test
Iodine/Starch Test

Procedure 1: Test for Reducing Sugars
1. Take a constant temperature bath from the Instruments shelf and place it onto the workbench.

2. Set the constant temperature bath to 100 °C.

3. Take a test tube from the Containers shelf and place it onto the workbench.

4. Take the 5% glucose solution from the Materials shelf and add 6 mL to the test tube. Record the color of the solution in the table below.

5. Take Benedict's solution from the Materials shelf and add 6 mL to the test tube. Record any color change in the table below.

6. Move the test tube into the constant temperature bath. Wait a few moments to see if the color changes. The color change usually begins at temperatures greater than 60 °C, but will finish by the time the solution reaches 100 °C. Take a thermometer from the Materials shelf and attach it to the test tube to monitor its temperature. Record any color changes.

7. Empty the test tube in the waste bin, then place the empty test tube in the sink.

8. Repeat steps 3 – 8 two more times, replacing the 5% glucose solution in step 4 with:

· 5% sucrose

· water

9. Clear your station by dragging all instruments back to the Instruments shelf and by emptying all containers in the waste bin and then placing the empty containers in the sink.

Solution

Initial Color

Color with Benedict’s Solution

Color after Heating

5% Glucose

5% Sucrose

Water

Which solution(s) did not contain reducing sugars?

What was the purpose of the water?

Procedure 2: Test for Starch
1. Take two test tubes and a 250 mL beaker from the Containers shelf and place them onto the workbench.

2. Add 90 mL of water and 10 mL of Lugol’s iodine from the Materials shelf to the 250 mL beaker. Rename the beaker “diluted Lugol’s iodine” by double-clicking on it.

3. Take the 2% starch solution from the Materials shelf and add 6 mL to one test tube.

4. Take the water from the Materials shelf and add 6 mL to the other test tube.

5. Record the color of the solutions in the test tubes in the table below.

6. Add 6 mL of the diluted Lugol’s iodine solution in the 250 mL beaker to each test tube.

7. Observe what happens in each test tube. Record your observations.

8. Empty the two test tubes in the waste bin, then place the empty test tubes in the sink. Keep the beaker of diluted Lugol’s iodine on the workbench to use in Experiment 5.

Starch Test Results

Solution

Initial Color

Color with Iodine Solution

Starch

Water

Which solution(s) reacted with the iodine solution?

Which solution is considered to be the negative control?

Based on your knowledge of what potatoes are made of, develop a hypothesis for testing whether they contain starch or not. What is your prediction for the outcome of this experiment?

Procedure 3: Test for Proteins
Proteins are polymers made up of amino acids. A peptide bond forms between the amino acid and the carboxyl group of an adjacent amino acid and is identified by a Biuret test. Specifically, peptide bonds in proteins complex with Cu2+ in Biuret reagent and produce a violet color. A Cu2+ must complex with four to six peptide bonds to produce a color; therefore individual amino acids to not react positively. Proteins have many peptide bonds and produce a positive reaction.

Biuret Test for Proteins
1. Take two test tubes from the Containers shelf and place them onto the workbench.

2. Take the 35% egg albumin from the Materials shelf and add 6 mL to the first test tube.

3. Take the water from the Materials shelf and add 6 mL to the second test tube.

4. Record the colors of both solutions in the table below.

5. Take the biuret solution from the Materials shelf and add 6 mL to each test tube.

6. Observe any color changes in the two test tubes. Record your observations.

7. Empty the test tubes in the waste bin, then place the empty test tubes in the sink.

Biuret Test Results

Solution

Initial Color

Color with Biuret Solution

35% Egg Albumin

Water

Which solution(s) contained protein?

Which solution is the negative control?

Procedure 4: Test for Lipids
Lipids include a variety of molecules that dissolve in non-polar solvents such as ether, acetone, methanol, or ethanol, but not as well in polar solvents such as water. Triglycerides are abundant lipids made of glycerol and three fatty acids. Tests for lipids are based on a lipid’s ability to selectively absorb pigments in fat-soluble dyes such as Sudan III.

Sudan III Test for Lipids

1. Take two test tubes from the Containers shelf and place them onto the workbench.

2. Take the corn oil from the Materials shelf and add 6 mL to one test tube.

3. Take the water from the Materials shelf and add 6 mL to the other test tube.

4. Record the initial colors of the liquids in the test tubes in the table below.

5. Take the Sudan III solution from the Materials shelf and add 6 mL to each test tube.

6. Observe any potential color changes in the two test tubes. Record your observations.

7. Empty the test tubes in the waste bin, then place the empty test tubes in the sink.

Biuret Test Results

Solution

Initial Color

Color with Sudan III Solution

Corn Oil

Water

Which solution(s) contained lipids?

Which solution was the negative control?

Procedure 5: Test the Contents of Foods
For each food sample, do the following:

1. Take four test tubes from the Containers shelf and place them onto the workbench. Based on steps 2 and 3, label each test tube with the material and test being performed. For example, label the test tube used to test onion juice for the presence of reducing sugars as “Onion Juice Benedict’s Solution”.

2. Add the reagents to the test tubes as directed in the table below. Be sure to use the diluted Lugol’s iodine you made in Experiment 2.

Test Tube

Reagent

1

6 mL Benedict’s solution

2

6 mL diluted Lugol’s iodine solution

3

6 mL Sudan III solution

4

6 mL biuret solution

3. Take the potato juice from the Materials shelf and add 6 mL to each of the four test tubes. Observe and record all color changes in the table below. Remember that the test tube with the Benedict's solution requires heating in a 100 °C constant temperature bath for the color change in the presence of reducing sugars to occur. Recall that you can use a thermometer to monitor the heating.

4. Empty the test tubes in the waste bin, then place the empty test tubes in the sink.

5. Repeat steps 1 – 4 for the onion juice, whole milk, and skim milk. Be sure to record all of your observations.

6. After you record the color changes in the 4 foods. Record whether each macromolecule was present or absent from each of the four foods by recording a plus (+) sign when the macromolecule is present. Record a negative ( - ) sign if the macromolecule is absent from the food group.

7. Clear the bench of all materials, containers, and instruments, then return to your course page to complete any assignments for this lab.

Results of Food Test

Food

Reducing Sugars

Starch

Lipids

Proteins

Color

+/-

Color

+/-

Color

+/-

Color

+/-

Potato Juice

Onion Juice

Whole Milk

Skim Milk

Why did you run each test separately before running tests on different types of foods?

What results would you expect from a biuret test of steak solution?

What results would you expect from a Sudan III test of olive oil?

Iodine solution is added to a cake solution. The color changes to bluish black. Based on this result, which biological molecules are present in the cake solution?

What is the relationship between monosaccharides and disaccharides?

Benedict’s solution is added to maple syrup and heated. The color changes from blue to orange. Based on this result, which biological molecules are present in the maple syrup?