Biological Molecules: Carbohydrates, Lipids, Proteins

By the end of this lab, the student should be able to:

  • Identify the functional groups for each of the biomolecules that react in the following biochemical tests: Benedictís test, Iodine test, Brown Bag test, Sudan III/IV test, and the Biuret's Test.
  • Describe the mechanism of reaction for: Benedictís test, Iodine test, Sudan III/IV test, and the Biuret's Test.
  • Interpret the results when presented with data for each of the biochemical tests.
  • Design experiments to identify biomolecules using biochemical tests.

Introduction

Biological Molecules contain specific chemical structures called functional groups, which can be distinguished by biochemical tests, such as Benedict's Iodine, and Sudan III/IV. The Benedict's test identifies reducing sugars (monosaccharide's and some disaccharides), which have free ketone or aldehyde functional groups).  The groups reduce CuSO4, a component of Benedicts, resulting in a color change in the benedicts solution from a turquoise to a brick to rusty-brown color. Another class of carbohydrates called starches, a polysaccharide, can be detected using the Iodine's test. Having a high molecular weight, iodine attaches to the helical structure of starches  yielding a blue-black precipitate. The Biuret's test identifies proteins by oxidizing 4-6 peptide bonds of a protein using CuSO4 and NaOH resulting in a color change in the CuSO4 solution from purple to a darker purple color.  Finally, the Sudan III/IV test is used to detect the hydrocarbon chains of lipids.  Sudan is a red, non-polar, dye that forms hydrophobic interactions with the hydrocarbon chains of lipids.  Alternatively, the Brown Bag test can also be used to identify lipids due to the oily nature of hydrocarbon chains. 

Carbohydrates:  Reducing sugars and starches

Reducing Sugars

Some sugars such as glucose are called reducing sugars because they are capable of transferring hydrogens (electrons) to other compounds, a process called reduction. When reducing sugars are mixed with Benedicts reagent and heated, a reduction reaction causes the Benedicts reagent to change color. The color varies from green to dark red (brick) or rusty-brown, depending on the amount of and type of sugar.


Increasing amounts of reducing sugar


green orange red brown

  • In a typical Benedicts Test (shown below), approximately 1 ml of sample is placed into a clean test tube along with10 drops of Benedict's reagent (CuSO4).  The reactions are heated in a boiling water bath for 5 minutes.

Below: The test solutions and Benedict's reagent are boiled in a water bath for five minutes.

Benedicts_test_for_sugars.jpg (16546 bytes) Click on the image to view an enlargement. Press the "Back" button to return.

Below: Results of several solutions tested with the Benedict's test

Results_of_Benedicts_test.jpg (48929 bytes) Click on the image to view an enlargement. Press the "Back" button to return.

Activity #1: Draw a large chart containing four equally divided columns and seven rows.  In the first row, label the first column "Unheated Reaction"; the second column "Color of Heated Reaction"; the third column "positive (+) reaction and negative (-) reaction"; and the fourth column "Structure of Reacting Functional Group"

In the first column, label rows 1-7 with the appropriate samples below:  

Test tube #1: distilled water + Benedicts
Test tube #2: glucose solution + Benedicts
Test tube #3: sucrose solution + Benedicts
Test tube #4: starch solution + Benedicts
Test tube #5: onion solution + Benedicts
Test tube #6:  Potato solution + Benedicts

In the second column labeled "Color of Heated Reactions", indicate the results of the Benedict's test after the reaction was heated.  In the third column, indicate a (+) for positive benedict's reaction and a (-) for negative benedict's reaction.  In the fourth column, draw the reacting functional group(s) of the samples that reacted with the benedict's reagent.


Starches

Iodine solution (IKI) reacts with starch to produce a dark purple or black color. KI Reagent: Iodine is not very soluble in water, therefore the iodine reagent is made by dissolving iodine in water in the presence of potassium iodide. This makes a linear triiodide ion complex which is soluble. The triiodide ion slips into the coil of the starch causing an intense blue-black color. If you were to set up an Iodine test, you would perform the following:

  • Use a wax marker to mark two test tubes 1 cm from the bottom.

  • Fill one of the tubes to the 1 cm mark with water and fill the other to the 1 cm mark with a 1% starch solution. Be sure to stir the starch before filling your tube.

  • Add two drops of IKI solution to each tube and note any precipitation or color change.

Activity #2:Design a chart and record your predictions for the starch test for reaction tubes 3-5. Include in your chart the following headings: Reactants, Predicted Color Results, Structure of Reacting Functional Group(s).  The results of a positive and negative control have been included in reaction tubes 1 and 2, respectively, for your convenience.

Below left: starch solution and IKI - Iodine turns dark in the presence of starch (dark: Positive control)

Below right: distilled water and IKI  ( clear: negative control)

starch_solution_with_IKI.jpg (12900 bytes) Click on the image to view an enlargement. Press the "Back" button to return.

Test tube #1: starch + IKI
Test tube #2: distilled water + IKI
Test tube #3: glucose solution + IKI (hint: Is glucose a starch or reducing sugar?)
Test tube #4: rice solution + IKI (hint: Is rice a starchy food or reducing sugar?)
Test tube #5: onion solution (hint: see diagram 2b below, then refer to the results of the benedict's test using onion)
Test tube #6:  Potato solution (hint: see diagram 2a below, then refer to the results of the benedict's test using starch)

Draw a potato cell in the space provided. Label the cell wall and starch granules.

2a) Which macromolecule are the dark granules within the potato cells composed of?  [Hint Ė What caused the iodine to turn dark?]

potato_stained_with_IKI_100x.jpg (79175 bytes) potato_stained_with_IKI_200x.jpg (41814 bytes) Click on the images to view an enlargements. Press the "Back" button to return.
Potato cells stained
with IKI X 100
Potato cells stained
with IKI X 200
 

2b) Does onion store food as starch?

onion_stained_with_IKI_100X.jpg (69822 bytes) Left: Onion stained with IKI X 100 - The nuclei of these cells are light brown in this photograph. The numerous starch granules seen in potatoes are absent.

Click on the image to view an enlargement. Press the "Back" button to return.

Lipids

 

Brown Paper Test

Certain kinds of paper such as a piece of brown paper bag can readily absorb lipids and can be used to test for the presence of lipids. 

  • Below: A drop of vegetable oil was placed on a brown bag (paper) on the left and a drop of water was placed on the paper on the right. The paper was photographed after approximately one minute. Notice the migration of the oil.  Which spot do you think will disappear in time?

     

    paper_test_for_lipids.jpg (26161 bytes)

    Activity #3: Try this at home, draw 10 circles spaced 2 inches apart on a 8" x 5" piece of brown paper, paper bag, or brown envelope.  Gather the materials below.  Then do a paper bag test on each item by either placing a drop of the sample on the brown bag or use another small piece of bag to crush the solid samples into the circle to release its liquid content.  Allow paper to dry for 15 min, then turn over the paper to the opposite side to review your results.  Do you see a stain (oil) for the below samples.  Create a chart and record your observations.

    Record your results in a chart. Include the headings Sample, positive (+) reaction and negative (-) reaction for Brown Bag test, and Structure of Reacting Functional Group in your chart.

Circle #1: oil
Circle #2: water
Circle #3: a small piece of potato chip
Circle #4: small piece of cheese
Circle #5: a small dab of salad dressing
Circle #6:  vinegar
Circle #7:  catsup or mustard
Circle #8:  Your choice
Circle #9:  Your choice
Circle #10: Your choice

Proteins

Biuret Test

The copper atoms of Biuret solution (CuSO4 and NaOH) will react with several peptide bonds on polypeptides, producing a color change from blue to a deep violet or blue color.  Oftentimes a light pink color may result in the presence of small peptide chains.

Color Indication
Blue No protein or peptides
Violet        Protein

In the below experiment, a biuret test has been performed on water, egg albumin, a protein found in chicken eggs, and starch.  The following procedure was used:

  • Three test tubes were labeled at 2 cm as follows:  one of the tubes to the 2 cm mark with water, the second one to the 2 cm mark with albumin solution (a protein), and the third one to the 2 cm mark with starch solution.

  • 5 drops of 3% copper sulfate solution (CuSO4) was added to each tube.

  • 10 drops of 10% sodium hydroxide solution (NaOH) was then added to each tube. 

Activity #4:  Design a chart and record the final color of each test tube. Include the headings Biochemical Test, Color results, and Reacting Group in your chart.

Below:

Tube 1: Water (control)

Tube 2: Albumin (protein)

Tube 3: Starch

biuret.jpg (90640 bytes)
 

Activity #5:  Your grandmother is a 65 year-old diabetic who is maintaining healthy blood-sugar levels through watching her diet.  Lately, she has been craving imitation crab meat.  She snacks on it throughout the day and adds them to several dishes she prepares for herself.  You suspect as a diabetic, she should not be eating this product. Design an experiment to test your hypothesis. You have the following materials at your disposal although you may not use all of them:

  • Brown Paper Bag
  • Benedict's solution
  • Biuret's solution
  • KI
  • Sudan III/IV
  • Hot plate
  • Water bath
  • A small homogenizer
  • Diabetic glucose testing strips
  • Blood pressure cuff

Questions:

1.  What is the importance of heating samples when performing the Benedict's test?

2.  Reagents bind to the functional group of biological molecules by covalent or non-covalent bonding or interactions.  Explain this interaction between each reagent and its prospective functional group.

3.  What results would you expect if KOH or NaOH were omitted in the Benedict's test?  Why?

4.  What difference in results would you expect if you performed in a test tube the Biuret's test on a protein solution and a solution of glycine?

5.  Explain why (molecular reason) polysaccharides do not give a positive reaction for reducing sugars?

6.  Why is it important to include negative and positive controls when performing a test?

 

References and credits
http://faculty.clintoncc.suny.edu/faculty/michael.gregory/files/Bio%20101/Bio%20101%20Laboratory/Chemical%20Composition%20of%20Cells/Chemical%20Composition%20of%20Cells.htm