Laboratory Notes for BIO 1003

© 30 August 1999, Mary Jean Holland

Revised 12 August 2019, Krista Dobi

ORGANIC MOLECULES—LABORATORY TESTS

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Organic compounds contain carbon atoms linked together to form chains or rings. Four classes of organic compounds—carbohydrates, lipids, proteins and nucleic acids—are found in large amounts in living organisms. The chemical properties of the different classes depend on the presence of specific functional groups. Today we will carry out tests for carbohydrates, lipids, and proteins. Each group will be given a different “mystery sample” containing one of these organic molecules; based on the results of the tests you do today, you should be able to identify what type of molecule you have been given.

General Instructions to Students

  1. Supply solutions and dry chemicals must be kept pure. Never pour extra fluid back into a stock bottle. This direction applies to all labs with chemical experiments.
  2. In this laboratory period you will learn about different classes of organic compounds by doing chemical tests for lipids, carbohydrates, and proteins. You will be learning about organic molecules in lecture and will also have the opportunity to discuss the results of today's tests in the lecture/recitation section.
  3. For this laboratory period, work in groups of three to four students. Each group will do all the tests and record results in the following tables:
    Table I. Results of Solubility Test for Lipids
    Table II. Results of Grease Spot Test for Lipids in Foods
    Table III. Results of the Biuret Test for Protein
    Table IV. Results of Benedict's Test for Reducing Sugars
    Table V. Results of Lugol's Test for Starch
  4. Because it takes time for water to boil on the hotplates, turn on your hotplates as soon as your instructor tells you to begin. REMEMBER: Bring goggles to your seat before you start heating the water. Once water is boiling, everyone in its vicinity must wear goggles.

I. Lipids

Lipids are organic molecules that are insoluble in water and other polar solvents. Lipids are readily soluble in nonpolar solvents, such as chloroform, benzene, and ether.

Note: Because it takes time for the spots in the Grease Spot Test to dry, a pair of students in each group should set this test up while the other pair of students sets up the solubility test. Everyone should observe the results and record them.

Tests for Lipids

Solubility in Polar and Nonpolar Solvents: Lipids are insoluble in polar solvents and soluble in nonpolar solvents. For this test, the polar solvent is water; the nonpolar solvent is corn oil:

  1. Set up and number the tubes.
  2. Add 1 mL (20 drops) each of the pair of liquids indicated below.
  3. Mix the contents of each tube using the vortex genie.
  4. Wait 2 minutes.
  5. Examine each tube carefully. Has the sample dissolved in the solvent or do you see two separate layers in the tube?
  6. Record your observations in Table I below.

Table I. Results of Solubility Test for Lipids

Tube number Tube
contents
How many layers do you see? (One or two?) Is the second substance soluble in water, a polar solvent? Is the second substance soluble in oil, a non-polar solvent ?
1 Water mixed with water      
2 Water mixed with corn oil      
3 Corn oil mixed with olive oil      
4 Mystery sample mixed with water      
5 Mystery sample mixed with corn oil      

"Grease Spot" Test: You perform this test every time you buy muffins or doughnuts in a paper bag. Lipids make unglazed paper (brown paper, writing paper) translucent:

  1. Put a drop of each sample on a piece of unglazed paper.
  2. Draw a circle around the spot with a soft pencil.
  3. Write the name of the sample in pencil next to the spot.
  4. Allow all spots to dry thoroughly.
  5. Hold the paper in front of a light source and observe the spots.
  6. Record your observations in Table II below.

Table II. Results of Grease Spot Test for Lipids in Foods

Spot number Samples Is the spot translucent? (yes or no?)
1 Water  
2 Corn oil  
3 Olive oil  
4 Mystery sample  

II. Proteins

Proteins are complex, specialized molecules composed of carbon, hydrogen, oxygen, and nitrogen. Many proteins also contain sulfur. The building blocks of proteins are the amino acids.

Biuret Test for Proteins: Biuret reagent is a light blue solution which turns purple when mixed with a solution containing protein. When the copper ions of the Biuret Reagent react with peptide bonds in the polypeptide chains, a purple color complex is formed.

Note: All groups will set up tubes 1-4 in Table V below. Groups 1, 3, and 5 should also set up tubes 5-8 for a total of eight tubes. Groups 2, 4, and 6 should also set up tubes 9-13 for a total of 9 tubes. Everyone is responsible for recording and knowing all results, so you should share information.

  1. Label a set of tubes (see note above) with a wax pencil.
  2. Add 2 mL (40 drops) of sample to each tube.
  3. Add 2 mL of biuret reagent to each tube. What color is the biuret reagent?
  4. Mix the contents of each tube using the vortex genie.
  5. Wait 2 minutes.
  6. Examine each tube carefully. Note the color.
  7. Record your observations in the Table V.

Table III. Results of the Biuret Test for Proteins

Tube
number
The color of Biuret Reagent is:
_______________
Initial color at mixing Final color after 2 minutes at room temperature Is this a positive test? Are peptide bonds present (Yes or no?)
1 Negative control: water      
2 Positive control: serum albumin      
  Amino acids:      
3      Glycine      
4      Lycine      
5 Mystery sample      

Exercise:

Note: Each member of the group should complete this exercise separately.

  1. Draw a table (in your notebook or on the computer) summarizing the results your group got for their mystery sample analysis. Don't forget to label your table with a title and description. (Since you are the scientist and this is your data, the format and content of this table is up to you. What would be the clearest way of presenting your data?)
  2. What type of organic molecule was your mystery sample? How did your results lead you to this conclusion?
  3. What results would you expect from the Biuret test if you had one sample containing eggs, and another sample containing honey?
  4. On what tests would you expect positive results if you were testing a potato chip?

III. Carbohydrates

Carbohydrates are the main energy-storage molecules in most organisms. Polysaccharides are formed by linking many monosaccharides together by a series of dehydration synthesis reactions.

Tests for Carbohydrates

Benedict’s Test for reducing sugars: Benedict’s reagent (a blue colored solution containing copper ions) is used to test for the presence of reducing sugars. When a solution containing Benedict’s reagent and a reducing sugar is heated, the copper (II) ions in the Benedict’s reagent are reduced to copper (I) ions and the solution changes from blue to green to orange to red-orange to brick-red. A brick-red precipitate (solid), copper (I) oxide (Cu2O), may appear in the bottom of the tube. The more reducing sugar present in the mixture, the more precipitate will form.

The half-reaction for Benedict’s Test for Reducing Sugars can be shown as:

2 Cu+2 + 2 e- arrow 2 Cu+1

Each copper (II) ion, Cu+2, is reduced to a copper (I) ion, Cu +1, by an electron from the reducing sugar. The reducing sugar is oxidized as a result of giving up its electron.

How to Proceed to Test for Reducing Sugars

  1. Set up tubes. Label each tube with a wax pencil.
  2. Add 2 mL (40 drops or one full dropper) of the sample to be tested.
  3. Add 2 mL of Benedict’s reagent. What color is the reagent?
  4. Mix the contents of each tube using the vortex genie.
  5. Record the color of the contents of each tube in the table as soon as you mix them.
  6. Place all the tubes in a beaker of boiling water for 1 or 2 minutes.
  7. Remove the tubes and allow to cool for 1 or 2 minutes.
  8. Observe each tube carefully. Record in Table III the color of any precipitate formed (it may take a few minutes for the precipitate to be visible at the bottom of the tube). Place a star next to the tube with the most precipitate.

Table IV. Results of Benedict's Test for Reducing Sugars

Tube Number The color of Benedict's Reagent is: __________ Initial color at mixing Final color after heating Is this a positive result? Is a reducing sugar present?
1 Negative control: Water      
2 Positive control: Glucose      
  Mono- or disaccharides:      
3      Fructose      
4      Lactose      
5      Sucrose      
  Polysaccharides:      
6      Starch      
7 Mystery sample      

Lugol’s Test (Iodine solution) for Starch: Lugol’s reagent contains iodine and potassium iodide (I2KI). It changes from a yellowish-brown to blue-black in the presence of starch.

How to Proceed to Test for Starch

  1. Set up a porcelain spot plate; number each depression in the plate with a wax pencil.
  2. Add 3 drops of each sample to be tested to a different well in the spot plate, and make a list of what sample corresponds to what number.
  3. Stir with a toothpick.
  4. Add 1 drop of Lugol’s reagent to each sample.
  5. Record the final color of the contents of each well in Table IV.

Table V. Results of Lugol's Iodine Test for Starch

Tube Number The color of Lugol's Reagent is: __________ Final color after mixing Is this a positive result? Is starch present?
1 Negative control: water    
2 Positive control: starch (a polysaccharide)    
  Mono- or disaccharides:    
3      Fructose    
4      Lactose    
5      Sucrose    
  Polysaccharides    
6      Starch    
7 Mystery sample    

Exercise:

Note: Each member of the group should complete this exercise separately.

  1. Draw a table (in your notebook or on the computer) summarizing the results your group got for their mystery sample analysis. Don't forget to label your table with a title and description. (Since you are the scientist and this is your data, the format and content of this table is up to you. What would be the clearest way of presenting your data?)
  2. What type of organic molecule was your mystery sample? How did your results lead you to this conclusion?
  3. What results would you expect from the Biuret test if you had one sample containing eggs, and another sample containing honey?
  4. On what tests would you expect positive results if you were testing a potato chip?


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Revised 8/12/19 (jhw)