Laboratory Notes for BIO 1003

© 30 August 1999, Mary Jean Holland


ORGANIC MOLECULES

Organic compounds contain carbon atoms linked together to form chains or rings. Four classes of organic compoundsócarbohydrates, lipids, proteins and nucleic acidsóare found in large amounts in living organisms. The chemical properties of the different classes depend on the presence of specific functional groups. In general, the larger molecules in each class are formed by joining one or more building block molecules together in a dehydration synthesis reaction during which a molecule of water is formed for each building block added. Large molecules are broken down into the smaller building block molecules by a reverse reaction called hydrolysis during which water is added. In this exercise you will learn about the structure and properties of carbohydrates, lipids and proteins and how to test for the presence of these organic molecules.

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.

I. Carbohydrates

Carbohydrates are the main energy-storage molecules in most organisms. They are also important structural components for many organisms. The building blocks of carbohydrates are small molecules called sugars, composed of carbon, hydrogen and oxygen. Carbohydrates are classified according to the number of sugar molecules they contain. Monosaccharides, such as glucose, fructose, ribose, and galactose, contain only one sugar molecule. Disaccharides, such as sucrose, maltose and lactose, contain two sugar molecules linked together. Polysaccharides, such as starch, glycogen, cellulose and chitin, contain many sugar molecules linked together.

Monosaccharides have the molecular formula (CH2O)n, where n may be any integer from 3 to 8. Monosaccharides contain hydroxyl groups and either a ketone or an aldehyde group. These polar functional groups make sugars very soluble in water. Glucose, the sugar found in the blood of most vertebrates including humans, has the molecular formula C6H12O6. Fructose, the sugar found in many fruits, has the same molecular formula as glucose, but the atoms of carbon, hydrogen and oxygen are arranged a little differently in the two monosaccharides. Glucose has an aldehyde group; fructose has a ketone group. This difference in structure gives the two monosaccharides slightly different chemical properties.

Disaccharides are formed by linking two monosaccharides together by a dehydration synthesis reaction. A molecule of water is formed in the process. Maltose (malt sugar) is formed by joining two glucose molecules together. Sucrose (cane sugar) is formed by combining glucose and fructose. Lactose (milk sugar) is formed by combining glucose and galactose.

Maltose, sucrose and lactose have the same molecular formula, C12H22O11, but slightly different structural formulae and slightly different chemical properties.

C6H12O6 + C6H12O6arrowC12H22O11 + H2O
glucose + glucosearrowmaltose + water
glucose + fructosearrowsucrose + water
glucose + galactosearrowlactose + water

Polysaccharides are formed by linking many monosaccharides together by a series of dehydration synthesis reactions. For each monosaccharide added to the polysaccharide chain, a molecule of water is formed. Polysaccharides are used as energy storage compounds by both plants and animals. Plants produce a polysaccharide called starch. Vertebrate animals, including humans, produce a polysaccharide called glycogen, which is stored in liver and muscle cells. Glycogen is sometimes called animal starch. Polysaccharides are also important as structural components in many organisms. Plant cell walls contain a polysaccharide called cellulose. Fungi cell walls and the exoskeletons of arthropods contain a polysaccharide called chitin.

Tests for Carbohydrates

Benedictís Test for reducing sugars: Benedictís reagent (a blue colored solution containing copper ions) is used to test for the presence of reducing sugars. When a solution containing Benedictís reagent and a reducing sugar is heated, the copper (II) ions in the Benedictís 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 (II) oxide (Cu2O), may appear in the bottom of the tube. The more reducing sugar present in the mixture, the more precipitate will form in the bottom of the tube.

The half-reaction for Benedictís 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.

A positive Benedictís test requires an aldehyde or ketone group that is located near a hydroxyl group. All monosaccharides are reducing sugars. Some disaccharides are reducing sugars, and some are not. If the reactive aldehyde or ketone groups of both monosaccharides are involved in the bond holding the two units together, these groups are not free to react with the copper ions in the Benedictís solution, and the disaccharide formed is not a reducing sugar. Polysaccharides do not test positive for reducing sugars unless they undergo a hydrolysis reaction (by heating or digestion) during which the polysaccharides are broken down to form monosaccharides.

How to Proceed to Test for Reducing Sugars

  1. Set up a row of 7 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 Benedictís 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.
  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 the Table the color and amount of any precipitate formed. For the amount of precipitate use symbols: 0, +, ++, +++. If there is no precipitate present, record the color of the solution.

Lugolís Test for Starch: Lugolís reagent (I2KI) changes from a yellowish-brown to blue-black in the presence of starch. Monosaccharides and disaccharides do not react with Lugolís reagent.

How to Proceed to Test for Starch

  1. Set up a row of 7 tubes. Label each tube with a wax pencil.
  2. Add 5 drops of the sample to be tested.
  3. Record the color of the contents of each tube in the Table.
  4. Add 1 drop of Lugolís reagent to the sample.
  5. Mix the contents of each tube using the vortex genie.
  6. Record the color of the contents of each tube in the Table.

Results of Tests for Carbohydrates

Tube
contents
Benedictís Test Lugolís Test
Original color before boiling Final color
after boiling
Original color before I2KI Final color
after I2KI
Water        
Glucose        
Fructose        
Sucrose        
Maltose        
Lactose        
Starch        


II. 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. Lipids include fats and oils (important as energy storage compounds), phospholipids and glycolipids (part of the structure of cell membranes), waxes (protective surface coatings on many plants and animals), and steroids (found in some cell membranes and many hormones).

Fats and oils have similar structures, and both serve as energy storage molecules. At room temperature oils are liquid and fats are solid. Both are triglycerides formed by combining a molecule of glycerol with three molecules of fatty acid. The properties of a triglyceride depend upon the structures of the fatty acids it contains. Fatty acids are long chains containing carbon and hydrogen with a carboxyl group (COOH) on one end, which makes the molecule an acid,. The carboxyl group is involved in bonding each fatty acid to the glycerol molecule. Fatty acids differ in the length of the chain and the number of double bonds between adjacent carbon atoms. If all the carbon atoms in a fatty acid chain are bonded to four different atoms (no double bonds between carbon atoms), the fatty acid has a straight chain without bends or kinks. Double bonds between carbon atoms cause fatty acids chains to bend or kink. The fatty acids of saturated fats (such as lard, bacon fat, or butter) contain no double bonds, a maximum of hydrogen atoms, and the straight fatty acid chains pack closely together. The fatty acids of unsaturated fats contain at least one double bond, fewer hydrogen atoms, and the fatty acid chains cannot pack as closely together because at least one of the chains has a kink or bend. Monounsaturated fats, such as olive oil, have one double bond. Polyunsaturated fats, such as corn oil, have two or more double bonds. The less "orderly" structure of unsaturated fats is responsible for their lower melting point.

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 mineral oil (a mixture of hydrocarbons):

  1. Set up two rows of tubes. Label each tube with a wax pencil.
  2. Add 1 mL (20 drops) of water to the first row of tubes.
  3. Add 1 mL of mineral oil to the second row of tubes.
  4. Add 1 mL of the sample to be tested to one tube in each row.
  5. Mix the contents of each tube using the vortex genie
  6. Wait 2 minutes.
  7. Examine each tube carefully. Has the sample dissolved in the solvent or do you see two separate layers in the tube?
  8. Record your observations in the Table. Soluble (+) or Insoluble (-).
  9. Save your tubes from the first row (water is the solvent) for the Sudan red test described below.

Sudan Red Test: Sudan red is a lipid soluble dye. When Sudan red is added to a mixture of lipids and water, the dye will move into the lipid layer coloring it red:

  1. Add one drop of Sudan red dye (dissolved in alcohol) to each tube from the water solubility exercise described above, and
  2. Mix the contents of each tube using the vortex genie
  3. Wait 2 minutes.
  4. Examine each tube carefully. Where is the red color found?
  5. Record your observations in the Table.

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

  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 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 the Table.

Results of Tests for Lipids

Tube
contents
Solubility Test Sudan red
dye test
Grease spot
test
Is it soluble in a polar solvent (water)? How many layers? Is it soluble in a non-polar solvent (oil)? How many layers?
Water        
Mineral oil        
Corn oil        
Olive oil        
Starch        


III. 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. There are twenty different amino acids commonly found in proteins. All of these amino acids have a similar structure. At the center of the molecule is the alpha carbon which is bonded to four different groups: (1) an amino group (NH2), (2) a carboxyl group (COOH), (3) a hydrogen atom and (4) the R group (also called the side chain). The different amino acids have different R groups; otherwise the twenty amino acids have identical structures.

Proteins are composed of one or more polypeptides. Polypeptides are formed by linking amino acids together in a long, unbranched chain. The amino acids are linked together by peptide bonds formed when the carboxyl group of one amino acid reacts with the amino group of the next amino acid in a dehydration synthesis reaction. As each amino acid is added to the growing polypeptide chain, a molecule of water is formed. Polypeptides have a free (unreacted) amino group located at one end of the molecule called the N-terminus and a free (unreacted) carboxyl group at the other end of the molecule called the C-terminus. Biochemists describe the structure of a specific polypeptide by writing down the sequence of amino acids starting at the N-terminus and proceeding along the chain to C terminus.

Proteins have many important roles in living organisms. Structural proteins, such as elastin and collagen, provide support. Regulatory proteins control cell processes. Storage proteins produced in reproductive structures are a source of amino acids for developing organisms, e.g., casein in milk, albumin in egg whites, various proteins in plant seeds. Contractile proteins are responsible for movement of cells and organisms. Transport proteins carry substances from one place to another, e.g., hemoglobin carries oxygen throughout the human body. Proteins also serve as antibodies, hormones, receptors, and enzymes.

Tests for Proteins and Amino Acids

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

  1. Label a set of tubes 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.
  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. Indicate presence (+) or absence (-) of peptide bonds in each sample.

Ninhydrin Test for Amino Acids: Because amino acids contain a free amino group, they are readily detected with ninhydrin reagent which reacts with free amino groups to form a purple or violet colored substance. Ninhydrin reagent can also be used to detect proteins, but the proteins must be heated or digested to hydrolyze the protein into free amino acids.

  1. Put a drop of each sample on a piece of filter paper.
  2. Draw a circle around the spot with a soft pencil.
  3. Write the name of the sample next to the spot.
  4. Allow all spots to dry thoroughly.
  5. Ask your instructor or lab assistant to put a drop of ninhydrin reagent on each spot.
  6. Wait for at least 20 minutes.
  7. Observe each spot carefully.
  8. Record your observations in the Table. Indicate presence (+) or absence (-) of free amino groups in each sample.

Results of Tests for Proteins and Amino Acids

Tube
contents
Biuret reaction Ninhydrin reaction
Color after two minutes Is protein present? Final color of spot Amino groups present?
Water        
Albumin        
Lysine        
Alanine        
Proline        
Glucose        


IV. Testing Foods for Carbohydrates, Lipids and Proteins

Results of Tests for Carbohydrates

Tube
contents
Benedictís Test: Lugolís Test:
Original color before boiling Final color
after boiling
Original color before I2KI Final color
after I2KI
Water        
Skim milk        
Whole milk        
7 UP soda        
Diet 7 UP        
Beer        
Egg white        
Egg yolk        
Potato        
Navy beans        
Onion juice        


Results of Tests for Lipids

Tube
contents
Solubility Test Sudan red
dye test
Grease spot
test
Is it soluble in a polar solvent (water)? Is it soluble in a non-polar solvent (oil)?
Water        
Skim milk        
Whole milk        
7 UP soda        
Diet 7 UP        
Beer        
Egg white        
Egg yolk        
Potato        
Navy beans        
Onion juice        


Results of Tests for Proteins and Amino Acids

Tube
contents
Biuret reaction Ninhydrin
Color after 2 min. Is protein present? Final Color of Spot Amino group?
Water        
Skim milk        
Whole milk        
7 UP soda        
Diet 7 UP        
Beer        
Egg white        
Egg yolk        
Potato        
Navy beans        
Onion juice        



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Wahlert & Holland (Rev. 7/8/99jj)

Last updated 30 August 1999 (JHW)