IDENTIFICATION OF ORGANIC MOLECULES
A cell may be compared to a living chemistry laboratory. Most functions within the cell take the form of interactions between organic (carbon-containing) molecules. Organic molecules found in living systems can be classified as carbohydrates, fats, proteins, or nucleic acids. Each of these classes of molecules is made of smaller units and both the smaller and larger units have specific properties that can be identified by simple chemical tests. In this laboratory investigation, you will learn to identify three of the four major types of organic molecules: carbohydrates, fats, and proteins and some of their smaller subunits.
The tests for the three types of organic molecules will be done:
- on water (to demonstrate negative results)
- on one or two substances which contain the molecules
being tested for (to demonstrate positive results)
- on several substances of unknown composition
Answer the questions in the laboratory exercise.
EXERCISE A: TESTING FOR CARBOHYDRATES
The basic structural unit of carbohydrates is the monosaccharide (single or simple sugar). Monosaccharides are classified by the number of carbons they contain: for example, trioses have three carbons, pentoses have five carbons, and hexoses have six carbons. Monosaccharides may contain as few as three or as many as ten carbons.
Monosaccharides are also characterized by the presence of
a carbon-oxygen bond. If found at the end of the molecule it is called a terminal aldehyde group or if found in the interior it would be called a ketone group. Both of these groups contain a double-bonded oxygen that reacts with Benedict's solution to form a colored precipitate.
When two monosaccharides are bonded together, they form a disaccharide. If the reactive aldehyde or ketone groups are involved in the bond between the monosaccharide units (as in sucrose), the disaccharide will NOT react with Benedict's solution. If only one group is involved in the bond (as in maltose), the other is free to react with the Benedict's reagent. Sugars with free aldehyde or ketone groups, whether monosaccharides or disaccharides, are called reducing sugars. These sugars are oxidized (lose electrons) to the copper ions in the Benedict's reagent which becomes reduced (gains electrons), hence the name reducing sugar. The color of the precipitate (material that settles to the bottom of the tube) varies dependent on the strength of the reducing sugar present. Scroll down in this animation to see the various color changes for Benedict's solution. In this exercise, you will use Benedict's reagent to test for the presence of reducing sugars.
Monosaccharides may join together to form long chains (polysaccharides) that may be either straight or branched. Starch is an example of a polysaccharide formed entirely of glucose units. Starch does not show a reaction with Benedict's reagent because the number of free aldehyde groups (found only at the end of each chain) is small in proportion to the rest of the molecule. Therefore, we will test for the presence of starch with Lugol's reagent (iodine/potassium iodide, I2KI).
Objectives:
Identify reducing sugars using Benedict's reagent
Identify polysaccharides using Lugol's reagent
PART 1. BENEDICT'S TEST FOR REDUCING SUGARSCheck out the animation showing the results.
When Benedict's reagent is heated with a reactive sugar,
such as glucose or maltose, the color of the reagent changes
from blue to yellow to reddish-orange, depending on the
amount of reactive sugar present. Orange and red indicate
the highest proportion of these sugars. Benedict's test will
show a positive reaction for starch only if the starch has
been broken down into maltose or glucose units by excessive
heating.
PART 2. LUGOL'S TEST FOR STARCHCheck out the animation showing the results.
Lugol's reagent changes from a brownish or yellowish color
to blue-black when starch is present, but there is no color
change in the presence of monosaccharides or disaccharides.
EXERCISE B: TESTING FOR LIPIDS
The word lipid refers to any of the members of a rather diverse group of organic molecules. Lipids are soluble in non-polar solvents such as chloroform, but are insoluble in water. Although lipids include fats, steroids, and phospholipids, this exercise will focus primarily on fats.
Triglycerides, a popular topic of discussions in diet and nutrition, are the most common form of fat. They consist of three fatty acids attached to a glycerol molecule. Triglycerides are found predominantly in adipose tissue and store more energy per gram than any other molecule.
At room temperature, some fats are solid (generally those found in animals) and are referred to as fats, while others are liquid (generally those found in plants) and are referred to as oils. Vegetable oil, a liquid fat, is a mixture of triglycerides.
Since both solid and liquid fats are nonpolar, we will test for their presence by using Sudan IV, a nonpolar dye that dissolves in nonpolar substances like fats and oils but not in polar substances like water. Sudan IV reacts with the fatty acid portion of the molecules and causes a red-orange color to be evident.
Objectives:
Distinguish between lipid and nonlipid substances using the
Sudan IV test.
EXERCISE C: TESTING FOR PROTEINS AND AMINO ACIDS
Proteins are made up of one or more polypeptides which are linear polymers of smaller molecules called amino acids. Amino acids derive their name from the amino group and the carboxyl group (acidic) that each possesses. Polypeptides are formed when amino acids are joined together by peptide bonds between the amino group of one amino acid and the carboxyl group of a second amino acid.
The Biuret reagent reacts with peptide bonds and will, therefore, react with proteins, like egg albumin. The reagent will not react with free amino acids like glycine or alanine. On the other hand, the reagent Ninhydrin will react with the amino group of free amino acids, but will not react with polypeptides.
Objectives:
Distinguish between free amino acids and proteins (polypeptides) on the basis of their ability to react with either Biuret reagent or Ninhydrin.
PART 1. TESTING FOR PROTEIN WITH THE BIURET REAGENTCheck out the animation showing the results.
The Biuret reagent is light blue. In the presence of proteins it turn violet. Other types of molecules may cause other color changes, but only the violet color indicates the presence of polypeptides.
PART 2. TESTING FOR AMINO ACIDS WITH NINHYDRIN
Ninhydrin reagent turns purple or violet in the presence of free amino acids. In the presence of proline, however, it turns yellow. The difference in the reaction is due to the fact that in proline the amino group is not free but is, instead, part of the ring structure of the molecule.
Summary Questions:
1. Define the term "positive control." What is the pupose of a positive control? Give an example of a positive control in the Benedict's Test.
2. Explain the limitations of the Benedict's Test in determining whether or not sugar is present in a certain food product.
3. Specifically, what does the Biuret reagent detect? Why would this test NOT detect an amino acid?
4. Why do fat droplets float, rather than mix with the water?
5. Why was water used as a test solution in each of the tests? What was its function or role in the exercises?
