Identifying Organic Compounds in Plants
Introduction: The purpose of this lab is to introduce some simple qualitative methods for identifying basic types of organic compounds in commonly encountered plant tissues, and to illustrate where in these tissues they are concentrated.
I. Carbohydrates
A. Simple Sugars
The basic formula for simple sugars is (CH2O)n: where "n" is three or some greater number. For some of the most common sugars n = 6 and, hence, their formula is C6H12O6. Sugars with this formula include both glucose and fructose. Both of these sugars react with Benedict's solution as do all "simple "sugars.
Procedure:
1. Take 5 ml of dilute corn syrup and add 1 ml of Benedict's solution in a test tube. Heat this tube in beaker of boiling water. What do you observe? This is a positive test for simple sugars such as glucose and fructose.
2. Repeat this test using a solution of sucrose (table sugar). Do you get a positive reaction?
3. Now add 5 ml of distilled water to a test tube. Place a piece of apple into the tube and crush it with a stirring rod. Pour the water into a clean test tube and test with Benedict's. Does apple have simple sugars?
B. Starches
Starches are long chains of the simple sugar glucose. Starch is easily identified using a solution of iodine and potassium iodide (I2KI).
Procedure:
1. Add 5 ml of distilled water to a test tube. Now add 5 drops of starch suspension. Swirl the tube and then add one drop of I2KI. The resulting color is diagnostic for the presence of starch.
2. Cut a thin slice of potato and make a wet mount using distilled water. Observe the tissues under low and high power. Remove the slide. Put a drop of I2KI on one edge of the cover slip and blot water from the other edge using tissue paper. Put the slide back under the microscope and observe any changes.
II. Lipids
Lipids are not one class of molecules like carbohydrates. However, all lipids are nonpolar: they do not mix in water and they will dissolve certain nonpolar substances that will not dissolve in water. Triglycerides, phospholipids, waxes, and steroids are all examples of lipids. In this exercise we will consider only the triglycerides, which are commonly known as fats and oils.
Procedure:
Take a piece of peanut seed, cut a thin slice and make a wet mount of the tissue. Observe the tissue under both low and high power. Now add I2KI as described previously and observe the distribution of starch in this tissue. Now add Sudan IV stain to your wet mount using the same procedure previously described for adding I2KI to a wet mount. This stain is nonpolar and will move into the lipid droplets residing in the tissue.
Questions:
Can you think of why it is adaptive for a plant to store food in the form of oils in a seed and not in a tuber?
Why don't animals lay down long-term energy stores in the form of starch?
III. Proteins:
There are twenty different amino acids found in living systems. Each of these has a different "R" group. A huge number of different proteins can be formed using different combinations of these twenty. One test for proteins uses concentrated nitric acid. The acid reacts with the "R' groups of certain amino acids.
Procedure:
Take two pieces of dry corn kernel that have been cut longitudinally. Take one half- kernel place it in a glass petri dish and add I2KI to its cut surface. Note where starch is located in the kernel. Take the second half and place it in a glass petri dish. Add two drops of concentrated nitric acid to the cut surface: be careful not to breath the fumes!!! Wait three minutes and check for a yellowish coloration indicative of proteins.
Compounds other than proteins will turn yellow after this treatment. To specifically test for the presence of proteins, add two drops of concentrated ammonium hydroxide to the yellowish tissue. Proteins should turn an orangish color after this check step.
WARNING: Both nitric acid and ammonium hydroxide are extremely caustic. Protect your eyes! Use safety glasses while working with the reagents, and avoid rubbing your eyes after using them until after you rinse your hands. Avoid breathing the fumes of either.
Questions and Speculation for Discussion:
In what tissue is the starch concentrated?
In which tissue is the protein concentrated?
Why are starch and protein located in different regions of a corn kernel?