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Introduction: One of the most sensitive techniques in Biochemistry for separating mixtures of different molecules is the technique of Chromatography. This technique depends on the ability of a mixture of solvents ("solvent system") to separate tiny amounts of a substance, called the solute, from the other solutes present in the mixture. Because each substance (solute) in the mixture is more or less soluble in the solvent than another.

Even though there are many forms of Chromatography (ex: gas chromatography and thin layer chromatography) the form used in this lab is called "Paper Chromatography". This method is sensitive and can separate as many as a dozen or so molecules one from the other.

Purpose:

To see how the process of chromatography can be used to separate the light absorbing, photosynthetic, pigments of spinach leaves.

Materials:

• Spinach leaves
• 1,000mL beaker
• Sheet of chromatography
• paper
• pencil
• aluminum foil
• chromatography solvent system (8% acetone, 92% petroleum ether
• ruler
• hair dryer

Procedure:

1. Pour approximately 1/2" chromatography ether into the beaker.
2. Prepare a sheet of Chromatography paper by cutting a length 6 to 10 inches long
3. At one end cut it to a point
4. Measure up the paper approximately 1"
5. Mark you origin by marking with a pencil or notch out two triangles on either side
6. Using a leaf start at the point of origin and rub the pigments from the leaf into the paper. Use a coin or the edge of scissors to do this.
7. You need to break the cell walls and put a green stain on the paper.
8. Place the paper into the beaker.
9. Allow the paper to soak up the ether. This is called letting it "run" your chromatogram.
10. After approximately 10-15 minutes the solvent should reach about 1/2 to 2/3 of the way up the paper strip.
11. Remove the chromatogram from the beaker and immediately mark the solvent front with your pencil at the points of color change.

Results:

In this exercise you used a technique to separate the colored pigments found in the leaves of spinach. As you conducted this lab you saw several colors; Two bands of green- one blue-green band, and one yellow-green band, which correspond to the two kinds of chlorophyll which is used to absorb light energy from the sun. You also saw a band of yellow which corresponded to a group of pigments called xanthophylls and a band of orange that corresponded to a group of pigments called "carotenes".

During this lab you may also may have seen a band of faint gray, between the carotenes and the xanthophylls, called pheophytin. All of the pigments are involved, in some way, to the photosynthetic process. The non-chlorophyll pigments are called "accessory pigments" and play an indirect yet important role in photosynthesis. The pigments found closet to the origin at the bottom of the piece of paper are the least soluble, and the one which moved the farthest up the paper are the most soluble in the solvent system.

         
           

©Copyright 1998 Elizabeth Beckett, Holly Bernitt, and Vishwa Chandra.