Exercise 1 – Separation of leaf pigments by paper chromatography
Chromatography is a technique used to separate substances in a mixture. It can be used to determine what pigments are present in a leaf. Paper chromatography can separate different pigments only if those pigments (a) have different solubilities, and/or (b) differ in the degree to which they adhere to the paper. Paper chromatography involves placing a drop of leaf extract near the bottom of a strip of paper. The bottom edge of the strip is then dipped into a solvent. As the leading edge of solvent moves up the paper and across the spot of leaf extract, the pigments dissolve and are carried along with the solvent. The more soluble a pigment, the farther it will travel up the strip. Once separated, the pigments can be identified and studied. Methods 1. You will need the following materials: chromatography paper coin (25-cent piece) wax pencil ruler chromatography jar and cork scissors plant leaves (geranium, spinach, etc.) test tube rack solvent (mixture of petroleum ether & acetone) 2. Cut a strip of chromatography paper about l inch longer than the length of the chromatography jar (a flat bottomed test tube). Trim the paper so that it slides easily into the jar. Remove the paper and place your initials on the jar with the wax pencil.
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3. Add about l cm of solvent (petroleum ether and acetone) to the empty jar and immediately cork the jar to reduce fumes. 4. Transfer some of the pigment from a spinach leaf to a piece of chromatography paper. To do this (a) place a strip of chromatography paper flat on the desktop, (b) place a leaf over one end of the paper strip, and (c) roll the edge of a coin (a 25-cent piece) back and forth across the leaf so that the pigment squeezes out and makes a thin line across the paper about 2-3 cm from the end of the strip. When you stand the paper upright inside the chromatography jar, the line should be horizontal.
5. Carefully insert the paper into the test tube, so that the paper is touching the solvent but the drop of leaf extract is not submerged in the solvent. Cork the test tube (the end of the paper will stick out past the cork). Observe the separation of pigments at 5-10 minute intervals for 30-40 minutes. Results 1. On the next page, under “Species 1”, sketch your chromatogram (paper strip) as it appears after 30- 40 minutes. Indicate the color of each of the spots for your species. Look for the presence of chlorophyll a (bluish green), chlorophyll b (pale green), and auxiliary pigments like xanthophyll (yellow) and carotene (orange, at the solvent edge). 2. Under “Species 2”, sketch and label a chromatogram obtained by someone who used a different species of plant. Chromatograms of leaf pigments from two kinds of plants Name of Species 1: Name of Species 2:
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