Chromatography Lab

Problem

How can you tell pigment separation by using Chromatography?

Objective

To prepare a chromatogram, separate pigments in a leaf and interpret the chromatogram.

Hypothesis

If I am to put a chromatography paper into a solvent, then it would separate the pigments depending on their Rf value. I think the pigments will separate in this order: Chlorophyll A, Chlorophyll B, Carotene, and Xanthophyll.

Introduction

Chlorophyll is the molecule that absorbs sunlight and uses its energy to synthesize carbohydrates from CO2 and water. This process is known as photosynthesis and is the basis for sustaining the life processes of all plants. Since animals and humans obtain their food supply by eating plants, photosynthesis can be said to be the source of our life also. As the chlorophyll in leaves decays in the autumn, the green color fades and is replaced by the oranges and reds of carotenoids. Xanthophyll is the yellow pigment in plants that, like chlorophyll, is responsible for the production of carbohydrates by photosynthesis.

Chromatography is any of various techniques for the separation of complex mixtures that rely on the differential affinities of substances for a gas or liquid mobile medium and for a stationary adsorbing medium through which they pass, such as paper, gelatin, or magnesia. (Dictionary.com)

The Rf values for each pigment are calculated to establish the relative rate of migration for each pigment. This value represents the ratio of the distance a pigment traveled on the chromatogram relative to the distance the solvent front moved. Scientists use the Rf value of a sample to identify the molecule. Any molecule in a given solvent matrix system has a uniquely consistent Rf value.

Materials

 1 large test tubes

 1 piece of roam rubber

 1 small test tube with chlorophyll

 1 strips of chromatography paper

 1 plastic test tube rack

 1 cork stoppers with clips

 1 spinach leaf

 1 dime

Procedure

1. Wrap the foam rubber around a large test tube (to keep it steady) and then place it into the plastic rack.

2. Using forceps or holding it by its edge, obtain a paper strip. DO NOT place fingers on the flat surface of the paper or it will interfere with the formation of the chromatogram.

3. Place the strip on a piece of paper towel when working on it. Using scissors, trim one end of the strip so that when clipped to the cork, it extends to the bottom of the test tube without touching the bottom. DO NOT fold or bend the paper strip. When extending from the clip into the test tube, the paper strip and the test tube must remain as straight as possible.

4. Using a metric ruler, draw a light pencil line across the width of the paper 2 centimeters from the bottom edge.

5. Carefully place the paper strip on the clip at the end of the test tube and place it down into the tube.

6. Using a wax pencil, draw a line on the test tube Ð... centimeter below the pencil line on the paper strip.

7. Remove the paper strip from the tube and place it back on the paper towel.

8. Take the spinach leaf and place it over the line drawn on chromatography paper. Roll dime over leaf until the color has been seen on the paper.

9. Add solvent to the large test tube to the line marked by the wax pencil.

10. Attach the prepared strip to the clip on the cork stopper and slowly lower it into the test tube containing the solvent. Be sure that the test tube has been tightly secured with the foam in the test tube rack. NOTE THE TIME THAT YOU PLACED THE PAPER INTO THE SOLVENT.

11. The solvent will immediately travel up the paper strip. When the solvent reaches the top of the paper strip (about 1 cm from the clip), remove the cork, pulling the paper out of the solvent and immediately mark the point that the solvent reached.

12. NOTE THE TIME IT TOOK FOR THE SOLVENT TO REACH THAT POINT.

13. Return the solvent to your teacher and return the test tube to the rack without washing it.

Lab Data

Some things that were observed during the lab were the color pigments moving up the strip of paper. The colors that were observed were green, yellow, and light tint of orange. Also the solvent moved up the strip fairly quickly.

Start Time: 1:10

Finish Time: 1:19

Time Elapsed: 9 minutes

Distance moved by solvent (in mm): 80

Substance Distance Moved (mm) Rf Rate of movement (mm/minute)

Chlorophyll B 70 .875 7.78

Chlorophyll