Thin Layer Chromotagraphy on Spinach Leaf Pigment Extracts

Introduction

The objective of this experiment is to extract pigments from spinach leaves and perform Thin Layer Chromatography (TLC) on the spinach leaf extract. We will also determine the best solvent mixture to use to separate the pigments. This experiment is designed to find the most efficient way to separate molecules, in this case pigments, so that they can be studied.

Thin Layer Chromatography is a technique that is uses a solvent to separate small amounts of material. Chromatography takes a mixture of material and causes them to move along a specific path at different rates, which results in their separation. For this lab, we consider the interaction of the pigments with the mobile phase. As TLC is being performed the solvent will move past the sample, and interact with the components of the substance. The stronger the interaction a substance has with the solvent, the farther down the path it will move.

In the extract we will be observing the behavior of two types of pigments, chlorophylls and carotenoids. These are two of the “light-trapping” molecules utilized by plants for photosynthesis. The chlorophyll pigments are often recognized as a green color, while the carotenoids are recognized as a yellow/orange color.

Procedures

Materials

All materials were provided by the preproom prior to the experiment.

Techniques

For extraction:

1. Dry the sample
2. Add the spinach leaves to the mortar
3. Add the abrasive, in this case sand, to the mortar
4. Add 1:1 Acetone/Hexane solvent to the mortar
5. Grind everything in the mortar
6. Decant the solvent

For TLC:

1. Obtain 8 chromatography plates, keep them clean
2. Mix 8 solvents of different ratios of hexane and acetone
3. Add one solvent to each test tube, about 1cm of depth
4. Use a capillary tube to apply pigment about 1cm from the end of the plate, and mark the starting position.
5. Cover the test tube and allow the TLC to run
6. Once the pigment runs to about 1cm from the top of the plate, remove the plate from the test tube
7. Mark the ending pigment locations and solvent lines

Results

Thin Layer Chromatography Plate Diagrams (not drawn to scale)

Figure 1

Solvent Ratios and Corresponding RF Values, Table 1

Solvent Number Ratio (Acetone/Hexane) Acetone Hexane RF

1 0:1 0 mL 1 mL 0.24

2 1:0 1 mL 0 mL 0.90

3 2:1 2 mL 1 mL 0.55

4 1:2 1 mL 2 mL 0.96

5 1:3 1 mL 3 mL 0.15

6 3:1 3 mL 1 mL 0.96

7 1:4 1 mL 4 mL 0.04

8 4:1 4 mL 1 mL 0.71

The RF value, or retention factor, is the ratio of the change in distance of the pigments to the change in distance of the solvent. This ratio is constant for any given component and solvent, and can be used to identify the spots. The closer the RF value is to 1, the higher the retention factor, and the better the separation.

Here are some examples of the calculations for RF values in this experiment:

RF = (distance of pigment change)/(distance of solvent change)

RF Value Calculations, Table 2

Solvent # Distance of Pigment Change Distance of Solvent Change RF Value

3 3.3 cm 6.0 cm 0.55

4 4.9 cm 5.1 cm 0.96

5 0.8 cm 5.5 cm 0.15

6 5.3 cm 5.5 cm 0.96

Conclusion

When beginning this experiment, we were presented with the problem of finding what solvent mixture provides the best separation of the spinach leaf pigments. After running the experiment with 8 mixtures of different acetone/hexane ratios, we have determined that the best solvent mixtures are a 1:2 ratio of acetone/hexane and a 3:1 mixture of acetone/hexane.

There were several problems that we encountered during this experiment. The first problem was during the extraction phase of the experiment. When attempting to decant the solvent, we found it difficult to obtain a highly concentrated amount of pigment without any solid pieces being mixed in. In attempt to remove all the solid pieces, we were left with an extract that was not highly concentrated. This problem led to a failed attempt at running the TLC plates. We then had to find another extract to use to run the plates again. After running a second set of TLC plates, the pigments appeared to separate as originally expected. Given the problems we encountered and failed attempts at running the experiment, I do not have complete confidence in our answer. While it is true that we did reach a conclusion, the reliability of this conclusion is doubtful.

It is practical to use TLC to separate leaf pigments because the different pigments have different reactions with the solvents, and have varied reactions with the different phases of the chromatography plate. Due to the different colors that the pigments produce it is easy to identify them on the TLC plate, and observe their separations based on the solvent.

The carotenoid pigments are more attracted to the mobile phase of the plate than the chlorophyll pigments. If you look at the Thin Layer Chromatography Plate Diagrams, it is evident that the spots that represent the carotenoids travel either the same distance as the chlorophyll, or farther in almost every solvent (excluding solvent #2). This can also be determined from the RF values. The RF values for Solvent #4 and Solvent #6 are both equal to 0.96. This is because the carotenoid pigment travelled almost the exact same distance as the solvent. These corresponding RF values can be found in the Solvent Ratios and Corresponding RF Values table, and the distances travelled by the pigment and the solvent can be found in table 2, RF Value Calculations.