Thin Layer Chromatography of Vegetables

Introduction:

Thin layer chromatography (TLC) is one type of chromatography. In TLC, the stationary phase is a thin layer of adsorbent particles attached to a solid plate. A small amount of sample is applied (spotted) near the bottom of the plate, and the plate is placed in the mobile phase. This solvent is drawn up by capillary action. Separation occurs as each component, being different in chemical and physical composition, interacts with the stationary and mobile phases to a different degree, creating the individual bands on the plate. The retention factor, Rf value, is used to characterize and compare components of various samples.

Rfvalue = distance from origin to component spot

distance from origin to solvent front

The pigments in vegetables, flowers, and leaves can be separated and identified by using thin-layer chromatography. Green pigments, known as chlorophylls, serve as the main photoreceptor molecules of plants. Carotenoids, yellow pigments, aid the plant in the photosynthesis process. In addition, xanthrophylls are contained in the chloroplasts which can be isolated and identified using chromatographic techniques.

Pigment / Color / Rf value
carotene / yellow-orange / 0.95
pheophytin a / Grey / 0.50
pheophytin b / lt. grey (may not be visible) / 0.45
chlorophyll a / blue-green / 0.45
chlorophyll b / Green / 0.42
xanthophylls / Yellow / 0.42
xanthophylls / Yellow / 0.33
xanthophylls / Yellow / 0.20

Purpose:

The purpose of this laboratory experiment is to isolate the pigments from spinach and carrots by using thin layer chromatography.

Equipment/Materials:

TLC silica gel platescapillary tubes

chromatography chambers70:30 hexane-acetone solvent

spinachmortar

shredded carrotspestle

hexanethin stemmed pipets

acetonetest tubes

centrifuge

Safety:

  • Always wear goggles in the laboratory.
  • Avoid contact with all chemicals.
  • Avoid open flames.
  • Dispose of chemicals as instructed by the teacher.

Procedures:

  1. Obtain a spinach leaf, and tear it into small pieces. Place the spinach pieces in a mortar.
  1. Add approximately 2 mL of acetone and carefully grind the spinach and acetone together with the pestle. Continue grinding until the acetone becomes dark green in color. More acetone may be added to compensate for evaporation.

3. Decant the liquid into a four-inch test tube and centrifuge. Be sure to

counterbalanceeach test tube with a test tube filled with water to approximately the

same volume as the sample and spin for approximately two minutes.

4. Repeat steps 2 and 3 using the same spinach remaining in the mortar and combine

theextracts.

5. Add approximately 2 mL of hexane to the acetone extract.

6. Stopper the test tube and shake vigorously.

7. Allow the two layers to separate.

8. Repeat steps 1-7 using shredded carrots.

9. With a pencil, draw a line approximately 1 cm from the short edge of the TLC plate.

Be careful not to scrape the coating of the plate. Mark the line with two tic marks

approximately 1/3 from either side. See diagram.

10. With a capillary tube, draw up a sample of the upper hexane layer from the test tube

containing the spinach sample and spot the sample on one of the tic marks on the

TLC plate. Be sure to label the spot at the top of the TLC plate.

11. Reapply the sample to the same place at least 6 times or until the spot is clearly

visible.

12. Repeat steps 10 and 11 using the carrot extract, but reapply the sample at least 12

times.

13. Fill the chromatography chamber to a depth of approximately 0.5 cm with the 70:30

hexane-acetone mobile phase.

14. Place the TLC plate in the chromatography chamber with the sample spot toward the

bottom. Be sure the sample spot is above the level of the solvent. Close the

chamber.

15. Allow the plate to remain undisturbed until the solvent reaches to within 1 cm of the

top.

16. Remove the plate from the chamber and immediately mark the solvent front using a

pencil.

17. Measure and record the distance from the spotting line (origin) to the center of each

spot and from the spotting line to the solvent front.

18. Identify each component (spot).

Data Table:

Distance solvent moved from the spotting line (origin) ______

Spinach

Color of spot / Distance moved / Rf value / Identity

Carrot

Color of spot / Distance moved / Rf value / Identity

Calculations:

1. Calculate the Rf value for each spot observed.

Questions:

1. Explain the color of the vegetable based on the results of the chromatography.

2. Which pigments, if any, were present in both vegetables? Explain.

3. Why was acetone used?

Thin Layer Chromatography of Vegetables

Teacher Notes

Lab Time: 40 minutes

Answers to Questions:

1. Explain the color of the vegetable based on the results of the chromatography.

Spinach is green due to the chlorophylls. The carrot is orange due to the

presence of carotenoids and the lack of chlorophylls.

2. Which pigments, if any, were present in both vegetables? Explain.

Carotenoids are present in both spinach and carrots. The colors and Rf value are

the same in each sample.

3. Why was acetone used?

Acetone was used to extract the pigments from the vegetables.

Considerations:

The time for the lab can be shortened by having lab groups extract the pigments from different vegetables and sharing the extracts. Additional vegetables may be used, such as red cabbage, parsley, etc. An extension of this lab would be to have students separate the pigments using an alumina prep-sep or packed alumina column. These extracts could be analyzed using a spectrophotometer to determine the absorption curve of each pigment to confirm its identity.

Updated 2/14/17