Dyes in Spinach

 

The green in spinach is mainly due to chlorophyll a and chlorophyll b.

Pheophytin a and pheophytin b are decomposition products in which chlorophyll a and b have lost their magnesium ions.

The yellow dyes in spinach include beta carotene and xanthophylls.

beta carotene

Xanthophylls are derivatives of beta carotene that contain oxygen. The xanthophyll in Spinach is about 96% lutein and 4% zeaxanthin.

lutein

 

zeaxanthin

(Differs from lutein in the position of a double bond in the ring on the right.)

 

A typical separation of dyes in spinach looks like this:

 

Separation of Plant Pigments by Thin Layer Chromatography

From: Quach, H. T.; Steeper, R L.; Griffin, G. W. J. Chem. Educ. 2004, 81, 385-7.

 

Introduction

The principal pigment in higher plants is chlorophyll a. Chlorophyll b, carotenes and xanthophylls play a secondary role by transferring the energy they absorb to chlorophyll a for use in photosynthesis. In the following experiment, the pigments found in the leaves of spinach (Spinacia oleracea) or kale (Brassica oleracea acephala) are separated by means of thin layer chromatography.

Chromatography is an analytical method permitting the separation of a mixture into its molecular components. The basic principle upon which it works is that a mixture first adheres to the dry chromatography plate. A developer or solvent is then passed through the coating on the plate in a fixed direction moving the pigment molecules of the mixture along at different rates. The greater the attraction between the molecules and the absorbing medium (coated on the plate), the slower the molecules ascend the coating. The greater the solubility of the components in the developer, the greater the distance the molecules move.

The developer used in this experiment contains a mixture of nonpolar solvents. No open flames -solvents are extremely flammable. The solvent molecules contain non-polar covalent bonds and any net charges are equally distributed within the molecules. (Water, a common solvent, shares electrons unequally such that the oxygen end is negative and the hydrogen ends are positive. Such molecules are called polar molecules.) As the nonpolar solvents move up the chromatography plate, the pigments move along with it. The more nonpolar the pigment, the more soluble it is in the nonpolar organic solvents, the faster it will move and the greater distance it will proceed up the film.

Chromatography was discovered by M. Tswett in 1906. He dissolved a mixture of plant pigments in petroleum ether and passed the solution through a column of calcium carbonate. Yellow and green zones always formed in the same relative positions in the column. .

Experimental Procedure

  1. On a balance weigh out 0.5 grams of fresh spinach and combine with 0.5 grams of anhydrous magnesium sulfate and 1.0 grams of sand. Transfer these materials to a mortar and using a pestle grind the mixture until a fine dry powder is obtained (grind the mixture really well). The anhydrous magnesium sulfate will remove the water from the leaves.
  2. Transfer the powder (2.0 grams total) to a small test tube and combine with 2.0 mL of acetone. Stopper the test tube and shake vigorously for approximately one minute. You need to make sure that the solid and solvent are well mixed.
  3. Allow this mixture to stand for 10 minutes, then using a pipet carefully transfer the solvent above the solid into a small microcentrifuge tube. Use care not to transfer any of the solid material. The solvent extract should be green. Cap the microcentrifuge tube to minimize solvent evaporation.
  4. Repeat the first three steps with a sample of frozen spinach. Repeat yet again with a sample of canned spinach.
  5. Obtain a TLC chamber (a 400 mL glass beaker covered with parafilm or aluminum foil) and add developing solvent (a mixture of petroleum ether, acetone, cyclohexane, ethyl acetate and methanol). The solvent should completely cover the bottom of the chamber to a depth of approximately 0.5 cm. Keep the chamber covered so that evaporation doesnt change the composition of the solvent.
  6. Obtain a TLC plate (a silica gel coated plastic sheet) which has been precut (5.5 x 9.0 cm) and make four dots equal distance apart with a pencil (why not an ink pen?) on the coated side approximately 1.0cm from the bottom of the strip. The dots should be parallel with the bottom of the strip. Label the first dot with the letter e (leaf extract), the second dot f (frozen spinach), the third dot c (canned spinach), and the last dot b (beta-carotene).
  7. Fill a capillary tube (TLC applicator) by placing it in the leaf extract (it will fill by capillary action) Keep your finger off the end of the capillary tube. Apply the extract to the center of the first dot (e) on the TLC plate by quickly touching the end of the TLC applicator to the plate. Allow to dry (you can gently blow on the strip). Repeat several times to make a concentrated dot of extract. Be sure to let dry between applications. In a similar manner, place an additional dot on each side of the first dot to make a short line of extract. Try to make the spots as small as possible but dark enough to see the color clearly.
  8. Repeat steps 5 & 6 with the extracts of frozen spinach, canned spinach, and beta-carotene. Place on appropriate pencil mark. You will be able to determine the positions of the pure pigments (or, at the beta-carotene) in your leaf separation by comparing them to the positions of the pure extracts.
  9. Carefully place the TLC plate in the TLC chamber. The TLC plate should sit on the bottom of the chamber and be in contact with the solvent (solvent surface must be below the extract dots). Cover the TLC chamber.
  10. Allow the TLC plate to develop (separation of pigments) for approximately 10 minutes. As the solvent moves up the TLC plate you should see the different colored pigments separating.
  11. Remove the TLC plate from the chamber when the solvent front is approximately 1.0cm from the top of the TLC plate. With a pencil, mark the level of the solvent front (highest level the solvent moves up the TLC plate) as soon as you remove the strip from the chamber (the solvent evaporates and disappears quickly). Also measure the pigment distances quickly as some pigments (especially the beta-carotene) may fade over time.
  12. Record the results of the separation on the data sheet.

 

Results

  1. Tape your chromatography plate to the data sheet in space provided. Draw arrows to the locations of the solvent front and the colored bands. Label each band as to its pigment. (Beta-carotene is the most non-polar pigment (highest Rf) and its band will be yellow. Chlorophyll a has a larger Rf than does chlorophyll b.
  2. For the following calculations mark the center of the initial pigment dot; this will be the starting point for all the following measurements. Also mark the middle point of each pigment band and the solvent front.
  3. The rate at which a pigment moves up the plate is reported as an Rf value which is defined as the ratio of the distance moved by the spot to the distance moved by the solvent. Determine the Rf values for each of the pigments you observe using the formula provided below.

 

  1. The literature gives Rf values of 0.61 and 0.49 for pheophytin a and pheophytin b. Use these values to help identify which spots are due to those compounds. Use the pure extract of -carotene to identify the location of that band.

 

Chromatography Apparatus


Separation of Plant Pigments by Paper Chromatography

Report Sheet

Name: ____________________________ Partner: ____________________________

 

 

distance, mm

 

Rf value

spinach

 

 

spinach

 

fresh

frozen

canned

-carotene

 

fresh

frozen

canned

-carotene

_____

_____

_____

_____

solvent front

 

 

 

 

_____

_____

_____

_____

-carotene

_____

_____

_____

_____

_____

_____

_____

 

pheophytin a

_____

_____

_____

 

_____

_____

_____

 

pheophytin b

_____

_____

_____

 

_____

_____

_____

 

chlorophyll a

_____

_____

_____

 

_____

_____

_____

 

chlorophyll b

_____

_____

_____

 

_____

_____

_____

 

lutein

_____

_____

_____

 

 

Tape TLC Plate Here


Questions

1.      Based on their absorption spectra, would you expect chlorophylls a and b to be the same color? Explain.

 

 

 

2.      What evidence, if any, do you see for the presence of other xanthophylls besides lutein in fresh spinach?

 

 

 

3.      Which of your pigment molecules was the most nonpolar? ______________________

 

4.      Which of your pigment molecules was the most polar? ______________________

 

5.      Why should you not use ink on the coating to mark your pigment placement?

 

 

 

 

6.      In the TLC experiment, why must the spot be applied to the TLC plate above the level of the developing solvent?

 

 

 

7.      What will be the result of applying too much compound to a TLC plate?

 

 

 

8.      Arrange the following in order of increasing Rf on a TLC plate:

benzoic acid

naphthalene

benzophenone

.

Highest Rf:

Intermediate Rf:

Lowest Rf: