Separation of Spinach Pigments by Column Chromatography

Theoretical Background

Chromatography is the science which studies the separation of molecules based on differences in their structure and/or composition [1]. Chromatography involves a sample (or sample extract) being dissolved in a mobile phase (which may be a gas, a liquid or a supercritical fluid). The mobile phase is then forced through an immobile, immiscible stationary phase. The phases are chosen such that components of the sample have differing solubilities in each phase. A component which is quite soluble in the stationary phase will take longer to travel through it than a component which is not very soluble in the stationary phase but very soluble in the mobile phase. As a result of these differences in mobilities, sample components will become separated from each other as they travel through the stationary phase [2].

Chromatographic separations can be carried out using a variety of supports, including immobilized silica on glass plates (thin layer chromatography), volatile gases (gas chromatography), paper (paper chromatography) and liquids which may incorporate hydrophilic, insoluble molecules (liquid chromatography) [1]. Column chromatography is another of the isolation and purification techniques used extensively by organic chemists to obtain pure samples of chemicals from natural sources or from reaction sequences [3].

If the compounds to be separated in a column chromatography are colored (as is the case for the pigments to be isolated in this experiment) then the progress of the separation can simply be monitored visually. However, it is more common that the compounds to be isolated from a column chromatography are colorless. In that case, several means for monitoring the separation progress have been devised. One of the simplest of these involves the collection of relatively small fractions of the eluent in labeled tubes and the analysis of the component(s) of these fractions by the technique of thin layer chromatography [4].

Column Chromatography of Spinach Pigments

Two categories of molecules are primarily for photosynthesis in plants: the chlorophylls and the carotenoids. Chlorophylls, the green pigments, absorb certain wavelenghts of light that are then converted into chemical energy. Examples of chlorophylls are chlorophyll a, chlorophyll b, pheophytin a, and pheophytin b. The structure of chlorophyll a is shown below. In chlorophyll b, the starred (*) methyl group is replaced by a –CHO group. Pheophytin a and b are identical to chlorophyll a and b, respectively, except that in these molecules, the Mg2+ ion is replaced by 2H+ ions [4].

Figure 1. Chlorophyll a

Carotenoids, the yellow pigments found in spinach, are also involved in photosynthesis. The structure of β-caretone is given below. α-Caretone differs from β-caretone in the placement of the double bond on one of the rings. Xanthophylls are oxygen-containing derivatives of the carotenes [4].

β-caretone

Carotene is the compound which gives butter and margarine their characteristic yellow color. Cows eat the caretone-containing green grass, but do not metabolize the carotene entirely, and it ends up in their milk and, thus, in the butter made from the milk. At certain times of the year, the food which cows eat does not contain carotene, and the butter made from this milk is white. Since the consumer prefers yellow butter (and margarine), the dairy companies used to add a dye called Butter Yellow to white butter. However, Butter Yellow (an azo dye) was found to be carcinogenic. Currently, butter and margarine are colored with synthetic carotene [5].

In this column chromatography experiment, you will separate the pigments from the spinach. An aliquot experiment of the extracted spinach will be applied to an silica column. Although there are many different components in the extract, they separate into two main bands on the column. The fastest-moving band (yellow) contains the carotenes and the slower-moving band (green) contains the chlorophylls.

APPARATUS

Equipment

Chromatography column Funnel

Erlenmeyer flask 50 mL * 6 Pipette 10 mL * 4

Mortar and pestle Beaker 400 mL

Beaker 100 mL Clamps, Ring

Stand Suction bulb

Chemicals

Silica Gel

Acetone, CH3COCH3

Petroleum ether

Sodium chloride, NaCl

Calcium carbonate, CaCO3

Sodium sulfate, Na2SO4

PROCEDURE

Preparation of the column

Fill the column with dry silica.

Put 1-2 mL 7:3 petroleum ether and acetone solution into the column to create a gel.

Collected excess solvent can be reused to place the column.

Preparation of spinach sample

Put the spinach into a mortar; add 3 mL acetone, 1 mL petroleum ether and spatula tip of calcium carbonate, CaCO3.

Grind the leaves until the pieces are too small to be seen and then filter the pigment extract if needed.

Put the filtrate into a separatory funnel and add 20 mL petroleum ether and 20 mL 10% NaCl solution.

Shake the separatory funnel and drain the lower layer into a beaker.

Wash the upper layer twice with 5 mL distilled water.

Then put the extract into a 50 mL erlenmeyer flask and add a spatula tip of sodium sulfate, Na2SO4, then decant the solution in another 50 mL erlenmeyer flask. Evaporate the excess solvent if needed.

Performing the column chromatography

Using a pipette, add 1-2 mL leaf extract to the top of the column.

Let the elute pass through the column slowly. As the eluent passes down the column, the sample is developed.

When the yellow band reaches the bottom of the column, collect layer in a test tube.

Add more of your solvent system to elute the remaining fraction.

Change the test tube as the eluent changes color. Separation of the sample is achieved.

If needed, evaporate the excess solvent in the sample, close the sample test tubes with parafilm and store.

TLC analysis

Perform a TLC analysis of your mixture and each fraction you’ve collected to confirm adequate separation

Calculate the Rf value for each spot

Results

Create a table containing following information about each spot: appearance, distance traveled, Rf value

Sketch your TLC plate

Attempt to identify each of the spots

Post-Lab Questions

Why are the chlorophylls less mobile than the carotenes on the TLC plate?

Very polar compounds are sometime purified by reverse phase chromatography where the stationary phase is very nonpolar while the solvent is very polar. Why might one want to use this technique with extremely polar compounds?

REFERENCES

Mckay, P., An Introduction to Chromatography, The National Health Museum http://www.accessexcellence.org/LC/SS/chromatography_background.html

Sheffield Hallam University, School of Science and Mathematics, Chromatography http://www.shu.ac.uk/schools/sci/chem/tutorials/chrom/chrom1.htm

Norman, R.O.C., Waddington, D.J., “Modern Organic Chemistry”, Mills and Boon, London, 1974.

Column Chromatography, Lab Techniques, Colorado University, 2005 http://orgchem.colorado.edu/hndbksupport/colchrom/colchrom.html

Nichols, M.A, Introduction to Chromatography, 2003

http://chemistry.jcu.edu./nicholsweb/ch223f01/spinachextraction.pdf