Experiment 3: 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
isolatiom 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 Mg 2+ ion is replaced by
2H+ ions [4].
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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].
Figure 2. 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.
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The fastest-moving band (yellow) contains the carotenes and the slower-moving band (green)
contains the chlorophylls.
APPARATUS
Equipment
Chromatography column
Erlenmeyer flask 50 mL * 6
Mortar and pestle
Beaker 100 mL
Stand
Funnel
Pipette 10 mL * 4
Beaker 400 mL
Clamps, Ring
Suction bulb
Chemicals
Silica Gel 40
Acetone, CH3COCH3
Petroleum ether
Sodium chloride, NaCl
Calcium carbonate, CaCO3
Sodium sulfate, Na2SO4
PROCEDURE
Preparation of the column
1. Place the glass wool at the bottom of the column.
2. Put 10 mL 7:3 petroleum ether and acetone solution into the column.
3. Weigh 20 g silica gel 40 in a 50 mL erlenmeyer flask, add some amount of petroleum ether
acetone solution slowly while mixing the slurry with a glass rod.
4. Place the slurry in the column with a pipette continuously, then place an erlenmeyer flask
under the column and allow the excess solvent to drain. Be careful : Never let silica to dry in
the column.
5. Collected excess solvent can be reused to place the column.
Preparation of spinach sample
1. Put the spinach into a mortar; add 22 mL acetone, 3 mL petroleum ether and spatula tip of
calcium carbonate, CaCO3.
2. Grind the leaves until the pieces are too small to be seen and then filter the pigment extract
if needed.
3. Put the filtrate into a separatory funnel and add 20 mL petroleum ether and 20 mL 10%
NaCl solution.
4. Shake the separatory funnel and drain the lower layer into a beaker.
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5. Wash the upper layer twice with 5 mL distilled water.
6. 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
1. Using a pipette, add 10 mL leaf extract to the top of the column.
2. Open the bottom outlet of the column, let the elute pass through the column slowly. As the
eluent passes down the column, the sample is developed.
3. When the yellow band reaches the bottom of the column, collect layer in a test tube.
4. Change the test tube as the eluent changes color. Separation of the sample is achieved.
5. If needed, evaporate the excess solvent in the sample, close the sample test tubes with
parafilm and store.
REFERENCES
1. Mckay, P., An Introduction to Chromatography, The National Health Museum
http://www.accessexcellence.org/LC/SS/chromatography_background.html
2. Sheffield Hallam University, School of Science and Mathematics, Chromatography
http://www.shu.ac.uk/schools/sci/chem/tutorials/chrom/chrom1.htm
3. Norman, R.O.C., Waddington, D.J., “Modern Organic Chemistry”, Mills and Boon, London,
1974.
4. Column Chromatography, Lab Techniques, Colorado University, 2005
http://orgchem.colorado.edu/hndbksupport/colchrom/colchrom.html
5. Nichols, M.A, Introduction to Chromatography, 2003
http://chemistry.jcu.edu./nicholsweb/ch223f01/spinachextraction.pdf
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