Chromatography of Plant Pigments
INTRODUCTION:
One of the main jobs of biochemists is to unravel the complexities of chemical compounds and reduce them to their individual
components. The term chromatography comes from two Greek words, "chromat" meaning color and the word "graphon" meaning to
write. Separation of the components of chemical compounds can be done by using several methods. Liquids can be separate by High
Performance liquid Chromatography (HPLC), while the components of gases are separated by Gas Chromatography. Chromatography is a
method for analyzing complex mixtures (such as ink) by separating them into the chemicals from which they are made. Chromatography is
used to separate and identify all sorts of substances in police work. Drugs from narcotics to aspirin can be identified in urine and blood
samples, often with the aid of chromatography.
Chromatography was first used to separate pigments (colors) in leaves, berries, and natural dyes. Paper chromatography is a technique used to
separate, isolate, and identify chemical components of a compound. In paper chromatography, the solid surface is the cellulose fibers in the
chromatography paper. A solvent or developer (water, alcohol, or acetone) is placed in the bottom of the chromatography chamber. The paper
acts as a wick to pull the solvent up the paper. The solvent front will "wick" up the chromatography paper by capillary action. A minute drop
of the ink or chemical mixture to be separated is placed near the bottom of the strip of chromatography paper, but slightly above the level of the
solvent in the chamber. As the solvent passes over the drop of ink, the components of the ink dissolve in the solvent. Because the components
of the ink do not all dissolve at the same rate, as the components of the mixture move upward, they show up as colored streaks. The separated
substances on the chromatography paper form a color pattern called a chromatogram.
To determine the rate of migration for each pigment or component of the ink, the Rf value for each pigment must be calculated. The Rf value
represents the ratio of the distance a pigment moved on the chromatogram relative to the distance the solvent front moved. Each pigment or
compound will have a unique Rf value that scientists can use to identify the substance. The Rf value is calculated using the following formula:
Rf = distance traveled by the compound / distance traveled by the solvent
In plants, chlorophyll often hides the other pigments present in leaves. In Autumn, chlorophyll breaks down, allowing xanthophyll and
carotene, and newly made anthocyanin, to show their colors.
The mix of pigments in a leaf may be separated into bands of color by the technique of paper chromatography. Chromatography will separate
plant pigments into individual components" The separation takes place by absorption and capillarity. The paper holds the substances by
absorption; capillarity pulls the substances up the paper at different rates. Pigments are separated on the paper and show up as colored streaks.
The pattern of separated components on the paper is called a chromatogram. Pigments separate because they have different solubility in the
solvent use to create the liquid medium and because the attraction forces between pigments and filter paper vary.
Plants contain chlorophyll, a green pigment, as well as carotenoids, pigments that range in color from red to orange to yellow.
PRELAB PREPARATION:
Gather leaves from several different plants. CAUTION: Avoid poisonous plants. Autumn leaves from deciduous trees are especially
interesting. Sort the leaves by kind (maple, etc.) and color. Review a diagram of a plant cell . Find the grana and the chloroplasts of the cell.
MATERIALS:
 Safety goggles
 Chromatography solvent (9 parts Petroleum ether to 1 part
acetone)
 Chromatography paper (or filter paper) about 1 cm x 15 cm
 Ethyl alcohol (alternative)
 Fresh spinach
 Test tube (alternative)
 Test tube rack (Alternative)
 Scissors and Ruler
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Chromatography Tube
Fresh leaves of plants
Glass stirring rod (Alternative)
Paper clip (Alternative)
Cork (to fit test tube) (Alternative)
Mortar and pestle (Alternative)
Sand (optional) (Alternative)
Syringe (5ml)
PROCEDURE:
Leaves should be grouped by kind (maple, etc.) and color. Work with a spinach leaf and with one or more other types.
CAUTION: Chromatography solvents are flammable and toxic. Have no open flames; maintain good ventilation; avoid inhaling fumes.
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Cut a strip of filter paper or chromatography paper so that it just fits inside the tube you will use. The sides of the strip should not touch the
glass. Cut a point at one end (triangular shape) Draw a faint pencil line as shown in figure 1. If using chromatography tube, simply attach
this strip to the top of the tube. If using test tube, bend a paper clip and attach it to a cork stopper. Attach the paper strip so that it hangs
inside the tube, as shown. The sides of the strip should not touch the glass.
Remove paper from tube, place the waxy side of the leaf facing outward towards the paper (over it). Use the ribbed edge of a quarter or
dime to crush the cells by rubbing the leaf along the light pencil line on the paper strip until you make a dark green line. This DO NOT
RUB THE LEAF ABOVE OR BELOW THE LINE. RUB THE LEAF ON THE LINE ONLY. Make sure the pigment line is on top of the
pencil line. Repeat procedure 8-10 times, using a new portion of the leaf each time to make a darker strand over the pencil line.
Use syringe to transfer 5 ml chromatography solvent into the tube. Fit the paper and cork assembly inside. Adjust it so that the paper point
just touches the solvent (but not the sides of the tube). The pencil line should not be submerged. The pigment must be above the level of
the solvent. Watch the solvent rise up the paper, carrying and separating the pigments as it goes. At the instant the solvent reaches near
the top, remove the paper and let it dry. Observe the bands of pigment. You can see carotenes (orange), xanthophylls (yellow),
chlorophyll a (yellow-green), chlorophyll b (blue-green), and anthocyanin (red). Identify and label the pigment bands on the dry strip.
Write the species of leaf on the strip as well.
Record the species, external color, and chromatogram pigments in the DATA TABLE of your report sheet.
Each pigment has an Rf value, the speed at which it moves over the paper compared with the speed of the solvent.
Rf = Distance moved by the pigment / Distance moved by the solvent
Measure the distance in cm from the starting point (pencil line) to the center of each pigment band. Then measure the entire distance
traveled by the solvent. Remember, the starting point for the solvent is also the pencil line and the ending point for the solvent is the top
edge of the paper. Do the required divisions and record your Rf values in the
Repeat steps 1 through 6 for each species you wish to test.
Alternative pigment preparation procedure (in case quarter method does not work):
Tear leaf into pieces about the size of a postage stamp. Put them into a mortar along with a pinch or two of sand to help with grinding.
Add about 5 ml ethyl alcohol to the leaf pieces. Crush leaves with the pestle, using a circular motion, until the mixture is finely
ground. The liquid in which the leaf pigments are now for paper chromatography dissolved is called the pigment extract. Use a glass
rod to touch a drop of the pigment extract to the center of the pencil line on the paper strip. Let it dry. Repeat as many as 20 times, to
build up the pigment spot. NOTE: You must let the dot dry after each drop is added. The drying keeps the pigment dot from spreading
out too much. Wash the mortar and pestle thoroughly, using a little alcohol to remove any remaining pigment.
Alternative:
This procedure can also be used to separate colors in pen ink or to compare different pens in the same way we compared different leaves.
Sometimes, water works as the solvent (or alcohol).
Results:
Chromatography Data
Leaf Type (species)
External color
Colors from the Top
Chromatogram Pigments
Pigment Names
Rf Values