Paper Chromatography of Spinach Pigments
Chromatography is a common laboratory technique used to separate macromolecules.
Perhaps the most well known type of chromatography is paper chromatography in which
separation is based upon the movement of molecules on a piece of filter paper. The
molecular mixture is spotted or streaked in a narrow line near one end of the paper (Figure
1). Spotting or streaking of the pigment is repeated a number of times to ensure an
adequate amount of material on the paper for subsequent extraction and characterization
of the separated molecules. For ascending paper chromatography, the filter paper is placed
into a sealed container (a chromatography jar) into which an appropriate solvent has been
added; the atmosphere inside the chromatography jar is saturated with solvent vapor.
When the end of the filter paper is placed into the solvent, the solvent moves up the paper
by capillary action, and the solutes separate from each other based on their relative
solubility in the solvent and water phases. (Chromatography paper typically contains 6-10%
water). Each solute has characteristic water: solvent partition coefficient. Each dissolved
solute travels up the paper until it reaches a point at which it is no longer soluble; here, the
solute remains behind.
The separation of solute molecules can be measured by the R f value, which is
Rf = distance traveled by solute from the origin
distance traveled by solvent from the origin
This laboratory exercise will use paper chromatography to separate photosynthetic
pigments extracted with acetone from fresh spinach leaves. The solvent employed to
separate the pigments will be a 9:1 mixture of petroleum ether: acetone. Following
chromatography, five distinct pigment bands will be resolved on the filter paper. The major
pigments appear, in order, from the origin to the solvent: chlorophyll b (olive-green),
chlorophyll a (blue-green), violaxanthin (yellow), lutein (yellow),
Their molecular structure is found in Figure 2. Following measurement of the relative
positions of each band on the chromatogram, the pigments will be eluted separately from
the paper using acetone. The eluate of each photosynthetic pigment will then be assayed to
determine its absorption spectrum using selected wavelengths from 400 to 700 nm.
You will also determine the concentration of the two chlorophyll pigments in spinach
leaves. For this, you will use the Beer-Lambert equation, A=cl. The absorption coefficient,
a, in acetone has already been empirically determined. For chlorophyll a, at 663 nm is
equal to 80.17cm /mg, while for chlorophyll b at 645 nm is 50.93 cm /mg. A is equal to the
absorbance measured from the spectrophotometer at the above wavelengths, the maximum
absorbance for each pigment. L is the light path length, typically 1 cm. Therefore, you will
be solving for the concentration or c. Units of concentration are expressed in mg/mL.
Following these calculations, you will also determine the ratio of chlorophyll a to chlorophyll
b ([chl a]/[chl b]), a number that is plant species-specific.
Preparation of the Chromatography Jar
1. Add about 100 mL of solvent (9 volumes petroleum ether: 1 volume acetone) to the
chromatography jar and place the lid on the jar After about 30 minutes, the
atmosphere in the jar will become saturated with solvent vapor. The chromatography
jar is now ready to use.
Preparation of the Spinach Extract
1. On a triple beam balance, weigh 3 grams of fresh spinach leaves. Remove any stems or
major veins before weighing.
2. Using scissors or a razor blade, cut the spinach leaves into small pieces. Add the leaves
to a chilled mortar. Add 15 mL ice-cold acetone to the mortar and a sprinkling of sea
sand. Grind the leaves with the mortar on ice for about 1 minute.
3. Decant or pour the liquid into a 50 mL tube. With the lid securely fastened, shake the
liquid vigorously for about 10 seconds, and then place the tube in the refrigerator for 10
4. Using a glass Pasteur pipet, transfer a portion of the extract from the upper layer of
the liquid to a small glass test tube. Cover the test tube with a piece of Parafilm to
prevent evaporation. Return the remaining extract to your instructor.
Preparation of the Chromatography Paper
1. Cut a strip of Whatman 3MM chromatography 5 cm wide. Do not touch the paper with
your bare hands; wear gloves to prevent transfer of oils from your skin to the
2. Using a pencil, draw a line about 3 cm from one of the short edges of the
3. Using a microcapillary tube, make 20 streaks of pigment extract along the line that was
drawn. It will not damage the pigment extract or line if you apply pigment directly to
the line. The streaked pigment should be no more than 3 mm in width. Allow each
streak to dry completely before addition of the next.
4. Place the chromatography paper carefully into the chromatography jar. Make sure that
the edges of the paper do not touch anything inside the jar. Replace the lid. Allow the
solvent to move up the paper until the solvent front is approximately 1 cm from the top
of the paper.
5. Remove the chromatogram from the jar. Using a pencil, make a small mark on the
chromatogram to indicate the farthest distance traveled by the solvent, and then allow
the chromatogram to dry. Determine the location of each of the five pigments.
Measure the distance each pigment traveled in mm from the origin to the approximate
center of each band. Measure the distance from the origin to the solvent front. Record
your results in Table 1.
Elution of the pigments and determination of their absorption spectra
1. Turn on the spectrophotometer and set the wavelength to 400 nm.
2. Using tape and a permanent marker, label 4 screw-cap test tubes as follows: chl a, chl
b, xanthophylls (violaxanthin and lutein), and
3. Add 4 mL acetone to each test tube.
4. Using scissors, cut out each pigment band. Further cut each band into smaller pieces
and place the strips into the appropriately labeled test tubes. Allow the pigments to
elute from the paper for 5 minutes. Occasionally swirl the tubes.
5. Using tape and a permanent marker, label 5 cuvettes as follows: chl a, chl b,
xanthophylls, -carotene, and blank. Do NOT mark directly on the cuvettes! Add 4 mL
acetone to a cuvette that is the blank.
6. Using a glass Pasteur pipet, remove as much of the acetone containing the eluted
pigment from the test tubes as possible. Place the eluate into the appropriately labeled
cuvettes. You need only enough liquid to fill each cuvette to the white line.
7. Determine the absorbance of each pigment using the wavelengths listed in Table 2.
Adjust the spectrophotometer with the reference blank each time you change
wavelengths. Record the absorbance values in Table 2.
1. Complete Table 1 by calculating the Rf values for each pigment.
2. For the absorbance data, you will be constructing two graphs:
Absorbance of chlorophyll a and chlorophyll b (y-axis) as a function of the
wavelength (x-axis). Label this graph “Figure 1. Absorption spectra of
chlorophyll a and chlorophyll b extracted from spinach leaves.”
Absorbance of the two xanthophylls and -carotene (y-axis) as a function of the
wavelength (x-axis). Label this graph “Figure 2. Absorption spectra of the
xanthophylls violaxanthin and lutein and -carotene extracted from spinach
3. Using the Beer-Lambert equation, calculate the concentration of chlorophyll a and
chlorophyll b in spinach. Show your work.
4. What is the [chl a]/[chl b] ratio in spinach?
Questions to answer:
1. What do the Rf values indicate about the relative solubilities of the pigments in the
2. Explain the different Rf values of the two chlorophylls on the basis of their molecular
structure. Be specific.
3. Explain the different Rf values of the three carotenoids on the basis of their
molecular structure. Be specific.
4. At what wavelength(s) is/are the absorption maxima for each of the pigments? Does
this agree with the known absorption maxima for each pigment? If you look up this
information, you must provide appropriate references.
5. What colors and wavelengths of light are absorbed best by each of the four pigments
(chlorophyll a, chlorophyll b, xanthophylls, and -carotene)?
6. What colors and wavelengths of light are not absorbed by each of the four pigments
(chlorophyll a, chlorophyll b, xanthophylls, and -carotene)?
7. What is one of the main advantages of plants possessing pigments with different
absorption maxima? How does this relate to the relative rate of photosynthesis?
What to turn in:
Figure 1 and 2
Full lab report
Table 1. Relative mobility (Rf) values of five photosynthetic pigments extracted from
spinach and subjected to paper chromatography.
Distance from origin Distance from origin
Pigment to solute band (mm) to solvent front (mm) Rf value
Table 2. Absorbance readings of photosynthetic pigments extracted with acetone from
spinach, separated by paper chromatography and eluted with acetone.
Wavelength (nm) Chlorophyll a Chlorophyll b Xanthophylls -carotene