IDENTIFICATION AND SEPARATION OF PHOTOSYNTHETIC PIGMENTS BY

THIN LAYER CHROMOTOGRAPHY (TLC)

Paper chromatography is widely used to separate and identify photosynthetic pigments but it is difficult to obtain
meaningful results. Thin-layer chromatography (TLC) enables a more complete separation of these pigments and
opens up the possibilities of a wide range of investigations into the nature of the pigments present in different
organs of a plant. TLC uses silica gel strips instead of chromatography paper. In paper chromatography, the
distance the pigments move is more or less determined by their relative solubilities; on a TLC strip, another effect
comes into play, which is the relative adsorption of the pigments to the TLC plate.
The technique is no different from paper chromatography, except TLC strips are used. In any chromatography
investigation, you must work quite quickly, as the pigments are rapidly broken down. If possible keep the
pigments in the dark, and do not allow them to get hot (holding tubes in your hands, for instance).

TLC CHROMOTOGRAPHY INVESTIGATION FOR PLANT PIGMENTS

Apparatus and equipment
Leaves to be investigated freshly picked
10 cm3 acetone in a stoppered bottle
20 cm3 chromatography solvent (2 parts petroleum ether : 1 part propanone) HIGHLY FLAMMABLE
Chromatography jar
Small beakers
Small stoppered tube (test tube? flat bottomed specimen jar?), covered by foil
Centrifuge tube
Pestle and mortar
Clean pipette
Silica gel strip, to fit the chromatography jar
Fine paint brush
Cling film
Access to a hair drier and centrifuge

Method
1. Pour chromatography solvent into a chromatography jar to a depth of about 5mm. This will mean
that there is sufficient for the silica gel strip to stand in, but without the solvent actually touching the
line of pigment extract. (See 8, 9 & 10 below) Put the lid on the jar and put it aside to allow the inside
air to become saturated with the solvent. Do not put the silica gel strip into the jar yet.
2. Cut out the petioles and any other big veins from the leaves.
3. Transfer some of the leafy material to a mortar and grind it to a thick paste.
4. Add approximately 1cm3 acetone to the paste and continue to grind, then add a further 2cm 3 acetone,
grinding as you add the solvent.
5. Pour off the liquid extract into a small beaker and then into a centrifuge tube.
6. Centrifuge the extract for a minute or two.
7. Use a clean pipette to extract the liquid extract which is on top of any solid material, and store this in
a small tube, which is covered from light. This extract contains the pigments.
8. Mark a pencil line on the silica gel strip, about 1 cm from the bottom.
9. Using the paint brush, apply the leaf extract in a thin straight line, along the pencil mark you have
drawn.
10. Make several applications, allowing 1 minute or more for each application to dry before applying the
next.
11. Gently place the silica gel strip into the solvent in the chromatography jar, with the marked part
down, and lay the strip against the side of the jar. Do not move the jar from now onwards.
12. Replace the lid on the jar.
13. Observe the chromatogram and remove it from the solvent when the solvent is about 1 cm from the
top. Mark the solvent top before it becomes invisible.

GETTING RESULTS
1. Make drawings of the two chromatograms, using coloured pencils to show the distribution of the
different pigments. Label the pigments and the line from which they were drawn (origin) and the
final reach of the solvent (solvent front) in each chromatogram. (Scale drawings may help you later to
work out the Rf values.)
2. Attempt to calculate the Rf value of each pigment:
Distance moved by the pigment (from their origin line)
Rf

=
Distance moved by the solvent (from the origin line)

3. Compare the Rf values you have calculated with the values given below and see if you can identify
some pigments correctly.
Pigment colour
pigment
Rf value
Orange yellow
carotene
0.96
Grey
phaeophytin
0.70
Blue green
chlorophyll a
0.58
Green
chlorophyll b
0.48
Deep yellow
xanthophyll
0.44

ASSESSMENT
Q.1
Draw the chromatogram which you obtained. Make sure that the chromatogram is fully labelled
to show:
(i)
the point (or line) on which the pigment mixture was spotted
(ii)
the final reach of the solvent front
(iii)
the point to which each pigment has reached.
Q.2
Calculate out the Rf values and compare them to the given Rf values. In this way try to identify
each of the pigments which you obtained. Show your calculations. Present your results in the form of a
simple drawing of the chromatogram, with each pigment labelled, showing its Rf value.
Q.3
Make a simple conclusion from the data which you have obtained, referring to the green plant
which you have investigated, and evaluate the investigation procedures, errors, etc. and recommend
suggestions for improving the investigation.

John Osborne
May 2015