The British School, Quito

Year 12 Diploma BiologyIDENTIFICATION 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.Two quite different techniques are described here, and both could be attempted. 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).

CHROMOTOGRAPHY INVESTIGATION 1

This technique is similar to the one we used previously, but uses silica gel strips instead of paper.

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 2cm3 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 lie 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 RESULTS1. 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 (HL only)Make a labelled drawing of a chloroplast. Annotate your drawing to show where each stage of the photosynthesis reaction occurs. Also give a scale for the drawing.

Q.2Describe the role of chlorophyll in the photosynthesis reaction. (HL students should refer to the other pigments, including the role of satellite [or antennae] pigments.)Q.3Draw the two chromatograms which you obtained. Make sure that the chromatograms are 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.4Calculate 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.5Make 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 2015Apparatus 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