John Schollar and Dean Madden
National Centre for Biotechnology Education, University of Reading
2 Earley Gate, Reading RG6 6AU | D.R.Madden@reading.ac.uk
Thin layer chromatography
Photosynthetic pigments from kiwi fruit
Aim
The aim of this practical activity is to extract, separate and identify
photosynthetic pigments from kiwi fruit. Unlike similar methods, it
uses a very small volume of solvent to extract the chloroplast pigments,
and is therefore safer and cheaper to carry out.
Introduction
Many fruits contain chloroplasts but these, and the chlorophylls they
contain, are usually broken down as the fruit ripens. Green-fleshed
kiwi fruit of the variety ‘Hayward’ (the main variety sold in the UK) are
unusual however, because even when they are ripe, their chloroplasts
remain intact. In contrast, the newer yellow-fleshed cultivars (so-called
‘Golden kiwi fruit’) degrade the chlorophyll during ripening and the
consequent loss of the green color uncovers the underlying yellow
carotenoid pigmentation.
Photosynthetic pigments can be extracted from kiwi fruit
chloroplasts by breaking up the fruit tissue in a suitable solvent. The
different pigments can then be separated by thin layer chromatography,
using a different solvent mixture.
You should be able to separate five or more different pigments, and
by calculating Rf values, it should be possible to identify each pigment.
FROM CROWHURST, ET AL (2008)
Fruit diversity in the genus
Actinidia. Only green-fleshed
varieties retain chloroplasts and
photosynthetic pigments in the ripe
fruit.
From: Crowhurst, et al (2008)
www.ncbe.reading.ac.uk 1 Copyright © Dean Madden, 2015
NCBE, University of Reading TLC of photosynthetic pigments
Rf values
Solvent
front
Rf values can be used to identify the pigments on a chromatogram.
An Rf value is a ratio, calculated as follows:
distance moved by pigment Distance Pigment
distance moved by solvent moved by spots
solvent
Rf values always lie between 0 and 1 (0 being a pigment that doesn’t
move at all and 1 being a pigment that is so soluble, it moves the same
distance as the solvent). Distance
moved by
Because for a given pigment, the Rf value will vary according to the
pigment
solvent (or mixture of solvents) used, Rf values are often written with the Origin
name(s) of the solvent(s) and the proportions of them after the number.
For example:
Rf = 0.24 (60 % ethanol)
Rf = 0.78 (cyclohexane : propanone : ethoxyethane [5 : 3 : 2])
Safety guidelines
The extraction and chromatography solvents are both highly
flammable, so they MUST NOT be used near naked flames.
Splashes of the solvent can cause SEVERE EYE DAMAGE, so you MUST
wear eye protection throughout the practical procedure.
Some people are allergic to kiwi fruit. If you are, you should NOT carry
out this practical activity. Alternative suggestions are given by Science
and Plants for Schools (see Teacher’s and Technician’s notes).
Equipment and materials
Needed by each person
Equipment
• Eye protection (safety spectacles or goggles)
• Plastic pipette tip OR a fine-tipped glass Pasteur pipette
• 1 cm3 plastic pipette (not a syringe)
• 1.5 cm3 microcentrifuge tube
• Plasticine®, BluTack ® or a rack for standing the microcentrifuge tube
vertically
• Knife The extraction solvent consists
• Pair of forceps of 3 parts propanone (also called
• Ruler propan-2-one or acetone) plus 2
• Sharp pencil with a fine point parts ethoxyethane (also called
diethyl ether or ether).
Materials
• Slice of kiwi fruit, in a plastic bag to stop it from drying out The chromatography solvent
• Approximately 1 cm3 of extraction solvent in a closed glass container consists of 5 parts cyclohexane,
• Approximately 2 cm 3 of chromatography solvent in a closed glass 3 parts propanone (also called
container propan-2-one or acetone) and 2
• Strip of thin layer chromatography (TLC) sheet, cut to fit inside the parts ethoxyethane (also called
bottle that contains the chromatography solvent. diethyl ether or ether).
www.ncbe.reading.ac.uk 2 Copyright © Dean Madden, 2015
NCBE, University of Reading TLC of photosynthetic pigments
Procedure
1 Before you start, put on the eye protection (safety glasses or
goggles).
2 Using the 1 cm3 pipette, carefully add extraction solvent to the
microcentrifuge tube to the level shown in Figure 1 (just above
the tapered part of the tube).
Fig. 1
DANGER
3 Close the microcentrifuge tube and the extraction solvent bottle
tightly to prevent evaporation of the solvent.
4 Take the kiwi fruit out of the plastic bag. Place the kiwi fruit on
the bag then use the knife to cut, from the kiwi fruit, a strip of
skin at least 30 mm long with a layer of green fruit flesh attached.
The fleshy layer attached to the skin should be approximately 1 mm thick.
5 Cut the kiwi fruit strip into two rectangles, each approximately
5 mm x 15 mm.
6 Use the forceps to place the two pieces of kiwi fruit in the
microcentrifuge tube.
7 With the forceps, squeeze the skin and flesh against the side of
the tube. Also squeeze the kiwi fruit pieces between the forceps.
The liquid will slowly develop a green colour.
8 After approximately a minute of squeezing, use the forceps to
push the fruit pieces to the bottom of the tube.
9 Close the tube and stand it upright on a piece of Plasticine®, BluTack ®
or in a rack. After about a minute the denser liquid will sink to the bottom
of the tube and a less dense, bright green layer will rise to the top of the
tube. Between the two layers of liquid you will see debris from the kiwi flesh.
www.ncbe.reading.ac.uk 3 Copyright © Dean Madden, 2015
NCBE, University of Reading TLC of photosynthetic pigments
10 Stand the bottle of chromatography solvent on a level surface.
Without opening the bottle, measure and record the depth of the
solvent in the bottle.
11 IMPORTANT: In this step, you must be VERY careful not
to damage the surface of the thin layer chromatography
(TLC) strip. Treat it with TLC (tender, loving, care). If you
do damage the surface, however, ask for a new TLC strip.
Try not to touch the white layer on the TLC strip with your fingers
— hold it by its edges. Very carefully, without pressing, draw the
pencil tip across the strip, 5 mm higher than the depth of the
chromatography solvent, making a line as shown in Figure 2.
Fig. 2
TLC strip
DANGER
Pencil line
(Origin)
5 mm
Depth of chromatography
solvent
12 Next, take the Pasteur pipette or plastic pipette tip. Open the
microcentrifuge tube and place the pipette tip just below the
surface of the top green layer of solvent. Some of the green liquid
will be drawn into the tip. Do not let the tip enter the liquid
below the top green layer. Close the microcentrifuge tube.
13 Hold the tip of the pipette vertically and briefly touch the middle
of the pencil line on the TLC strip with the pipette tip so that a
single, small drop soaks onto the white coating. Remove the tip
immediately.
14 When the spot has dried, place a second drop of the green kiwi
extract on the same spot. Continue adding spots of liquid, waiting
for each spot to dry after application until you have a small
green ring 3–5 mm diameter. You will need to apply about 60–100
drops of kiwi extract.
www.ncbe.reading.ac.uk 4 Copyright © Dean Madden, 2015
NCBE, University of Reading TLC of photosynthetic pigments
15 Remove the cap from the bottle of chromatography solvent.
Holding the top of the TLC strip (the end furthest from the
green ring you have produced) with forceps, lower the strip very
carefully into the bottle. As you do so, check that the ring and
pencil line will lie above the level of the solvent (see Figure 3). If
it looks as if the ring will be submerged, stop and use the plastic
pipette to remove a small amount of the chromatography solvent
and try again.
Fig. 3
Pencil TLC strip
line must
be above
solvent
16 Ensure that the TLC strip is not touching the sides of the bottle,
except where it leans against the bottle at the top. Replace the cap
on the bottle and check that the solvent is soaking up the strip.
17 When the solvent has reached approximately 2 mm from the end
of the strip (this will take several minutes), use forceps to remove
the strip from the bottle and immediately mark the exact position
of the solvent front on the TLC strip with a pencil.
18 Replace the cap on the bottle to prevent the solvent from
evaporating.
19 As quickly and accurately as you can, use a sharp pencil to mark
the position of the centre of each different pigment on the TLC
strip (see Figure 4).
Fig. 4
Origin Pigment spots Solvent
front
www.ncbe.reading.ac.uk 5 Copyright © Dean Madden, 2015
NCBE, University of Reading TLC of photosynthetic pigments
20 Measure the distance from the origin to the solvent front. Record
your result to the nearest 0.5 mm. Record your answer to 1 decimal
place.
21 Measure the distances from the origin to the centre of each
pigment spot to the nearest 0.5 mm. Record the colour of each
spot as well. Write your results in a copy of Table 1; if you see
more pigment spots than there is space for, extend the table
appropriately.
Table 1 Pigment Pigment Distance from origin
code colour (mm)
A
(closest to origin)
B
Increasing
C distance from
origin
D
22 From your results, calculate the Rf values for each pigment as follows:
Rf = distance moved by pigment
distance from origin to solvent front
23 Record your results in a copy of Table 2, below, to 2 decimal places
(extend the table if necessary).
Table 2
Your
pigment Rf value
code
A
(closest to origin)
www.ncbe.reading.ac.uk 6 Copyright © Dean Madden, 2015
NCBE, University of Reading TLC of photosynthetic pigments
24 Table 3 shows Rf values for several different photosynthetic
pig ments. T hese values were obta ined using t he same
chromatography solvent as the one you have used. Use the Rf
values in Table 3 and your own calculated Rf values to complete
a copy of Table 4.
Table 3
Pigment name Rf value
Xanthophyll 0.40
Chlorophyll B 0.42
Chlorophyll A 0.47
Phaeophytin 0.55
Carotene 0.93
Table 4
Pigment code Your Rf value Suggested pigment
A
(closest to origin)
Additional investigations
1 Compare chromatograms produced by the green variety of kiwi
fruit ‘Hayward’ with that produced by a golden variety such as
‘Gold’ or ‘SunGold’.
2 The Science and Plants for Schools (SAPS) website has numerous
suggestions for thin layer chromatography of plant pigments
(including anthocyanins from coloured leaves, etc) see: ‘TLC of
plant photosynthetic pigments’: www.saps.org.uk/secondary/
teaching-resources/1347-a-level-set-practials-tlc
www.ncbe.reading.ac.uk 7 Copyright © Dean Madden, 2015
NCBE, University of Reading TLC of photosynthetic pigments
Teacher’s and technician’s notes
Timing
This practical activity can easily be completed in 40–60 minutes.
Troubleshooting
To obtain good results, it is important that students produce a small,
concentrated spot of pigment on the TLC sheet. They will also need to
mark the solvent front immediately on removing the TLC sheet from
the bottle, as the solvent will evaporate quickly. The pigment spots can
also fade rapidly, so the position of these should be marked as soon as
possible after marking the solvent front.
Safety
Solvents
In contrast to similar protocols, only small volumes of the solvents are
used here, and almost always in containers that are or can be closed.
The solvents used in this activity are highly flammable however and
there is a serious risk of the liquid or vapour catching fire. Therefore
the solvents MUST NOT be handled where there are naked flames.
Ensure that the room is well ventilated and that teachers and
technicians are familiar with appropriate ways of putting out fires.
In addition, the solvent can cause severe eye damage, so eye
protection MUST be worn. Teachers must assess whether students
should wear eye protection at all times while in the laboratory (for
example, if there is a risk of students who are still carrying out the
practical activity splashing solvent onto those who have finished the
work).
The solvent bottles supplied to students should be marked with
the signal word ‘DANGER’, a ‘Flammable’ pictogram (GHS02) and an
‘Exclamation mark’ pictogram (GHS07).
After the activity, the solvents should be disposed of in accordance
with the institution’s usual procedures. The solvents MUST NOT be
emptied down a drain.
Schools in England, Wales and Northern Ireland should refer to
CLEAPSS Hazcards 45A and 85 for further guidance on handling
and disposing of the solvents. Similar advice is offered to schools in
Scotland by SSERC.
Kiwi fruit allergy
Some people are allergic to kiwi fruit. Such people should NOT
carry out this practical activity. Alternative suggestions are given
on the Science and Plants for Schools (SAPS) website (see page 10 of this
document).
Anaphylaxis campaign has more information about kiwi fruit allergy:
www.anaphylaxis.org.uk (look under ‘food’ in the ‘Factsheet’ section).
www.ncbe.reading.ac.uk 8 Copyright © Dean Madden, 2015
NCBE, University of Reading TLC of photosynthetic pigments
Equipment and materials
Needed by each student
Equipment
• a glass, screw-capped ‘universal’ bottle (sometimes called a ‘wide-
mouthed McCartney bottle’) or similar. Note: some plastics will dissolve
in the solvent used for chromatography, so it is important to use a glass
container, with a lid made of metal or a suitable plastic (one that does not
dissolve in the solvent).
• a newly-sharpened pencil with a fine point (HB is suitable).
• a pair of blunt/coarse forceps (metal, not plastic).
• a plastic (polypropylene) pipette, for handling the chromatography
solvent. Note: A 1 cm3 pipette or similar is suitable: candidates do not need to
measure out exact volumes. The tip of the pipette must be able to fit inside the
container of chromatography solvent. A plastic syringe is unsuitable for this
task because the solvent will dissolve the ink markings on the syringe barrel.
• a clean 1.5 cm3 microcentrifuge tube (‘Eppendorf’ type).
• a piece of Plasticine® or BluTack ® or a rack so that the microcentrifuge
tube can be stood vertically.
• a clean plastic (polypropylene) micropipette tip (e.g., a non-graduated
100 µL tip) OR a clean fine-tipped glass Pasteur pipette OR a drawn
melting point tube.
• a knife (a plastic ‘disposable’ picnic-type knife is suitable).
• a ruler for measuring millimetre lengths.
• eye protection (safety spectacles or goggles).
Materials
• a transverse slice of freshly-cut, ripe, kiwi fruit, approximately 6
mm thick, provided in a plastic bag. Note: This MUST be of the species
Actinidia deliciosa, var. ‘Hayward’. Almost all kiwi fruit sold in the UK are
of the ‘Hayward’ variety, and several supermarkets label them as such. It
is essential to use this particular variety as it is the only one that contains
intact chloroplasts in the fruit. Once the fruit has been cut, the photosynthetic
pigments in the tissue degrade rapidly, so it is important to slice the fruit
no more than an hour or so before the practical task starts. Recently-bought
kiwi fruit or those that have been stored in a fridge for a week or so will be
fine for this task, but if the uncut kiwi fruit are very hard to the touch, they
should be left for a few days at room temperature to ripen.
• a strip of thin layer chromatography (TLC) sheet, cut to fit inside the
glass screw-capped container. Note: Typically, a rectangle of TLC sheet
15 mm x 75 mm is required. The long edges of the strip must NOT touch the
inside of the glass container. The white coating on the TLC plate can easily
be scratched off, so it must be cut and handled with care. It is an important
precaution to wear disposable gloves when cutting the sheet, to prevent oils
from your skin from contaminating it. (The students do not need gloves: they
will be told to handle the strip by its edges only.)
• approximately 1 cm3 of freshly-prepared extraction solvent in a
closed glass container labelled with appropriate safety warnings*.
The extraction solvent consists of 3 parts propanone (also called
propan-2-one or acetone) plus 2 parts ethoxyethane (also called
diethyl ether or ether). Note: No alternative solvents should be used, as
the procedure will not give the expected results if they are. For example, do
not use petroleum ether.
• approximately 2 cm3 of freshly-prepared chromatography solvent in
a closed glass container, labelled with appropriate safety warnings*.
www.ncbe.reading.ac.uk 9 Copyright © Dean Madden, 2015
NCBE, University of Reading TLC of photosynthetic pigments
The chromatography solvent consists of 5 parts cyclohexane, 3
parts propanone (also called propan-2-one or acetone) and 2 parts
ethoxyethane (also called diethyl ether or ether).
* it is advisable to use paper labels on the bottles, as the solvent may wash
away ink applied directly to the glass or lid of the bottles. See page 8 of this
document for safety guidance.
Specimen results
The photograph on the right shows a typical chromatogram obtained
using this method.
Suppliers
TLC Sheets
To obtain the same Rf values as those in Table 3, it is essential to use
Macherey-Nagel DC Polygram® SIL G/UV254 TLC sheets. These are available
from Fisher Scientific in packs of 50 sheets (Product code: 10640493).
Each sheet measures 50 x 200 mm.
Fisher Scientific UK Ltd
Bishop Meadow Road
Loughborough T: 01509 555 500
LE11 5RG W: www.fisher.co.uk
NCBE TLC Pack
The NCBE provides a pack of materials with TLC sheets, microcentrifuge
tubes and pipette tips that can be used for this practical activity. See:
www.ncbe.reading.ac.uk
Additional information
Crowhurst, R.N. et al (2008) Analysis of expressed sequence tags from
Actinidia: applications of a cross-species EST database for gene
discovery in areas of flavor, health, color and ripening. BMC Genomics
9, 351. The electronic version of this article can be accessed free-of-charge at:
www.biomedcentral.com/1471-2164/9/351
Tomkins, S.P. and Miller, M.B. (1994) A rapid extraction and fast
separation of leaf pigments using thin layer chromatography. School
Science Review 75 (273) 69–72.
Science and Plants for Schools (SAPS) has numerous suggestions for TLC of
pigments from other plant materials: ‘TLC of plant photosynthetic
pigments’: www.saps.org.uk/secondary/teaching-resources/1347-
a-level-set-practials-tlc
www.ncbe.reading.ac.uk 10 Copyright © Dean Madden, 2015
