Group Members: ______________________________________________________________________________

Grade & Section: __________________________ Date: ____________ Score: ________

SEPARATING MIXTURES (LEAF PIGMENTS) USING PAPER CHROMATOGRAPHY

I. Background Information
Paper chromatography is a process that uses chromatography paper to separate and
identify the different substances in a mixture. Chromatography means “to write with
color.” The substances in the mixture dissolve in the alcohol and move up the paper.
The heavier substances move up the paper more slowly. The lighter substances move
up the paper more quickly. So heavy and light substances get separated from one
another on the paper.
Plants contain chlorophyll, a green pigment, as well as carotenoids, pigments that
range in color from red to orange to yellow.

II. Objectives
The aim of this activity is to extract photosynthetic pigments from leaves and to
separate them using thin layer chromatography in order to see multiple different
pigments from a single extract and to allow the identification of the pigments through
the calculation of their Rf values and observations of their colour.

Safety Notes: The solvents used to extract the pigments from the leaves and run the
chromatogram are flammable so ensure that there are no naked flames or other sources
of ignition in the laboratory. Wear eye protection when using the solvent. Avoid inhaling
vapour as much as possible. Ensure laboratory is well-ventilated. Wear lab gown during
the activity.

III. Materials
Isopropyl alcohol acetone
Mayana leaves spinach leaves
Mohr pipette ruler
scissors chromatography
paper pencil tape
beaker mortar and pestle
capillary tube Pasteur pipet

IV. Procedure
1. Obtain a strip of chromatography paper.

2. Use a ruler to measure and draw a light pencil line 2-cm above
the bottom of the paper strip.

3. Place plant material (ripped or cut up into small pieces if whole

leaves are too big) in a mortar and grind with the pestle.
4. Add 1-2 mL of the extraction solvent (acetone) and continue to
grind until the liquid appears dark green in color.
5. Dip a capillary tube/fine pipette into the liquid and transfer a tiny
drop of the extract onto the middle of the pencil line on the
chromatography paper (see figure 1). Touch it briefly to ensure
your spot gets no bigger than 3mm diameter.
6. Allow the spot to dry and then add another drop. Continue
adding drops and drying until the spot is dark green (5-10 drops Figure 1. Adding a
is usually enough). drop of extract

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7. Tape the top of the paper strip to a pencil so that the end of the strip hangs down. The
pencil should be able to sit across the top of the beaker with the
bottom of the paper strip just touching the bottom of the beaker. Cut
off any excess paper from the TOP of the strip if it is too long.

8. Remove the pencil/paper strip contraption from the beaker for the
moment.

9. Carefully add running solvent (isopropyl alcohol) to the beaker until it

reaches a depth of 1-cm in the beaker. Repeat Steps 2-9 for the setup
using acetone as solvent.

10. Lay the pencil across the top of the beaker with the paper strip
extending into the alcohol. Make sure that the level of the
alcohol/acetone is below the line on your paper strip!

11. Observe as the alcohol gets absorbed and travels up the paper by
capillary action. This may take up to 5-20 minutes. Do not touch your
experiment during this time. Have a time-lapse recording of this to Figure 2. Model
observe the capillary action. for step 10

12. When the solvent front (the furthest the alcohol/acetone has travelled) has absorbed to
approximately 1-cm below the pencil line, you may remove the pencil/paper strip from the
beaker to dry on your counter.

13. Immediately mark the location of the solvent front using a pencil before the solvent
(alcohol) evaporates.

14. Photograph the chromatogram (for analysis of the photograph) and mark the location of
each colored spot. (The colored spots can fade and become difficult to locate as time goes
by).

15. Record the color of each spot and how

far each spot has travelled from the
original location (use mm).

16. Record your results in the data table.

17. Using a ruler and the following

formula, measure the Rf values of
each pigment spot (use the center of
the spot). Use the Rf values to identify
the plant pigment.

Since the fastest molecules will travel

the greatest distance, or to the highest
point along the strip, the relative
distances can be measured, and the
flow rate (migration) of the molecules
(Rf) can be calculated by using the Figure 3: Measurements needed to calculate an Rf value
following formula:

Use the following Rf values in Table 1 and 2 to identify the plant pigment.

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Table 1: Information to help identify pigments
Pigment Rf value range Color Relative position
Carotene 0.89-0.98 Yellow Very close to the solvent front
Pheophytin a 0.42-0.49 Grey Below the top yellow, above the greens

Pheophytin b 0.33-0.40 Brown Below the top yellow, above the greens

Chlorophyll a 0.24-0.30 Blue green Above the other green, below the grey

Chlorophyll b 0.20-0.26 Green Below the other green

Xanthophylls 0.04-0.28 Yellow Below, or almost at the same level of, the
highest green

Note: Rf values are given as a range due to the fact that the components of the solvent mixture
evaporate at different rates. So the solvent mixture you are using might be in slightly different
proportions to other times this chromatography is carried out. It is particularly dependent on
environmental temperature. Also note that the solvent evaporates very quickly so the solvent front
must be identified as soon as the plate is removed from the glassware – slight errors in the
measurement of the solvent front or of the center of the pigment spot will change the calculated Rf
value.

Table 2: Information to help identify possible Xanthophylls

Pigment Rf value range Color Relative position
Lutein 0.22-0.28 Yellow Below, or almost at the same level of, the
highest green
Violaxanthin 0.13-0.19 Yellow Below, or almost at the same level of, the
highest green
Neoxanthin 0.04-0.09 Yellow Below, or almost at the same level of, the
highest green

V. Data
Spinach Chromatogram (Attach the chromatogram here or its picture. Put labels.)
Chromatogram (Isopropyl Alcohol) Chromatogram (Acetone)

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 Mayana Chromatogram (Attach the chromatogram here or its picture. Put labels.)
Chromatogram (Isopropyl Alcohol) Chromatogram (Acetone)

Pigments in Spinach Leaves (Isopropyl Alcohol)

Distance Alcohol Traveled (Spinach): ___________ mm

Color of Pigment Distance Traveled (mm) Rf value Pigment’s Identity

Pigments in Spinach Leaves (Acetone)

Distance Acetone Traveled (Spinach): ___________ mm

Color of Pigment Distance Traveled (mm) Rf value Pigment’s Identity

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 Pigments in Mayana Leaves (Isopropyl Alcohol)

Distance Alcohol Traveled (Mayana): ___________ mm

Color of Pigment Distance Traveled (mm) Rf value Pigment’s Identity

Pigments in Mayana Leaves (Acetone)

Distance Acetone Traveled (Mayana): ___________ mm

Color of Pigment Distance Traveled (mm) Rf value Pigment’s Identity

VI. Analysis Questions (You may use the back part of this paper for your answers.)
1. Did the leaf you test contain different pigments? Compare and contrast the pigments in
spinach and Mayana. Refer to your results to support your answer.

2. Why did the separation of pigments in the spinach/mayana extract occur as it did? (i.e.
How does paper chromatography work?)

3. Why are spinach leaves green even though other pigments are present? What are the
factors contributing to the color of mayana leaves?

4. Cite other applications of paper chromatography in real life.

Reference:
Columbia University. (2022). Chromatography of spinach.08 - columbia.edu. Retrieved October 14, 2022,
from
http://www.columbia.edu/~mvh7/STEP/Regents%20Bio/Photosynthesis/Chromatography%252Bof
%252BSpinach.08.pdf
Symonds, A. (2022, June 15). A-level set practicals - TLC of plant photosynthetic pigments. Science & Plants
for Schools. Retrieved October 14, 2022, from https://www.saps.org.uk/teaching-
resources/resources/1347/a-level-set-practicals-tlc-of-plant-photosynthetic-pigments/

Documentation: Attach pictures of your experiment.

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