1) OBJECTIVES

1.1 SCIENTIFIC OBJECTIVES:

To apply the technique of paper chromatography as a method of separating
individual photosynthetic pigments contained in plant tissue.
To find out the solubility of plant pigments in two different types of solvent.
To find the best solvent ratio for separating of pigments.
2) INTRODUCTION
2.1 THEORY AND CONCEPT OF CHROMATOGRAPHY
Chromatography is a technique used to separate molecules on the basis of differences in
size, shape, mass, charge, solubility and adsorption properties. The term chromatography is
derived from Greek words Chroma (colour) and Graphe (write). In the other words,
Chromatography is a technique for separating and identifying mixtures of compounds based
upon their different rates of adsorption. There are several types of chromatography includes
paper chromatography, thin layer chromatography and others. All types of chromatography work
on the same principle. They all have a stationary phase (usually a solid, thick liquid or bonded
coating) and a mobile phase (usually a liquid or a gas). In any chromatographic technique, a
stationary stays fixed in one place, while a mobile phase or eluent moves trough it or across it.
Chromatography of leaves is an experiment conducted to separate the photosynthetic
pigment in the leaf. A pigment is simply a molecule that absorbs and reflects light.
Photosynthetic pigments are usually present in chloroplasts and influence the colour of leaves.
Plant leaves contain several common types of photosynthetic pigments, which are chlorophyll
includes chlorophyll a (dark green) and chlorophyll b (yellowish-green), xanthophylls (yellow)
and carotenoids (orange). Chlorophyll are greenish pigments that captures the energy of sunlight.
Leaves, which are green in colour are said to have

abundance of chlorophyll pigment.

Carotenoids are usually called accessory pigment, which cannot transfer sunlight energy directly
to photosynthetic pathway, but must pass their absorbed energy to chlorophyll. Other than that,
xantrophyll is also an accessory pigments that are known as light-absorbing compounds, found in
photosynthetic organisms that work in conjunction with chlorophyll a. Some leaves contain
another accessory pigment called anthocyanin. If the leaves have abundance of this pigment, the
leaves may appear in red of purple in colour according to its pH. This pigment is water soluble
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while other pigment are not. In photosynthetic tissues, anthocyanins have been shown to act as a
"sunscreen", protecting cells from high-light damage by absorbing blue-green and ultraviolet
light, thereby protecting the tissues from high-light stress.
The method of separation used is paper chromatography, which means that the pigments
are separated based on rates moved by each pigment. The extract is also separated based on the
solubility of the various pigments presence in the leaf. In this experiment, normally, the
stationary phase is a solid such as paper, starch, alumina, or silica and the mobile phase is a
liquid such as water, common organic solvents such as ethanol, or solvent mixtures. The basis for
this paper chromatography is the fact that porous paper, cellulose, has an enormous surface area
to which molecules or ions of substances are attracted (adsorbed) and then released (desorbed)
into the solvent as an aqueous solution passes over the paper.
Separation of components occur, that is, they will travel at different speeds in a moving
solvent because the varying attractions between these pigment components and the paper. This
method of separation is known as partitioning. Also, the pigments will be carried along at
different rates because they are not equally soluble. Therefore, the less soluble pigments will
move slower up the paper than the more soluble pigments. This condition will produce a
developing chromatogram.
The identity of the components can be deduced by comparing a chromatogram of the
unknown mixture with chromatograms of mixtures with known composition (standards). By the
way, the distance traveled by a particular compound can also be used to identify the compound.
The ratio of the distance traveled by a compound to that of the solvent front is known as the R f
value. Retention factor or Rf value is applied in chromatography to make the technique more
scientific than a mere analysis. Rf is defined as the distance travelled by the compound divided
by the distance traveled by the solvent as shown in the formulae:

2.2 HYPOTHESES

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1. Anthocyanin soluble in water and insoluble in any non-polar solvent while

carotene, xantrophyll and both chlorophyll a and b insoluble in water and soluble in
non-polar solvent.
2. The more soluble the photosynthetic pigment, the further the movement of that
photosynthetic pigment.
3. The higher the rate of adsorption between pigment particle and chromatography
paper, the further the movement of the pigment particle above the chromatography
paper.
4. Carotene is the most soluble pigment in non-polar solvent, followed by xanthophyll,
chlorophyll a and finally chlorophyll b.
2.3 VARIABLES
Manipulated variable :

Chromatography solvent.

Responding variable :

Retention factor (Rf value).

Constant variable

Types of leaves used.

3) METHODOLOGY
3.1

CHEMICALS

30mL of propanone, 30mL of water, 3 or 4 fresh spinach leaves.

3.2

APPARATUS

a pair of scissors, a pencil, a ruler, pestle and mortar, 50mL beakers, chromatography paper, pin,
glass tube, chromatography chamber.

3.3

DIAGRAM

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3.4

PROCEDURE

PART A: PREPARATION OF PIGMENT EXTRACT.

1.
2.
3.
4.
5.
6.

Finely, cut up some spinach leaves and fill a mortar to about 2cm depth.
Mash the leaves using pestle and mortar.
Pour 15ml of propanone into the mortar.
After 10 minutes, the chlorophyll will dissolve in the propanone.
Remove the leaves from the mortar.
Repeat step 1-5 by using water as solvent.

PART B: PREPARATION OF CHROMATOGRAPHY CHAMBER AND PAPER.

1. Use a thin strip of chromatography paper, making sure that it is not longer than the
chromatography chamber.
2. On the chromatography papers, draw a pencil line 2cm from the bottom.

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Avoid using pen when drawing the line on the paper as the ink will dissolve and the

line will slowly move up the paper.

3. Use a fine glass tube to put liquid from leaves extract onto the center of the line on that two
chromatography papers separately. Keep the spot as small as possible.
4. Allow the spot to dry, and then add another same spot on top. The idea is to build up a very

concentrated small spot on the paper.

Make sure that there is enough transferring of pigments onto the chromatography
paper in order to achieve clear separation.

5. Pin paper to the hook so that when placed in the chromatography chamber, the paper is just
clear of its base.
6. Make a solvent with ratio 50:50 of propanone and water.
7. Pour the mix solvent of propanone and water into the chromatography chamber.

Make sure that propanone is keep away from sparks or open flame.
The experiment must be done under vent hood, and beaker containing propanone

must remain covered and avoid direct inhalation of the propanone.

8. Repeat step 1-6 with ratio of propanone and water 100:0, 0:100, 70:30 and 30:70.
9. Hang the paper so that it dips in the solvent.
Immerse the chromatography paper just below the pencil line.
10. Cover the chromatography chamber with its lid.

Avoid moving the chromatography chamber in any way once the chromatography has
started.

11. Leave the experiment until the solvent has soaked near to the top, and then remove the
paper from the chamber.
12. Mark how high the solvent gets on the paper with a pencil and let the chromatography dry.
13. The results obtained are observed and recorded in a table.
4) RESULTS AND ANALYSIS
4.1

TABULATION OF DATA
SUBJECT

Chlorophyll

Chlorophyll b

Xanthophyll

Carotene

Anthocyanin

Color of pigment

a
Blue-green

Yellow-green

Yellow

Orange

Red/Purple

Pigment origin

2.0

2.0

2.0

2.0

2.0

(cm)
Pigment distance

5.1

4.9

6.4

7.8

from origin (cm)

Solvent distance

8.0

8.0

8.0

8.0

from origin (cm)

Rf value

0.63

0.61

0.80

0.98

(calculated)

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TABLE 1: DATA FOR EXTRACTION OF SPINACH LEAF USING SOLVENT RATIO OF

PROPANONE:WATER, 100:0
SUBJECT

Chlorophyll

Chlorophyll b

Xanthophyll

Carotene

Anthocyanin

Color of pigment

a
Blue-green

Yellow-green

Yellow

Orange

Red/Purple

Pigment origin

2.0

2.0

2.0

2.0

2.0

(cm)
Pigment distance

7.7

from origin (cm)

Solvent distance

8.0

from origin (cm)

Rf value

0.96

(calculated)

TABLE 2: DATA FOR EXTRACTION OF SPINACH LEAF USING SOLVENT RATIO OF

PROPANONE:WATER, 0:100.

SUBJECT

Chlorophyll

Chlorophyll b

Xanthophyll

Carotene

Anthocyanin

Color of pigment

a
Blue-green

Yellow-green

Yellow

Orange

Red/Purple

Pigment origin

2.0

2.0

2.0

2.0

2.0

(cm)
Pigment distance

4.0

3.4

4.5

5.0

2.3

from origin (cm)

Solvent distance

8.0

8.0

8.0

8.0

8.0

from origin (cm)

Rf value

0.50

0.43

0.56

0.63

0.29

(calculated)

TABLE 3: DATA FOR EXTRACTION OF SPINACH LEAF USING SOLVENT RATIO OF

PROPANONE:WATER, 70:30.
SUBJECT

Chlorophyll

Chlorophyll b

Xanthophyll

Carotene

Anthocyanin

Color of pigment

a
Blue-green

Yellow-green

Yellow

Orange

Red/Purple

Pigment origin

2.0

2.0

2.0

2.0

2.0

(cm)
Pigment distance

1.5

1.3

1.9

2.2

5.4

from origin (cm)

Solvent distance

8.0

8.0

8.0

8.0

8.0

from origin (cm)

Rf value

0.19

0.16

0.24

0.28

0.68

(calculated)

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TABLE 4: DATA FOR EXTRACTION OF SPINACH LEAF USING SOLVENT RATIO OF

PROPANONE:WATER,30:70.

SUBJECT

Chlorophyll

Chlorophyll b

Xanthophyll

Carotene

Anthocyanin

Color of pigment

a
Blue-green

Yellow-green

Yellow

Orange

Red/Purple

Pigment origin

2.0

2.0

2.0

2.0

2.0

(cm)
Pigment distance

2.6

2.5

3.2

3.6

3.8

from origin (cm)

Solvent distance

8.0

8.0

8.0

8.0

8.0

from origin (cm)

Rf value

0.33

0.31

0.40

0.45

0.48

(calculated)

TABLE 5: DATA FOR EXTRACTION OF SPINACH LEAF USING SOLVENT RATIO OF

PROPANE:WATER, 50:50.

4.2

DIAGRAM

Anthocyanin

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5) DISCUSSION

Adsorption is the process by which atom, molecules, or ions from a substances either a gas is
being attracted or dissolved solid and liquid held to the surface of another substances which an
adsorbent. Absorption is the process by which a fluid is dissolved by a solid (absorbent) or a
liquid. Paper chromatography is a process in which pigments are separated from an initial
concentrated solution through the process of capillary action. A solvent is placed at the bottom of
the paper. As the solution travels up the paper, like soluble pigments will travel with the solvent
until the bonds between the solvent and pigment become so weak that it must break attraction
and imprint itself a certain height up the paper.
In this experiment, propanone and water were used as solvent to decolourize the leaf. The
separation into constituent parts is dependent on the differences in solubility in propanone and
water. Polarity is when one end is positively charged while the other end is negatively charged.
Water molecules are polar in nature while propanone is made of non-polar molecules. Only other
polar molecules can dissolve in water because polar molecules dissolve only in polar solvents
and non-polar molecules dissolves only in non-polar solvents. Therefore, chromatography paper
was used to determine the pigments that present in the leaf. Theoretically, there are five types of
pigment present in the leaf which were chlorophyll a and b, xanthophyll, carotene, and
anthocyanin. Carotene has the highest pigment distance from origin. Therefore, from this

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experiment, it is proved that there are five types of pigment present in the leaf which that have
been stated before.
All pigments have their own functions. Chlorophyll a works to absorb light for used in
photosynthesis. It readily absorbs violet/blue and red light but not much lighter blue, green and
yellow light. Chlorophyll b is structurally only slightly different from chlorophyll a but its
absorption spectrum is somewhat different. Chlorophyll b absorbs more in the blue and orangered ranges. Thus, chlorophylls appear green because the pigments absorb light in all of the other
colour ranges and only green is transmitted to our eyes. Carotenoids act as accessory pigments
which absorbs wavelengths of the light that chlorophyll cannot absorb effectively, enabling the
plant to use more suns energy. As a result, carotenoids appear in various shade of yellow or
yellow-oranges to our eyes. Xanthophyll also acts as accessory pigments that work with
chlorophyll a. For anthocyanin, it does not participate in photosynthesis. It occurs widely among
higher plants and generally gives colour to flower, but also occur in leaves and fruits. These
pigments help to protect against excessive sunlight that can damage some leaf tissues.
From the results of extraction of spinach leaf using propanone,:water, 50:50 it shows that
carotene has the highest pigment distance from origin which was 7.2cm followed by xanthophyll
with 6.4cm. The highest pigment distance from origin showed that carotene was the most nonpolar and it was observed that carotene has orange colour of pigment while xanthophyll pigment
has yellow colour. Chlorophyll a has a higher pigment distance from origin than chlorophyll b
which was 5.1cm. It was observed that chlorophyll a has blue-green coloured while chlorophyll b
has yellow-green colour. Chlorophyll b has the lowest pigment distance from origin which was
4.9cm because it is the most polar of the pigments. Carotene is the most soluble because it has no
polar group because of its molecular structures. Xanthophyll is classified in two polar groups
while chlorophyll a is in five polar groups and chlorophyll b is in six polar groups that make it
least soluble in the solvent.
Anthocyanin which has red or purple colour of pigment shows no changes in pigment
distances and solvent from origin when it was extracted in propanone. So, anthocyanin does not
have Retention factor,

Rf

. The reason why there are only chlorophyll a and b, xanthophyll and

carotene extracts from propanone solvent is because carotene and xanthophyll are hydrophobic
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due to the large hydrocarbon chains and are completely insoluble in water and soluble in nonpolar solvent. Chlorophyll a and b have hydrophobic tails which make them insoluble too.
Chlorophyll a and b, xanthophyll, and carotene have the same value of solvent distance from
origin which is 7.2cm. However, the

Rf

value for each pigment was not same. It was

calculated by dividing the distance travelled by the component with distance travelled by solvent.
Rf

Carotene has the highest

value which was 1.00 followed by xanthophyll with 0.89,

chlorophyll a with 0.71 and chlorophyll b with 0.68. Anthocyanin does not have result because
there was no change in distance from origin. Besides, data for extraction of spinach leaf using
water, only Anthocyanin shows the changes while chlorophyll a and b, xanthophyll, and carotene
does not have any changes in pigment distance and solvent distance from origin and

Rf

value. For anthocyanin, there was a change in pigment distance from origin which was 7.7cm
while the solvent distance from origin is 8.0cm. The
the same formula, so the

Rf

Rf

value has been calculated by using

value for anthocyanin was 0.96.

Anthocyanin is soluble in water because they occur as glycosides involving sugarssuch as

glucose, rhamnose, occasionally pentose. They are water soluble vacuolar pigments that may
appear red, purple, or blue depending on the pH indicators because their color changes with pH;
they are pink in acidic solutions (pH<7), purple in neutral solutions (pH~7), greenish- yellow in
alkaline solutions (pH>7), and colourless in very alkaline solutions, where the pigment is
completely reduced. The reds, the purples, and their blended combinations that decorate autumn
foliage come from anthocyanin. Thus, the colour of spinach leaves still in green as chlorophyll is
dominated the leaves but it may turns to purple when it comes to summer as anthocyanin
pigments are actively produced towards the end of the summer.
There are few precautions should be taken in this experiment which are handling the paper
with dry hands so it will not allow for additional uncontrolled moisture to the paper which will
interfere with the separation of components. Next, avoid using pen when the line is drawn on the
paper as the ink will dissolve and the line will slowly move up the paper, make sure that there is
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enough transferring of pigments onto the chromatography paper in order to achieve clear
separation, the propanone is made sure to be kept away from sparks or open flame, the
experiment is done under a vent hood, but beakers containing propanone must remain covered
and avoid direct inhalation of the solvent. Finally, the chromatography paper is immersed just
below the pencil line.

6) CONCLUSIONS

In conclusion, we have identified that there are five types of pigments present in spinach leaf
which are xanthophyll, anthocyanin, carotene, chlorophyll a and chlorophyll b. Each types of
pigments are able to be identified as they produce different types of colour spot on the
chromatography paper. From our experiment, we find that the colour of spot of chlorophyll a is
dark-green while chlorophyll b is yellowish-green, xanthophylls are yellow, caratenoids are
orange and in this experiment, antocyanin comes in pink as the water that we used may be in
acidic condition.
Based on our results, we can conclude that our hypothesis are all accepted. Carotene has the
highest Rf value, followed by xanthophylls, chlorophyll a and chlorophyll b in propanone. Thus,
carotene is the most soluble pigment in non-polar solvent as it has furthest movement in
chromatography paper and highest rate of adsorption between pigment particle and
chromatography paper followed by xanthophyll, chlorophyll a and b. These four pigments are
soluble in non-polar solvent and insoluble in water. Yet, anthocyanins are soluble in water and
insoluble in non-polar solvent.
Thus, our objectives of the experiment are well achieved. The objectives of the experiment
are to apply the technique of paper chromatography as a method of separating individual plant
pigments contained in plant tissues, we are also need to identify the changes of colour of spot in
chromatography paper and finally we insist to find out the solubility of plant pigments in two
different types of solvent, which are water and propanone.

7) REFLECTIONS
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The tauhidic objective of conducting this experiment is actually to explore the uniqueness of
different types of pigment contained in a leaf created by ALLAH S.W.T by paper
chromatography. Alhamdulillah, special thanks to our Creator, Allah The Almighty, in His
Infinite Wisdom has created different types of pigment in chloroplast in order to sustain the
process of photosynthesis in plants for the benefits of all living organism.
We can view our experiment from the Islamic perspective which is it is Allah, the best
planner of all things. Allah (SWT) ordained that plants, people and animal receive their food
from what plants produce in green factories. These green factories are built up by the plant at the
beginning of its growth and are called chloroplasts which contain chlorophyll. In Al- Quran,
chlorophyll is called Al-Khadir (green substance), where the plant utilizes the light energy and
changes it into chemical energy leading ultimately to production of various kind of grain, fruit
and other parts of vegetation that we see in gardens and fields. The Quran draws our attention to
these facts in the Saying of Allah (SWT),

It is He who sends down water (rain) from the sky. With it We produce vegetation of
all kinds from which (water or plants) We produce green substance (Khadir), out of which We
produce grain in clusters. And out of date palms, from their spathes come forth clusters of
dates hanging low and near, and (We produce) gardens of grapes, olives and pomegranates,
each similar (in leaves and shape), yet different (in fruit and taste). Look at (and think over)
their fruits when they begin to bear fruit, and (look at) the ripeness thereof. Behold! In these
things there are sign for people who believe
(AL-ANAAM:99)

Next, based on our experiment, we find that every creation of Allah comes in different
special characteristics. As we can see, anthocyanins are the only pigments in spinach that soluble
in water instead of other four pigments and this pigment also do not soluble in non-polar solvent
like the others. In the verse of Yunus, Allah said:

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Say, who provides sustenance for you from the heaven or earth? Or who is it that has
power over the ears and the eyes? And who brings forth the livingfrom the dead and brings
forth the dead from living? And who regulates all affairs? They will say ALLAH! then they
say, will you not seek HIS protection.....
(YUNUS:32)

The verse above explain about the Universe and the nature of creation of Allah. There are
many instances in the Al-Quran, which Allah explain of His Wisdom in creating things in
Universe with its variety and diversity, a Universe in which it is possible for life to exist.
Everything that is created by Allah is very significant and special that cannot be taken for
granted. According this experiment, we find out that there are many types of crucial pigment
presence in plant instead of chlorophyll in order to ensure the plant able to adapt with
surrounding environment.

It is Allah Who splits open the seed and the pits. He causes the living to issue from the
dead, and the dead to issue from living. Behold the works of Allah! How can you deny?
(AL-ANAAM:95)
For instance, anthocyanin pigment will change the colour of green spinach to purple or
red during summer. Yet, we in Malaysia do not have that season, so we just can see that spinach
in green only eventhough anthocyanin is among of the pigment presence in that green spinach.
Alhamdulillah, Praise to Allah, The Almighty and The Sustainer of All Mankind.

8) REFERENCES

a. BOOKS
Nazira, Z., Haliza, H., Zahala, G., Kamaril, A. T., Rosmarina, J., Saadah, M. N.
(2011). Photosynthesis. (ms; 130-136)
Malaysia: Pearson Malaysia Sdn. Bhd.
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Biology Text Book. Kuala Lumpur,

 Liew, S. L., Sudani. S., Kamaludin. A. R., Less. S. C., Azlina. A. A., Pariza. Z.
(2014). Photosynthesis. (ms;284-320) Biology For Matriculation Semester 1
Fourth Edition Updated. Shah Alam, Selangor DarulEhsan: Oxford FajarSdn.
Bhd.
b. JOURNAL
Eduard, R. (2014). Journal of chromatography & separation techniques, 5: e124
c. ELECTRONIC SOURCES
Bite Sci-zed. (Oct 29, 2012). Leaf Color Chromatography [Video file]. Retrieved
from https://www.youtube.com/watch?v=qH-AJDqsSII
Science Buddies Staff. (2015, June 15).Paper Chromatography: Advanced
Version 2.Retrieved July 3, 2015 from http://www.sciencebuddies.org/sciencefair-projects/project_ideas/Chem_p010.shtml
Dave,

A.

(2010).

Chlorophyll

Chromatography.

Retrieved

from

http://www.thenakedscientists.com/HTML/experiments/exp/chlorophyllchromatography/
Novanet. (Apr 27, 2013). Chlorophyll chromatography[Video file]. Retrieved
from https://www.youtube.com/watch?v=jiPd5CkCkkU

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