Organic Chemistry Practical Report

Title: Thin-layer Chromatography: Separation of Plant Pigment

By: Lim Wei Gene
ID: 12085189
Sunway Imail: 12085189@imail.sunway.edu.my
Lecturer: Miss Jane Gew Lai Ti
Sunway University
Faculty of Science and Technology
Biological Science Department
BSc. (Hons) Medical Biotechnology

Title:
Thin-layer Chromatography: Separation of Plant Pigment
Objective:
Objective of this experiment is to study the thin-layer chromatography of extracted
leave pigment from spinach and -carotene from tomato paste, and the tomato
juice rainbow.
Introduction:
Thin-layer Chromatography, TLC, is a technique where components of the
mixtures separated by differential migration through a planar bed of the stationary
phase. It is a form of planar chromatography which is similar to the paper
chromatography, but the only different is that the stationary phase of TLC is finelydivided sorbent spread as thin layer on the supporting flat surface such as plastic,
aluminum or glass plate. Separation of sample solute can be done by applying
(spotting) small amount of the sample near the end of the plate and place in the
mobile phase. Mobile phase is a phase when the base of the plate is inserted inside
a particular solvent and allows the solvent to move upwards by capillary action.
Solvent used in mobile phase is depends on the type of sample solute that will be
undergoing chromatography; example, for thin-layer chromatography of vegetable
leaf pigment, solvent used will be the mixture of hexane and acetone with the ratio
70:30 (to prepare 100mL of solvent), for thin-layer chromatography of -carotene
from tomato paste, carrot paste or other type of either fruit or vegetable that
contain -carotene , solvent used will be the mixture of petroleum ether: toluene:
acetone with the ratio 45:1:4 (to prepare 100mL of solvent).
As the separation occurs, each component present in the solute will interact
with the stationary and mobile phase to a various degree which creates the
individual bands of the plate. It is consider as the best separation of the component
when the solvent polarity is adjusted to allow the retardation factor, Rf value, of the
compound that has been separated in the range of 0.4 to 0.6. Rf value is the ratio of
distance traveled by a particular spots from baseline, compared to the distance
traveled by the solvent front which is measured from the baseline. Rf value that has
been calculated is helpful in identifying compound.

Rf value formula:
Rf =

Distance travelled by solute ( pigment )

Distancetravelled by solvent

Plants convert light energy to chemical energy by

photosynthesis with the present of carbon dioxide, CO 2, water,
H2O, and chlorophyll. Photosynthesis of plant occurs in the
organelles called chloroplast. Chloroplast present in plants contain
of several colored pigments, which can be classified into two
different categories; the chlorophylls and carotenes. Chlorophyll
present in the plant can be further differentiated into two types
which is chlorophyll- a (R=CH3) and chlorophyll- b (R=CHO).

Figure 1.0 structure of Chlorophyll a and Chlorophyll-b

As the chlorophyll-a and chlorophyll-b losses the magnesium

ion, Mg2+ ion, which attaches with four nitrogen atom, it forms

the decomposition products which is the pheophytin-a and

pheophytin-b.

Figure 1.1 Structures of Pheophytin A and Pheophytin B

Both chlorophyll and carotene that is present in the plant has
capability of absorbing most of the light wavelength except for
green wavelength which is the only reason why chloroplast is
green in color.

Figure 1.2 Absorption Spectra of Chlorophyll-a and Chlorophyll-b

In fruits such as tomato and carrot, there are two main

components that can be found; the -carotene (yellow-orange in
color) and also the lycopene (red in color) pigment. Both carotene and lycopene pigment are classified as alkenes.

Figure 1.3 Structure of -carotene

Figure 1.4 Structure of Lycopene

When bromine water is added into the tomato juice, it
allows the bromine atom to react with the double bond of the
tomato pigment breaking the double bond in it. Bromine water is

added into the cylinder filled full with tomato juice, allowing the
bromine water to penetrate through the tomato juice. This causes
more and more double bond present in the tomato pigment
started to react with the bromine water and eventually most of
the double bond in tomato pigment will be broken. Slow stirring
while adding bromine water will cause a series of color change in
the cylinder which looks like rainbow.

Procedure:
Part A: Extraction of the leaf pigment.
A small mortar and pestle were used to grind several spinach
leaves thoroughly in a mixture of 4 mL petroleum ether and 2 mL
ethanol. The liquid extracts were transferred to the test tube by
using Pasteur pipet and were swirled gently with an equal volume
of water. Aqueous layer were removed and discarded by using
gravitational filtration method and the petroleum ether extract
were transferred to the Erlenmeyer flask. Several spatula-tips full
of anhydrous sodium sulfate were added. The solution were
decant from the drying agent after 5-10 minute by using
gravitational filtration together with the cotton wool. The steps
above were repeated by replacing the spinach leave to spinach
stem.
Part B: Thin-Layer Chromatography
Preparation of TLC Chamber

A developing chamber was prepared by placing a folded filter

paper lengthwise in a glass container. 100 mL of a 70:30 mixture
of hexane and acetone were prepared to use as eluent. The
eluent was added into the developing chamber until it forms a 1cm layer on the bottom of the container. The glass container was
then covered with the glass container cover.
Preparing and developing a plate
A 2 cm x 10 cm strip of TLC plate was obtained. The pencil dot
were then placed in the middle of the plate about 1 cm from the
bottom end. A capillary pipet was used to apply a spot of pigment
solution over the pencil dot by lightly applying the tip of the pipet
to the surface of the plate. The spotting process was performed
additionally four to five times, allowing the solvent of each drop to
be evaporated before adding the next drop of solvent. As the spot
have thoroughly dried, the strip was then placed in the
developing chamber containing the correct eluent. The solvent
front was then allowed to move within 2-3 mm of the top of the
plate and the plate was removed from the chamber. Position of
the solvent front was then marked with a pencil and the plate was
allowed to air-dry. Rf values for each of the pigment observed was determined
by using the formula provided below.
Rf =

Distancetravelled by solute ( pigment )

Distancetravelled by solvent

Part C: Extraction of -carotene from tomato paste.

A small mortar and pestle were used to grind the tomato and
about 10g of the grinded tomato were transferred into the 50mL
beaker. 15mL of 95% ethanol were added into the beaker
containing grinded tomato and the mixture were stirred
vigorously with a spatula until the grinded tomato will not stick on
the spatula. A small amount of glass wool was placed in a small
funnel blocking the funnel exit. The funnel was then placed into

the 50mL Erlenmeyer flask and the tomato grinded-ethanol

mixture was poured into the funnel. After the completion of the
filtration, the glass wool was squeeze lightly with the spatula. This
step is conducted is to remove the water from the tomato paste
and the aqueous components. To extract the pigment, the residue
in the glass wool will be used.
The residue and the glass wool were then placed in a 50mL
beaker. 10mL of petroleum ether were added and the mixture was
stirred for 2 minute to extract the pigment. The extract was
filtered as before through a new funnel with glass wool blocking
the exit, into the new and clean 50mL beaker. The beaker was
then placed under the hood on a hot plate. Low heat were used
and the solvent were taken care of not to evaporate all. The
beaker was then covered with aluminum foil after the
evaporation.
Part B was then repeated by using petroleum ether: toluene:
acetone with the ratio of 45:1:4 as an eluting solvent. The
separate capillaries was used, one for the tomato paste extraction
and one for the -carotene solution.

Part D: Tomato juice Rainbow

A small mortar and pestle were used to grind the tomato and
about 15g of the tomato paste were transferred into the beaker.
30 mL of water were added into the beaker containing the tomato
paste and stirred. The tomato juice was then transferred into a
50mL graduated cylinder and the pipet was used to add 5 mL of
saturated bromine water (drop wise). The solution was stirred
with a glass rod gently. The color and their positions in cylinder
were observed. Observation was recorded.
Result and calculation:

Components

Distance travelled by Spinach leaf and Stem (cm)

Solvent (hexane and

acetone)

1st Spot (Leaf)

Length
Rf value
(cm)
14.30
2.40
0.16

Pigment 1st

2nd Spot (Leaf)

Length
Rf
value
14.30
2.40
0.16

1st Spot (Stem)

Length Rf value
14.30
2.20

0.15

2nd Spot (Stem)

Length
Rf
value
14.30
2.15
0.15

Pigment 2nd

2.60

0.18

2.60

0.18

2.50

0.17

2.50

0.17

Pigment 3rd

14.30

1.00

Table 1: Result of TLC plate of Separation of spinach leaf and stem

Calculation step:
Distance travelled by Spinach leaf:
1st pigment:
Rf = distance moved by the solute (pigment)
= 2.40cm 14.30cm
= 0.17cm

distance moved by solvent

Components

Distance travelled by Tomato and -carotene

Solvent (Petroleum ether, toluene and

acetone)

1st Spot (carotene)

2nd Spot (carotene)

1st Spot
(Tomato)

2nd Spot
(Tomato)

Pigment 1st

Length
(cm)
12.50
3.25

Rf
value
0.26

Length
(cm)
12.50
3.20

Rf
Lengt
Rf
value h (cm) value
12.50
0.25
1.60
0.13

Length
(cm)
12.50
1.40

Rf
value
0.11

Pigment 2nd

3.30

0.26

12.00

0.96

4.20

0.34

4.00

0.32

Pigment 3rd

12.00

0.96

12.10

0.97

11.90

0.95

Pigment 4th

12.30

0.98

12.20

Table 2: Result of TLC plate of Separation of Tomato and carotene

Calculation step:
Distance travelled by Tomato:
1st pigment:
Rf = distance moved by the solute (pigment) distance moved by solvent
= 3.25cm 12.50cm
= 0.26cm

TLC Plate Diagram:

0.98

Figure 1.5 TLC Plate of Separation of Spinach pigments of leaf and

stem

Figure 1.5 TLC Plate of Separation of -carotene and Tomato

pigments
Discussion :
In the TLC Plate that contains separation of the spinach;
three pigments were able to be identified. While for the spinach
stems, only two pigments were able to be identified. On the TLC
plate, color of the pigments wasnt really visible to be identified.
Thus, to identified the pigment on the TLC plate, obtain
experiment Rf value are calculated can compared with the
theoretical Rf value of the pigment which is stated in table 3
below.
Spinach Pigments
Carotene
Chlorophyll a
Chlorophyll b
Xanthophyll I

Rf value
0.98
0.59
0.42
0.28

Xanthophyll II
0.15
Table 3 Theoretical Rf value of the spinach pigment
Based on the table 1 above, Rf values of the first pigment
(spinach leaves) are 0.16 respectively. But in table 3, R f value of
0.16 does not falls into any of the theoretical R f value. Thus we
can conclude that this pigment was not one of the spinach
pigments. Rf value of the second pigment (spinach leaves) are
0.18 and 1.00 respectively. Once again, in table 3, R f value of 0.18
and 1.00 does not falls into any of the theoretical R f value. Thus
we once conclude that event this pigment (stem) was not one of
the spinach pigments.
As for the stem sample, Rf value of the first pigment are 0.15
respectively. In table 3, Rf value of of 0.15 falls into a spinach
pigment called Xanthophyll ll. Thus we can conclude that this
pigment was one of the spinach pigments which is the
Xanthophyll ll. Rf value of second pigment (stem) are 0.17. But in
table 3, Rf value of 0.17 does not falls into any of the theoretical
Rf value. Thus we can conclude that this pigment was not one of
the spinach pigments.

In the TLC Plate that contains separation of the tomato and carotene
(Vitamin); four pigments were able to be identified in tomato and
three pigments were able to be identified in the -carotene
(Vitamin). As the same thing that has occurs in the spinach TLC
plate, it also occurs in the tomato and -carotene TLC plate. Thus,
to identified the pigment on the TLC plate, obtain experiment Rf
value are calculated can compared with the theoretical R f value of
the pigment which is stated in table 4 below.
Pigment name
-carotene
-carotene

Rf value
0.97
0.94

Lycopene
0.81
Leutein
0.75
Violaxathin
0.66
Neoxathin
0.28
Table 4 Theoritical Rf values of the carotenoid plant pigment
According to the table 2 above, Rf values of the first pigment
(-carotene) are0.26 respectively. Rf values of 0.26 in table 4 does
not show any of the carotenoid plant pigment. Thus, we can
conclude that high chances that this pigment of R f values of 0.16
are not one of the -carotene pigment. As for the second pigment
(-carotene), Rf values are also 0.26, which is the same as the
first pigment of the -carotene. Thus has the same conclusion as
the first pigment. Rf values of the third pigment (-carotene) are
0.96. But in table 4, Rf value of 0.96 does not falls into any of the
theoretical Rf value. Thus we can conclude that this pigment was
not one of the -carotene pigments.
As for the tomato sample, Rf value of the first pigments
(tomato) are 0.13 and 0.11 respectively. Both Rf value of the first
pigments does not falls into any of the theoretical R f value in table
4 above. Thus we can conclude that these two pigments was not
one of the tomato pigments. In the second pigment (tomato
pigment), the Rf value are 0.34 and 0.32 respectively. Once
again, in table 4, Rf value of 0.34 and 0.32 does not falls into any
of the theoretical Rf value. Thus we once conclude that these two
pigments was not one of the tomato pigments.
Rf value of the third pigment are 0.97 and 0.95
respectively. In table 4 above, Rf value of 0.97 falls into one of the
carotemoid plant pigment which is
-carotene. Thus we can conclude that this particular pigment is
one of the carotenoid plant pigment called as -carotene. On the
other hand, Rf value of 0.95 does not falls into any of the
carotenoid plant pigment base on the table 4 above. Thus we can
conclude that this pigment with the Rf value of the 0.95 was not
one of the tomato pigment. Last but not least, R f value of pigment

fourth (tomato) are 0.98 respectively. But in table 4 above, R f

value of 0.98 does not falls into any of the carotenoid plant
pigment. Thus we can once conclude that, this particular pigment
which has Rf value of 0.98 was not one of the tomato pigment.
Question:
1) The spot should not be immersed in the solvent in the
developing chamber is because as the spot is immersed, it
will be able to dissolve into the solvent used. Furthermore, as
the spot is immersed into the solvent, it will not separate
inside the solvent used.
2) Solvent must not be allowed to evaporate from the plate
during development is because solvent that are used in this
experiment are volatile which simply meant that it is easily
evaporated. If the solvent evaporated, there will not enough
solvent to move the spot upwards of the TLC plate for
separation to occur.
3) Diameter of the spot should be as small as possible is
because to prevent any mixing occurs during the separation
process. Furthermore, small spot are able to move more
independently with interfering with the surrounding spot
during separation process.
4) As we know that the solvent used in TLC helps separation of
the pigment found in the either plant or fruits, and also it is
easily evaporates. Hence, once we remove the TLC sheet,
solvent that has been absorbed by the finely-divided sorbent will
immediately evaporated together with spot, causing no spot were able to be
observed. However, the evaporated spot still can be seen by using UV lamp.
The separated pigments will actually fluoresce when exposes to UV light.
5) Rf is the retention value. It is determined by measuring the distance of the
pigment travels divided by the total distance solvent travelled. Formu la of
Rf is as followed:
Rf =

Distance travelled by solute ( pigment )

Distancetravelled by solvent

Conclusion:
In conclusion, TLC, Thin-Layer chromatography technique is
widely used because it is useful and cheap to identify a particular
component inside the mixture. TLC technique is also very useful
in identify the polarity of a particular component present in a
mixture.

Reference :
Chatwal, & Gurdeep, R. (n.d.). ebrary:
Document Viewing Options. Retrieved
from
http://site.ebrary.com/id/10416169?
ppg=39

Experiment 6: Separation of Plant

Pigments by Thin Layer
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Harcourt, J. (n.d.). Isolation of

Carotenoids.

John, M. C. (n.d.). Thin Layer and Column

Chromatography.

Pavia, D. L., Lampman, G. M., Kriz, G. S.,

& Engel, R. G. (2004). Isolation of
Chlorophyll and Carotenoid Pigments
from Spinach. A Microscale Approach
3rd Edition, 81, 385-387.

Rodriguez-Amaya, D. B. (1999). A Guide

to Carotenoid Analysis in Foods.