Separation of Plant Pigments of Komote Tops by
Thin Layer Chromatography
Adapted from: Quach, H. T.; Steeper, R L.; Griffin, G. w. J. Chem. Educ.2004,g1. 3g5-7 and
Department of Chemistry, Troy University
Chromatography is widely used for the identification of components as well as its
separation into molecular components of a mixture. It was discovered by M. Tswett in 1906,
where he dissolved a mixture of plant pigments in petroleum ether ani passed the solution
through a column of calcium carbonate. He was able to demonstrate that chromatography was
applied only to coloured compounds, and the results of separation could be followed by visual
Chromatography is an analytical method permitting the separation of a mixture into its
molecular components. The basic principle upon which it works is that a mixture first adheres to
the dry chromatography plate. A developer or solvent is then passed through the coating on the
plate in a fixed direction moving the pigment molecules of the mixture along at different rates.
The greater the attraction between the molecules and the absorbing medium (coated on the
plate), the slower the molecules ascend the coating. The greater the soiubility of the components
in the developer, the greater the distance the molecules move.
The developer commonly used contains a mixture of nonpolar.or polar solvents. (Ensure
th,at there are no open flames in the laboratory because solvents are extremely
The solvent molecules contain non-polar covalent bonds and any net charges are equally
distributed within the molecules.
Chromatography has been the very suitable method for separation of pigments of plant
extracts, especially when a small amount of material is available. In higher forms of planti, the
principal pigment is chlorophyll a. Chlorophyll b, carotenes and xanthophylls play a secondary
role by transferring the energy they absorb to chlorophyll for use in photosynthesii.
Chlorophyll a is colored dark green to blue green, whereas, chlorophyll b is yillow green. B-
carotene is colored light yellow
ln the following experiment, the pigments found in the leaves of kamote tops, Ipomoea
batotas, are separated by means of thin layer chromatography.
*Fresh Kamote tops (violet colored,
mm), lab gown, surgical masks,
PER SECTION) pairs of gloves
+*Lab Kit (shoebox with section and (to be provided by the PSHS LAB)
names of GROUP members) (l) TLC plate (3.0 x 6.0 cm) and
- 2 rags, paper towels or toilet paper, (l) TLC chamber
hand soap, liquid detergent, dish (l) set mortar and pestle (small)
sponge, test tube brush, pair of (l) 4.0 mL test tube
scissors, masking tape, fine-tip (l) 1.5 mL eppendorftube
marker, wooden pencil, ruler (w/ (l) capillary tube
Science and Technologt Research I PSHS-MC Sy 2010-20t 1
(l) 2.0 mL measuring pipet Acetone
Parafilm (as needed) Petroleum ether
(l) spatula Isopropanol
( l) pair of forceps Distilled water
l. On a balance weigh out 1.0 gram of kamote tops. Manually tear/cut the leaves into small
pieces and transfer to a mortar and using a pestle, grind the leaves very well. Transfer the
crushed leaves to a small test tube and add 2.0 mL of acetone. Cover the test tube with a
parafilm and shake vigorously for approximately one minute between hands.
2. Allow this mixture to stand for about l0 minutes, then using a pipet carefully transfer L0
mL of the solvent above the solid into a small 1.5 mL eppendorf tube. Use care not to
transfer any of the solid material. The solvent extract should be green. Cap the tube to
minimize solvent evaporation .lDispose remaining extract to an orgonic waste jar!l
Obtain a TLC chamber with developing solvent: a mixture of 25.0 mL petroleum ether,
2.5 mL and 5 drops distilled water (to be prepared by the instructor).The solvent should
completely cover the bottom of the chamber to a depth of approximately 0.3 cm (-2.0
mL). Keep the chamber covered so that evaporation doesn't change the composition of
4. Obtain a TLC plate (a silica gel coated plastic sheet) and draw a line measuring 0.5 cm
from the bottom as well as from the top. Place two small dots of equal distance apart with
a lead pencil on the coated side approximately 0.5 cm from the bottom of the strip. The
dots should be parallel with the bottom of the strip. Label the first two dots with the
numbers."" l and 2" to indicate two trials
5. Fill a capillary tube (TLC applicator) by placing it in the leaf extract (it will fill by
capillary action). Place one finger at the end of the capillary tube to control application of'
extract to the TLC plate. Apply the extract to the center of the first dot on the TLC plate
by quickly touching the end of the TLC applicator to the plate. Allow to dry. Repeat
several times (-20 times) to make a concentrated dot of extract. Be sure to let dry
between applications. Try to make the spots as small as possible but dark enough to see
the color clearly. lDispose used capillary tube at designated waste beaker!l
6. Using a pair of forceps, careflully place the TLC plate in the TLC chamber. The TLC
plate should sit on the bottom of the chamber and be in uniform contact with the solvent
(solvent surface must be below the extract dots). Cover the TLC chamber.
7. Allow the TLC plate to develop (separation of pigments) for approximately 10-15
minutes. As the solvent moves up the TLC plate you should see the different colored
Science and Technologt Research I PSHS-MC SY 20 1O-201 1
8. Remove the TLC plate from the chamber when the solvent front reaches the 0.5 cm from
the top of the TLC plate using a pair of forceps. With a*pencil, mark the level of the
solvent front (highest level the solvent moves up the TLC plate) as soon as vou remove
the strip from the chamber (the solvent evaporates and disappears quickly). Using a
ruler, also measure the different pigment distances (in mm) quickly as some pigments
may fade over time.
9. Record the results of the separation on the data sheet.
t. Tape your chromatography plate to the data sheet in space provided. Draw arrows to the
locations of the solvent front and the colored bands. Label each band as to its type of
2. For the following calculations mark the center of the initial pigment dot; this will be the
starting point for all the following measurements. Also mark the middle point of each
pigment band and the solvent front.
J. The rate at which a pigment moves up the plate is reported as an Rs value which is
defined as the ratio of the distance moved by the spot to the distance moved by the
solvent. Determine the Rrvalues for each of the pigments you observe using the formula
distance moved by solute (pigment)
distance moved by solvent
( h,tnrl)rr I irl ot P.r t af ilrrt ", _-
Hr.r rli,ping (.h,rrnht,t_
tolraql"f tqt3t - rto'r1 lrr;ri
:p:r:olirnn:*lt c.5 lrr: f:r:rtt t*11
flrr eloping oh ent *.3*t I 1t1q1
Science and Technologt Research I PSHS-MC SY 20IO.2O11
Separation of Plant Pigments by TLC Plate Data Sheet
Project Code: Names:
State your own objective/s.
II. Materials and Methods:
Construct a flowchart or a schematic diagram to summarizethe materials and
methods used in the experiment.
III. Data and Results
Distance (mm) Rf Value
Trial I Trial2 Trial 1 Trial2
solvent front .
Tape, Draw, and Label TLC Plate Here
Science and lechnologt Research I PSHS-MC SY 20]O'2OI I
ry. Discussion and Analyses:
Discuss the results of the experiment by answering the following questions
completely and concisely.
t. Based on the results of the experiment, what evidence, if any, do you see for the presence of
other pigments aside from chlorophyll a and b? Identify these pigments and label properly in
the data sheet.
2. Which among the pigment molecules, chlorophyll a, b and B-carotene was the most
nonpolar? . The most polar pigment?
Explain your answer briefly.
3. Why shoutd you use pencil on the coating to mark your pigment movement?
4. In the TLC experiment, why must
a) the TLC plate be coated thinly with silica gel?
b) the spot to be applied to the TLC plate be placed above the level of the developing
c) you wear a mask when handling solvents like petroleum ether?
5. Consider three compounds to be separated on a TLC plate:
benzoic acid naphthalene
Which compound corresponds to the:
Explain your answer.
State your conclusion in answer to your objective/s.
List references used to complete this report. Observe corect APA format for the
bibl iographical entries listed.
Science and Technolog,, Research I PSHS-MC Sv 2010-201 I