INTRODUCTION

The dictionary's description of chromatography explains that an analytical method used by the
chemical substances to separate a mixture into their compounds. In the laboratory, different
forms of chromatography such as thin-layer chromatography, planar chromatography, column
chromatography, gas chromatography, and others are used. However, the thin layer
chromatography (TLC) is the most common form used as shown in figure 1. The thin layer
chromatography principle consists of two phases which are the stationary and mobile phase. A
thin layer of silica ( SiO 2) is coated on plastic slides during the mobile phase. This is because the
particles are smaller that it can behave like dust when not viewed to a surface. The sample
mixture is added as a small spot near the bottom of the plate, then placed into a jar containing a
few ml of solvent. By capillary action the solvent climbs up the plate, carrying the sample
mixture along with it. The compound mixture that will move along with the stationary phase that
went through the mobile phase and it separates for each component in the sample or compound
mixture depending on the widely different degree of adhesion. So, every part of the spotted
mixture will go up at a different rate. When the solvent gets close to the top of the plate,
evaporation is allowed, leaving the components of the mixture at different distances from the
point of origin behind. The RF value (retention factor) is the ratio of the distance a substance
moves to the distance the solvent moves in. Hence, this value features the compound, solvent,
and stationary phase.

Distance component travelled

Moreover, flow rates are calculated in terms of the values of the Rf (retention factor). A Rf is a
relative distance a sample component has moved compared to the distance moved by the solvent
in the mobile phase. The equation shown above states that Rf is calculated by division of the
distance of component travelled over the distance travelled by the solvent. So, the value of Rf
can never be greater than 1. In that solvent, the Rf value for a component is characteristic of that
component. It will therefore still be the same (whereas the mobile and stationary phases are
identical) and can be found in other mixtures.
Finally, using the thin layer chromatography process, the plant leaf pigment can be separated by
and identified. Plants use several different pigments for their light harvesting processes. Both
compounds fell into the chlorophyll and carotenoid groupings. The representative members of
these groups with chlorophyll a and b, and β-carotene are shown in Figure 2. Chlorophyll is
known to have green pigment which serves as the plant leaf 's main photoreceptor molecules. In
addition, carotenoids are known to have yellow pigment which can assist the plants in
photosynthesis. Other non-photosynthetic pigments such as anthocyanins or other flavonoids that
help determine the colour of flowers. Whereas pigments have the main purpose of attracting
birds and insects for pollination. On a piece of TLC paper, we will be able to observe the
pigment of the plant and measure the Rf value of each mixture of components.
ANALYSIS
Materials:

Chromatography jar with lid, pestle and mortar, funnel, filter paper, sand, beakers, vials, pencil,
scale, thread, scissor, chromatography paper, capillary tube, petroleum ether: acetone (9:1), plant
leaf.

Procedure:

1. Two to four plant leaves are taken, and a scissor is used to cut the leaves into pieces and
remove the midrib.
2. To support the grinding process, a mortar and pestle is used to grind the leaves and a
small amount of acetone, petroleum ether and some pre-washed fine sand is applied.
3. Using filter paper and funnel the extract is filtered, and the supernatant is collected into a
small container.
4. The paper and chamber chromatography are prepared. A strip of Whatman filter paper is
taken, then pre-cut by 20 cm to about 5 cm.
5. A pencil is used to draw a line about 1 cm from the paper above and it has been cut into a
V shaped under the pencil line.
6. The chromatography jar is taken. A thread was stretch across the diameter of the mouth
of the jar and was use a sellotape to secure it.
7. The solvent of 9:1 petroleum ether and acetone are poured into the chromatography jar.
8. 8. A fine capillary tube is used on chromatography paper to add micro droplets of the left
extract in the middle of the pencil line. Allowed it to air dry and added a second drop
further. Repeat the drop and air dry for up to 5-10 drops.
9. A fold was made on the other end of the paper and the paper was put onto the thread.
10. The paper's pointed tip was lying beneath the jar's solvent surface, avoiding the center of
the leaf extract labelled.
11. When the solvent front reaches within 1 cm of the top paper the chromatography paper is
removed. The pigment spots are circled, and the distance is marked at the furthest.
APPENDIX

Figure 1
(Source Khan Academy)

Figure 2 (a) Chlorophyll a, (b) chlorophyll b, and (c) β-caroteneand each pigment has (d) a
unique absorbance spectrum.
(Source:https://bio.libretexts.org/Bookshelves/Introductory_and_General_Biology/Book
%3A_General_Biology_(Boundless)/8%3A_Photosynthesis/8.2%3A_The_LightDependent_Rea
ctions_of_Photosynthesis/8.2B%3A_Absorption_of_Light)

Figure 3

(Source: https://slideplayer.com/slide/3715510/)
REFERENCES
Aryal, S., Rafat, A., Malakar, A. C., & Singh, D. S. (2020). Chromatography- definition, principle, types,
applications. Retrieved June 2020, from https://microbenotes.com/chromatography-principle-types-
and-applications/

Colour, chlorophyll and chromatography. (n.d.). Retrieved June 2020, from

EXP.6, Thin Layer Chromatography, Retrived June 2020, from

Thin Layer Chromatography Definition, Principles, Procedure and Theory. (2017). Retrieved June 2020,
from https://chemdictionary.org/thin-layer-chromatography/

Video reference: https://www.youtube.com/watch?v=iPpy4khqtks