SBL1023

TECHNIQUES IN BIOLOGICAL AND

BIOCHEMISTRY LABORATORY

PLANT PHYSIOLOGY

NAME: FARAH WAHIDAH BINTI JEPPRI

MATRIC’S NUMBER: E20161013726
GROUP: A
LECTURER’S NAME: ASSOCIATE PROF DR. SHAKINAZ BINTI DESA
 LAB 6 – PLANT PHYSIOLOGY

INTRODUCTION
Plants consists of two categories of pigment that used for photosynthesis which are
primary and accessory pigment. Chlorophyll is a primary pigment in organelle chloroplast with
two types of chlorophylls which are chlorophyll a and chlorophyll b. The chlorophyll are green
pigment that absorb blue, red, orange and yellow but reflect green light as light contains
different colours of different spectrum. Besides, accessory pigment are red, yellow or orange
that absorb all the other colours.
Paper chromatography is a porous paper that used to separate the plant pigments from
plant using hydrophobic solvent. Different molecules from a mixture have different solubility.
With the aid of the solvent, the rate of solubility of the molecule can be known from “molecular
race track” that are created. Some compounds are hydrophobic that do not like to dissolve in
water (water fearing) and some compounds are hydrophilic that like to dissolve in water (water
loving). Chromatography is a method of separating and isolating molecules based on their level
of hydrophobic or hydrophilic properties. Different plants pigments will move at different rates
through the piece of paper as the liquid solvent is absorbed upward. From the band or distance
of different movement of pigment in the paper chromatography, the solubility of pigment can
be known.

OBJECTIVES
 To separate the plants pigments that are present in leaves using chromatographic
technique.
 To determine the Retention factor (Rf) of different pigment based on distance travelled
by pigment carried by solvent in paper chromatography.

METHADOLOGY

1) The chloroplast extract was dropped on the prepared chromatography paper by using
pin head as the dropper.
2) The extract was dropped at about 1.0 cm from the pointing end of the paper. The drop
was dried with a hair dryer and the process was repeated for 3-4 times until one small
dot of thick pigment available.
3) The paper strip was attached at the cork stopper by using pin. The strip was placed
vertically and straight into the test tube which contain solvent.
4) The solvent was let to move and the paper was removed before the solvent reaches the
top of the chromatography paper.
5) The last range of the solvent was marked with pencil.
RESULT

𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒕𝒓𝒂𝒗𝒆𝒍𝒍𝒆𝒅 𝒃𝒚 𝒄𝒐𝒎𝒑𝒐𝒖𝒏𝒅,𝑹𝒔

Rf =
𝑫𝒊𝒔𝒕𝒂𝒏𝒄𝒆 𝒕𝒓𝒂𝒗𝒆𝒍𝒍𝒆𝒅 𝒃𝒚 𝒔𝒐𝒍𝒗𝒆𝒏𝒕,𝑹𝒑

Leaf A
Pigments / colour Colour Rf

Pigment 1 Green 4.3 cm/ 13 cm =

0.33 cm

Pigment 2 Light yellow 4.8 cm/ 13 cm =

0.37 cm

Figure above shows the separation of two pigment of leaf A.

Leaf B
Pigments / colour Colour Rf

Pigment 1 Purple 2.5 cm/ 13 cm =

0.19 cm

Pigment 2 Green 7.2 cm/ 13 cm =

0.55 cm

Figure above shows the separation of two pigments of leaf B.

Figure above shows the movement process in system of pigment in leaf A and leaf B.
DISCUSSION

From the experiment, there are two pigments that exist from the separation of
photosynthetic pigment from two leaves, which were leaf A and leaf B at the paper
chromatography. The separation of pigment from leaf A can be seen based on their colour
which pigment 1 was green and pigment 2 was light yellow. The separation of pigment from
leaf B was observed and pigment 1 was purple while pigment 2 was green in colour. The
separation of pigment on the paper chromatography was with the aid movement of developing
solvent mixture that was prepared fresh before use. This is because the developing liquid phase
comprises of a pure solvent but more often it is a mixture of two or more solvents in specified
proportions. In case solvents are mixed and stored for long periods there could be loss of
volatile component which will alter the mixing proportions. The paper chromatography with a
line that contain a dot of leaf extract was placed in the test tube and it is important to keep the
dye spots above the solvent level because the dye spots will dissolve in the developing solvent
that preventing the spots contain pigment from separating out and no observation and
measurements could be taken.

During the paper development, it is necessary to cover the test tube to prevent the
evaporation of the eluting solution from the paper. The paper development was let for few
minutes without disturbing or shaking the system and it is important to stop the chromatogram
before the solvent front reaches the top of the chromatography paper because to measure the
Rf value, the starting and front of solvent must be known. The stop line should be done below
the top of the paper so that Rp value can be measured from the starting line to the stop line and
thus, Rf value can be calculated. As the solvent reach the stop line, the paper was removed
from the test tube and the separation spots observed was immediately marked to able us
measure how far it travelled.

After several observation, measurement and calculation, The Rf value of pigment 1 was
0.33 cm and pigment 2 was 0.37 cm for leaf A while for leaf B, it was observed to have 0.19
cm for pigment 1 and pigment 2 was 0.55 cm in distance. The pigment which move the furthest
was pigment 2 by its distance for both leaves. This indicate that the soluble pigment 2 dissolve
in the solvent easiest and thus move the furthest. Our result satisfied the hypothesis as the more
soluble the pigment, the further the movement of the pigment, thus the higher the Rf value.
Pigment 2 was the most soluble pigment based on the Rf value as it was very soluble in the
solvent and because it form no hydrogen bond with cellulose. The Rf value of pigment 2 also
indicate that the pigment has the smallest molecular size among the two pigment as it dissolve
the most easiest. The smaller the molecular size of pigment, the higher the Rf value and it prove
that the molecular size is also inversely proportional to the Rf value.

The chromatography successfully separating the primary pigment which is green

pigment and accessory pigment of the leaf used in the experiment which is yellow and purple
pigments. In this experiment, the factor that affecting the movement of pigment was the
porosity of the chromatography paper and the solubility of the solvent as well as the molecular
size of the solute. There were several limitations that made the pigments not separate very well
as the separation seen on the paper was not too visible to see. The system should not be shaken
that make the solvent move as the separation process that was ongoing will disturbed. The leaf
extract that was taken was too concentrated and in very little amount. In this experiment, avoid
the chromatogram run too long in the test tube as the solute and solvent will travel too far and
run out of the plate. Therefore, the Rf value cannot be calculated without a measured solvent
front. Extra attention and awareness should be taken while doing this experiment to minimize
the error and to get the best result.

REFERENCES
1. Archived on 26 December 2017 from
https://quizlet.com/68396986/1lab-15-flash-cards/

2. Archived on 26 December 2017 from

https://www.wisdomjobs.com/e-university/chromatography-interview-questions.html

3. Lecture note, Plant physiology.

4. Laboratory Manual SBL 1023