SBL1023

TECHNIQUES IN BIOLOGICAL AND

BIOCHEMISTRY LABORATORY

PLANT PHYSIOLOGY: CHROMATOGRAPHY

NAME: NUR IZZATIE BINTI KHAIZAAR

MATRIC’S NUMBER: E20161013729
GROUP: A
LECTURER’S NAME: ASSC. PROF DR. SHAKINAZ BINTI DESA
INTRODUCTON
Chromatography is one of a method to separate and analyse a mixture of
chemical compound. The separation including the uses of adsorbent; for example
paper, clay or gel. The spot will be detected on the paper in different distance and
colour. There are two types of chromatography which are gas and liquid
chromatography. For our experiment, we did more focusing on liquid chromatography
since we use the plant extraction to study about colour pigment in the plant extraction.
For liquid chromatography, Thin Layer Chromatography (TLC) is used to determine
the separation of chemical compound. In TLC, we have to prepare the right amount or
of solvent, good extraction of chemical compound and measurement of distance spot
that existed on the paper. One of the reason we use TLC is because the result will be
given in rapidly and simplicity.

OBJECTIVE
To determine the separation of plant extraction through Thin Layer Chromatography.

METHODOLOGY
1. The chromatography paper is lined with pencil 1 cm from the end of the paper.
2. The line is marked with ‘X’ to put the extraction of plant.
3. Two plants; green spinach and red spinach are extracted.
4. The extraction of plant is taken by the pin to the ‘X’ marked. The extraction is
dried and the step is repeated until three times.
5. The solvent is prepared by adding in the test tube.
6. The paper is placed in the test tube and the paper is made sure that the solvent
is not pass through the line of the paper.
7. The adsorption is observed and the distance of each spot that existed is
measured.
RESULTS

Type of plant Observation Explanation

Red Spinach There are two spots

(pigment) that existed on the
chromatograph paper; green
spot and yellow spot.
The first spot is green and
after that is yellow.

Green There is one spot (pigment)

Spinach that existed on this paper.
Only green spot detected.
CALCULATION
distance travelled by compound
Rf =
distance that tracelled by solvent
For Red Spinach:
Green pigment
4.3 𝑐𝑚
Rf =
4.9 𝑐𝑚
= 0.88
Yellow pigment
4.9 𝑐𝑚
Rf =
4.9 𝑐𝑚
= 1.0

For Green Spinach:

Green pigment
2.5 𝑐𝑚
Rf =
7.2 𝑐𝑚
= 0.35
DISCUSSION
Based on this experiment, chromatography is one of the method to separate
the pigments that contain in these two plants; Red Spinach and Green Spinach.
Results obtained shows that Red Spinach have two pigments that can be detected;
yellow and green pigments. Meanwhile, Green Spinach only have one pigment which
is green pigment. From the calculation, for Red spinach, the Rf of yellow pigment is
greater than green pigment. This is show that yellow pigment has lighter mass than
green pigment to travel due to the paper. That is the reason the green pigment is lower
Rf since the green pigments used to move slow due to its large mass.
For Green spinach, there is only one pigment that contain in this plant. I think
there are many pigments that have in this plant, but maybe my fault that put the
extraction to the paper too small in amount or I did not make a good pure extraction.
The Rf of green is 0.35. This value also can be assumed that the green pigment is the
slowest pigments to move in chromatography.
The preparation of solvent, we have to prepare it freshly because if the solvent
is stored for too long, maybe the compounds in the solvent already loss of volatile.
There is important to keep the extract of leaf above the solvent level because to avoid
the extract leaf submerge in the solvent that will cause no spot produced. The test
tube is supposed to be covered during the paper development because to make sure
there is pressure in the test tube so that it help the movement of solvent. It is also
important to mark the solvent level on the chromatography paper when we remove it
from the test tube because so the point at which the solvent stopped could be noted
in case the solvent kept advancing when removed.

REFERENCES
1. Archived on 4 January 2018
https://www.chemicool.com/definition/chromatography.html

2. Archive on 5 January 2018

http://lab-training.com/2014/12/05/10-common-interview-questions-paper-
chromatography/

3. Archived on 5 January 2018

https://www.coursehero.com/file/p3qealq/C-Why-is-it-important-to-keep-the-
dye-spots-above-the-solvent-level-It-is/