LABORATORY REPORT

TECHNIQUE IN BIOLOGY AND BIOCHEMISTRY LABORATORY

(SBL1023)

STUDENT NAME ALISYA SYAZRIN ADILA BINTI

MOHD NASIR
REGISTRATION NO. E20161015898
GROUP B
EXPERIMENT EXPERIMENT 6: PLANT
PHYSIOLOGY
LECTURER NAME ASSOCIATE PROF. DR SHAKINAZ
DESA
 PRACTICAL 6: PLANT PHYSIOLOGY

INTRODUCTION
The field of plant physiology includes the study of all the internal activities of plants—those
chemical and physical processes associated with life as they occur in plants. This includes
study at many levels of scale of size and time. At the smallest scale
are molecular interactions of photosynthesis and internal diffusion of water, minerals, and
nutrients. At the largest scale are the processes of
plant development, seasonality, dormancy, and reproductive control. Major subdisciplines of
plant physiology include phytochemistry (the study of the biochemistry of plants)
and phytopathology (the study of disease in plants). The scope of plant physiology as a
discipline may be divided into several major areas of research.

A pigment is a molecule that absorbs light. White light contains all of the different colors of
the visual spectrum. This can be observed in a simple rainbow during a rain storm or by
using a prism that splits white light into its various colors.
In plants, there are two categories of pigments used for photosynthesis: primary pigments
and accessory pigments. The chlorophylls are the primary pigments of photosynthesis, with
two types called chlorophyll a and chlorophyll b. The chlorophylls are green pigment
molecules that absorb blue, red, orange, yellow, etc., but reflects green light. On the other
hand, the accessory pigments are red, yellow or orange – they absorb all of the other
colors.

In this experiment, we have used paper chromatography technique to separate the plant
pigments from a plant using a hydrophobic solvent. Chromatography is a technique that
allows us to separate different molecules from a mixture based on differences in solubility.
Some compounds do not like to dissolve in water. These are called hydrophobic
compounds. On the other hand, some molecules are hydrophilic, meaning they like to
dissolve in water. Chromatography is a method of separating and isolating molecules based
on their level of hydrophobic or hydrophilic properties. Those pigment molecules that have
the highest solubility in the liquid solvent used will be carried along through the paper the
fastest. Those pigments that are least soluble in the solvent will move more slowly or not at
all. The various plant pigments have differing degrees of hydrophobicity. Therefore, if we use
a liquid solvent that is hydrophobic, different plant pigments will move at differing rates
through the piece of paper as the liquid solvent is absorbed upward.

OBJECTIVE
To separate pigments of different compounds/substances using paper chromatography
method.
MATERIALS
Solvent (petroleum ether)
Sample substance (red and blue ink)
Cork stopper
Pin
Mortar
Leaf extract (green and red spinach)
Test tube
Strips of filter paper

PROCEDURE
1. Using the microbiurette/pin head as the dropper, the chloroplast extract was dropped
on the prepared chromatography paper.
2. The extract was dropped at about 1.0 cm from the pointing end of the paper. The
drop was drying with a hair dryer and the process is repeated for 3-4 times until one
small dot of thick pigment available.
3. The paper strip was attached at the cork stopper using a pin. The strip was placed
vertically and straight into the test tube which contained solvent.
4. The solvent was moves and the paper is removed before the solvent front reaches
the top of the chromatography paper.
5. The last range of the solvent was marked with pencil
6. Step 1 to 5 were repeated using extract of red spinach, blue ink and red ink.
7. Each pigment obtained from the experiment was identified and recorded.
8. The Rf value for each pigment was observed and calculated
RESULTS

Green spinach:
Colour of pigment Distance travel by Distance travel by Rf Value
compound (cm) solvent (cm)
Red 2.5 6.4 𝟐.𝟓 𝒄𝒎
Rf =
𝟔.𝟒 𝒄𝒎

= 0.39 cm

Pale yellow 6.2 6.4 𝟔.𝟐 𝒄𝒎

Rf =
𝟔.𝟒 𝒄𝒎

= 0.97 cm

Figure 1.0 The chromatogram of green spinach

Red spinach:

Colour of pigment Distance travel by Distance travel by Rf Value

compound (cm) solvent (cm)
Pink 1.2 8.7 𝟏.𝟐 𝒄𝒎
Rf = 𝟖.𝟕 𝒄𝒎

= 0.14 cm

Green 5.6 8.7 𝟓.𝟔 𝒄𝒎

Rf = 𝟖.𝟕 𝒄𝒎

= 0.64 cm
Yellow-green 6.1 8.7 𝟔.𝟏 𝒄𝒎
Rf = 𝟖.𝟕 𝒄𝒎

= 0.70 cm

Figure 1.1 The chromatogram of red spinach

DISCUSSION
The purpose of this experiment is to observe how chromatography can be used to separate
mixtures of chemical substances. Chromatography serves mainly as a tool for the
examination and separation of mixtures of chemical substances. Chromatography is using a
flow of solvent or gas to cause the components of a mixture to migrate differently from a
narrow starting point in a specific medium, in the case of this experiment, filter paper. It is
used for the purification and isolation of various substances. A chromatographically pure
substance is the result of the separation. Because purification of substances is required to
determine their properties, chromatography is an indispensable tool in the sciences
concerned with chemical substances and their reactions.

Chromatography is also used to compare and describe chemical substances. The

chromatographic sequence of absorb substances is related to their atomic and molecular
structures. A change in a chemical substance produced by a chemical or biological reaction
often alters the solubility and migration rate. With this knowledge, alterations or changes can
be detected in the substance.

In all chromatographic separations, there is an important relationship between the solvent,

the chromatography paper, and the mixture. For a particular mixture, the solvent and the
paper must be chosen so the solubility is reversible and be selective for the components of
the mixture. The main requirement, though, of the solvent is to dissolve the mixture needing
to be separated. The porous paper used must also absorb the components of the mixtures
selectively and reversibly. For the separation of a mixture, the substances making up the
mixture must be evenly dispersed in a solution, a vapour, or a gas. Once all of the above
criteria have been met, chromatography can be a simple tool for separating and comparing
chemical mixtures.

The number of the spots or bands tells us how many compounds are in the substance. Their
colour and the distance they travel might help us to identify those compounds. We can try to
find out which dyes were used in black markers using pencil from the same package as a
reference samples. If certain reference colour sample will travel the same distance (Rf) that
one of the black marker colours both of them likely to be the same chemical compound. We
can't identify the chemical substance by paper chromatography, but we can roughly analyze
the mixture with this simple and neat technique.

CONCLUSION
The proposed hypothesis was correct. The paper chromatography did show that black ink
could be separated into various colours. The black ink gets its colour from a mixture of
various coloured inks blended together. The first colour of ink to appear on the filter paper
was red followed by pale yellow. The second colour of ink is pink followed by green and
yellow-green. The colours separated the way they did because of the differences in their
molecular characteristics, specifically, their solubility in water and their rate of absorption by
the paper. The most soluble and readily absorbed ink colour was the yellow.

REFLECTION
For my main experiment, I did the chromatography of a leaf.
Chromatography is important because it helps separate molecules from each other by
molecular mass, allowing us to look observe the different substances that make up a whole.
Factors that allow chromatography to happen include the adhesion of the molecules to the
chromatography paper, the varying masses of the different molecules, and the variety of
solubility levels of the molecules in the solvent. The chromatography paper used in this
experiment pulled the solvent and allowed the leaf pigments to move up. The separation of
the pigments on the paper allows us to observe the various pigments easily, as they can be
differentiated by their different colours. The solvent is important to this experiment because it
moves the leaf pigments up the paper through capillary action, due to both the properties of
adhesion and cohesion, in order to separate the different pigments. Pigments are important
to photosynthesis because they absorb different wavelengths of light, specifically visible light
waves, which has enough energy to cause chemical change but not damage cells.

REFERENCES
http://www.biologyjunction.com/paper_chromatography_report.htm
https://en.wikipedia.org/wiki/Plant_physiology
http://www.scienceprojectlab.com/paper-chromatography-experiment.html