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EXPERIMENT OF PLANT PHYSIOLOGY (CHROMATOGRAPHY)

INTRODUCTION

Plant leaves contain a number of important pigments including chlorophylls,


carotenes, and xanthophylls. During the summer when leaves contain large
amounts of chlorophyll (and are thus green), the presence of the other pigments is
not obvious to the eye. During the fall, however, after most of the chlorophyll has
been degraded, these other pigments can be more readily observed, and the leaves
of many plants take on the variety of beautiful colors that are typical of fall foliage.
During this lab you will have a chance to collect leaves of various colors and
analyze the different pigments that are present by using chromatography method.

Chromatography is a technique used to separate complex mixtures between


a stationary phase and a mobile phase. The concept behind this process is that the
smaller the affinity a molecule has for the stationary phase, the faster it migrates.
The stationary phase varies depending on the type of chromatography used. Most
of the time, chromatography is used for purification techniques. The process is also
used in separation of substances, an example of which is plant pigments.

In this experiment, chromatography was used to separate pigments


extracted from green spinach and purple spinach.

METHODOLOGY

1. Firstly, a few of green spinach and purple spinach are extracted.

2. Using the microbiurette/ pin head as the dropper, the extract chloroplast is
dropped on the prepared chromatography paper (see Figure 1).

3. The extract is dropped at about 1.5 cm from the pointing end of the paper.
Then, the drop is dried with a hair dryer and the process is repeated for 3-4
times until one small dot of thick pigment available.

4. The paper strip is attached at the cork stopper using a pin. The strip is
placed vertically and straight into the test tube which contained solvent.

5. Let the solvent moves and the paper is removed before the solvent front
reaches the top of your chromatography paper.

6. Last range of the solvent is marked with pencil.


RESULTS

Type of leaf Pigment name Colour Distance Distance


compound, cm solvent, cm
Green spinach Chlorophyll B Yellow 7.3 8.2
green
Chlorophyll B Yellow 6.9
Purple spinach green 8.0
Anthocyanin Purple 2.6

i) Chlorophyll B (yellow green) pigment (green spinach)

7.3 𝑐𝑚
𝑅𝑓 = = 0.89 𝑐𝑚
8.2 𝑐𝑚
ii) Chlorophyll B (yellow green) pigment (purple spinach)

6.9 𝑐𝑚
𝑅𝑓 = = 0.86 𝑐𝑚
8.0 𝑐𝑚
iii) Anthocyanin (purple) pigment (purple spinach)

2.6 𝑐𝑚
𝑅𝑓 = = 0.33 𝑐𝑚
8.0 𝑐𝑚

DISCUSSION

In this experiment, chromatography technique is used to separate the


pigment extracted from green spinach and purple spinach.

From this experiment, the developing solvent mixture is prepared fresh


before used because as we know the developing liquid phase comprises of a pure
solvent but more often it is a mixture of two or more solvents in specified
proportions. So, in this case, the solvents are mixed and stored for long periods,
there could be loss of volatile component which will alter the mixing proportions.
That’s why we are prepared the fresh solvent before used. Then, it is important to
keep the dye spots (leaf extract) above the solvent level because because if the dye
spots is submerged in the solvent, then the spots would dissolve into the solvent
preventing them from separating out and no measurements or observations could
be made. Next, it is necessary to cover the test tube during the paper development
because open test tube will cause the solvent mixture to more quickly diverge from
the intended mixture for the chromatography, which is it will detract the
reproducibility and likely give effect towards the process of the separation of
pigment.
After that, it is important to stop the chromatogram before the solvent front
reaches the top of your chromatography paper because we cannot determine the Rf
value if the solvent reaches to the top of chromatography paper as we need to
measure the distance from the start to the front of solvent. We can be able to see
where it stops even if we let it go as high as we want but not until it reaches the top
of the paper. Then, it is important to mark the solvent level on the chromatography
paper when we are removed it from the test tube because the point at which the
solvent stopped could be noted if in the case, the solvent kept advancing when
removed. The pigment migrated the farthest is anthocyanin because it most soluble
in the solvent. Certain pigments have a harder time to being moved along the
chromatography paper by solvents than others did. If a pigment is made up from
larger molecules than it, it will move at a slower speed than the other pigments
causing a separation in the colors. On the other hand, we want to discuss if we
allowed our chromatogram to develop too long in the solvent, it would travel too far
and possibly run of the plate. So, we would not be able to calculate the Rf values
because we don’t have the values of distance travelled by solvent.

CONCLUSION

Paper chromatography proved to be an accurate successful method of


separating and observing the various colors of plant pigments. The pigments
dissolved in the solvent and migrated upward. The colours were observed and their
migration distances were measured and recorded. The Rf value of each pigment was
determined by dividing its migration by the migration of the solvent. It was
determined that two pigments were present in the spinach leaves which is
anthocyanin and chlorophyll b. anthocyanin pigment is the most soluble, while
chlorophyll b was the least soluble.

REFERENCES

https://www.ukessays.com/essays/biology/characterization-of-photosynthetic-
pigments-using-paper-biology-essay.php

https://students.ga.desire2learn.com/d2l/lor/viewer/viewFile.d2lfile/1798/12844
/photosynthesis18.html

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