ACTIVITY 5

PAPER CHROMATOGRAPHY: Chromatographic Separation of Plant Pigments

Valiente, K., Apostol, D., Palaya, J.,
Jimenez, F., Banganan, K., Dolcho, K.
Department of Biology, School of Natural Sciences, Saint Louis University
October 9, 2019
ABSTRACT (5 points)
Objective of the experiment was to correlate the affinity of the different plant pigments to the mobile and
stationary phase by separation and identification based on each of their color and through computation
and comparison of flow rate of the plant pigments. The varying pigments became more visible when it
latched onto the chromatography paper. Chromatography paper was the medium used to absorb the
different pigments in the plant which made comparison and elaboration easier. RESULTS. In
conclusion,

INTRODUCTION (10 points)

Chromatography is a widely used technique with various types that are used to analyze each
components of a certain substance. There are two main factors in which the sample mixture was done, the
mobile phase and stationary phase. Mobile phase is a solvent which carries the sample through the
stationary phase while the latter on the other hand, is the object responsible for the separation of compounds
in the sample by taking advantage of a molecules ability to react with another one to form a chemical
compound or known as the affinity.

In this experiment, the experimenters used Paper Chromatography Technique wherein, the
chromatography paper acted as the stationary phase and was suspended to a suitable solvent which is the
mobile phase. Sample mixture would be inked or spotted at the bottom part of paper which would allow
mobile phase to flow in the stationary phase wherein the compounds in the sample are separated based on
their affinity. Compounds that had greater affinity to the mobile phase have travelled faster compared to
the compounds that had greater affinity to the stationary phase.

This study is performed so that experimenters, through separation, could identify the different plant
pigments based on its color, and illustrate the structure of the different plant pigments. The objective of the
experimenters in this experiment is to correlate the affinity of plant pigments to the mobile and stationary
phase, compute and compare the flow rate value of different plant pigments.

PROCEDURE (10 points)

The experimenters covered the 1000 mL beaker with a black paper so that light would not penetrate
through. They grounded the leaves using mortar and pestle with a small amount of distilled water that was
added to extract the liquid needed. After, they added three spots of ink from the plant’s juice and dipped it
to the chromatography paper using capillary tube at the lower part of the paper (1 cm starting from the
bottom) where they have marked with circles and lines, each of three columns. Placed the chromatography
paper in the covered 1000 mL beaker and added a solvent vapor and waited for 2 minutes before plunging
the lower part of the chromatography paper to the solvent vapor where it was instantly covered by an
aluminum foil. The experimenters waited for an hour and some minutes (Time Start: 1:57 pm, Time Ended:
3:06 pm). Chromatography paper was removed from the beaker and measured. Results were documented
and lab report was made.

RESULTS AND DISCUSSIONS (30 points)

Pigments are defined as substances in plants that are able to absorb visible light. These can be classified
into three basic groups.

The first are the chlorophylls which are greenish pigments containing a porphyrin ring. The ring contains
several double bonds which makes it stable, and at the same time allows free migration of electrons. In
this way, when sunlight strikes the plant surface, electrons in the pigment molecules of the chloroplast
thylakoid become excited which in turn pass on this excitation to the photosystems that begins the series
of cellular events that generate oxygen and sugar.

Four species of chlorophyll – a, b, c, and d – are known. Chlorophyll a is the primary photosynthetic
pigment in all higher plants, algae, and the cyanobacteria. Chlorophyll b is found virtually in all higher
plants and green algae, differing from the former only in that a formyl group substitutes for the methyl
group in ring II. Chlorophyll c is meanwhile found in the diatoms, dinoflagellates, and brown algae and
lacks the phytol tail of chlorophyll a. Lastly, chlorophyll d is found only in the red algae and has an (-O-
CHO) group in place of the (-CH=CH2) group on ring I of chlorophyll a.

The second class of pigments are the carotenoids. They are usually red, orange, or yellow pigments
composed of two small six-carbon rings connected by a chain of carbon atoms. Their high carbon content
prevents them from dissolving in water and as such they must be attached to membranes within the cell.
They have several functions, including the broadening of the spectrum of colors able to drive
photosynthesis (especially in seasons with shortened days such as fall and winter), and in photoprotection;
they are able to absorb and dissipate excessive light energy that can otherwise damage chlorophyll or
interact with oxygen to produce reactive oxidative molecules that can damage the cell.

The third class of pigments are the phycobilins. They are water-soluble pigments found in the chloroplast
stroma or the cell cytoplasm. Occurring only in the Cyanobacteria and Rhodophyta, they are efficient in
absorbing light wavelengths that are not well absorbed by chlorophyll a. These pigments are bound to
phycobiliproteins which pass on the absorbed light energy to chloroplasts for photosynthesis.

Knowledge of which types of pigments are present in a plant is useful in the field of agriculture. Using
this information, lights that promoting the optimal growth of plants having certain pigments can be
developed, increasing their yield. Additionally, pigments extracted from plants can be used as dyes in
scientific research.

One way of determining the pigments present in a plant sample is through paper chromatography. Paper
chromatography separates pigments present in the plant sample based on their solubilities in the solvent;
compounds which are very soluble move along with the advancing solvent front, while less soluble
compounds travel slowly through the paper, well behind the solvent front. Chlorophyll a is slightly
soluble in a 3:1:1 mixture of petroleum ether, acetone, and water (which was the resulting mixture used in
the study), while carotenoids are very soluble in this system. This difference in solubility should allow the
separation of chlorophyll a from the carotenoids and chlorophyll b, which is less soluble than chlorophyll
a.

The following chromatogram was obtained from the chromatography done in the study.

The “solvent front” is the position of the liquid solvent on the chromatography
paper at any given time. the solvent will gradually move from the bottom toward
the top of the paper, carrying dissolved pigments with it. The chromatogram was
stopped before the solvent front reaches the top of the paper and marked the
location. The researchers - will use this distance to calculate Rf
Chlorophyll B

Chlorophyll A

carotenoid

origin (where the pigment extract was applied)

Obtained chromatogram from the paper chromatography of leaf extracts. Only one trial was used in the
identification of pigments, with the topmost band determined to be a carotenoid, followed by chlorophyll
A and chlorophyll B.

The first pigment band was identified as a carotenoid due to its yellow-orange color, the second band
identified as chlorophyll A due to its darker green color, and the third band identified as chlorophyll B
due to its yellow-green color. The study is said to be a success in this respect as the results obtained
matched the theoretical results.

Additionally, pigments can also be identified based on the value of their retention factor; the retention
factor (Rf) is calculated as the distance the pigment travels (in centimeters) divided by the distance the
solvent travels (in centimeters). Standard values of the Rf are compared to the calculated values and the
closest standard value that matches the calculated value is used to identify the pigment. This step
however, was no longer done in the study.

Spectrophotometry can be utilized for measuring the chlorophyll content of a leaf by measuring the
absorbances of the plant extract at red and far red regions of the visible light spectrum. The absorbance of
the extract is directly proportional to its chlorophyll content. The experiment measured the absorbances of
old and young leaves coming from the same plant in order to compare their chlorophyll content. In the
experiment, the pigments from old and young plants were subjected to spectrophotometry to identify
which wavelength would yield the highest absorption therefore identifying the pigments present, and also
compare at which level of maturity plants would yield more chlorophyll. The former is possible because
plant pigments participate in photosynthesis by absorbing light, and there is the optimal wavelength
wherein they can absorb the most amount of light and can therefore enhance the process of
photosynthesis.

Pigments in seed plants may be present as chlorophyll a, b, and carotenoids, all with varying abundance.
For the old and leaf samples, it can be seen that the measured absorbances peaked at two wavelengths
(Figure 2). The first peak is around 450-470 nm while the second peak is around 663 nm. This data
implies that most of the pigment extracted must be from chlorophyll a and b, since theoretically, these
pigments peak at 430-450 nm and 640-660 nm. It should also be remembered that peaks in an absorbance
vs. wavelength pigment spectra means that these pigments absorb and utilize light best in these
wavelengths. In Figure 3 below, the other pigments and corresponding peak wavelengths can be seen.

Aside from knowing the pigments present in the leaves, the graph could also show the relative amount of
chlorophyll present in the leaves. Theoretically, older leaves contain much more chlorophyll than younger
leaves; this is contrary to the results. These results can only make sense if the plant from which the extract
was taken from has a magnesium deficiency, assuming that no methodological error was committed. Plant
with Mg deficiency tend to sequester Mg from old leaves by degrading chlorophyll and then transporting
the retrieved Mg to the younger leaves which have higher photosynthetic needs.

Other methods of measuring the pigment content of leaves include using chlorophyll content meters,
which do not require an extract to be prepared, and the more superior technique known as chlorophyll
fluorescence where the ratio of chlorophyll fluorescence at certain wavelengths give a linearly
proportional estimation of the chlorophyll content.

The experimenters covered the whole body of 1000 mL beaker with a black paper because light
would easily penetrate through the apparatus and would have made the experiment meaningless because
chlorophyll and chromoplast would easily react with light in which they would easily disappear, having
low visibility would make it hard to compare the raw pigments and would take the whole experiment
longer.
 The beaker was filled with 15 mL chromatographic solvent and was saturated with the solvent
vapor by covering it with an aluminum foil for 2 minutes. After that, it is then opened and the
chromatography paper with the dipped plant’s juice is placed inside with a string at the top and the bottom
part is dipped on the chromatographic solvent where the part of the dipped juice is soaked. It is again
covered by an aluminum foil and placed in the dark corner between the cabinets where light is not
present.

CONCLUSION (10 points)

Paper chromatography was used for separating and identifying the color pigments of the Iresine
Herbstii leaves. It shows in the experiment that pigments moved upward. The colors were observed,
measured and recorded. The experimenters started at 1:57pm and ended at 3:06 pm. There are 3 colors
divided in filter paper. The color at the top was light green which is the chlorophyll has a measurement of
(*) for both sides and (*) in the middle, the yellow-orange or indicated as carotenoid has a measurement of
(**) in both side and middle which travel the farthest, the red-violet color has a measurement of (*) wherein
it is at the bottom which is called anthocyanins. These three pigments has a big role for the plants. The
polarity of the pigment of the plant leaves was observe, on how it interact with the filter paper and those
pigment that was separated through chromatography. The overall result was it is polar because the outcome
was 10,5,10. The plant pigments will be absorb by the chromotographic solvent who act as the mobile phase
that was sticked in the stationary phase which is the filter paper. The hree of them will interact with each
other, the pigment will be separated because of the solvent used depending on the polarity. Polarity of the
compounds dictates their affinities towards the stationary and mobile phases.

LITERATURE CITED (5 points)

Author (s). Year Published. Title of Book. Place of Publication: Publishing Company, Inclusive pages.
[Book source]

Author (s). Year of Publication. Title of Journal. Publisher Volume (Number): Inclusive Pages. [Journal
source]

Webpage URL, date last retrieved/accessed. [Internet source]