Lab Report: Observations on Plant Chromatography Experiments
Landasan, Guenez, Carson, Navarez, Olan
Abstract
The purpose of the experiment is to differentiate the pigmentations present in
multiple leaves as the subject and deduce whether only one pigment or multiple are
present in the leaf. The experiment utilized materials such as 70% Isopropyl Rubbing
Alcohol, an assortment of leaves with observable different pigmentations, glass dishes
for containing the leaves in alcohol, a metal food tray, coffee filters, and boiling hot
water. We are to incline that leaf pigmentation is what the eye generally sees
everyday. This results in us thinking that red leaves are simply composed of
anthocyanins, a pigment that has a range of colors from red, purple, blue, and black,
or that green leaves mainly possess chlorophyll only, a green pigment that is
commonly recognized in its role in photosynthesis. Several leaves are gathered from
various garden sources and brought alongside the aforementioned materials These
range from green, red, and brown leaves. They are cut into several smaller pieces and
placed into three different glass dishes, assorted based on their color. They are then
submerged in alcohol and ground further with a pestle in order for the alcohol to get
in and do its work. They are then placed in a metal food tray and hot water is added
around them in order to keep the temperature up for the experiment. The alcohol and
leaves are swirled occasionally every five minutes and the hot water is replaced with
freshly boiled hot water in order to keep a constant high temperature all throughout
the experiment. They are left to do the reaction for 30 whole minutes with the
occasional stirring and water replacement and then opened to be checked. After 30
minutes, the solutions have noticeable tints of color and produced an earthy odor.
Coffee filter strips are then prepared and put inside the solutions in order to gather the
tinted liquid for better observation, and left from 30 to 120 minutes to cool and dry.
From the experiment, we can conclude that the brown leaves produced the expected
reddish-brown pigment as a result of tannins, however, the red and green leaves
leached out a surprising amount of yellow pigment. This may be due to the presence
of xanthophylls in the leaves, which were more pronounced than the pigments they
were expected to release.
Keywords - pigmentation, xanthophyll, tannins, chlorophyll
Introduction
The purpose of the experiment is to observe the different pigments present in
various plants, and compare the leaching pigments to the original pigmentation in said
leaves.
Any colorful material generated by a plant is referred to as a plant pigment. A
pigment is a chemical substance that absorbs visible light between the wavelengths of
380 nm (violet) to 760 nm (ruby-red). Plant pigments come in a variety of shapes and
sizes, and they may be found in a variety of chemical compounds. Plant pigments are
responsible for the color of leaves, flowers, and fruits, as well as governing
photosynthesis, growth, and development. Plant pigment chromatography is a useful
technique in the separation and identification of different plant pigments.
Plant pigment chromatography is useful for identifying unknown compounds often used in crime scene investigations to match ink, lipstick, or colored fibers.
Plant pigments have a key role in photosynthesis, plant growth, and
development (Sudhakar et al., 2016). Insects, birds, and mammals are attracted to
pigments as visual signals for pollination and seed dissemination. It has been found
that pigments protect plants from UV and visible light damage (Tanaka et al., 2008).
All fruits contain chlorophyll, which serves as the primary pigment for capturing
yellow and blue light for photosynthesis, which produces energy for plant
development and growth. Carotenoids and anthocyanins, unlike chlorophylls, are
accessory pigments (usually with a complementary absorbance spectrum to
chlorophylls) and secondary metabolites with far more diversified structures and
activities in plants, as well as greater potential nutritional and health advantages in the
diet (Chen, 2015). The human diet includes a variety of pigment-rich fruits. Fruit
pigment content has been researched for decades, not only because of the aesthetic
appeal of mature fruit colors, but also because of the diverse spectrum of compounds
that are present and responsible for the distinctive green, yellow, orange, red, blue,
and purple hues.
Ripening is the process through which a fruit undergoes physiological and
biochemical changes to achieve desired color, flavor, fragrance, sweetness, texture,
and hence eating quality. The ripening process normally takes place after a fruit has
reached full maturity. When a fruit achieves complete maturity, it reaches its
maximum size and weight, and its growth rate slows. Depending on the species, fruit
ripening can occur on the plant or after harvest (Li, 2012). It is the process of
pigments which help us understand these stages of phenomena of not only fruit but all
varied plant life in general.
Plant leaves include three types of pigments, and their retention or creation
impacts the color of leaves before they fall away, molecules, which go beyond basic
chemical formulae that specify the amount of atoms of different elements that make
up the molecule. The most prominent example is glucose, which is a common sugar.
Glucose, which is a disaccharide, can be purchased as a sweetener. It is
usually one half of typical table sugar (sucrose). Chlorophylls, carotenoids, and
anthocyanins are the three types of pigments that are present during the aging of
leaves. More elaborate diagrams will be presented to demonstrate the architecture of
these pigments. Carotenoids are water-repelling pigments with a very long chain that
are produced in the plastids of plant cells. A common carotenoid, ß-carotene, is
synthesized in the chromoplasts of the ray flowers of the sunflower, resulting in vivid
yellow-orange hues. These pigments absorb preferentially in the blue wavelengths,
scattering the longer wavelengths and resulting in the yellow hue. Carotenoids are left
over in the chloroplasts in fall leaves and are exposed by the loss of chlorophyll. Most
common examples of them are lycopene (commonly found in tomatoes) and
xanthophylls (yellow hued pigment in plants). The pigments of photosynthesis are
chlorophylls a and b. They're made in the photosynthetic tissues of the leaf's
chloroplasts. Because of the long phytol tail of the molecule, chlorophyll molecules
are particularly water repellent. The molecule's closed ring is comparable to
hemoglobin in human blood, except instead of iron, it carries a magnesium ion.
Because each ring includes four nitrogen atoms, it is a huge and costly chemical to
produce. Chlorophyll is generally broken down at the end of the leaf's life cycle, and
the plant resorbs much of the nitrogen. Anthocyanins are water-soluble pigments
generated in the cytoplasm of colored plant cells via the flavonoid pathway. The sugar
molecule's connection makes them highly soluble in the vacuole's sap, which is where
these molecules are store once they are launched. During the fall, they are responsible
for the pink-red hue of most flower petals, most red fruits (such as apples), and
practically all red foliage. Anthocyanins absorb blue-green wavelengths of light,
enabling red wavelengths to be dispersed by plant tissues, making these organs visible
to humans as red.
Objectives of the study
Upon completion of the experiment, we should:

As the subject, be able to distinguish the pigmentations present in various
leaves.

Determine if the leaf has only one pigment or many pigments.
The purpose of these experiments is to:

Differentiate the pigmentations present in multiple leaves as the subject

Deduce whether only one pigment or multiple are present in the leaf
Materials and Methods
List of Materials

70% Isopropyl Rubbing Alcohol

An assortment of leaves with observable different pigmentations

Glass dishes for containing the leaves in alcohol

a metal food tray

coffee filters

boiling hot water

pestle
Experimental Procedure (minute 30)
Leaves are
assorted by color,
torn into pieces
and dropped into
the dishes
They are then
submerged in
alcohol and
ground further
with a pestle.
Add more hot
water to keep
temperature hot.
As color
separates, add
coffee strips.
Experimental Procedure (Hour 1 and minute 30)
Leave for 30-120
minutes and then
dry the strips.
Results and Discussion
I.
Result
Observe color of
dried coffee
strips.
Add hot water to
the metal food
tray to keep
temperature up
Leave for 30
minutes to warm
up. Swirl the
leaves in the jar
every 5 minutes.
After two hours of drying, green leaves yielded this yellow-green color. It was
somewhat surprising since one would anticipate the color to be predominantly
green, but there were glimpses of yellow in there as well.The yellow-green tint
on the coffee filter was significantly more noticeable and crisper with Red
Leaves. It was expected to create a predominantly red pigment many hours
after the experiment, therefore this was unexpected. The reddish-brown hue on
the coffee filter was formed by the brown leaves. The pigment's color closely
mimics the brown leaves utilized in the experiment, therefore this was the
least surprising result.
II.
Discussion
Prior the experiment, it is common to assume that the color of the leaves that
the naked eyes perceive is the same with the actual pigment of the plant. Thus,
this experiment is conducted to clarify if this hypothesis is true and if there is a
presence of one or multiple pigments in a leaf. As aforementioned above, the
green leaves which were expected to yield green pigments actually showed
yellowish pigments as well. Since chlorophyll gives the plant a bright green
color, it blocks out some yellow hues from the xanthophyll. It's actually
present in the leaf but is only showed when the dominant green color starts to
disappear. But that's not the case when it comes to the red pigment of the leaf
caused by anthocyanins since it is only produced before the leaves fall off
from the branches. Thus, we can conclude that the leaf's seemingly one color
is a mixture of more than one pigment which became more pronounced in a
certain stage of the leaf's life in a particular season.
But still it is important to take note of the type of leaf used in the experiment.
To make more conclusive findings, other researchers might also try
performing leaf pigment chromatography on the leaves of varying colors from
same tree or plant, though this might sound challenging, to test if the leaves of
that particular plant changes color from green to orange, yellow, red, and
brown respectively.
Conclusion
We may deduce from the experiment that tannins created the predicted reddish-brown
pigment in the brown leaves, but that the red and green leaves leached off a surprising
quantity of yellow pigment. This might be owing to the presence of xanthophylls in
the leaves, which were more noticeable than the pigments predicted to be released.
Appendix
References
Latha, P., Reddy, P.V., &Sudhakar, P. (2016)Phenotyping Crop Plants for
Physiological
and
Biochemical
Traits:
Plant
Pigments
Retrieved
from:
https://www.sciencedirect.com/book/9780128040737/phenotyping-crop-plants-forphysiological-and-biochemical-traits
Ohmiya, Akemi,Sasaki,Nobuhiro&Tanaka,Yoshikazu(2008) Biosynthesis of Plant
Pigments:
Anthocyanins,
Betalains
and
Carotenoids
Retrieved
from:https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-313X.2008.03447.x
Chen,
Chunxian
(2015)
Overview
of
Plant
Pigments
Retrieved
from:
https://www.researchgate.net/publication/283871822_Overview_of_Plant_Pigments
Li Li (2012) Fruit Pigment Changes During Ripening Retrieved from:
https://www.sciencedirect.com/science/article/pii/B9780081005965216569