Mindanao State University

College of Agriculture
General Santos City

Chromatography of Plant Pigme nts: Utilizing of Chili Extract

A Written Report Submitted to:

Dr. Ange m Descallar

Subject Instructor

A Partial Fulfillment of the Requirement in Organic Chemistry Laboratory

Submitted by:
Mendez, Maria Cheska M.

December 2023
Introduction:

Chromatography is a process utilized for separating complex mixtures into their individual
components. This essential tool was first used in the 19 th century, where a complex pigment is
dissolved using a sheet of paper which contacts with a container filled with water or alcohol,
then pigments are separated and this also where the chromatography gets its na me. Furthermore,
chromatography undergoes phases to separate mixtures. In the mobile phase, the mixture is
dissolved in a substance. Then, the different components of the mixture will travel at different
speed in the stationary phase, causing them to separate from one another. The solvent used
carries the dissolved pigments as it moves up the paper. The largest molecules of the mixture
will travel more slowly while the smallest ones race ahead; this causes the stationary phase to
develop discrete bands of color corresponding to each component of the mixture.
Chromatography can also be used to separate substances based on its characteristics such as
polarity and solubility and one of its application is the separation and analysis of plant pigments.

Plant pigments consist a variety of pigments and these pigments plays important roles in
photosynthesis and other biological processes. Chlorophyll is the most common plant pigment in
and it is responsible for the green color of most plants. Aside from chlorophyll we have
anthocyanins and carotenoids. Anthocyanins constitute an important group of plant pigments that
are soluble and belong to the family of flavonoids. They are also responsible for the red, pink,
purple, and blue colors to plants. Moreover, Carotenoids are plant pigments responsible for the
yellow, bright red and orange color, and plays a key role in plant health.

In this laboratory experiment, we are expected to separate and identify the different pigments
present in a plant extract utilizing the chromatography process. Considering also the polarity and
solubility of the solvent used, we will be able to separate the pigments into distinct bands on the
chromatography paper and identify what kind of pigment present based on its characteristic
color. This experiment will help in deepening our understanding of the principles behind
chromatography as well as give us a hands-on experience with one of the most important
techniques in modern analytical and organic chemistry.

Review of Related Literature:

The value of chromatography depends on its ability to separate the components of mixtures with
large number of either similar or dissimilar analysts. Its result can be presented in a
chromatogram. It could be paper chromatography where series of spots are vis ible in the
chromatography paper and computer-generated print out comprising a series of peaks from a
baseline drawn on a time axis (Robards & Ryan, 2019). Furthermore, the methods utilized in the
production of extracts as well as obtaining a high-quality of bioactive fractions or compounds of
medicinal plants includes the selection of a suitable solvent, extraction techniques,
physiochemical screening procedures, fractionation methods, and identification techniques
(Abubakar, & Haque, 2020). Additionally, the natural red pigments in plants such as carotenoids,
anthocyanins, and betacyanins are most widely used in the food industry and the purpose of these
pigments was to improve the health effects of the diet (Fernández-López, et.al. 2020).
Furthermore, the photosynthetic pigments of the three green algae isolated from freshwater
ecosystem that were investigated could have a potential use in the nutritional application. It
contains pigments of chlorophyll a, chlorophyll b, carotenoids, xanthophylls, β-carotene and
lutein. Chlorophyllide B, and a type of xanthophyl (Aditi, et.al. 2020). Moreover, the UV–vis
spectrophotometric method was utilized for the rapid measurement of chlorophylls, carotenoids,
and pheophytins. In addition, the substances that were separated on a Thin- Layer
Chromatographic plate were identified and quantified using spectral analysis and the standard
calibration curve method (Hynstova, et.al. 2018).

Amaranthus edible stems and leaves are convenient and abundant source of minerals,
carotenoids, digestive fiber, and protein that contains lysine and methionine. It also has abundant
antioxidant pigments, such as anthocyanin, carotenoids, chlorophylls, etc. that are essential
components in the food industry as well as in the remedy of diseases (Sarker & Oba, 2020).
Furthermore, Aside from the high-performance liquid chromatography, Raman micro-
spectroscopy can be used to determine carotenoid pigments or several carotenoids in an
admixture and unknown biological system (Osterrothová, et.al. 2019). Moreover, It has recently
been found that, in a mixed deciduous broadleaf forest, leaf chlorophyll content serves as a more
accurate proxy for leaf photosynthetic capacity than leaf nitrogen content (Chou, et.al. 2020).
A fundamental biophysical technique for separating, identifying, and purifying the constituents
of a qualitative and quantitative blend study is chromatography. The process is divided into
many categories depending on the chromatographic mode, process geometry, and separation
theory. In the stationary bed-based separation technique: column, thin layer and chromatography
on paper are included. In addition, metal affinity of the biomolecules is frequently used in
affinity chromatography. While the High-performance liquid chromatography (HPLC) is a
significant qualitative and quantitative method and it is used for pharmaceutical and biologic
sample estimation. Lastly, Quality control of product components chromatography technique is
the most flexible, easiest, efficient and quickest type (Patil, et.al. 2020). Moreover, paper
chromatography is a type of analytical tool which is utilized for the separation of colored
component of paper fibers (stationary phase) and solvent (mobile phase) in the atmosphere. In
addition, this technique has been used from the ancient times for the separation of organic and
inorganic compounds (Ganesh, et.al. 2021).

Materials:
1. Fresh Malabar spinach leaves (or other plant material, better if it contains more colors)
2. Mortar and Pestle
3. Filter Paper
4. Beaker
5. Acetone
6. Glass Vial
7. Chromatography Paper
8. Pencil
9. Ruler
10. Test tube
11. Toothpick
Methods:
Important: Wash your hands thoroughly with soap and water before proceeding.

1. Crush a few spinach leaves using a mortar and pestle and add a small amount of acetone
to make an extract.
2. Filter the extract to remove any solids, and then pour a small amount of the extract into a
glass vial.
3. Cut a piece of chromatography paper to a size that will fit inside the test tube, and draw a
line using a pencil about 1 cm from the bottom of the paper.
4. Using a toothpick, place a small drop of the spinach extract on the pencil line, about 1 cm
from the edge of the paper.
5. Place the chromatography paper in the test tube with the pencil line above the level of the
acetone in the tube. Allow the paper to sit in the acetone until the solvent has migrated up
the paper, carrying the pigments with it.
6. Remove the paper from the test tube and allow it to dry completely.
7. Once dry, mark the distances traveled by each pigment from the pencil line, and identify
the different pigments based on their characteristic colors.
8. Submit the chromatography paper to your instructor.

Result and Discussion:

The purpose of the experiment was to separate and identify the different pigments present in a
plant extract using chromatography. In this experiment, we utilized chili extract, and methane as
our solvent, which is responsible in the separation of pigments. It carries the dissolved pigments
as it moves up the paper. After the experiment, we successfully separate and identify the kind of
pigment present in the chili extract based on its characteristic color in the chromatography paper.
The result only suggests that chromatography is an effective process of separating and
identifying pigments in a plant, supporting the previous research on the chromatography of plant
pigments.
The pictures below shows the result attained upon the conduction of chromatography of a chili
extract with the usage of methane as the solvent.

Based on the observation, the pigments present in the chili extract were primarily carotenoids,
namely capsanthin and beta-carotene. Capsanthin is responsible for the intense red color of chili
peppers, while beta-carotene contributes to the yellow and orange hues. These pigments were
identified through the chromatography technique, which separates the different pigments based
on their solubility and molecular size. The chromatogram revealed distinct bands of red and
orange, indicating the presence of capsanthin and beta-carotene, respectively. Therefore, the
characteristics colors of the pigments in the chili extract were vibrant red and yellow-orange.
Furthermore, the reason behind why the pigments separated differently than the expected is due
to the solvent used. Since the pigments are carried up, it travels at different rates depending on
the solvent utilized and it may also because they are not equally soluble. A pigment that is the
most soluble will travel the greatest distance and a pigment that is less soluble will move a
shorter distance.

REFERENCES:

Abubakar, A. R., & Haque, M. (2020). Preparation of medicinal plants: Basic extraction and
fractionation procedures for experimental purposes. Journal of Pharmacy and Bioallied
Sciences, 12(1), 1. https://doi.org/10.4103/jpbs.jpbs_175_19

Aditi, N., Jaishini, C., Raisa, F., Aamna, K., & Rupali, B. (2020). Photosynthetic pigments, lipids
and phenolic compounds of three green algae isolated from freshwater ecosystem. J.
Algal Biomass Utilization, 11(1): 68-83.
https://storage.unitedwebnetwork.com/files/521/8f072f204b860818d68b9535eaa64de1.p
df

Chou, S., Chen, B., Jing, C., Wang, M., Wang, S., Croft, H., & Shi, Q. (2020). Estimation of leaf
photosynthetic capacity from the photochemical reflectance index and leaf pigments.
Ecological Indicators, 110, 105867. https://doi.org/10.1016/j.ecolind.2019.105867
Fernández-López, J. A., Fernández-Lledó, V., & Angosto, J. M. (2020). New insights into red
plant pigments: more than just natural colorants. RSC Advances, 10(41), 24669–24682.
https://doi.org/10.1039/d0ra03514a

Ganesh, S. S., Rao, P. S., Jadhav R. S., Piyusha, K., & Diksha, A. (2021). A review on
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of Pharmaceutical Analysis, 11(1), 45-48.
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Hynstova, V., Štěrbová, D., Klejdus, B., Hedbávný, J., Húska, D., & Adam, V. (2018).
Separation, identification and quantification of carotenoids and chlorophylls in dietary
supplements containing Chlorella vulgaris and Spirulina platensis using High
Performance Thin Layer Chromatography. Journal of Pharmaceutical and Biomedical
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Osterrothová, K., Culka, A., Němečková, K., Kaftan, D., Nedbalová, L., Procházková, L., &
Jehlička, J. (2019). Analyzing carotenoids of snow a lgae by Raman microspectroscopy
and high-performance liquid chromatography. Spectrochimica Acta Part A: Molecular
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DOCUMENTATION:

Pouring of the methane solvent Pouring of the ethyl acetate

into the graduated cylinder into the solvent
Placing a small drop of chili extract Placing the chromatography paper that
into the chromatography paper contains the chili extract into the
beaker filled with the mixed mixture

Observing the chromatography paper Watching the chromatography paper in

until the solvent has migrated up in the the chromatogram after the solvent
paper, carrying the pigments with it migrated up in the paper