Name: Paul Matthew F. Gomez
Section: 2ChE-A
Date Performed: 11/19/2022
Date Submitted: 12/8/2022
Experiment No. 4
Column Chromatography of Plant Pigments
Abstract
A qualitative determination and dialectric analysis of the organic compositions of Capsicum
frutescens also known as, Siling labuyo, using column chromatography technique. It was
prepared by extracting the outer layer of the Capsicum frutescens and grinding it using sand,
mortar, and pestle. Proceeding with mixing the powdered sample with 10mL of
Dichloromethane which was then filtered out using a filter paper discarding the solid residue.
10mL of saturated sodium chloride solution and a pinch of anhydrous sodium sulfate was then
added to create the pigment extract. The column was then prepared by pouring 0.75g of silica gel
in a Pasteur pipette and then adding the first eluent, 1:1 Dichloromethane/Hexane mixture to the
Pasteur pipette enough to soak the whole silica gel. Subsequently, 0.5mL of pigment extract was
added to the pipette which was then followed by the chromatography extraction phase. The
results yielded a yellow-greenish, strong orange, and opaque orange pigment for the first,
second, and third eluents respectively.
Keywords: Qualitative, Dialectric, Capsicum frutescens, Chromatography, Eluent, Pigments
I.
Introduction
Column chromatography is a technique of separating substances based on their affinities on their
mobile and stationary counterparts. The principle of column chromatography basically revolves
around the polarities of different pigments to the eluents thus, as the pigments flow through the
Pasteur pipette at different rates, separating them in fractions is possible. The component with
the lowest affinity and adsorption to the mobile phase usually flows at a faster rate compared
with those with higher affinity thus, the components that flow faster are extracted first rather than
those which flow slower.
Basically, the mobile phase that transports the components is called as the eluent, and the
mixture that exits the Pasteur pipette is called as eluate which is a mixture of the mobile phase
and the analyte which in this case, the pigments. Since, the adsorption of the pigments in the
column occurs in a reversible manner, the rate of the movement of the components can be
expressed using the retardation factor:
𝑅𝑓 =
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒 (𝑃𝑖𝑔𝑚𝑒𝑛𝑡)
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 (𝐸𝑙𝑢𝑒𝑛𝑡)
1
EQ 1.
Column chromatography has a lot of applications, especially in the field of chemical engineering,
and chemistry in general. One of these is isolating active ingredients and analyzing them
qualitatively just like in the case of determining the organic composition of Capsicum frutescens.
Through this technique, the researcher can estimate the contents and impurities in a certain
substance. Therefore, purification is made possible using the chromatography technique.
Subsequently, column chromatography can be used to separate specific molecules from the
compound and use it to form a new substance. Although column chromatography has a lot of
benefits, it is also lengthy and resource demanding to use compared to other separation techniques
let alone automating the process which can compromise the cost effectiveness of the technique.
Image taken from: https://bitesizebio.com/29947/basics-column-chromatography
Fig 1. The basic principle of column chromatography
illustrated above is the observable phenomena that happens during a column chromatography
technique. As mobile phase was added, a component separation occurs due to the difference of
flow rates of these specific components in terms of the eluent added. The polarity of eluting
agents determines the rate at which the components of the mixtures move through column. The
more polar a component is to the eluting agent. The faster it moves through the column. The
polarity of a component can be determined using dielectric constants.
2
Table 1. Dielectric constants of common solvents at room temperature and atmospheric pressure
Dialectic Constant, 𝜺
8.93
6.0
1.99
32.6
2.38
Solvent
Dichloromethane
Ethyl Acetate
Hexane
Methanol
Toluene
As shown in the table above, there are different dielectric constants in a particular eluent thus,
these dialectic constants can be further utilized in order to extract certain pigments in column
chromatography. The dielectric constant of a mixture of solvents can be expressed as the sum of
the products of the dielectric constant of each solvent and its corresponding concentration.
𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑓𝑖𝑟𝑠𝑡 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 (𝑚𝐿)
𝜀𝑚𝑖𝑥𝑡𝑢𝑟𝑒 = 𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑚𝑖𝑥𝑡𝑢𝑟𝑒 (𝑚𝐿) (𝜀𝑓𝑖𝑟𝑠𝑡 𝑠𝑜𝑙𝑣𝑒𝑛𝑡) +
𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑒𝑐𝑜𝑛𝑑 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 (𝑚𝐿)
𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑡ℎ𝑒 𝑚𝑖𝑥𝑡𝑢𝑟𝑒 (𝑚𝐿)
(𝜀𝑠𝑒𝑐𝑜𝑛𝑑 𝑠𝑜𝑙𝑣𝑒𝑛𝑡)
EQ 2.
II. Materials
The experimental scope was conducted through the utilization of solutions and equipment
available in the UST Chemical Engineering Laboratory. Thus, all the resources were not
limited and not virtually gathered.
Sample. Capsicum frutescens, red pepper, was chopped into pieces wherein, the seeds were
removed. Approximately 0.5 grams were used. Coarse sand was also used as the grinding
medium for the sample.
Solutions. 10mL of Dichloromethane, 10mL of saturated sodium chloride solution, and a
pinch of anhydrous sodium sulfate was used to prepare the pigment extract. 0.75g of silica
gel and 1:1 dichloromethane/hexane solution was also utilized to prepare the column. Lastly,
1:1 dichloromethane/ hexane solution, dichloromethane, and 1:1 dichloromethane/methanol
solution were used as eluents.
Instruments. Vessels and materials such as mortar, pestle, Pasteur Pipette, three-pronged
clamp, iron stand, test tubes, spatula and cotton were used to prepare, store and execute the
experiment.
3
III.
Experimental
a. Extraction of Plant Pigment
To prepare the pigment extract, approximately 0.5 grams of Capsicum frutescens was
chopped into smaller pieces removing the seeds in the process. The chopped Capsicum
frutescens was grinded into fine powder using coarse sand, mortar and pestle. Subsequently,
the fine powder was then mixed with 10mL dichloromethane and stirred up until it looks
reddish. The liquid was then filtered out using filter paper and transferred into a test tube to
discard the solid residue. 10mL of saturated sodium chloride was then added to the filtrate
and the filtrate was covered with a cork in order to invert it three times. This allows the
formation of two layers in the test tube. The upper layer was then removed using Pasteur
pipette leaving the dichloromethane layer in the test tube. a pinch of anhydrous sodium
sulfate was then added and decanted thus, forming the pigment extract to be used for the next
phases.
b. Preparation of Column
Subsequently, a clean Pasteur pipette was prepared by removing the stopper and inserting a
piece of cotton in the bottom of the pipette. This was done in order to prevent the silica gel
from escaping the pipette. The Pasteur pipette was then attached to an iron stand in
preparation for the later steps. The column was then filled with approximately 0.75 grams or
up until the mark on the pipette with silica gel. The column was then filled with 1:1
dichloromethane/hexane solution up until all the silica gel inside the column was soaked.
Making sure that the silica in the column is an important aspect in column chromatography.
Once the silica gel is soaked with the solvent, it is prone to cracking when it is left to dry.
This cracking produces microcavities in the stacked silica gel thus, the flow of the eluent in
the column would not be able to easily reach the pigments in the column due to its faster flow
rate.
c. Separation of the Pigments
Thereafter, 0.5mL of Pigment extract was added to the column. Each drop was allowed to
settle in the silica gel before adding the next drop. Once the pigment extract was settled, 1:1
Dichloromethane/Hexane was continuously added to the column up until the color band moves
down the column. The eluate was then collected for this eluant. Once the color band becomes
colorless. Dichloromethane was then added to the column continuously up until the color band
moved down the column. The eluate was then collected for this eluant just like the previous
one. Lastly, dichloromethane/methanol eluant was then added to the column extracting the last
color band. The pigment from this eluant was then collected. Ultimately, there should be three
pigment samples that should be gathered in this experiment.
4
IV.
Results
Eluent
Color band/s obtained
hexane/dichloromethane
Yellow- Greenish Pigment
dichloromethane
Strong Orange
dichloromethane/methanol
Opaque Orange
Table 1: Color band obtained from each eluent used
This table shows that the 1:1 hexane/dichloromethane eluent extracted the yellow-greenish
pigment from the sample, Pure dichloromethane eluent extracted a strong orange pigment.
Lastly, 1:1 Dichloromethane/ methanol eluent extracted an opaque orange pigment.
V.
Discussion
This experiment was conducted in order to analyze the different compositions of Capsicum
frutescens qualitatively and relate the gathered results with the concept of dielectric constants.
This was done by applying EQ 2. with the specific eluent and pigments. The pigments gathered
in the experiment were a yellow-greenish pigment from 1:1 hexane/dichloromethane eluent, a
strong orange pigment from the dichloromethane eluent, and an opaque orange pigment from 1:1
dichloromethane/methanol eluent. Based on its color bands, the yellow-greenish pigment
vaguely signifies the chlorophyll content of the Capsicum frutescens. Chlorophyll is one of the
most important organic compounds that a plant possesses. It absorbs sunlight and converts water
and carbon dioxide into glucose and oxygen as its by product. Moreover, it is also responsible for
the green appearance of plant leaves. Chlorophyll is also evident in unripe red pepper thus, as we
can observe it also exemplifies green color when the red pepper is not yet matured. The ripening
process decomposes this chlorophyll into violaxanthin and lutein. Violaxanthin provides the
yellow coloration of unripe red pepper in this stage as the red pepper approaches maturity. As the
red pepper approaches maturity, it also turns red. This is due to the reason that it produces
carotenoids as it approaches its peak maturity. (Takemura et al., 2022).
Table 2. Dielectric constants of the eluents
Dielectric Constant, 𝜀
5.46
8.93
Eluent
Dichloromethane/hexane
Dichloromethane
5
Dichloromethane/methanol
20.77
The dielectric constant of a substance is directly related to its polarity. Thus, from the dielectric
constants shown above, the DCM/methanol eluent is the most polar, followed by the pure DCM,
and the least polar is the DCM/hexane eluent. Since the DCM/hexane eluent is the least polar, it
implies that the compound it separated, chlorophyll, has the lowest polarity. On the other hand,
the DCM/methanol eluent separated the opaque orange pigment, proving that those compounds
are the most polar among the pigments present in the siling labuyo. The second compound,
strong orange pigment, was separated by DCM, and hence has a polarity greater than the yellowgreenish pigment, but lower than the opaque orange pigment. With this the flow of each
component can easily be predicted using dielectric constants.
VI.
Conclusion
Ultimately, Column Chromatography is an excellent separation technique in order to
qualitatively analyze the components of a specific substance . In the case of this experiment. The
Capsicum frutescens pigments exemplified three color bands in the final product these are Yellow
pigment found in the first eluent, strong orange pigment found in the second pigment and lastly opaque
orange pigment from the third eluent, These pigments were gathered through utilizing the different
dielectric constants of the eluents. Relating the previously discussed dielectric constants. It can be said
that the organic component which was extracted using the dichloromethane/methanol is the most polar
while the component which was extracted using dichloromethane/hexane is the least polar. As we can
observe from this picture. These color-bands qualitatively showcases the presence of these organic
compounds through its pigment. Since column chromatography is generally a qualitative separation
process, we can vaguely analyze the exact contents of the carotenoids, chlorophyll and in the
experiment proper.
VII.
References
Abesamis, M. F., Acosta, L. J. F., Agustin, F., Aquitana, M. C., & Bagsican, M. J. (n.d.). Column
and thin layer chromatography. Column and Thin Layer chromatography. Retrieved November
28, 2022, from https://www.scribd.com/doc/29411801/Column-and-Thin-LayerChromatography#:~:text=of%20the%20siling%20labuyo%20were%20extracted%20with%20the
,was%20determined%20by%20using%20thin%20later%20chromatography%20%28TLC%29.
Admin. (2022, May 31). Column chromatography - principle, procedure, Applications & Elution
in chromatography. BYJUS. Retrieved November 28, 2022, from
https://byjus.com/chemistry/column-chromatography/
6
Torres, W. by D. J. (2021, September 29). Column chromatography made simple: An easy to
follow guide. Bitesize Bio. Retrieved November 28, 2022, from
https://bitesizebio.com/29947/basics-column-chromatography
Vedantu, A. (2022, April 27). Column chromatography. VEDANTU. Retrieved November 28,
2022, from https://www.vedantu.com/chemistry/column-chromatography
Takemura, Sahara, & Misawa. (n.d.). Violaxanthin: Natural function and occurrence,
biosynthesis, and heterologous production. Applied microbiology and biotechnology.
Retrieved November 24, 2022, from https://pubmed.ncbi.nlm.nih.gov/34338805/
VIII.
Appendix
Figure 2: The actual pigments extracted from chromatography experiment
7
Figure 3: Set-up for the column chromatography
Dielectric constants computations
1
1
ε (of 𝐷𝐶𝑀/ℎ𝑒𝑥𝑎𝑛𝑒) = ( ) (8.93) + ( ) (1.99) = 𝟓. 𝟒𝟔
2
2
ε (of 𝐷𝐶𝑀) = 𝟖. 𝟗𝟑
1
1
ε (of 𝐷𝐶𝑀/𝑚𝑒𝑡ℎ𝑎𝑛𝑜𝑙) = ( ) (8.93) + ( ) (32.6) = 𝟐𝟎. 𝟕𝟔𝟓
2
2
8