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BIOORGANIC
CHEMISTRY
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BioOrganic Chemistry Laboratory CH205 (2015-2016) Experiment 4

Separation of Colored Constituents from the Fruits of

Capscium frutescens through Column Chromatography
Mary Bernardine G. Bagalay, Julie Ann Kim F. Berdonado,
Jeanne Isabelle B. Bilasano, and Assumpta Minette C. Burgos*
Department of Speech Language Pathology, College of Rehabilitation Sciences
University of Santo Tomas, Espana Street, Manila 1008
Date Submitted: April 4, 2016
Abstract:
Column chromatography was used to separate the colored constituents of the Capsicum frutescens. Afterwards, a UV/VIS
Spectrophotometer was used to determine the maximum observance of each of colored constituents. Results yielded a
0.50mL yellow eluate with a maximum observance of 446.0nm; 0.90mL orange eluate; 0.90 mL golden yellow eluate with a
max of 484.91nm; and 0.40mL rusty orange eluate with max of 452.72 nm for hexane, dichloromethane in hexane,
dichloromethane, and methanol in dichloromethane respectively.
Keywords: Capscium frutescens, red pepper, eluate, column chromatography, spectrophotometry

Introduction
The Capsicum frutescens is a member of the Solanaceae (Nightshade family). Capsicum frutescens
is also known as cayenne pepper, or locally known as siling labuyo. Cayenne pepper is a small shrub
with alternate and has oval to lanceolate leaves (World of Chilles, n.d) . The flowers are white. Its fruits
change its color from green to yellow to orange then to red. A red cayenne pepper indicates a mature
cayenne pepper. They have a hot pungent taste; the size of its fruit is contrary to the level of its
spiciness: the smaller the cayenne pepper, the hotter. Their seeds are hotter than their pods. And it is the
seeds where you can find capsaicin (carotenoid), which is believed to have an antibiotic properties.
Some hear-say of the indigenous people in the mountains say that cayenne pepper can make you voice
sound better. Moreover, Capsicum frutescens can be used as a food ingredient. In the Philippines, it is
among the common ingredients used by locals in cooking Bicol Express, insarabasab, and laing to
name a few.
The color of the Capsicum frutescens is influenced by the carotenoid present. Carotenoids are
class of phytonutrients (plant chemicals) that are found in a variety of plants, algae, and bacteria. These
are pigments responsible for the red, orange, and yellow hues of many fruits and vegetables. Besides
chlorophyll, carotenoids also help in the absorption of light energy in photosynthesis. In terms of health
benefits, carotenoids function as an antioxidant that fights off free radicals- single oxygen atoms that
*Assumpta Minette C. Burgos

ricochet around the cell thereby destroying cell structures and properties. They also serve as an antiinflammatory and aids in cardiovascular disease prevention.
Carotenoids can be classified into two classifications carotenes and xanthophylls. Xanthophylls
contain oxygen which gives them a vibrant yellow hue. Lutein and zeaxanthin are xanthophylls that are
mostly linked with eye health. Good sources of lutein and zeaxanthin are kale, spinach, paprika,
avocado, and yellow-fleshed fruits. Another example of a xanthophyll is beta-cryptoxanthin what
provides provitamin A. Beta- cryptoxanthin is believed to reduce lung cancer risk.
Carotenes are contains hydrocarbons and does not contain oxygen. Because of this, they exhibit
an orange color. Some example of carotene are -carotene, -carotene, and lycopene. -carotene can
produce twice as much vitamin A than -carotene and beta-cryptoxanthin (a xanthophyll). It is also
responsible for the orange color of some foods. Among the few sources of -carotenes are mangoes,
papaya, carrots, spinach, kale, and pumpkin. Second, -Carotene is believed to benefit longevity. People
with highest level of -Carotene in their blood are less likely to die of heart disease compared to those
with high level of -carotene (Ito Y, et. al, 2006). Pumpkin, carrots, tomatoes, collard, tangerines,
among other else has a good amount of -Carotene in them. Lastly, lycopene is a red pigment
responsible for the color of watermelon and tomatoes. Lycopene is the most effective in deactivating
free radicals. This characteristic of lycopene may be attributed to their molecular shape.
Carotenoids in Capsicum frutescens can be only be known after conducting column
chromatography. Subsequently, further study on the chemical characteristics of the Capsicum frutescens
can be made through spectrophotometry.
In this experiment, the groups objective was to separate the colored constituents of the red
pepper, through column chromatography then, determine the major and minor colored constituents. In
addition to that, the group had to characterize the separated colored constituents through their UV
spectra (max) and relate them with their structure.

Methodology
The materials used in the experiment were 5 pieces of red pepper (Capsicum frutescens), hexane,
dichloromethane (DCM), methanol, spatula of anhydrous sodium sulfate (Na2SO4), spatula acid
washed sand, and silica gel for column chromatography (Merck 7734). The following apparatuses
namely mortar and pestle, 6cm glass funnel, 50 mL beaker, microcolumn chromatography set up, vials,
and latex gloves were also used.
Fresh red pepper fruits (Capsicum frutescens) must be first obtained. Then, triturate them with 5
mL dichloromethane. Trituration of the pepper eliminates impurities. The residue will be discarded and
decantate will be collected. Anhydrous sodium sulfate (Na2SO4) was added to the decantate to remove
traces of water that may still be present in the extract. Originally, this part of the experiment should have
been done by the group but, due to time constraints one of the course professors did it on their behalf.
*Assumpta Minette C. Burgos

Afterwards, Capsicum frutescens extract was collected and placed in a beaker. Silica gel was
added to Capsicum frutescens. While swirling the mixture with a stirring rod, the solvent evaporated and
dry powder remained. The pigment mixture was then put aside.
Another course professor assisted the students in preparing the microcolumn. The microcolumn
was prepared by using a 5.75-inch Pasteur pipette. First, the pipette was plugged with cotton. The
cotton plugged the exit of the Pasteur pipette and thereby hold the column of stationary phase and let
only the solvent and the sample escape. Next, a stationary phase was placed on top of the cotton using a
microspatula. In this experiment, silica gel was used as a stationary phase. Lastly, a thin layer of sand
was poured over the silica gel using a spatula. This is to keep the top of the column level when eluent is
added. The thin layer of sand was also to minimize disturbances of the column and diluting the sample.
As each layer was added, tapping of the pipette was observed to prevent air bubbles, cracks and
channel formations. Formation and/or presence of air bubbles will lead to a poor separation. In addition
to that, if the surface is not on the same level, compound on one side will adsorb faster than the other. In
such cases, overlapping of bands may be observed. Pasteur pipette was clamped to an iron stand where it
suspends just enough for a test tube to stand at its end. The set aside pigment mixture was then tipped on
top of the sand.
Subsequently, eluents were then collected. Eluents are solvent or solvent system that carries the
component of a mixture through the stationary phase by gravity or pressure. In this experiment the
eluents used were hexane, 50% dichloromethane (DCM) in hexane, dichloromethane, and 50mL
methanol in DCM. These were loaded into the pipette one after the other in the order: hexane> 50%
dichloromethane (DCM) in hexane> dichloromethane>50mL methanol in DCM. Shifting from one
eluent to another was done after collecting the colored eluates in test tubes. The production of eluates is
plausible due to the process of column chromatography.
Then, collected eluates were transferred to a 10mL graduated cylinder to measure the volume.
Afterwards, the eluates were transferred back to its original test tube. The collected eluates were then
subjected to thin layer chromatography using dichloromethane/hexane (1:1) as a solvent system. For thin
layer chromatography, spectrophotometer was used. Spectrophotometer measures the UV spectrum of
the pure eluates. It also aids in determining the maximum absorbance ( max) of the pure eluates.
Again, due to limited time, the group had to surrender their samples to their course professor for
spectrophotometry. Results of spectrophotometry was given and the maximum absorbance (max) was
determined.
Results and Discussion
In previous separation experiments, extraction and distillation were used. As for this experiment
another way of separating compounds is introduced that is column chromatography.
Column chromatography is a method used for separation and purification of solids and liquid. If
distillation separates through the difference of boiling points, column chromatography separates through

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the difference of polarity from a non-polar to semi-polar then to polar. The principle of column
chromatography is based on differential adsorption of substance by the adsorbent.
There are three interactions that occur in column chromatography: the activity of the stationary
adsorbent phase, the polarity of the eluting solvent; and lastly, the polarity of the compounds that are
being chromatographed (Williamson & Masters, 2011).
Stationary phases are porous solids in which molecules of some substances adhere to the organic
compounds and interact with them through intermolecular forces of attractions namely, ion-dipole,
dipole-dipole, hydrogen bonding, dipole induced dipole, and van der Waals forces. Here are stationary
phases in order of increasing adsorptive power for molecules: alumina (Al 2O3), charcoal, florisil, silica
gel (SiO2), and inorganic carbonates (e.g. starch). Inorganic carbonates separates natural products and
magnesium silicate separates acetylated sugars, steroids, and essential oils. Silica gel is used to separate
compounds such as hydrocarbons, alcohols, ketones, acids, azo compounds, and amines. Alumina can
be any of the following forms: basic, acidic, and neutral. When separating carboxylic acid and amino
acid, acidic alumina may be used. Basic alumina separates amines, whereas neutral alumina separates
non-acidic and non-basic compounds. Among of them, alumina and silica gel are the most commonly
used laboratory stationary phases.
However for this experiment, it is important to note the quality of the collected product. Higher
specific area and the uniformity of the particles are requirements for better quality adsorbents (Martin &
Gilbert, 2011). Since silica gel has 500 g2/mL while alumina has only 150 g2/mL, then silica gel is more
preferable.
Polarity of eluents affect the relative rates at which compounds move through the column. A
general rule of rule the thumb is if the substance to be separated is polar, the developing solvent should
be less polar (Martin & Gilbert, 2011). Consequently, non-polar substance would require a slightly polar
solvent. So, highly polar solvent will better solvate polar constituents and move even highly polar
molecules rapidly through the column. Rapid movement will lead to overlapping of colored bands and
disrupt it. Hence, it is advisable to use eluents in an increasing polarity. The commonly used eluents in
order of increasing polarity are: petroleum ether or hexanes, cyclohexane, toluene, chloroform, ethyl
ether, ethyl acetate, acetone, propanol, ethanol, methanol, water and acetic acid. In addition to that,
heres the expected elution order of organic classes in increasing polarity (move more slowly): saturated
hydrocarbons, unsaturated hydrocarbons, ethers, esters, halides, ketones, aldehydes, amines, alcohols,
and acids and bases.
Following this rule, in this experiment, eluents were poured in a series of increasing polarity: hexane,
DCM, DCM in MeOH, and then DCM in hexane. The less polar eluents were first used to eluate a less
polar compound. Once colored bands are no longer visible and have been collected in the test tube, a
more polar solvent is added. Hexane yielded a 0.50mL yellow eluate. For DCM in hexane, it yielded a
0.90mL orange eluate; whereas, DCM generated 0.90 mL golden yellow eluate. Lastly, methanol in
DCM, produced 0.40mL rusty orange eluate. Results of the column chromatography is expressed in
Table 1.

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Table 1. Results of Column Chromatography.

Eluate
Hexane
Dichloromethane
(DCM) in hexane
Dichloromethane
Methanol in DCM

Color
yellow

Volume
0.50 mL

max
446.00 nm

orange

0.90 mL

inconclusive

golden yellow
rusty orange

0.90 mL
0.40 mL

484.91 nm
452.72 nm

However, it was observed that more bands separated in hexane which is theoretically
contradictory. It can be inferred that there was failure to properly label the eluents used and/or
contamination of an eluent with another eluent.
Each of the collected eluate were subjected to spectrophotometry. Spectrophotometer consists of
two instruments, spectrometer and a photometer. A spectrometer produces light of any wavelength,
whereas a photometer measures the intensity of light. When put together, spectrophotometer indicates
the absorbance of a particular wavelength. Certain ranges of wavelength corresponds to a specific color
that may or may not be visible to the naked eye. These colors are those that can be spotted in the
rainbow: red, orange, yellow, green, blue and violet. Table 2 shows wavelength corresponding to their
color.
Table 2.
Color that corresponds a particular wavelength
WAVELENGTH
COLOR
violet
blue
green
yellow
orange
red

COLOR
ABSORBED
380450 nm
450495 nm
495570 nm
570590 nm
590620 nm
620750nm

The color that we see in a sample is due to the selective absorption and transmittance of certain
wavelengths. If a sample absorbs all the wavelengths, it will appear black; but if it does not absorb any
wavelength, it will appear white or colorless. The color absorbed by the spectrophotometer are those that
are not visible to the naked eye. On the other hand, the color that are transmitted, those that are not
absorbed-and those that pass through- are the colors that can be seen by the naked eye. The color
perceived is complimentary to the color absorbed. Table 3 will show the complementary colors of
absorbed color based on wavelength.

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Table 3.
Complementary colors of the absorbed color based on
wavelength

COLOR
violet
blue
green
yellow
orange
red

WAVELENGTH
COLOR
COLOR
ABSORBED
OBSERVED
380450 nm
yellow-green
450495 nm
yellow
495570 nm
purple
570590 nm
blue
590620 nm
greenish blue
620750nm
bluish green

Results of the spectrophotometer of hexane, DCM in hexane, DCM, and MeOH in DCM can be
seen in Figures 1, 2, 3, and 4. It is expressed in a graph where the x-axis represents the wavelength and
the y-axis for absorbance.

Figure 1:
Spectrophotometer result of hexane

Figure 2.
Spectrophotometer result of DCM eluate

Figure 3.

Figure 4.

Spectrophotometer result of DCM only eluate

Spectrophotometer result of DCM MeOH eluate

*Assumpta Minette C. Burgos

The maximum absorbance (max ) of hexane was 446.00nm. For DCM in hexane, it yielded an
inconclusive max , as seen in figure 2. The maximum absorbance was not determined by the software.
Next, DCM generated a max of 484.91 nm as seen in figure 3. Lastly, methanol in DCM, produced a
max of 452.72nm, as seen in figure 4. However, the quotation for the hexane cannot be fully concluded
since the max was only measured by a ruler compared to the results of the other two where the max was
determined. If the hexane has a wavelength 446.00nm, then the absorbed color is violet. Likewise,
DCM in hexane has an undetermined absorbed color due to inconclusive data. DCM has an absorbed
color of blue and MeOH in DCM with blue, as well.
It is said that the more conjugated the structure is, the higher the maximum absorbance (Burgess
& Mielenz, 2012). Hence, the major yellow pigment- -carotene, will precede capsanthin, the major red
pigment.

Conclusion
The Capsicum frutescens were separated into four eluates by column chromatography. Results of
spectrophotometer shows that maximum observance in wavelength. Certain ranges of wavelength
corresponds to a specific color. The color perceived is complimentary to the color absorbed by the
spectrophotometer. The maximum observance is relative to the structure of the compound. The more
conjugated the structure of a compound, the higher the maximum observance.

*Assumpta Minette C. Burgos

References
Burgess, C., & Mielenz, K. D. (2012). Advances in standards and methodology in spectrophotometry.
Philadelphia: Elsevier.
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Koffi-Nevry, R., Kouassi, C., Nanga, Z. Y., Koussmon, M., & Yao Loukou, G. (2010). Antibacterial
activity of two bell pepper extracts: Capsicum annuum L. and Capsicum frutescens.
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Massachusetts, USA: Cengage Learning
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World of Chillies. (n.d). Chilli plants: Capsicum frutescens. Retrieved on February 2, 2016 at
www.worldofchillies.com/Chilli-plant-varieties

*Assumpta Minette C. Burgos