Separation of the colored compounds of Capsicum frutescens fruits

Rosheen Angel Villaraza, Dana Young, Dana Fay Yu, Ray Allen N. Zafra*

Department of Biology, College of Science

University of Santo Tomas, Espana Street, Manila 1008

Abstract:

The technique known as column chromatography was utilized to segregate the colored
constituents of the specimen, Capsicum frutescens. A Spectrophotometer was then used to
determine the maximum observance or lambda max for the colored constituents. The experiment
bore a result that displayed the following: 4.2 ml yellow colored eluate bearing a maximum
observance of 451 nm for hexane; 1.2 ml yellow eluate for dichloromethane-hexane with a
maximum observance of 456 nm; 3.3 ml orange eluate with a maximum observance of 485 nm
for dichloromethane an finally, 0.3 ml orange colored eluate for dichloromethane-methanol
bearing a maximum observance of 450 nm.

Introduction

Capsicum frutescens is a member of the Solanaceae or also known as the Nightshade

family. One name for Capsicum frutescens is Cayenne pepper or known here in the Philippines
as siling labuyo.

The mentioned pepper is a short, tiny shrub and has oval and leaves of lanceolate and
alternating nature [1].. These peppers have a hot, spicy taste and the size of the fruit is contrary
to the spice level, so in other terms, the smaller the fruit, the spicier and hotter it is. Another
feature is that its seeds are hotter than its pods and it is within the seeds where one can find
capsaicin, the carotenoid component of the pepper.

Capsicum frutescens is commonly used as ingredient or enhancer in numerous cuisine

worldwide. In the Philippines, dishes such as gising-gising, laing and the famous Bicol Express
are only few of which contain Capsicum frutescens as a main ingredient.

The carotenoid present within Capsicum frutescens determines the color of the fruit itself.
To expound further, carotenoids are kinds of phytonutrients that can be found in various plant
life and protists. These are that give off the yellow, red and/or orange color variations of any and
all fruits and vegetables in existence. In conjunction with chlorophyll, the carotenoids aid in the
absorption of light in the process of photosynthesis.

Carotenoids may be further classified into xanthophylls and carotenes. Xanthophylls

harbor oxygen which in turn give them a somewhat yellow hue. Some examples of xanthophylls
are lutein and zeaxanthin which are commonly related to eye health. Another example is beta-
cryptoxanthin which is widely reported to greatly lessen the risk of lung cancer.

Carotenes contain hydrocarbons at the lack of oxygen and due to this, they are orange.

Noteworthy examples of carotenes are -carotene, -carotene, and lycopene. -carotene has the

ability to produce twice as much vitamin A than -carotene and beta-cryptoxanthin. A research
produced a result in which those with the highest level of -Carotene in the blood are less likely
to suffer and die of any heart disease as opposed to those with relatively high levels of -carotene
[2]. Finally, lycopene is the pigment that provides the red color of tomatoes and even
watermelons.

The carotenoids in Capsicum frutescens may only be determined after column

chromatography. Furthermore, spectrophotometry may be performed so that additional studies
may be conducted on the chemical characteristics of the Capsicum frutescens.
 In this here experiment, the objective was to separate the colored constituents of the red
pepper though the technique called column chromatography and then afterwards determine the
colored constituents. Aside from the mentioned primary objective, the group also had to
characterize the separated colored constituents though their corresponding UV spectra, also
called as the lambda max (max).

Results and Discussion

Unlike the previous experiments concerned with separation which utilized extraction and
distillation, this experiment made use of yet another separating technique, one called column
chromatography.

To define, column chromatography is a method utilized for the separation and subsequent
purification of both solids and liquids. Distillation separates through the boiling point difference
of the compounds, whereas column chromatography separates through polarity differences.
Differential adsorption of substance by the adsorbent is the basis of column chromatographys
principle.

In column chromatography, there are interactions: The stationary adsorbent phases

activity, the eluting solvents polarity and finally, the polarity of the chromatographed
compounds [3].

Stationary phases are solids in which the molecules of substances adhere to the present
organic compounds and further interact with them through the different forces of attraction
which are, ion-dipole, dipole-dipole, hydrogen bonding, dipole induced dipole, and van der
Waals forces. The following are some examples of stationary phases: alumina, charcoal, florisil,
silica gel and some other inorganic carbonates.

In this experiment, silica gel was used due to its higher specific area and the uniformity
of its particles as compared to the aforementioned stationary phases. This is so due to the fact
that for this experiment, the quality of the collected products must be duly noted and is of utmost
importance [4].
The polarity of eluents influences the rates at which the compounds travel and move
through the column. An established rule is that if a substance to be separated is polar, hence the
dissolving solvent must be lesser in polarity. Consequently, non-polar substances require polar
solvents. In relation, highly polar solvents will solvate polar constituents better and even move
highly polar molecules hastily through the column. Hasty or quick movement will undoubtedly
result to overlapping of the bands and cause disruption. Therefore, it is highly recommended to
make use of eluents in increasing polarity.

In accordance to the general rule stated, in the experiment, eluents were poured in a series
of increasing polarity: hexane, DCM-Hexane, DCM and lastly, DCM-MeOH. First used were the
eluents of lesser polarity to eluate less polar compounds. At the moment that the color bands
were no longer visible and have been collected in the test tubes, a more polar eluent was
subsequently added. Hexane produced a 4.2 ml yellow eluate. DCM-Hexane generated a 1.2 ml
yellow eluate. DCM yielded 3.3 ml orange colored eluate and lastly, DCM-MeOH generated
0.30 ml orange eluate. Results of the column chromatography is displayed in Table 1.

Table 1. Results of Column Chromatography.

Eluate Color Volume Lambda Max
Hexane Yellow 4.2 ml 451 nm
DCM-Hexane Yellow 1.2 ml 456 nm
DCM Orange 3.3 ml 485 nm
DCM-MeOH Orange 0.3 ml 450 nm

Afterwards, the collected eluates were subjected to spectrophotometry.

A spectrophotometer is composed of two instruments, mainly a spectrometer and a

photometer, hence the name. The spectrometer produces light of any wavelength while the
photometer measures light intensity. In combination, a spectrophotometer specifies the
absorbance of a specific wavelength. There are various ranges of wavelength that adheres to
colors that may or may not be visible by the naked eye. These colors are nothing else but the
ones that make up the rainbow: red, orange, yellow, green, blue, indigo and violet. The second
table shows the wavelength corresponding to their colors.

Table 2.
Wavelength-Color correspondence

Wavelength
Color Color Absorbed
Red 650-800
orange 590-640
yellow 550-580
Green 490-530
Blue 460-480
indigo 440-450
violet 390-430

The colors seen in the sample are due to the selective transmittance and/or absorption of
wavelengths. The colors invisible to the naked eye are those that were absorbed by the
spectrophotometer. The colors than are observable are transmitted, unabsorbed and pass through.
The third table shows the color absorbance of the eluates in the test tubes.

Table 3
Colors absorbed by the eluents

Eluates Visible Color Lambda max Absorbed color

Hexane Yellow 451 nm Blue
DCM-Hexane Yellow 456 nm Blue
 DCM Orange 485 nm Blue
DCM-MeOH Orange 450 nm Blue

Finally, spectrophotometer results of all the eluates have been compiled into one graph
for reference. The X-axis represents the wavelength while the Y-axis represents the absorbance.

Figure 1

Hexane eluate yielded to a lambda max of 451 nm, DCM-Hexane eluate resulted a
lambda max of 456 nm. The DCM eluate generated a 485 nm lambda max and lastly, the DCM-
MeOH produced a lambda max of 450 nm. All results were generated by a program.
Experimental

The Capsicum frutescens was prepared by first deseeding it and subsequently placed
under freeze drying treatment. Afterwards, it was cut into small pieces, weighing 5g each and
further segregated into batch groupings. These were then grinded along with sand with the use of
a mortar and pestle set. The resulting ground fruit was then applied with 3 ml of dichloromethane
or as mentioned repeatedly, DCM. The mixture produced was then pipetted and further filtered
into a beaker of 50 ml and then silica gel was allowed to absorb the filtered mixture. The DCM
component was allowed to evaporate, thus producing the samples required for the
commencement of the experiment.

Five test tubes were used, one of them labeled Test Tube C, was utilized in switching
between the solvents. For the first solvent, hexane, a white substance was observed in the silica
gel, the segregation of this substance was done by placing it in test tube C. After some drops of
hexane, a yellow colored substance was extracted and this in turn was contained inside the test
tube labelled 1. An aspirator was used to push down the eluting solvent until the yellow reached
the bottom column where a cotton was situated.

The next solvent, DCM-Hexane was used. The remaining residues left by the first eluate
was collected into test tube C, this was then exchanged with test tube 2 when another yellow
substance reached the cotton below.

The eluate was then switched to a third one after extraction, this time the eluate was
DCM. An orange substance was extracted from this. The yellow residue left by the DCM-
Hexane solvent was collected by test tube C before test tube 3 was used to collect the new color.

After the orange color was extracted, the final solvent, DCM-MeOH was introduced.
Like previously performed, the residue left by the previous solvent was collected in test tube C
until the last of the orange color reached the cotton. The fourth test tube was then used to collect
the final orange color. All test tubes were then placed in a beaker of 100 ml and wrapped with
foil or paper to protect the pigments from contact with light.
The test tubes holding the pigments were then put through UV-Vis spectroscopy for latter
characterization. In the process of UV-Vis spectroscopy, cuvettes were used to hold the sample
to be tested upon. A reagent blank must first be mounted prior to loading the sample, the reason
for this treatment was to ensure that only desired substances would be put through the process.
Eluting solvents were used as the reagents. Following this, a reagent blank of hexane was used
for test tube one prior to loading the test tubes contents. The procedure was repeated for the
subsequent test tubes. Afterwards, a graphical figure displaying the lambda max of all eluates
was formulated.
Conclusion

Column Chromatography was used to separate the compounds of Capsicum frutescens

into four colors. UV-Vis spectroscopy was then performed to characterize the colors by their
respective wavelengths. Test tube 1, hexane, bearing a yellow eluent, yielded a lambda max of
451 nm. Another yellow eluent, DCM-Hexane, test tube 2, generated a lambda max of 456 nm.
Test tube 3, DCM, with an orange eluent, resulted in a lambda max of 485 nm. Test tube 4
containing DCM-MeOH, bearing yet another orange eluent, yielded a lambda max of 450 nm.
Both major and minor components were determined by volume of the pigment extracted. The
yellow eluent extracted from hexane was established as the major component, bearing a volume
of 4.20 ml. On the other hand, the orange eluent extracted from DCM-MeOH was established as
the minor component, bearing a volume of 0.30 ml.

References

[1] World of Chillies. (n.d). Chilli plants: Capsicum frutescens. Retrieved on February 2,
2016 at www.worldofchillies.com/Chilli-plant-varieties.

[2] Ito, Y., et al. (2006). Cardiovascular disease mortality and serum carotenoid levels: A
Japanese population based follow-up study. Journal of Epidemiology, 16(4), 154-160..

[3] Williamson, K. & Masters, K. (2011). Organic Experiments: Macroscale and

Microscale, Sixth Edition. Belmont, California, USA: Brooks/Cole, Cengage Learning

[4] Martin, S. & Gilbert, J. (2011). Organic Chemistry Lab Experiments Fifth Edition.
Boston, Massachusetts, USA: Cengage Learning.
References

Burgess, C., & Mielenz, K. D. (2012). Advances in standards and methodology in spectrophotometry.
Philadelphia: Elsevier.

Ito, Y., et al. (2006). Cardiovascular disease mortality and serum carotenoid levels: A Japanese
population based follow-up study. Journal of Epidemiology, 16(4), 154-160.

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. International Journal of Food Properties, 15(5), 961-971.

Martin, S. & Gilbert, J. (2011). Organic Chemistry Lab Experiments Fifth Edition. Boston,
Massachusetts, USA: Cengage Learning

Poole, C. F. (2003). The essence of chromatography. Philadelphia: Elsevier.

Sommer, L. (2012). Analytical absorption spectrophotometry on the visible and ultraviolet: The
principles. Philadelphia: Elsevier.

Williamson, K. & Masters, K. (2011). Organic Experiments: Macroscale and Microscale, Sixth Edition.
Belmont, California, USA: Brooks/Cole, Cengage Learning

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