ABSTRACT

Column chromatography was used to separate coloring components in plant samples specifically
Capsicum frutescens or 'Siling labuyo'. Results of column chromatography showed that from
Hexane and Hexane:DMC, yellow eluted solution was seen while from DCM and DMC:MeOH,
we can see an orange eluted solution.
INTRODUCTION
In this experiment, we used Chili pepper (Capsicum frutescens), a kind of cayenne pepper that is
commonly found in the Asia. It is conical and tapering in shape ranging from 1.5 to 3 cm long. It
turns from green or yellow to red as it ripens and it usually has a spicy or hot taste due to the
presence of vanilloid capsaicin. Column chromatography of the plant pigment aims to separate
the components of chili peppers.
III. RESULTS AND DISCUSSION
Each step in the preparation of every experiment plays a vital role in obtaining the correct and
expected results (Jones, 1997). For experiment 4B, Capsicum frutescens or 'Siling labuyo was
chosen because of the abundance of its different colored pigment. The following are the
available pigments: xanthophylls (yellow pigments), carotenes (orange pigments), and
anthocyanins (red pigments). One type of xanthophyll that is abundant in capsicum species is
capsanthin that gives its pink to red color. The chili was deseeded and cut into small pieces
before the addition of 3M Dichloromethane, cutting the chili and adding an organic chemical will
allow better extraction. For the column preparation, column pipette was not used because the
experiment was going to be a miniature version, so we used a dropper. Small cotton was pushed
down up to the tip to act as a filter. Silica gel is then placed; it is a highly activated adsorbent,
furnished in a wide range of mesh sizes to suit various industrial applications. It is non-corrosive,
odorless, tasteless, non-toxic, and chemically inert. It is a highly porous form of silica, with an
extremely large internal surface area. Some organic compound might bind to the silica gel but
the strength of their bond varies. Nonpolar compounds bind using van der Waals forces and they
easily dissolve while polar compounds bind using dipole-dipole and they dissolve last (Engel,
Kriz, Lampman, 2013). Adding eluting solvents after dropping 10 drops of extract will help
isolate the different colors and pigments present in the chili pepper. According to Engel et al.,
solvents should also be added drop wise to prevent formation of bubbles and it also prevents
cracking from the heat generated by the reaction. Table 2: Eluting solvents and the resulting
colors Based on table 2, the eluting solvents were added accordingly based on their polarity.
Non-polar solvents were added first and polar solvents were added last. Improper order of the
addition of eluting solvent will result to erroneous extraction. Silica gel will attract the polar
compounds due to its high polarity that is why DCM:MeOH was Eluting Solvent Color of Eluted
Solvent Hexane Yellow Hexane:DCM (1:1) Yellow DCM Orange DCM:MeOH (1:1) Orange the

last one eluted. Eluting with DCM however, produce insoluble compounds because of its nonpolar state. Based on our results we can tell that yellow eluate is the least polar because it
travelled faster and came out first while orange eluate is the most polar because it travelled
slower due to the attraction towards the very polar silica gel.
IV. EXPERIMENTAL
The Chile peppers were triturated using the mortar and pestle until delapitated, and then
eventually the group added 10mL DCM-hexane(1:1) was poured. The mixture is triturated
2-3 times and then the extract from it is collected using a beaker, covered and was set aside
first, to prepare the column chromatography set up.
Using the Pasteur pipette with the cotton in it for bed support, the group filled it with silica
up to the indented portion of the glass. The pipette is clamped to the iron stand and the test
tubes are prepared as receivers. The group also prepared the solvents. One test tube
contains 0.5mL of DCM-hexane(1:1), another for another 0.5mL DCM only, and another test
tube for 0.5ml DCM-methanol(1:1)
One milliliter of the extract was introduced to the pipette. As the liquid goes down, it is
eluated by the first solvent, the 2mL DCM-hexane, followed by the 2mL DCM and lastly the
2mL DCM-methanol. The group changes the receiver each time the color varies. The
number of drops per pigment is also noted. After collecting all the eluates, Thin Layer
Chromatography was performed.
The eluates were applied on the TLC plate(5cm x 8cm) by equidistantly spotting each
pigment ten times. Each spot was allowed to dry first before applying the succeeding spots.
It was ensured that the spots are made small so that when the plate develops, the colors
would not disarray.
The developing chamber was prepared by placing the approximate amount of DCMhexane to a beaker. The inner wall was lined by filter paper to allow the TLC plate to stand,
and then covered with watch glass for few minutes for equilibration.
After equilibrating, the developing plate was carefully introduced to the developing
chamber. The solvent system was allowed to rise up until it reaches just one centimeter
from the upper end. The developing plate was then removed from the chamber carefully,
and the group marked the solvent fronts of each pigment and then air-dried the plate. The
components were visualized and checked by the UV lamp. The retention factors were
measured after.

IV. CONCLUSION
Different techniques of chromatography were used in this experiment. From the thin layer
chromatography, calculated retention factor values showed that caffeine is the most polar

compared to other samples because of its low RF value while Ibuprofen is the least polar because
it has the highest RF value. Column chromatography showed that the yellow pigment of
Capsicum frutescens, xanthophyll, is the least polar because it travelled the fastest along the
silica gel-filled column. On the other hand, the orange pigment, carotene, is the most polar since
it travelled the slowest due to its high attraction against the very polar silica gel. References:
Brown, W. H., Foote, C. S., & Iverson, B. L. (2012). Organic Chemistry (6th ed.). Belmont, CA:
Thomson Brooks/Cole. Carey, F. A. (2008). Organic Chemistry (7th ed.). New York, NY:
McGraw Hill. Engel, R., Kriz, G. Lampman, G. (2013). A macroscale approach to Organic
Laboratory Techniques, 5th edition. United States of America: Cengage Learning. Jones, M.
(1997). Organic Chemistry. New York: Norton & Company Inc. Masters, K., Minard, R.,
Williamson, K. (2007). Macroscale and Microscale Organic Experiments, 5th edition. Boston,
MA: Houghton Mifflin Company. Masters, K., Williamson, K. (2012). Techniques Labs for
Macroscale and Microscale Organic Experiments, 6th edition. Belmont, CA: Cengage Learning.
History of Chromatography. (n.d.). Retrieved September 28, 2016, from
http://www.umich.edu/~orgolab/Chroma/chromahis.html Thin Layer Chromatography (TLC)
Guide [PDF]. (2012, January).