ORGANIC

CHEMISTRY
Organic Chemistry Laboratory CH 200 (2014 2015) Experiment 4

Chromatographic Analysis of Capsicum frutescens through Thin Layer

Chromatography and Column Chromatography
Raymond Michael S. Baladad, Kenny Bautista, Ayshee Capuchino, Charmaine Chan*
Group 2, 3Bio3
Department of Biological Sciences, College of Science
University of Santo Tomas, Espana Avenue, Manila

Date Submitted: September 4 2014

Abstract
Experiment 4 is divided into two parts. First is Experiment 4A: Chromatography of Commercial Analgesics. In
this experiment, a pre-coated TLC plastic sheet with fluorescent indicator is used to determine the composition of 8
commercial analgesics with the use of Ethyl Acetate-Methanol-Acetic Acid as its solvent with a ratio of 25:1:1. Spots on
the TLC sheet were seen by the use of the UV light chamber. The second part of this experiment is Experiment 4B:
Column Chromatography of Plant Pigments. In this experiment, Capsicum frutescens or the siling labuyo undergoes
extraction of plant pigment, column packing, column loading and column elution in order to separate its natural color
components.
Keywords: Chromatography, Thin Layer Chromatography (TLC), Column Chromatography, Ultra Violet Spectroscopy
and Retention Factor

Introduction
Chromatography is a technique used to separate mixtures. It is done in order to determine and
identify compounds in a solution. It involves samples turned into their mobile phase and making
them move through a stationary phase, which separates the samples into their different components
as they flow pass through the stationary phase. This stationary phase or also called the adsorbant is a
fine porous solid such as silica gel, which has the capability of retaining both solvents and solutes.
Chromatography can be preparative or analytical. Preparative chromatography is separating or
extracting components for other uses, such as purification. While Analytical chromatography is
separating compounds into components to identify its unknown substances or identification.
Chromatography is important and is used in daily lives of people. It is used to test every day products
such as food. Food samples are tested and to be identified if affected by contamination or not, to
distinguish if such products are safe to be distributed to the population.
Chromatography collectively refers to various techniques in separating mixtures. Two of its
known types are thin layer chromatography (TLC) and column chromatography. Thin layer
chromatography is an example of analytical chromatography wherein; the mixture or the organic
compound is extracted into components in order to identify its composition or the unknown. This
technique makes use of thin film silica coated on a sheet of glass or plastic, where a sample of the
compound is spotted onto the sheet. The solvent then runs upward through capillary action together

with the components. Some move fast up while some move slow or remain at the bottom, which
results to the separation of the components. The more polar the solvent, the faster the components of
the compound will move. After the TLC plate develops, the spots are then to be determined under the
ultra violet spectroscopy, which gives off energy to the organic molecules, which makes the structure
of the molecule visible.
Column chromatography is an example of preparative chromatography wherein, the organic
compound are extracted or separated into components to make further uses of the different
components. This technique is also very useful in identifying components of compounds, such as
color components of plants. Eluent or solvents that are responsible for the separation of the
components are dropped in prepared microcolumn, which contains sand and the compound extract
adsorbed in silica gel (Figure 1). As the eluent passes through the organic compound, the
components of this compound separates and flows together with the specific eluent and are then
collected. Different components are soluble to a specific eluent, such as hexane, methanol,
dichloromethane (DCM) and the like. This explains why different eluent are used and dropped to the
organic compound.

Figure 1: Column Chromatography set up

Methodology
After the extraction of caffeine from experiment 3, the characterization of caffeine was done
in the first part of experiment 4. First, the isolated caffeine from the previous experiment was
dissolved in 2mL methyl toluene. Second, the teacher spotted the TLC plate and each analgesic was
blotted on the designated spots. Using the capillary vessel, the isolated caffeine, standard caffeine,
acetaminophen, phenacetin, and mefenamic acid were blotted 3 times on the TLC plate while the
unknown, aspirin, and ibuprofen were blotted 5 times on the TLC plate. Next, the filter paper lining
was placed inside a 250 mL beaker, letting the bottom part submerge in Ethyl Acetate-MethanolAcetic Acid with a 25:1:1 ratio. Then the beaker was covered with a watch glass. The filter paper
absorbed the solvent and the level rose (Figure 2). This step is to be done up until the solvent reaches
the assigned marking line. Afterwards, it was visualized under the UV light to produce the
chromatogram sketch. Dark spots were seen on the paper and were traced and Rf was calculated.

In this experiment, 5 pieces of ripe fruits of Capsicum frutescens or the siling labuyo were
prepared. By the use of scissors, the fruit was cut into half, and each fruit was deseeded. After all the
seeds were removed, the fruits were cut into smaller pieces and weighed using the electronic gram
balance. The cut pieces of fruits were then placed in a mortar where it was triturate together with
spatula of sand. After crushing and mixing the small pieces together with the sand, 5 mL of
dichloromethane was added. After mixing the mixture, it was then decanted. The residue left in the
mortar was discarded, while the decantate was filtered in a glass funnel with a filter paper that has
spatula of anhydride Na2SO4. After filtration, the residue was discarded, while the filtrate was
transferred in a 100 mL beaker. In the beaker, spatula of silica gel was added. Using the glass rod,
the mixture in the beaker was mixed until it became powder like. In this part of the experiment, the
pepper extract were absorbed by the silica gel. Next is the preparation of the microcolumn. First,
close the possible holes, to prevent the exit of the substances placed in the microcolumn, such as the
silica gel, sand, and powdery pepper extract. Next, place the silica gel, filling of the microcolumn
followed by the addition of small amount of sand, then the placement of the pepper extract absorbed
in silica gel and lastly, the addition of small amount of sand on the topmost layer. After the
preparation of the microcolumn, is the preparation of the setup of the column chromatography for
column loading and column elution. The iron stand, clamp, and beaker were important for this setup.
Afterwards, drops of hexane were added to the microcolumn and were eluted. Hexane soluble
colored eluent were placed on a separate test tube. After the first set, for the second set, hexane:
DCM was added and then was eluted. Hexane: DCM soluble colored eluent was placed on a new test
tube. After the second set, for the third set, DCM was added and was eluted. DCM soluble colored
eluent was placed on another new test tube. After the third set, for the fourth set, DCM: methanol
was added and was eluted. DCM: methanol soluble colored eluent was placed on a new fourth test
tube. And lastly, after the fourth set, for the fifth set, drops of methanol was added and was eluted.
Methanol soluble colored eluent were placed on the last test tube and the microcolumn was
discarded.

Figure 2: Thin Layer Chromatography set up

Results and Discussion
In Experiment 4A, after obtaining the tracing of the spots made by the UV light, the
retardation values were obtained for, specifically, isolated caffeine and standard caffeine. The rf
value for isolated caffeine 0.38 and for the standard caffeine, the rf value is 0.36. The rf value is
solved by
having 2.1 cm for the distance travelled by the compound and 5.6 cm for the distance travelled by the
solvent.

Table 1: Commercial Analgesic and its Rf Value

Commercial Analgesic
Isolated Caffeine
Standard Caffeine

Rf Value
0.38
0.36

In Experiment 4B, as seen in Table 2, the group was able to extract 5 color components with
the help of 4 solvents namely Hexane, Hexane: DCM, DCM, DCM Methanol and Methanol with the
technique called column chromatography. The colors extracted are light yellow, yellow, orange,
cloudy orange and colorless or clear, respectively. Having the volume 1.2, 1.6, 1.4, 1.1, and 1.0 in
mL, respectively.
Table 2: Color Reactions and Volume Extracted
Solvent
Hexane
Hexane: DCM
DCM
DCM Methanol
Methanol

Color
Light yellow
Yellow
Orange
Cloudy orange
Colorless, clear

Volume (mL)
1.2
1.6
1.4
1.1
1.0

Conclusion
Based on the gathered results on the chromatography of commercial analgesic, it is evident
that the unknown value is phenacetin. With regards to isolated caffeine and standard caffeine, its
retardation factor values were 0.38 and 0.36, respectively. The difference between the two caffeine is
very minimal because it is of the same identity.
Aside from chlorophylls, other photosynthetic plant pigment is the carotenoid. Carotenoids
are known for its yellowish orange coloration. Based on the second experiment that was performed
by the group, the results obtained after the siling labuyo underwent column chromatography, are the
extraction of 5 color pigments with the support of the eluent or solvents. Light yellow is the color
soluble to hexane eluent. Yellow is the color soluble to hexane: DCM eluent. Orange is the color
soluble to DCM eluent. Cloudy orange is the color soluble to DCM: methanol eluent. Lastly,
colorless or clear is soluble to methanol eluent. Focusing on the extracted pigments released by the
red pepper, yellow pigments are xanthophylls and orange pigments are carotenes. And another color
pigment under carotenoid is red which are called the anthocyanins. But the most abundant type of
xanthophyll in the red pepper is called the capsanthin.
Appendix

Rf value of isolated caffeine = 2.1 cm

5.6 cm
= 0.38

Rf value of standard caffeine = 2.0 cm

5.6 cm
= 0.36
References
Klein, D (2012). Organic Chemistry. Hoboken New Jersey: John & Wiley Sons Inc.
McNair, H, & Miller, J. (2009). Basic gas chromatography (2nd ed.). New Jersey: John & Wiley Sons
Inc.
Smith, J. G. (2013). General, Organic and Biological Chemistry. (2nd ed.). Avenue of the Americas,
New York: McGraw-Hill.
Column
set
up.
Retrieved
September
http://www.pitt.edu/~ceder/apparatus/column_collect_firstfrac.jpg
TLC
setup.
Retrieved
September
2,
labs.leeds.ac.uk/brewing/images/TLC/tlc_expt2.jpg

2014

2,
from

2014

from

http://www.virtual-