Thin Layer Chromatography of Siling Labuyo Extract

Allen Acuna, Sharlin Ahmed, Nathan Alarcon, Carl Alvero, Kristoffer Antonio, Jan Eric Balete
Group1, 2C-Medical Technology, Faculty of Pharmacy, University of Santo Tomas
ABSTRACT
Chromatography is one the techniques used in chemistry to separate the components of a mixture. In the experiment, siling labuyo
extract was used as the mixture. Thin layer chromatography was then used to determine the purity of the components of the pigments
in siling labuyo, which were extracted using the eluates DCM hexane, DCM, and DCM MeOH. A UV lamp was used to illuminate the
developed TLC plate, and the retention factors of each colored component were measured. However, only the crude extract developed
properly, due to the choice of solvent used. The crude extract yielded two components. Component A had a retention factor of 0.63,
while component B had a retention factor of 0.13.
INTRODUCTION
Chromatography is a technique used for
separating the components, or solutes, of a mixture
based on the relative amounts of each solute
distributed between a moving fluid stream, called
the mobile phase, and an adjacent stationary phase.
The mobile phase may be either a liquid or a gas,
while the stationary phase is either a solid or a
liquid. The underlying principle of chromatography
is that different substances have different ratios of
attraction to the stationary and mobile phases. A
compound which is more attracted to the stationary
phase will move more slowly as compared to a
compound that is more attracted to the mobile phase
which results to a separation of the compounds
(Britannica, 2014). The type of chromatography
used is thin-layer chromatography (TLC) which is a
technique for identifying compounds, or
determining their purity. Thin layer chromatography
has a couple of advantages compared to column
chromatography, being that thin layer
chromatography requires only small quantities of
the sample, and is much faster to perform.
(University of California, Los Angeles, 2009) The
TLC plate used is an aluminium sheet covered on
one side with alumina, which acts as the stationary
phase. As for the mobile phase, a DCM (CH
2
Cl
2
)
hexane (C
6
H
8
) 1:1 solvent system is used.
Aside from the crude extract, three different
eluates resulted from the extraction of the siling
labuyo, which are then used as components for the
thin layer chromatography. The three eluates are a
mixture of the crude extract and with the addition of
dichloromethane hexane (DCM C
6
H
8
),
dichloromethane (DCM), and dichloromethane
methanol (DCM MeOH), with DCM hexane being
the least polar of the three, and DCM MeOH being
the most polar.
The objectives of the experiment are the
determination of the purity of the eluates extracted
from the siling labuyo using thin layer
chromatography, and the measurement of their
retention factors (R
f
). The retention factor is defined
as the ratio of the distance travelled by the centre of
the spot to the distance simultaneously travelled by
the mobile phase (IUPAC Gold Book, 2014). The
larger an R
f
of a compound, the larger the distance
it travels on the TLC plate. When comparing two
different compounds run under identical
chromatography conditions, the compound with the
larger R
f
is less polar because it interacts less with
the polar adsorbent on the TLC plate. (Guanxi
Normal University)
METHODOLOGY
Ten pieces of siling labuyo were grinded
and triturated using a mortar and pestle. DCM
hexane was then added to form the crude extract.
The components of the crude extract were then
separated by column chromatography. The eluents
used were DCM, DCM hexane, and DCM
methanol. The eluates were then collected in
separate test tubes.
The TLC plate was prepared by drawing a
straight line 1.5cm away from the bottom of the
TLC plate to serve as the base line, and another
straight line 1.5cm from the top of the TLC plate to
serve as the solvent front. The crude extract and the
eluates were then applied to the TLC plate by
spotting ten times on the base line. The spots were
made as small as possible and as equidistant to each
other as possible.













Figure 1. TLC Plate Preparation
The developing chamber was prepared by
lining the inside of a 250mL beaker with filter
paper, and filling it with an approximate ammount
of the mobile phase, DCM hexane, just enough so
that it wont reach the base line when the TLC is
placed inside. The beaker was then covered with a
watch glass and a piece of cotton was used to plug
the beaker spout. The beaker was then left to
equilibriate for ten minutes.
After the developing chamber was
equilibrated, the TLC plate was then placed inside
to lean against the inside of the beaker lined with
filter paper. The TLC plate was then taken out after
the DCM hexane solvent has reached the solvent
front, and left to air-dry. The developed TLC plate
was then put under UV light for better observation
of results.

Figure 2. Developing Chamber Setup
RESULTS AND DISCUSSION
TLC Plate Examination










Figure 3. Developed TLC plate
(Spots from left to right: Crude Extract, DCM hexane, DCM,
DCM MeOH)
In reference to figure 3, (from left to right on
the base line) the first spot is the crude extract from
the siling labuyo (colored dark orange), the second
is the DCM hexane eluent (colored yellow), and the
third is the DCM eluent (colored pink), while the
last spot is the DCM MeOH eluent (colored yellow-
orange). As seen in the figure, only the crude
extract developed properly, resulting to two
components labelled A and B, wherein component
A is invisible to the naked eye and needed exposure
to UV light in order to be seen. The other spots
however, are almost indistinguishable to the eluent
spots. They are too close to the base line, and this
means that the eluent is, in relation to the samples,
not polar enough. In future experiments, this can be
remedied either by increasing the ratio of the more
polar solvent of your eluent or by switching to a
more polar solvent (University of Alberta 2014).
Calculation of Retention Factor (R
f
)
The general formula for computing a
components retention factor is shown below:




The solvent front is 7.0 cm away from the
baseline. Component A travelled a distance of
4.4cm from the base line, giving us a retention
factor of 0.63.

Component B, however, travelled only a
distance of 0.9cm from the base line. This gives us a
retention factor of 0.13.

Table 1. Thin Layer Chromatography (Table of Results)
Component Distance of Component
from Base Line
Retention
Factor (R
f
)
A 4.4cm 0.63
B 0.9cm 0.13

REFERENCE
Chromatography. (January 29, 2014). In Encyclopdia Britannica
online. Retrieved from
http://www.britannica.com/EBchecked/topic/115917/chromatography
University of California, Los Angeles. (August 14, 2009). Thin Layer
Chromatography. Retrieved from
http://www.chem.ucla.edu/~bacher/General/30BL/tips/TLC1.html
Retardation Factor in Planar Chromatography. (February 24, 2014).
In IUPAC Gold Book online. Retrieved from
http://goldbook.iupac.org/R05353.html
Guangxi Normal University. (n.d.). TLC Retention Factor (R
f
).
Retrieved from
http://www.ce.gxnu.edu.cn/organic/net_course/content/tlc/Retention_
Factor.htm
University of Alberta. (September 20, 2014). Thin Layer
Chromatography. Retrieved from
http://www.chem.ualberta.ca/~orglabs/Techniques%20Extra%20Info
/TLC.html