TITLE

J. Sumagaysay, W. Sy, H.W. Tan, M.P. Tan, J.M. Tecson, M.A.M. Tumang
Group 7,2F-Ph,Faculty of Pharmacy, University of Santo Tomas, Manila

Abstract
Chromatography is the science which the studies the separation of molecules based on the
differences in their structures and composition. The objectives of the experiment are to separate the
colored components of siling labuyo and malunggay leaves using column chromatography, to determine
the purity of the components using thin layer chromatography, and to measure the Rf values of the
colored components in thin layer chromatography. There are two main types of chromatography: liquid
chromatography (LC) and gas chromatography (GC). Each has their own strengths and weaknesses. In
this experiment, pigments of siling labuyo were extracted using DCM- hexane, DCM, and DCMmethanol. Extracts were introduced into the column, and the eluates were collected. This process is
called the column chromatography method. The purity of the components were determined using thin
layer chromatography. In order to visualize the developed thin layer chromatography plate, a UV lamp
was used; the retention or retardation factor was measured.
interact strongly with the stationary phase will
move slowly. In the ideal case, each component
I. Introduction
of a mixture will have different partition
coefficient between mobile and stationary
Chromatography is a powerful technique
phases, and consequently each will move
for separating mixtures. It is the separation of a
through a system at a different rate, resulting in
mixture by passing it in solution or suspension or
complete
separations.
Practically,
as a vapor (as in gas chromatography) through
chromatography is used for forensic testing,
a medium in which the components move at
ebola immunization, performance enhancing
different rates. Chromatography uses phase
drug testing, and at the horse meat scandal.
equilibrium partitioning principles to separate
proteins, nucleic acids, or small molecules in
Thin-layer chromatography (TLC) is a
complex mixtures based on their differing
chromatography technique used to separate
interactions with a stationary phase and a
non-volatile
mixtures.
Thin-layer
mobile phase. There are two main types of
chromatography is performed on a sheet of
chromatography: liquid chromatography (LC)
glass, plastic, or aluminium foil, which is coated
and gas chromatography (GC).
Liquid
with a thin layer of adsorbent material, usually
chromatography
(LC)
is
an
analytical
silica gel, aluminium oxide(alumina), or
chromatographic technique that is useful for
cellulose. Similar to other chromatographic
separating ions or molecules that are dissolved
methods, thin layer chromatography is also
in a solvent. Gas chromatography is a type of
based on the principle of separation. The
chromatography used in analytical chemistry for
separation depends on the relative affinity of
separating and analyzing compounds that can
compounds towards stationary and the mobile
be vaporized without decomposition.Both LC
phase. The compounds under the influence of
and GC can be used for either preparative or
the mobile phase (driven by capillary action)
analytical applications. The underlying principle
travel over the surface of the stationary phase.
of chromatography is that different substances
During this movement, the compounds with
have different partition coefficients between the
higher affinity to stationary phase travel slowly
the stationary phase will spend most of its time
while the others travel faster. Thus, separation of
in the mobile phase and move rapidly through
components in the mixture is achieved. Once
the chromatographic system. Compounds that

separation occurs, the individual components

are visualized as spots at a respective level of
travel on the plate. Their nature or character are
identified by means of suitable detection
techniques. TLC system components consists of
TLC plates, preferably ready made with a
stationary phase: These are stable and
chemically inert plates, where a thin layer of
stationary phase is applied on its whole surface
layer. The stationary phase on the plates is of
uniform thickness and is in a fine particle
size.Another is the TLC chamber. This is used
for the development of TLC plate. The chamber
maintains a uniform environment inside for
proper development of spots. It also prevents
the evaporation of solvents, and keeps the
process dust free. A Mobile phase comprises of
a solvent or solvent mixture The mobile phase
used should be particulate-free and of the
highest purity for proper development of TLC
spots. The solvents recommended are
chemically inert with the sample, a stationary
phase. A filter paper is moistened in the mobile
phase, to be placed inside the chamber. This
helps develop a uniform rise in a mobile phase
over the length of the stationary phase. The
advantages of TLC are it being a simple process
with a short development time, it helps with the
visualization of separated compound spots
easily, it helps in isolating of most of the
compounds, the purity standards of the given
sample can be assessed easily, the method
helps to identify the individual compounds, the
separation process is faster and the selectivity
for compounds is higher (even small differences
in chemistry is enough for clear separation), and
it is a cheaper chromatographic technique. TLC
is used to check the purity of given samples, to
evaluate the reaction process by assessment of
intermediates, reaction course, and so forth, to
purify samples, i.e for the purification process, to
keep a check on the performance of other
separation processes, and for the identification
of compounds like acids, alcohols, proteins,
alkaloids, amines, antibiotics, and more.
In chromatography, the retardation
factor (R) is the fraction of an analyte in the
mobile phase of a chromatographic system.[1] In

planar chromatography in particular, the

retardation factor Rf is defined as the ratio of the
distance traveled by the center of a spot to the
distance traveled by the solvent front. [2] Ideally,
the values for RF are equivalent to the R values
used
in
column
chromatography.
In
chromatography the retardation factor, R, is the
fraction of the sample in the mobile phase at
equilibrium, defined as the quantity of substance
in the mobile phase over the total quantity of
substance in the system. Rf values are
necessary because every substance has a
characteristic Rf for a given solvent. If one
knows the solvent and the Rf, one could predict
fairly accurately what the material was that
migrated that distance relative to the solvent.
This is assuming that one does not know all the
materials that were in the original mixture, and
one is hoping to identify them by using
chromatography to separate them. The
objectives of the experiment are to separate the
colored components of siling labuyo and
malunggay
leaves
using
column
chromatography, to determine the purity of the
components using thin layer chromatography,
and to measure the Rf values of the colored
components in thin layer chromatography.
Conclusion
It can be concluded that two eluates were
yielded from the extraction of the colored
components of siling labuyo using Column
Chromatography. Dark yellow and light yellow
were yielded respectively. The color of the
developed plate was not visible by the naked
eye; hence, it was placed under UV light for
viewing. The developed plate was not able to
show completely the separation of colors. The
possible sources of error are from the spotting of
the TLC plate, When the extracted pigments of
siling labuyo were spotted on the plate, it was
not left completely dry before placing the
succeeding spots in addition to that; the spots
were not small enough which have caused color
the color to disarray.
Sources:

Chromatography | chemistry | Britannica.com.

(n.d.). Retrieved October 23, 2016, from
https://www.britannica.com/science/chromatogra
phy
Column and Thin Layer Chromatography Scribd. (n.d.). Retrieved October 23, 2016, from
https://www.scribd.com/doc/29411801/Columnand-Thin-Layer-Chromatography
K. (n.d.). Principles of chromatography | Khan
Academy. Retrieved October 23, 2016, from
https://www.khanacademy.org/testprep/mcat/chemical-processes/separationspurifications/a/principles-of-chromatography
Retardation factor | science | Britannica.com.
(n.d.). Retrieved October 23, 2016, from
https://www.britannica.com/science/retardationfactor

1. Fixing the Set-Up

a. The group placed silica gel and
cotton inside a Pasteur pipette
b. The group wrapped the pipette
using a tissue and placed it between
an iron clamp that is attached on an
iron stand.
c.

After, the group placed an

Erlenmeyer flask beneath it.
Figure 2.2 Set Up for Collection of Eluates

THIN LAYER CHROMATOGRAPHY chemguide - Main Menu. (n.d.). Retrieved

October
23,
2016,
from
http://chemguide.co.uk/analysis/chromatography
/thinlayer.html
II. Methodology
The group sliced the siling labuyo into half and
removed its seeds from the body. After this, the
group triturated the sili with 10 ml of solvent, the
DCM Hexane. The extract was then collected in
a test tube.

2. Preparing the Solvent System

a. The group prepared 1mL of DCM
hexane 1:1, DCM 1:1 and DCM
METHANOL 1:1 in 3 separate test
tubes
b. The group then filled placed the
extract on the column and gently
poured the solvent up to the brim
using the solvents respectively and
simultaneously until the 1 mL of each
is all used up.
Figure 2.3 Introducing Solvents to the

Figure2.1 Triturating Siling Labuyo

Pipette

c.

Every color of eluate was then placed

in different test tubes. Each color was
recorded and the number of drops
per color was also noted.

3. Preparation of Thin Layer Chromatography

a. The group placed a filter paper in a
beaker with DCM Hexane in it and
covered it with watch glass. The group
waited for it to equilibrate.

Figure 2.5 Spotting the Eluates Using a

Capillary Tube
c.

After this placed the TLC plate inside

the developing chamber and waited
until the solvent reached the 0.5 cm
mark on top.

Figure 2.6 TLC Plate inside the Developing

Figure 2.4 Equilibrating the Developing

Chamber
b. While waiting, one member from the
group drew 0.5 cm and 1 cm on each
side of the 5x8cm TLC plate and using
a capillary tube placed ten drops of
each color on the TLC plate.

Chamber
d. The group placed a dot in the middle
of the darkest shade of each color
produced. The plate was then taken to

the satellite to visualize components

using a UV lamp

stationary phase, silica gel (silicon dioxide)

and poured with the extract from the red
siling labuyo. It was then added with the
mobile phase, 3.00 mL of each 1:1 DCMhexane, DCM, and 1:1 DCM-methanol,
successively to avoid the column to run dry.
Different colored eluates were collected in
different vials; colorless eluates were
discarded.
(table 3.1) kay milan
Table 3.1 shows the number of drops of
each collected eluate. There were four
colored pigments collected from the column
chromatography, yellow, light yellow,
orange, and red.

Figure 2.6 TLC Plate under the UV Lamp

e. .The group measured the distance
travelled by the spot and computed for
its Rf values.

III. Results and Discussion

1. Extraction of the pigments of red siling
labuyo
The mesocarp of the red siling labuyo was
triturated in a mortar and pestle with 1:1
DCM-hexane. DCM-hexane was used in
the experiment because it is an organic
solvent where the components of the red
siling labuyo is soluble in it.
2. Extraction of pigments using column
chromatography
The Pasteur pipette served as the column
chromatography. It was packed with a

3. Paper chromatography
Ten drops of each eluate was plotted in the
TLC plate. A filter paper placed in the
solvent system 1:1 DCM-hexane until the
solvent reaches the top of the filter paper to
allow it to equilibrate. Then, the plotted TLC
plate was submerged in the solvent system
until it reached the solvent front.
a.) Observing the TLC plate under the UV
lamp
After the solvent phase reached the
solvent front, the TLC plate was
observed under a UV lamp. The
separated pigments in the TLC plate
that have glowed under the UV lamp
was encircled.
b.) Detemining the Rf value
The distance travelled by the spots in
the TLC plate was used to determine
the Rf value, it was divided by the
distance of the solvent front.
Table from milan
Table 3.3 shows the different Rf
values