1

Column and Thin Layer Chromatography Using Red Siling Labuyo

(Capsicum frutescens) Extract
Rada, R.A.P., Rojo, K.A.A., Roque, M.F.Y., Santos, R.S.S., See, K.A.A.*,
Silang, G.M.
Group 7, 2A Pharmacy
Department of Pharmacy, Faculty of Pharmacy
University of Santo Tomas
Abstract
Chromatography is a technique used to separate and to identify components
in a mixture. In this experiment, column chromatography and thin layer
chromatography (TLC) were performed. The experiment was conducted to
separate the different pigments in the pure extract of red siling labuyo
(Capsicum frutescens) through column chromatography, to determine the
purity of the different pigments obtained through thin layer chromatography,
and to calculate the retention factor (R f) of each pigment obtained, including
the pure extract using the data obtained from TLC. The colored eluates
collected from column chromatography are yellow (195 drops), red (97
drops), and red orange (18 drops). In TLC, the solvent front travelled 5.2 cm.
The Rf values computed were: red (pure extract): 0.85, yellow: 0, orange: 0,
and red orange: 0.13.
Introduction

components of the mixture being

Chromatography is defined

separated. Affinity is determined

as a method used to separate

by

components

Adsorption

of

mixture

by

adsorption

and

is

defined

solubility.
as

the

differential absorption between a

binding of molecules on a surface.

stationary

mobile

In chromatography, how well a

phase. The stationary phase is the

component of a mixture binds to

non-moving

phase

in

the stationary phase. Solubility, on

chromatography,

through

which

the other hand, is the amount of

the mobile phase flows, and where

substance that will dissolve in a

in the distribution of components

given amount of solvent. In this

between the two phases occurs.

case, it is how well a component of

The

phase

mobile

and

phase

carries

the

mixture

dissolves

in

the

components through the stationary

stationary phase.

phase.

absorptivity are closely related to

the

The principle of separation in

polarity

instance,

of
a

Solubility and
substances.
separation

of

chromatography is the differential

components

affinities

chromatography is being done, the

of

the

different

through

For

column

2
column being composed of a polar

Deseeded and chopped red

stationary phase, and the solvent

siling labuyo was grinded in a

being used is increasing in polarity.

mortar and pestle. After which,

Following

like

the extracting solvent, DCM-

eluates

hexane (1:1) was added to the

the

dissolves

principle

like,

of

the

collected will also be increasing in

grinded

sample.

The

pure

polarity, since the most non-polar

extract was collected using a

component of the mixture will go

Pasteur pipette.

with the most non-polar solvent,

and so on and so forth. Thin layer
chromatography also follows the
same

principle

as

column

chromatography (TLC). Retention

factor (Rf) value is the quantitative
measure of a components affinity
to the mobile phase. In normal
chromatography,

in

which

the

stationary phase is polar and the

mobile

phase

is

non-polar,

Figure 1: Extracting the sample

the

B. Column Chromatography
Figure 2 shows the column

lesser the polarity of a component,

chromatography

the higher the Rf value.

column

to

experiment
The
experiment

objective
is

to

of

separate

be
was

set-up.
utilized

The
in

the

prepared

by

this

placing a small cotton ball inside a

the

Pasteur

pipette,

and

filling

the

pigments of the red siling labuyo

pipette with silica gel up to its

using column chromatography, to

neck. The column was clamped to

use the eluates obtained for thin

the iron stand.

layer

chromatography,

and

to

compute for the retention factors

of each pigment from the values
obtained in TLC.
Experimental
A. Extracting the Sample

3
one of the lines. The pure extract
and each of the colored eluate
collected were spotted on the thin
layer

chromatography

times.

Then,

the

plate

10

developing

chamber was prepared by filling a

400 mL beaker with 30 mL of DCMhexane

(1:1).

The

beaker

was

covered with a watch glass and a

Figure 2: Column

tissue, and was left to rest for 15

chromatography set-up

DCM-hexane,
solvent,

was

the

run

extracting

through

the

column until it was wet. Then, 0.5

mL of the pure extract was placed
on top of the column. 3 mL of DCMhexane

was

introduced

to

the

column, and colored eluate was

collected.

Through

the

same

process, another batch of colored

eluate was collected in a different

minutes

to

allow

the

solvent

system to equilibrate.
After 15 minutes, the TLC plate
was placed inside the chamber. The
chamber was once again covered
with a watch glass, and the solvent
system was left to develop. After
reaching the 1.5 cm mark, the TLC
plate

was

removed

from

the

chamber to dry.

test tube using 3 mL of DCM.

Again, the process was repeated,
and

another

eluate

batch

of

colored

was collected, this time

using DCM: methanol (1:1) as the

solvent.
C. Thin Layer Chromatography
The thin layer chromatography
plate was prepared for use by

Figure 3: Development

After

drying,

the

distances

measuring and lining a 1.5 cm

travelled by the components were

space on both ends of the plate,

visualized under UV light, and their

and drawing 4 equidistant spots on

corresponding places were marked

4
with a pencil for the measurement

which is DCM-hexane, its affinity to

of

the stationary phase is the lowest

the

distance

that

they had

travelled.

of the all the three components.

Therefore, it is the least polar

D. Computation of the Retention

Factors of the Pure Extract and

applied in the explanation above, it

the Colored Eluates

The retention factor values

is

were computed using the

Rf =

distance travelled by the pigment

distance travelled by the solvent

Results and Discussion

In column chromatography,
the solvents used were increasing
polarity,

the

most

inferred

component

non-polar

solvent being used first. Table 1

from

chromatography.

Based

is

the

orange

intermediate

in

the most polar. In summary, the

eluates collected are increasing in
polarity.
Color of

Volume of

component
Yellow
Orange
Red orange

eluate (in drops)

195 drops
97 drops
18 drops

Table 1: Volume of the eluates

collected from Column

shows the volumes of each eluate

collected

that

polarity, and the red orange being

formula:

in

component. From the same logic

Chromatography

column
on

the

The

color

of

eluates

results, the yellow component is

collected

the most abundant, with 195 drops

chromatography can be explained

collected, followed by the orange

by the pigments present in the

component with 97 drops, and

pure extract of red siling labuyo.

lastly, the red orange component,

There are three pigments present

with 18 drops. From the obtained

in the extract, namely Carotene,

data, and the fact that the solvents

and Capsanthin, and Capsorbin.

used in the experiment used were

The yellow component obtained is

increasing in polarity, it is inferred

Carotene, the orange component

that the yellow component is the

Capsanthin, and the red orange

most non-polar of all the three.

being Capsorbin.

Since the yellow component flowed

with the most non-polar solvent,

from

the

column

5
Figure 4 shows the TLC plate

the

experiment

yielded

correct

after the experiment. Table 2 shows

results, the results obtained would

the distances travelled by the pure

have

extract and the yellow, orange, and

component

red

orange

eluates

development

shown

that

the

had

the

yellow
highest

during

the

retention factor value, since it is

and

the

the most non-polar component of

stage

computed Rf values.

the pigment. The second pigment

would have had the intermediate
retention factor value, because in
comparison with the other two
pigments present, it is intermediate
in non-polarity. The red orange
pigment would have had the lowest
retention factor, since it should
have had the lowest affinity to the

Figure 4: TLC plate after the

experiment

Color of

Distance

compone

of

nt

componen

Rf value

mobile phase among the three

components,

Chromatography is a method

origin (X)

of used to separate components of

in cm
4.4 cm
0 cm
0 cm
0.7 cm

a mixture based on differential

0.85
0
0
0.13

were

some

errors

during the experiment, because

results

distances

absorption.

In

column

chromatography, the last eluate

collected was the least non-polar of
all the components, since it was

There

obtained.

solvent

Conclusion

orange

the

the

system used is non-polar.

t from

Red
Yellow
Orange
Red

since

expected

Ideally,

were

none

travelled

of
by

not
the
the

component should be 0 cm. Had

used

with

solvent.

the

least

Among

non-polar

the

three

components, it would have had the

strongest

affinity

to

the

polar

stationary phase, explaining the

scarce

volume

collected.

The

of

the
thin

eluate
layer

6
chromatography (TLC) experiment

t-prep/mcat/chemical-p

performed

did

expected

results.

not

yield

the

rocesses/separations-

Had

the

purifications/a/principles-of-

experiment showed the expected

results,
would

the
have

yellow
had

component
the

highest

retention factor value computed

because it is the most non-polar of
all

the

components.

It

affinity

to

the

polar

stationary phase.

Khan Academy. (2016). Principles

chromatography. Retrieved
October

18,

2016

Thin

Layer Chromatography. Retrieved

October

18,

2016

from

http://www.chemguide.co.uk/analy
sis/chromatography/thinlayer.html
Boston College. (n.d.).

References
of

Clark, Jim. (2016, June).

was

expected that it would have the

lowest

chromatography

October

18,

2016

Retrieved
from

http://www.bc.edu/schools/cas/ch

from
emistry/academics/undergrad/org/

https://www.khanacademy.org/tes

fall/TLC.pdf