COLUMN AND THIN LAYER CHROMATOGRAPHY

Adolfo, Angela Nicole G., Alcaraz Frances Jillian F., Aliman, Stephanie T., Astor, Jashmin D.,
Bauzon, Franchesca D., Cabral, Danica Rose G.

Department of Pharmacy, Faculty of Pharmacy

University of Santo Tomas
ABSTRACT
Chromatography is a set of laboratory techniques in the separation of a mixture by passing
it in solution or suspension. This experiment aims to separate the colored components of red
Cayenne Pepper, or commonly known as the siling labuyo to determine the purity of the
components using thin layer chromatography; and measure the Rf values of the colored
components in thin layer chromatography. Column Chromatography was used to separate the
different colored pigments in the extraction of Cayenne Pepper (Capsicum annuum). Colored
eluate was collected through Column Chromatography and the purity of these eluates was
generously observed through a filter paper with a solvent of DCM-hexane.
INTRODUCTION

Compounds that interact strongly with the

stationary phase will move slowly. All forms of

Chromatography can be defined as a

laboratory technique that

chromatography work on the same principle.

separates

components within a mixture by using the

Diverse

types

of

differential affinities of the components for a

C h r o m a t o g r a p h y are possible, depending

mobile medium and for a stationary medium

on the physical states of the phases.

through which they pass. The underlying

Chromatographic separations can also be

principle of chromatography is that different

carried out using thin layer chromatography

substances

partition

(tlc) and column chromatography which a

coefficients between the stationary and

variety of supports, including immobilized

mobile phases. A compound that interacts

silica on glass plates. And these are the

weakly with the stationary phase will spend

chosen methods of Chromatography for the

most of its time in the mobile phase and move

experiment.

have

different

rapidly through the chromatographic system.

Chromatography

chromatographic techniques because it can

substance into its component parts, which is

be used in both analytical and preparative

very useful, as substances are often unique

applications.

in their composition. It can identify a

chromatography be used to determine the

substance and show how it differs from

number of components of a mixture, but it

others that may look alike on the surface. All

can also be used to separate and purify

types of chromatography are useful for

substantial quantities of those components

analytical

purposes. Under appropriate

for subsequent analysis. And this method is

conditions, all types of chromatography can

used for the extraction of the eluent of the

be

Cayenne pepper. The disadvantage of a

used for

separations.

separates

preparative
In

every

scale

only

can

column

of

column chromatography is that it is time-

chromatography there are three elements

consuming and tedious, especially for large

to be considered: the size of the sample

samples. If it is unnecessary to preparative

(Load),

of

separate

components (Resolution), and the Speed.

analytical

relative

type

Not

separat ion

large

quantities

methods

such

of

sample,

as

paper

chromatography may be more suitable and

In the experiment the method of

easier to perform.

Thin Layer Chromatography (TLC) and

Column Chromatography were chosen to
accomplish the objective.

Figure 1. Column Chromatography

Figure 1 shows the set up and process of a

Figure 2. Thin Layer Chromatography

Column Chromatographyand this type of

chromatography
advantageous

is
over

considered
most

other

Thin-Layer Chromatography (TLC)

involves the same principles as column

chromatography; it is also a form of solid

liquid adsorption chromatography. In this

DCM: hexane or Dichloromethane

case, however, the solid adsorbent is spread

hexane is the solvent system used to

as a thin layer on a plate of glass or rigid

elute

plastic. The solvent travels up by plate

column.

through a

chromatography

through capillary action. The set up for a Thin

Layer Chromatography is shown above

The solid phase (silica gel) is eluted

(Figure 2) where a drop of the solution to be

with this solvent system until fully solvated,

separated is placed near one edge of the

the compound to be purified is then loaded

plate, and the plate is placed in a container,

onto the solvated solid phase, and the column

called a developing chamber, with enough of

is eluted with the same solvent system until

the eluting solvent to come to a level just

your desired compound has come off the

below the point of origin. The solvent

column

migrates up the plate, carrying with it the

components of the mixture at different rates.
The result then, is a series of spots on the
plate, falling on a line perpendicular to the
solvent level in the container.

the following aspects as its advantages: It is

simple, fast, efficient to use and it requires
only small amounts of sample. TLC is
generally used as an qualitative analytic
technique, such as checking the purity of a
compound or determining the number of
in

mixture

or

column

chromatographic function. In addition, TLC is

useful for determining the best solvents for a
column chromatographic separation. It can
be used for an initial check on the identity of
an unknown sample. Preparative plates can
be carried out with special thick-layered TLC
plates.

(R value) is the ratio of the distance that the

spot travelled relative to the distance moved
by the solvent which in this case is the DCM-

Thin Layer Chromatography has

components

The Retention or Retardation Factor

hexane, the formula is shown below.

METHODOLOGY

cover the beaker with a watch glass and then

allow the set-up to equilibrate. Allow the

The materials used are mortar and

solvent to rise up leaving a 1cm space at the

pestle, iron stand and clamp, pasteur

upper end of the TLC. Then remove the

pipettes,

vials,

plates and mark the line immediately,

capillary tubes, precoated TLC plates, filter

because the unmasking of the TLC could

paper, silica gel, cotton and siling labuyo

cause the solvent to disappear instantly.

which

beakers,

is

watch

our

glass,

extract.

DCM

(dichloromethane), hexane, acetone.

RESULTS AND DISCUSSION

First,

set

up

the

Column

Chromatography apparatus and crush the

siling labuyo using the mortar and pestle. Get
the extract and using DCM as the first to

Plant Used: Cayenne Pepper (a.k.a. Siling

Labuyo)
Solvent System Used: DCM:hexane (1:1)

extract until it changes color. Take note that

a help of a rubber aspirator was used to help

Column chromatography:

the experiment extract quickly because of

The Cayenne Pepper was extracted

the short amount of time the students were

using the Pasteur pipette, and the number of

given. Next is the Hexane, and the method

drops were taken note of, as each pigment

or process is repeated until the eluent

were separated into layers. The summary of

changes in color. Also, be sure to change the

the findings can be seen in Table 1.

test tube every time the color changes. Dont

forget to record and dont let the extract dry.
Lastly, the process is once again repeated
with the application of acetone. Each of the
reagent has an amount of 5mL. After the
extraction, administer a generous amount of
the eluates on a 5cm 8cm TLC plate and

Table 1 - Volume Chromatography

Color of
Volume of
Component
eluate (drops)
1
Yellow
120
2
Clear
164
3
Orange
135
4
Pale Yellow
110
5
Dark Orange
44

start spotting 10 times using the capillary

tube. Do not forget to let it dry, make sure
that the spots are visible. And prepare the

Thin layer chromatography:

chamber for the Cayenne Pepper, trace the

In the experiment, four pigments

inner wall of the chamber with a filter paper,

were revealed as the color components of

the extracted form of red siling labuyo. The

2)

following

Rf =

colors

were

then

placed

Orange pigment
32

correspondingly on the filter paper and was

0.54

left to stand for several days. The results can

be seen in table 2, with the yellow pigment
having traveled 7 cm from the origin, orange

3)

traveled 32 cm, pale yellow could not be

Rf =

Dark orange pigment

4

0.83

seen, and lastly, the dark orange pigment

5

rose to 4 cm from the origin point.

CONCLUSION
Table 2 - Thin Layer Chromatography
Color of
Component
1
2
3
4

Yellow
Orange
Pale Yellow
Dark Orange

Distance of
component
from origin
(X) in cm
7 cm
32 cm
NA
4 cm

In the experiment, chromatography

Rf Value

was used to separate the different pigments

of Cayenne Pepper (or commonly called as

0.13
0.54
NA
0.83

the red Siling Labuyo) with the solvent

system of DCM:hexane. The distance of the
colored pigments and solvent system were
necessary to solve for the Rf value. This Rf
value shows how long did the solute and
solvent traveled to determine some of their
characteristic values. It was known that the

Computations:

lower the value of Rf, the more polar the

substance is. Thus, chromatography is a
Rf = distance traveled by compound
Distance traveled by the solvent

useful way to separate components of a

mixture as it travels at different rates.

REFERENCES
1)
Rf =

Yellow pigment
7
5

0.13

Website:
Chemistry: Chromatography. (2012,
August). Retrieved from http://www.marz-

kreations.com/Chemistry/Chromatography/
Dyes/RF-Values.html
Books:
Robards, K., Haddad,P.R., Jackson,P.E.,
(1994). Principles and Practice of Modern
Chromatographic Methods. San Diego,CA:
Academic Press Inc.

Williams, T. I., (1947). An Introduction to

Chromatography.New

York:

Chemical Publishing Co., Inc.

DOCUMENTATION