COLUMN AND THIN LAYER CHROMATOGRAPHY

Michael Dominique B. Allag, Eryll Joy H. Agojo, Camille A.

Baetiong, Greanne P. Danica Ballesta, Rachel Anne S. Barlao
Group 1 2C Medical Technology Organic Chemistry Laboratory

Abstract
Chromatography is a technique for separating mixtures into their components in order to analyze,
identify, purify, and quantify the mixture or components. There are different types of chromatography
and each has its own advantages and disadvantages. In this experiment, DCM-hexane was used to
extract the different pigments of the siling labuyo. Extract was introduced into the column and eluate
was collected, this process is the column chromatography (CC) method. The purity of the components
was determined by using thin later chromatography (TLC). UV lamp was used to visualize the
developed TLC plate and the Retention or Retardation Factor was measured.

I.

Introduction

Chromatography can be defined as a

laboratory technique that
separates
components within a mixture by using the
differential affinities of the components for a
mobile medium and for a stationary medium
through which they pass. The underlying principle
of chromatography is that different substances
have different partition coefficients between the
stationary and mobile phases. A compound that
interacts weakly with the stationary phase will
spend most of its time in the mobile phase and
move rapidly through the chromatographic
system. Compounds that interact strongly with
the stationary phase will move slowly. All forms of
chromatography work on the same principle.
Diverse types o f
Chromatography
are
possible, depending on the physical states of
the phases. Employing a gas the mobile
phase
is termed gas chromatography (gc) or vapor
phase chromatography (vpc). Separations using
gas chromatography involve vapor phase versus
adsorption
and/or
equilibria.
Liquid
Chromatography
(lc)
refers
to any
chromatographic process that employs a mobile
liquid phase. Chromatographic separations can
also
be
carried
out
using
thin
layer
chromatography (tlc) and column chromatography
which a variety of supports, including immobilized
silica on glass plates.

Chromatography separates a substance

into its component parts, which is very useful, as
substances are often unique in their composition.
It can identify a substance and show how it
differs from others that may look alike on the

surface. All types of chromatography are

useful for analytical
purposes.
Under
appropriate
conditions,
all
types
of
chromatography can be
used
for
preparative s c a l e s e p a r a t i o n s . In e v e r y
type of chromatography there are three
elements to be considered: the size of the
sample (Load), re lative se paratio n o f
co mpo ne nts (Resolution), and the Speed.

It would be ideal if all three elements could be

maximized so that complete separation of
samples of any desired size could be quickly
achieved. In practice, generally two of these
elements can be maximized at the expense of
the third. For routine analytical work,
resolution and speed are maximized at the
expense of the load. In preparative scale
separations, load,
and
speed
can
be
maximized but then separations are usually
incomplete. Complete separations of large
samples can be achieved but the overall
operation is likely to be slow and tedious, and
may involve the use of large quantities of
solvent that must be distilled for reuse, or
discarded.

In
the
experiment,
Chromatography
and
Chromatography were used.

Column
Thin

Column
chromatography
is
advantageous
over
most
other
chromatographic techniques because it can be
used in both analytical and preparative
applications.
Not
only
can
column
chromatography be used to determine the
number of components of a mixture, but it can
also be used to separate and purify substantial
quantities of those components for subsequent
analysis. This is in contrast to paper
chromatography, which is solely an analytical
method.
The disadvantage of a column
chromatography is that it is time-consuming
and tedious, especially for large samples. If it
is unnecessary to preparative separate large
quantities of sample, analytical methods such
as paper chromatography may be more
suitable and easier to perform.
Thin-Layer
Chromatography
(TLC)
involves the same principles as column
chromatography; it is also a form of solid liquid
adsorption chromatography. In this case,
however, the solid adsorbent is spread as a
thin layer on a plate of glass or rigid plastic.
The solvent travels up by plate through
capillary action. A drop of the solution to be
separated is placed near one edge of the plate,
and the plate is placed in a container, called a
developing chamber, with enough of the
eluting solvent to come to a level just below
the point of origin. The solvent 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.

TLC has a number of advantages: It is

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

DCM
hexane
or
Dichloromethane
hexane is the solvent system used to elute
through
a
chromatography
column. This means that the mobile phase
(solvent system) consists of 1:1 (ratio of
volume) mixture
of
dichloromethane
(DCM; CH2Cl2), and hexane (C6H14).
The solid phase (silica gel) is eluted with
this solvent system until fully solvated, the
compound to be purified is
then
loaded onto the solvated solid phase,
and the column is eluted with the same
solvent system until your desired compound
has come off the column
T he Retention or Retardation Factor (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-hexane.

The o b j e c t i ve s o f t h e e x p e r i m e n t
are the following: separate the colored
components of red siling labuyo using column
chromatography, t o predict the purity of
components using column and thin layer
chromatography (TLC) and lastly, to measure
the Retention/Retardation Factor (R values) of
colored components in TLC.

II.

Experimental

Pigments of the siling labuyo were extracted

by cutting it to pieces and by pouring DCMhexane and eventually triturating it by using a
mortar and pestle with the ratio of 1:1. The
extracted pigments were set aside for a
while.
Silica Gel Column was prepared by plugging
the column with cotton followed by
the
silica gel which was uniformly packed and
contained no holes or air bubbles until it
reached the indented part of the Pasteur
pipette.
0.5 ml of the extract was placed on top of
the column using Pasteur pipette. The
p i g m e n t m i x t u r e w a s e l u t e d u s i n g 10ml
DCM-hexane.
The s y s t e m s o l v e n t was
introduced in portions. The column was not
allowed to run dry and the colorless eluate
collected was discarded. Test tubes were
changed each time the color of the eluate
varies. The number of drops for each color
was noted.
After collecting the eluates from the
column, Thin Layer Chromatography was
performed.
The color of the eluate varies. The number
of drops for each color was noted.
After collecting the eluates from the
column, Thin Layer Chromatography was
performed.

The e l u a t e s w e r e a p p l i e d o n t h e
5cm X 8cm pre-coated TLC plate
by
equidistantly spotting each spot 10 times. The
spot was allowed to dry first before applying
the succeeding spots. It was ensured that the
spots made were small as possible so that
when the plate develops,
the colors would
not be disarray.
Developing Chamber was prepared by
placing the approximate amount of
DCM hexane. The inner wall of the chamber
was lined with filter paper to allow
the
TLC plate to stand. The developing chamber
was covered with watch glass and was allowed
to equilibrate.
The TLC plate was carefully introduced
in the developing chamber. The solvent
system was allowed to rise up until it
reaches just 1cm from the upper end. The
TLC plate was then removed carefully from the
chamber. The solvent front was immediately
marked and the plate was allowed to dry.
The
components
were visualized
using the ultraviolet lamp after the plate has
developed after elution process and this causes
substances to appear as colored spots.
The R values were measured and
chromatographic plates were documented.

III.

Results and
Discussion

Plant used: Siling Labuyo

Solvent System used: DCM-Hexane

Column Chromatography:
Two eluates were yielded from the extraction of
the colored components of siling labuyo using
column chromatography. Two different shades
of colors were obtained: Light yellow and light
orange. The volume of the light yellow eluate
collected from the column was 35 drops while
on the other hand, the volume of the light
orange was 85 drops.

Color
of
Componen
light yellow

light orange

Volume of eluate
(no. of
drops)
35
85

Table 1 Column Chromatography

(Table of
Results)

travelled 4.3 cm while the light orange

eluate travelled 0.5 cm.
The color of the developed plate
was not visible by the naked eye. It was
placed UV light for viewing.
The general formula for computing the R
value is shown below:

After measuring the distance

traveled for each spot, The R value (also
known as Retardation or Retention Factor
was computed) Retardation or Retention
Factor is the ratio of time spent in the
stationary phase relative to time spent in the
mobile phase.
Distance of solvent: 5cm

Thin Layer Chromatography

Table 2 Thin Layer Chromatography

(Table of Results)

With reference to Figure 4, (From left to

right) the first spot is the Crude Eluate; the
second spot is the first eluate collected from
the column and the third spot is the second
eluate collected from the Column
Chromatography.
The crude eluate travelled 4.0 cm
from the origin; the light yellow eluate

1
2
3

Color of
Component
Light Yellow
Light Orange
Crude

Distance of
Component from
origin (x) in cm
4.3 cm
0.5
cm
4.0
cm

Rf
Value
0.86
0.1
0.8

The developed plate wasnt 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 werent small

enough which have caused color the color
to disarray. Another source of error is not
covering completely the developing
chamber
during the development of TLC
plate.

V. References
BOOKS:
Fedessenden, R.J., Fedessenden, J.S., &
Feist
P.
(2001).
Organic
Laboratory
Techniques. Canada: Brooks/ Cole. Pg.
119-140
Robards,
K.,
Haddad,P.R.,
Jackson,P.E.,
(1994). Principles and Practice of Modern
Chromatographic Methods. San Diego,CA:
Academic Press Inc. Pg. 1-34, 36-225
Williams, T. I., (1947). An Introduction to
Chromatography.New
York:
Chemical
Publishing Co., Inc. Pg. 1-85

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