COLUMN AND THIN LAYER CHROMATOGRAPHY

Maria Feliza C. Abesamis, Marie Em Clarisse P. Acosta, Francheska M. Agustin,

Mary Christelle G. Aquitania and Marilu Jane H. Bagsican
Group 1 2E Medical Technology Organic Chemistry Laboratory

Abstract

Chromatography is a powerful technique for separating mixtures. There are different types
of chromatography and each has its own strengths and weaknesses. In this experiment, pigments
of the siling labuyo were extracted with the use of DCM-hexane, 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 versus adsorption and/or equilibria. Liquid

the separation of a mixture into various
fraction by distribution between two
phases, one phase being stationary and
essentially two dimensional (a surface),
and the remaining phase being mobile.
The underlying principle
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
chromatographic system. Compounds that
interact strongly with the stationary phase
will move slowly. In the ideal case, each
component of a mixture will have a
different partition coefficient between
mobile and stationary phases,
and consequently each will move through
a system at a different rate, resulting in
complete separations.
Various types of 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
Chromatography (lc) refers to any
chromatographic process that employs a
mobile liquid phase.
All types of chromatography are
of useful for analytical purposes. Under
appropriate conditions, all types of
chromatography can be used for
preparative scale separations. In every
type of chromatography there are three
elements to be considered. The Load (or
the size of the sample),The Resolution (or
the the relative separation of components),
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 TLC has a number of advantages: It
for reuse, or discarded. is simple, quick and inexpensive, and it
requires only small amounts of sample.
In the experiment, Column TLC is generally used a qualitative analytic
Chromatography and Thin technique, such as checking the purity of a
Chromatography were used. 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. TLC is
fast, efficient, and simple to use.

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. DCM hexane or Dichloromethane
hexane is the solvent system used to
The disadvantage of a column elute through a chromatography
chromatography is that it is time- column. This means that the mobile
consuming and tedious, especially for
phase (solvent system) consists of 1:1
large samples. If it is unnecessary to
(ratio of volume) mixture of
preparative separate large quantities of
sample, analytical methods such as paper dichloromethane (DCM; CH2Cl2), and
chromatography may be more suitable hexane (C6H14).
and easier to perform.
The solid phase (silica gel) is
Thin-Layer Chromatography (TLC) eluted with this solvent system until
is closely related to column fully solvated, the compound to be
chromatography. The adsorbent is coated purified is then loaded onto the
on one side of a strip or plate of glass, solvated solid phase, and the column is
plastic or aluminum. The solvent travels eluted with the same solvent system
up by plate through capillary action.
 until your desired compound has come
off the column.
The Retention or Retardation Factor
(Rf 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 objectives of the experiment
are the To separate the colored
components of siling labuyo using column
chromatography, To determine the purity
of the components using thin layer
chromatography and lastly is to Measure
the Retention/ Retardation Factor (Rf
values) of colored components in TLC.
II. Experimental
Pigments of the red siling labuyo
were extracted by pouring DCM- hexane
and eventually pounding it using mortar
and pestle with the ratio 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 pigment mixture was eluted using
10ml DCM-hexane. The system solvent
was introduced in portions. The column
was not allowed to run dry and the
colorless eluate collected was discarded.
The vials 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 eluates were applied on the
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 Developing 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 developing 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 With reference to Figure 4, (From
using the UV lamp. Rf values were left to right) the first spot is the Crude
measured and chromatoplates were Eluate; the second spot is the first eluate
documented. collected from the column and the Third
spot is the second eluate collected from
III. Results and Discussion the Column Chromatography.

Plant used: Siling Labuyo The Crude eluate travelled 5.5 cm

Solvent System used: DCM-Hexane from the origin; The Dark Yellow eluate
travelled 2.0 cm while the Light Yellow
Column Chromatography eluate travelled 2.8 cm.

Two eluates were yielded from the The color of the developed plate
extraction of the colored components of was not visible by the naked eye. It was
siling labuyo using Column placed UV light for viewing.
Chromatography. Dark Yellow and Light
Yellow were yielded respectively. The Calculation of Rf
Volume of the dark yellow eluate collected (Retardation/ Retention Factor):
from the column was 96 drops while on
the other hand, the volume of the light After measuring the distance
yellow was 61 drops. traveled for each spot, The Rf value (also
known as Retardation or Retention Factor
Color of Volume of eluate was computed) Retardation or Retention
Component (no. of drops) Factor is the ratio of time spent in the
stationary phase relative to time spent in
1 dark yellow 96
the mobile phase.
2 light yellow 61

Table 1 Column Chromatography The formula general formula for

(Table of Results) computing the Rf value is shown below:

Thin Layer Chromatography

Since Rf value is a ratio, Rf doesn’t have a

unit.
Computation of the Rf value has been Fedessenden, R.J., Fedessenden, J.S., &
provided below: Feist P. (2001). Organic Laboratory
Techniques. Canada: Brooks/ Cole. Pg.
Distance of solvent: 6cm 119-140

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

Chromatography.New York: Chemical
Publishing Co., Inc. Pg. 1-85

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

WEBSITES:
Table 2 Thin Layer Chromatography
(Table of Results) THIN LAYER CHROMATOGRAPHY
Distance of Retrieved August 21, 2009 , from
Color of Component from Rf http://www.wellesley.edu/Chemistry/che
Component origin (x) in cm Value
m211lab/Orgo_Lab_Manual/Appendix/Tec
1 Crude F 5.5 cm 0.91
hniques/TLC/thin_layer_chrom.html
2 Dark Yellow 2 cm 0.3
3 Violet 2.8 cm 0.3
COLUMN CHROMATOGRAPHY
The developed plate wasn’t able to Retrieved August 21, 2009, from
show completely the separation of colors. http://www.chemguide.co.uk/analysis/chr
The possible sources of error are from the omatography/column.html
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 weren’t 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:

Pastro, D. J., John, C. R., & Miller, M. S.

(1998). Experiment and Techniques in
Organic Chemistry. New Jersey: Prentice
Hall. Pg. 60-83