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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


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 Chromatography (lc) refers to any
fraction by distribution between two chromatographic process that employs a
phases, one phase being stationary and mobile liquid phase.
essentially two dimensional (a surface),
and the remaining phase being mobile. All types of chromatography are
The underlying principle of useful for analytical purposes. Under
chromatography is that different appropriate conditions, all types of
substances have different partition chromatography can be used for
coefficients between the stationary and preparative scale separations. In every
mobile phases. A compound that interacts type of chromatography there are three
weakly with the stationary phase will elements to be considered. The Load (or
spend most of its time in the mobile phase the size of the sample),The Resolution (or
and move rapidly through the the relative separation of components),
chromatographic system. Compounds that and the Speed.
interact strongly with the stationary phase
will move slowly. In the ideal case, each It would be ideal if all three
component of a mixture will have a elements could be maximized so that
different partition coefficient between complete separation of samples of any
mobile and stationary phases, desired size could be quickly achieved. In
and consequently each will move through practice, generally two of these elements
a system at a different rate, resulting in can be maximized at the expense of the
complete separations. third. For routine analytical work,
resolution and speed are maximized at the
Various types of Chromatography expense of the load. In preparative scale
are possible, depending on the physical separations, load and speed can be
states of the phases. Employing a gas the maximized, but then separations are
mobile phase is termed gas usually incomplete. Complete separations
chromatography (gc) or vapor phase of large samples can be achieved but the
chromatography (vpc). Separations using overall operation is likely to be slow and
gas chromatography involve vapor phase 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 color of the eluate varies. The number of
off the column. drops for each color was noted.

The Retention or Retardation Factor After collecting the eluates from the
(Rf value) is the ratio of the distance that column, Thin Layer Chromatography was
the spot travelled relative to the distance performed.
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 The eluates were applied on the
values) of colored components in TLC. 5cm X 8cm pre-coated TLC plate by
equidistantly spotting each spot 10 times.
II. Experimental The spot was allowed to dry first before
applying the succeeding spots. It was
Pigments of the red siling labuyo ensured that the spots made were small
were extracted by pouring DCM- hexane as possible so that when the plate
and eventually pounding it using mortar develops, the colors would not be
and pestle with the ratio 1:1. The disarray.
extracted pigments were set aside for a
while. Developing Chamber was prepared
by placing the approximate amount of
Silica Gel Column was prepared by DCM hexane. The inner wall of the
plugging the column with cotton followed chamber was lined with filter paper to
by the silica gel which was uniformly allow the TLC plate to stand. The
packed and contained no holes or air developing chamber was covered with
bubbles until it reached the indented part watch glass and was allowed to
of the Pasteur pipette. equilibrate.

0.5 ml of the extract was placed on The Developing plate was carefully
top of the column using Pasteur pipette. introduced in the developing chamber. The
The pigment mixture was eluted using solvent system was allowed to rise up
10ml DCM-hexane. The system solvent until it reaches just 1cm from the upper
was introduced in portions. The column end. The developing plate was then
was not allowed to run dry and the removed carefully from the chamber. The
colorless eluate collected was discarded. solvent front was immediately marked and
The vials were changed each time the 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

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

Table 2 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
1 Crude F 5.5 cm 0.91
2 Dark Yellow 2 cm 0.3
3 Violet 2.8 cm 0.3
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


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

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