Introduction

Column chromatography is a separation technique that is used to purify mixtures by

forcing it to run into a narrow tube. Column chromatography takes advantage of the
affinity of the compounds contained in the mixture to the stationary phase and to
the mobile phase. The stationary phase in column chromatography is the fixed part
where the mixture will be forced to pass through. The mobile phase or the eluent is
the phase that moves through the stationary phase carrying the mixture and its
compounds. This process, solutes are carried by the mobile phase through the
stationary phase, is called elution. The separation also uses an external force,
pressure or gravity, to separate the different compounds.
There are different types of column chromatographic techniques and they are
classified according to the type of mobile phase: gas chromatography, liquid
chromatography and supercritical fluid chromatography (Skoog, West, Holler &
Crouch, 2014). In this experiment, we will be using a liquid mobile phase and a
solid stationary phase. The main principle in liquid-solid chromatography is
adsorption. Adsorption is the adhesion of atoms or ions of a liquid or gaseous
mixture into a particular surface. The stationary phase helps in the separation of the
mixture by adsorbing the polar compounds hence, owing to its adsorptive power.
The stationary phase is commonly insoluble and immiscible with the mobile phase
and in liquid-solid chromatography, the common stationary phases used are
adsorbents.
The aim of this experiment is to apply techniques in the packing of the column; to
separate the different plant pigments contained in the sample, Carmona retusa
Merr.; and to determine if the said techniques efficiency.

Methods
Leaves of tsaang gubat, Carmona retusa Merr., were collected from the botanical
garden in the roof deck of University of the Philippines College of Pharmacy. The
leaves were removed from the stems then were triturated or cut into smaller
segments before undergoing the extraction procedure. Solvent used for extraction
of the pigments in the leaves was ethanol and of technical grade quality. The leaves
were then macerated with the ethanol, 95%, and left for at least 65 hours before
the experiment proper. The hexane and ethyl acetate used as mobile phase were of
technical grade also.
The column was then packed using three different adsorbents with varying degrees
of absorptive power. Each layer then is separated by a piece of filter paper. Alumina,
10ml, is packed on the first layer, magnesium carbonate, 10ml, on the second and
lastly, 40ml of powdered sucrose. The column was then subjected to equilibration
by saturating it with the mobile phase before the introduction of the extract. The

extract, 5ml of which, was put into the top of the column and is washed with
enough mobile phase, preventing the layers to dry, letting the sample to travel
down the packed stationary phase. This was done until a part of the pigments
reached the end of the column. No pigments or compounds of the extract were
eluted.

Results and Discussions

The chromatogram

References:
Skoog, D., West, D., Holler, J. and Crouch, S. (2014). Fundamentals of Analytical
Chemistry (9th ed.). Belmont, CA: Cengage Learning.