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I.

Introduction
Chromatography involves a variety of techniques that aim to separate or
isolate organic compounds from a certain mixture. The basis for the origin of the
word chromatography is color, since it comes from the Greek words chroma
meaning color and graphy meaning written. It includes the selective distribution
of the components between a mobile phase and stationary phase. The mobile phase
in the separation may either be a liquid or a gas and will carry the compounds along
a column. On the other hand the stationary phase, a selectively absorbing mixture,
may be composed of various types of materials. The selective and preferential
adsorption of the components of a mixture of organic compounds in the mobile phase
by the stationary phase is the principle behind the separation of these components.
There are two major classes of chromatography: gas chromatography (GC) and
liquid chromatography (LC). Gas chromatography makes use of a gaseous mobile
phase, usually helium or nitrogen, and a stationary phase that is either a liquid
adsorbed on a solid support, an organic compound bonded to a solid support, a solid,
or a nonvolatile liquid. It is most applicable for organic compounds that are
relatively volatile and thermally stable. Liquid chromatography utilizes a liquid
mobile phase which is usually a common organic solvent and a stationary phase
that may consist of a liquid adsorbed on a solid support, an organic species spread
over a solid support, a solid, or a resin (Shriner et. al., 1998). The latter type of
chromatography can be executed through adsorption chromatography and partition
chromatography.
Adsorption chromatography involves the utilization of the selective
adsorption of the components, which are to be separated in a mixture, from solution
by a suitable substance. This separation can be most relevant if the components are
colored. The procedure consists essentially in allowing a solution of the substance in
a suitable solvent (mobile phase) to pass slowly down a long column containing the
adsorbent material (stationary phase). The tenacity with which this material
adsorbs the various components of the mixture may vary noticeably, with the
outcome of a sharp separation of the components into colored zones or bands (Mann
& Saunders, 1960). Also, each solute (components of the mixture) has its individual
equilibrium between adsorption onto the surface of the solid and solubility in the
solvent.
Compound in stationary phase compound in mobile phase

The differences in the color of the column or band through which the mixture travels
indicate the solutes that have been separated The least soluble or best adsorbed
ones travel more slowly, suggesting that the individual components move at
different rates along with the mobile phase through the stationary phase. Eluent is
the term given for the solvent placed into a column, while the liquid that flows out of
the end of the column is called eluate (Royal Society of Chemistry, n.d.). The
polarity of the components of the mixture, the activity of the adsorbent, and the
polarity of the liquid mobile phase set the limit on the extent of adsorption on an
individual mixture substituent. Functional groups of compounds with a
characteristic polarity will have stronger adsorption affinity towards the surface of
the stationary phase. However, the relative values of the adsorption desorption
equilibrium constants, K, for each of the components of the mixture dictate the
actual process of separation.
One form of adsorption chromatography that uses a solid stationary liquid mobile
phases is thin layer chromatography (TLC). The former phase makes use of glass,
metal or plastic pates that are covered with a reedy layer of adsorbent while the
latter phase is a pure solvent or a mixture of solvents. The individual polarities of
the mixture substituents serve as the backbone of the appropriate mobile phase
composition. This type has become of significant utilization due to its simplicity,
efficiency, inexpensiveness, speed and most of all requirement of only small
quantities of material. It is specially carried out for determining the number of
compounds in a mixture, for aiding in determining whether or not two compounds
are identical, and for following the course of a reaction (Mohrig, Hammond, &
Schatz, 2010).
In contrast to adsorption chromatography, partition chromatography relies on the
phenomena of solubility. It consists of a liquid stationary phase reinforced on the
surface of a solid and a liquid or gas mobile phase that is insoluble in the former
phase. The most common type of this technique is the liquid liquid
chromatography wherein the stationary phase is physically adsorbed at the solid
support packing. The principle in this type is the differences in partition coefficients
of the components in the liquid liquid mixture. This coefficient is given by K,
defined by:

=Cs/Cm

where Cs is the concentration of solute in the stationary phase and Cm is the


concentration of the solute in the mobile phase.

Probably the first and the simplest type of chromatography that people meet is
paper chromatography. It is an example of partition chromatography that utilizes a
chromatography paper wherein a drop of solution or a mixture of dyes or inks are
inferiorly placed with respect to the paper and allowed to dry. Although if precision
is not required, a filter paper is frequently substituted. The edge of the paper with
the solution is immersed in a solvent, and then the solvent will eventually arise by
capillary action. The paper along with the bound water is the stationary phase
while the solvent is referred to as the mobile phase. The components of the mixture
are carried along with the solvent up the paper to varying degrees, depending on
the compounds ability to be adsorbed onto the paper versus being carried along
with the solvent. This action is efficiently completed due to the composition of the
paper. Filter paper is largely made up of cellulose, a polyhydroxy compound, to
which polar molecules are strongly adsorbed and retained, while the solvent is less
polar, usually composed of a mixture of water and an organic liquid. In execution,
this type works through the partition of the solutes between the bound water in the
papers fibers and the solvent. This happens due to the differences in the affinity of
the component of the mixture in the phases that differ in polarity as previously
mentioned. The water and the solvent facilitate an incessant exchange of solutes.
However, the components that are more soluble in the mobile phase remain for a
longer time and are carried up the paper rapidly. Paper chromatography is
effectually applied in component analysis and identification, usually of amino
acids and anions. It is also carried out in RNA fingerprinting and separating and
testing histamines and antibiotics.
As each solute distributes itself (equilibrates) between the stationary and the mobile
phase, the distance a solute moves is always the same fraction of the distance moved
by the solvent. As depicted by Oregon State University, this ratio is referred to as
the retention factor or relative mobility (Rf), which is assigned to each specific band
that also comprises the individual solutes characteristics. This factor is defined by:
From manual

where the origin is the midpoint of the original spot. This ratio should be a constant
that is characteristic of the solute(s) in a particular spot under a particular set of
chromatographic conditions such as the paper chromatogram and the solvent used
(2004).
The experiment requires the use of paper chromatography in the separation of plant
pigments and the identification of amino acids while the analysis of the component
dyes of different brands of black ink involves thin layer chromatography. Prior to
and during the execution of the procedures, there are several points that should be
clearly considered. First, the sample spot should be above the level of the solvent in
the developing chamber so that it will not wash away with the solvent. Second, the
sample spot should be minimized in order to maximize the measurement of the
distance moved of a particular solute. Third, the application of the sample should
only be minimal so that the sample will not spread out as it moves. Lastly, the
developing chamber should be covered tightly while the developing chromatogram is
being established because the paper or TLC medium may dry out as solvent
vaporizes and escapes.