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/Cmwhere 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.
