Column Chromatography as a Chemical Purification Technique
By Phill Rasnick
Introduction
This document is intended to provide students and professionals in
organic chemistry labs with a detailed description of the process of
column chromatography. Column chromatography is an extraordinarily
useful technique in organic chemistry labs because it provides a simple
and inexpensive means of isolating individual compounds from a
mixture. This process isolates components of a mixture based on the
polar properties of the components.
Polarity: Polarity refers to the distribution
of elections within a compound. Nonpolar
compounds have uniform electron density
while polar molecules have areas with
unequal electron distributions. For this
reason, polar molecules have poles which
are negative or positively charged relative
to one another.
Column Setup/Preparation
Column chromatography works through the “like dissolves like”
principle of organic chemistry. This principle describes the concept that
compounds with similar polarities will have higher affinities towards
each other and will be more soluble than compounds whose polarities
differ. Using this principle, column chromatography makes use of a
stationary crystalline phase and a nonpolar liquid mobile phase to
separate the substances in a mixture based on the differences in their
relative polarities. The stationary phase generally consists of either silica
or alumina, both of which are polar compounds. While the mobile
phase is nonpolar, it can have a range of polarities that it can be based
on its chemical composition.
For a mobile phase to be effective in isolating specific components, the
nonpolar behavior of the liquid must be able to elute one component
more rapidly through the column. For this reason it can often be
difficult for components with very similar polarities to be isolated via
column chromatography. Micro scale experimentation is the only
method which can effectively demonstrate a nonpolar solution’s ability
to separate the compounds of a mixture. Therefore, micro scale
experiments are useful in helping to choose the proper mobile phase
composition to separate a specific mixture.
The main column used is a glass cylinder which can vary widely in height
and diameter for purposes of both micro and macro scale use. On the
bottom of the cylinder a stopcock controls the outflow of liquid mobile
phase, and a filter is used to prevent the stationary phase from exiting
the tube or clogging the stopcock and disrupting flow through the
column.
Stationary Phase: The stationary phase is
the crystalline solid material which remains
within the column. For chromatography
purposes, the stationary phase is
composed of polar substances, either silica
or alumina.
Mobile Phase: The mobile phase is the
liquid component of the column setup, and
is nonpolar in nature. This fluid is
continuously eluted through the column
and is responsible for carrying compounds
to be isolated through the column.
Loading the Column
Once the apparatus is setup, the chromatography column is then
packed using one of two methods: either the dry method, or the slurry
method. When packing the column using the dry method, the
powdered stationary phase (silica or alumina) is first added to the
column, filling the majority of the tube. The mobile phase is then added
to the column so that the entire contents of the stationary phase are
saturated. In the slurry method, a slurry is first made by combing the
eluent (mobile phase) with the powdered stationary phase. The slurry is
then poured into the column avoiding the formation of any air bubbles
which would affect the flow through of the column. After the column is
packed with the stationary phase, the organic mixture to be separated is
layered on top. Often when carrying out column chromatography, a
layer of sand will be added over the organic layer to prevent any
disruption of the organic layer as new eluent is added throughout
running the column.
Running the Column
Funnel
Sand
Organic Solution
Stationary
Phase
Filter
Stopcock
Figure 1. The above image shows a basic
setup for column chromatography for
micro scale purposes.
As the column is run, the stopcock is used to control the outflow of
eluent from the column and collect the flow through in regular intervals.
The eluent that drains through the stopcock is collected in a series of
fractions of regulated volume to ensure that there is no impurities in
the fractions containing the desired product. Because the organic
compounds being isolated are often colorless, various analytic
techniques such as analytic chromatography, UV absorption, and
fluorescence are used to identify whether the desired product is
contained within the fraction. For fractions that contain only the desired
substance and mobile phase liquid, the eluent is evaporated or
separated using other techniques, yielding the pure product. While
running the column, it is also important to continuously add eluent as
the column is drained to help move substances through the column and
keep the stationary phase wet.
Separating Mixture Components
Polar molecules contain electronegative atoms or functional groups
which can either attract or withdraw electrons from regions on the
molecule, resulting in a non-uniform electron distribution. Because
electrons are negatively charged particles, regions with high electron
density will have a negative charge in relation to regions on the
molecule with low electron density. This separation of charges results in
a dipole moment and is the basis for the intramolecular forces between
adjacent polar molecules.
Electronegativity: electronegativity is the
tendency for atoms or functional groups
to attract electrons towards itself.
The oppositely charged poles of the dipole act analogous to a magnate
by attracting the oppositely charged pole of another molecule. These
attractive forces, with are collectively termed dipole-dipole interactions
and London dispersion forces, result in the strong intramolecular
attraction between polar molecules. Figure 3 below demonstrates how
dipoles interact via their opposite poles. The strength of these
intramolecular forces is affected by the strength of the dipole moment.
Molecules with functional groups or electronegative atoms that cause
the largest separation of charges are going to be the most polar
molecules and have the strongest dipole moments.
Column chromatography works on the basis that the more polar
substances in the mixture will contain stronger intramolecular forces
with the polar stationary phase. As the column is run and eluent is
drained from the tube, the organic molecules are washed down the
tube at varying rates. The rate that different compounds elute through
the tube is directly related to the level of polarization of the molecules
as well as the polarization of the mobile phase. Polar molecules will
elute from the column at a slower rate when compared to nonpolar
compounds because of the stronger intramolecular forces attracting
them to the stationary phase. Nonpolar compounds, having weaker
intramolecular forces, will not adhere as strongly to the stationary
phase enabling those compounds to be washed through the column
more rapidly (Figure 2). Because of these properties, polar substances
may often take a long period of time to run through the column without
changing the mobile phase. Therefore, if the more polar substance is
the desired component to be isolated, a more polar mobile phase is
often used to elute the polar substance after the nonpolar component
has been drained from the column in its entirety to prevent any
contamination of the product.
Organic
Mixture
More Polar
Compound
More Nonpolar
Compound
Figure 2. The above image represents
the separation of components of a
mixture (green) as column
chromatography is run. Because more
polar compounds (yellow) will have
stronger intramolecular forces with the
polar stationary phase, polar
substances will elute through the
column at a slower rate than the
nonpolar components (blue).
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Figure 3. This image displays the general concept between dipole-dipole interactions. Each oval represents
a molecule at its dipole moment. The red area represents the relatively positive pole of the molecule where
electron density is low. The blue region represents the negative pole with high electron density. The image
shows the positive pole of one molecule forming an attraction to the negative pole of an adjacent molecule.