Chromatography

What Is It??
Hello, everyone, and welcome to the wonderful world of
chromatography! What is chromatography, you ask?? Well, quite simply, it is a
broad range of physical methods used to separate and or to analyze complex
mixtures. The components to be separated are distributed between two phases:
a stationary phase bed and amobile phase which percolates through the
stationary bed.

How Does It Work? Like Magic!

A mixture of various components enters a chromatography process, and the
different components are flushed through the system at different rates. These
differential rates of migration as the mixture moves over adsorptive materials
provide separation. Repeated sorption/desorption acts that take place during the
movement of the sample over the stationary bed determine the rates. The
smaller the affinity a molecule has for the stationary phase, the shorter the time
spent in a column.

So, Why Is It So Special?

In any chemical or bioprocessing industry, the need to separate and purify
a product from a complex mixture is a necessary and important step in
the production line. Today, there exists a wide market of methods in
which industries can accomplish these goals. Chromatography is
a very special separation process for a multitude of reasons! First of all,
it can separate complex mixtures with great precision. Even very
similar components, such as proteins that may only vary by a single
amino acid, can be separated with chromatography. In fact,
chromatography can purify basically any soluble or volatile substance
if the right adsorbent material, carrier fluid, and operating conditions
are employed. Second, chromatography can be used to separate
delicate products since the conditions under which it is performed are
not typically severe. For these reasons, chromatography is quite well
 suited to a variety of uses in the field of biotechnology, such as
separating mixtures of proteins.

Chromatography - Equipment
The figure below will help you to follow along in our discussion.

The Column
Although there are other types of chromatography (e.g. paper and thin layer),
most modern applications of chromatography employ a column. The column is
where the actual separation takes place. It is usually a glass or metal tube of
sufficient strength to withstand the pressures that may be applied across it. The
column contains the stationary phase. The mobile phase runs through the
column and is adsorbed onto the stationary phase. The column can either be
a packed bed or open tubular column.

Packed Bed Column

A packed bed column is comprised of a stationary phase which is in

granular form and packed into the column as a homogeneous bed. The
stationary phase completely fills the column.
 Open Tubular Column

An open tubular column's stationary phase is a thin film or layer on the

column wall. There is a pasageway through the center of the column.

The Mobile and Stationary Phases

The mobile phase is comprised of a solvent into which the sample is injected.
The solvent and sample flow through the column together; thus the mobile
phase is often referred to as the "carrier fluid." The stationary phase is the
material in the column for which the components to be separated have varying
affinities. The materials which comprise the mobile and stationary phases vary
depending on the general type of chromatographic process being performed.

Gas Chromatography

The mobile phase in gas chromatography is generally an inert gas. The

stationary phase is generally an adsorbent or liquid distributed over the
surface of a porous, inert support.

Liquid Chromatography

The mobile phase in liquid chromatography is a liquid of low viscosity

which flows through the stationary phase bed. This bed may be
comprised of an immiscible liquid coated onto a porous support, a thin
film of liquid phase bonded to the surface of a sorbent, or a sorbent of
controlled pore size.

Chromatography - Basic Operation

Hey there! We have more interesting information about chromatography! Let's
see what actually takes place in a chromatographic separation.

You can skip this review paragraph. The process of a chromatographic

separation takes place within a chromatography column. This column, made of
glass or metal, is either a packed bed or open tubular column. A packed bed
column contains particles which make up the stationary phase. Open tubular
columns are lined with a thin film stationary phase. The center of the column is
hollow. The mobile phase is typically a solvent moving through the column
which carries the mixture to be separated. This can either be a liquid or a gas,
depending on the type of process. The stationary phase is usually a viscous
liquid coated on the surface of solid particles which are packed into the column
as discussed above, although the solid particles can also be taken as the
stationary phase. In any case, the partitioning of solutes between the stationary
and mobile phases lead to the desired separations.

Let's see a different sketch of the process:

1. Feed Injection
The feed is injected into the mobile phase. The mobile phase flows through the
system by the action of a pump (older analytical chromatorgraphy used
capillary action or gravity to move the mobile phase).

2. Separation in the Column

As the sample flows through the column, its different components will adsorb
to the stationary phase to varying degrees. Those with strong attraction to the
support move more slowly than those with weak attraction. This is how the
components are separated.

3. Elution from the Column After the sample is flushed

or displaced from the stationary phase, the different
 components will elute from the column at different
times. The components with the least affinity for the
stationary phase (the most weakly adsorbed) will
elute first, while those with the greatest affinity for
the stationary phase (the most strongly adsorbed)
will elute last.
4. Detection
The different components are collected as they emerge from the column. A
detector analyzes the emerging stream by measuring a property which is related
to concentration and characteristic of chemical composition. For example, the
refractive index or ultra-violet absorbence is measured.

Example
The figure below shows a simple separation by chromatography. A continuous
flow of solvent carries a solution of solutes A and B down a column. (a) As the
solvent carries the two solutes down the column, we begin to see some
separation of the solution. (b) At some later point in time, it can be seen that
solute B is moving at a much faster rate than A. (c) In (d), solute B emerges
first, while solute A finally emerges in (e). Thus, solute A has a greater affinity
for the stationary phase than solute B. By varying the pH of the solvent or
temperature of the column, the output of the column can be significantly
altered, such as the timing of when individual species emerge.
Chromatography - The Chromatogram
Let's consider the output from the detector: the chromatogram. The figure
below will help you to follow along in our discussion.

Since the sample is separated in the column, different peaks on the

chromatogram correspond to different components in the sample mixture. The
chromatograms above show the results of separations of protein mixtures by
ion exchange chromatography. The lettered peaks correspond to different
proteins (A = ovalbumin, B = conalbumin, C = cytochrome c, D = lysozyme).
The separation corresponding to the chromatogram on the left was performed at
pH 5.85, while the one on the right was performed at pH 6.5. It is evident that
operation conditions such as pH and temperature have a significant effect on
the output.

What Information Can Be Attained?

• The level of complexity of the sample is indicated by the number of
peaks which appear.
• Qualitative information about the sample composition is obtained by
comparing peak positions with those of standards.
• Quantitative assessment of the relative concentrations of components is
obtained from peak area comparisons.
• Column performance is indicated by comparison with standards.

Types of Chromatography

Adsorption Chromatography

Adsorption chromatography is probably one of the

oldest types of chromatography around. It utilizes a
mobile liquid or gaseous phase that is adsorbed onto
the surface of a stationary solid phase. The
equilibriation between the mobile and stationary
phase accounts for the separation of different solutes.
 Partition Chromatography

This form of chromatography is based on a

thin film formed on the surface of a solid
support by a liquid stationary phase. Solute
equilibriates between the mobile phase and
the stationary liquid.

Ion Exchange Chromatography

In this type of chromatography, the use of a

resin (the stationary solid phase) is used to
covalently attach anions or cations onto it.
Solute ions of the opposite charge in the
mobile liquid phase are attracted to the resin
by electrostatic forces.

Molecular Exclusion Chromatography

Also known as gel permeation or gel

filtration, this type of chromatography
lacks an attractive interaction between
the stationary phase and solute. The
liquid or gaseous phase passes through a
porous gel which separates the
molecules according to its size. The
pores are normally small and exclude the
larger solute molecules, but allows
smaller molecules to enter the gel,
causing them to flow through a larger
volume. This causes the larger molecules
to pass through the column at a faster
rate than the smaller ones.
 Affinity
Chromatography

This is the most

selective type of
chromatography
employed. It
utilizes the specific
interaction between
one kind of solute
molecule and a
second molecule
that is immobilized
on a stationary
phase. For
example, the
immobilized
molecule may be
an antibody to
some specific
protein. When
solute containing a
mixture of proteins
are passed by this
molecule, only the
specific protein is
reacted to this
antibody, binding it
to the stationary
phase. This protein
is later extracted by
changing the ionic
strength or pH.