SCHX1010 INSTRUMENTAL METHODS OF ANALYSIS UNIT-V

UNIT-V Chromatographic methods

Chromatography, 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 a mobile phase which percolates through the stationary bed.

The history of chromatography begins during the mid-19th century. Chromatography, literally
"color writing", was used—and named— in the first decade of the 20th century, primarily for the
separation of plant pigments such as chlorophyll. New types of chromatography developed
during the 1930s and 1940s made the technique useful for many types of separation process.

Some related techniques were developed during the 19th century (and even before), but the first
true chromatography is usually attributed to Russian botanist Mikhail Semyonovich Tsvet, who
used columns of calcium carbonate for separating plant pigments during the first decade of the
20th century during his research of chlorophyll.

Chromatography became developed substantially as a result of the work of Archer John Porter
Martin and Richard Laurence Millington Synge during the 1940s and 1950s. They established
the principles and basic techniques of partition chromatography, and their work encouraged the
rapid development of several types of chromatography method: paper chromatography, gas
chromatography, and what would become known as high performance liquid chromatography.
Since then, the technology has advanced rapidly. Researchers found that the main principles of
Tsvet's chromatography could be applied in many different ways, resulting in the different
varieties of chromatography described below. Simultaneously, advances continually improved
the technical performance of chromatography, allowing the separation of increasingly similar
molecules.

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.

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. 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.
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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 terms
 The analyte is the substance that is to be separated during chromatography.
 Analytical chromatography is used to determine the existence and possibly also the concentration
of analyte(s) in a sample.
 A bonded phase is a stationary phase that is covalently bonded to the support particles or to the
inside wall of the column tubing.
 A chromatogram is the visual output of the chromatograph. In the case of an optimal separation,
different peaks or patterns on the chromatogram correspond to different components of the
separated mixture.

Plotted on the x-axis is the retention time and plotted on the y-axis a signal (for example obtained
by a spectrophotometer, mass spectrometer or a variety of other detectors) corresponding to the
response created by the analytes exiting the system. In the case of an optimal system the signal is
proportional to the concentration of the specific analyte separated.
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 A chromatograph is equipment that enables a sophisticated separation e.g. gas chromatographic

or liquid chromatographic separation.
 Chromatography is a physical method of separation in which the components to be separated are
distributed between two phases, one of which is stationary (stationary phase) while the other (the
mobile phase) moves in a definite direction.
 The effluent is the mobile phase leaving the column.
 An immobilized phase is a stationary phase which is immobilized on the support particles, or on
the inner wall of the column tubing.
 The mobile phase is the phase which moves in a definite direction. It may be a liquid (LC and
CEC), a gas (GC), or a supercritical fluid (supercritical-fluid chromatography, SFC). A better
definition: The mobile phase consists of the sample being separated/analyzed and the solvent that
moves the sample through the column. In one case of HPLC the solvent consists of a
carbonate/bicarbonate solution and the sample is the anions being separated. The mobile phase
moves through the chromatography column (the stationary phase) where the sample interacts with
the stationary phase and is separated.
 Preparative chromatography is used to purify sufficient quantities of a substance for further use,
rather than analysis.
 The retention time is the characteristic time it takes for a particular analyte to pass through the
system (from the column inlet to the detector) under set conditions. See also: Kovat's retention
index
 The sample is the matter analysed in chromatography. It may consist of a single component or it
may be a mixture of components. When the sample is treated in the course of an analysis, the
phase or the phases containing the analytes of interest is/are referred to as the sample whereas
everything out of interest separated from the sample before or in the course of the analysis is
referred to as waste.
 The solute refers to the sample components in partition chromatography.
 The solvent refers to any substance capable of solubilizing other substance, and especially the
liquid mobile phase in LC.

The stationary phase is the substance which is fixed in place for the chromatography procedure. Examples
include the silica layer in Chromatography ,Thin layer chromatography.
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Chromatography - Equipment

Fig:5.1 Chromatography equipment

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
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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

Fig:5.2 Liquid Chromatography

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).
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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.
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Fig:5.3 Column chromatography

Chromatography - The Chromatogram

Fig: 5.4 Chromatogram.

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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.

Gas Chromatography
Gas chromatography makes use of a pressurized gas cylinder and a carrier gas, such as helium, to
carry the solute through the column. The most common detectors used in this type of
chromatography are thermal conductivity and flame ionization detectors. There are three types of
gas chromatography that will be discussed here: gas adsorption, gas-liquid and capillary gas
chromatography.

Gas adsorption chromatography involves a packed bed comprised of an adsorbent used as the
stationary phase. Common adsorbents are zeolite, silica gel and activated alumina. This method
is commonly used to separate mixtures of gases.

Gas-liquid chromatography is a more common type of analytical gas chromatography. In this

type of column, an inert porous solid is coated with a viscous liquid which acts as the stationary
phase. Diatomaceous earth is the most common solid used. Solutes in the feed stream dissolve
into the liquid phase and eventually vaporize. The separation is thus based on relative volatilities.

Capillary gas chromatography is the most common analytical method. Glass or fused silica
comprise the capillary walls which are coated with an absorbent or other solvent. Because of the
small amount of stationary phase, the column can contain only a limited capacity. However, this
method also yields rapid separation of mixtures.

Liquid Chromatography

There are a variety of types of liquid chromatography. There is liquid adsorption

chromatography in which an adsorbent is used. This method is used in large-scale applications
since adsorbents are relatively inexpensive. There is also liquid- liquid chromatography which is
analogous to gas-liquid chromatography. The three types that will be considered here fall under
the category of modern liquid chromatography. They are reverse phase, high performance and
size exclusion liquid chromatography, along with supercritical fluid chromatography.

Reverse phase chromatography is a powerful analytical tool and involves a hydrophobic, low
polarity stationary phase which is chemically bonded to an inert solid such as silica. The
separation is essentially an extraction operation and is useful for separating non-volatile
components.
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High performance liquid chromatography (HPLC) is similar to reverse phase, only in this
method, the process is conducted at a high velocity and pressure drop. The column is shorter and
has a small diameter, but it is equivalent to possessing a large number of equilibrium stages.

Size exclusion chromatography, also known as gel permeation or filtration chromatography does
not involve any adsorption and is extremely fast. The packing is a porous gel, and is capable of
separating large molecules from smaller ones. The larger molecules elute first since they cannot
penetrate the pores. This method is common in protein separation and purification.

Supercritical fluid chromatography is a relatively new analytical tool. In this method, the carrier
is a supercritical fluid, such as carbon dioxide mixed with a modifier. Compared to liquids,
supercritical fluids have solubilities and densities have as large, and they have diffusivities and
viscosities quite a bit larger. This type of chromatography has not yet been implemented on a
large scale

Ion Exchange Chromatography

Ion exchange chromatography is commonly used in the purification of biological materials.

There are two types of exchange: cation exchange in which the stationary phase carries a
negative charge, and anion exchange in which the stationary phase carries a positive charge.
Charged molecules in the liquid phase pass through the column until a binding site in the
stationary phase appears. The molecule will not elute from the column until a solution of varying
pH or ionic strength is passed through it. Separation by this method is highly selective. Since the
resins are fairly inexpensive and high capacities can be used, this method of separation is applied
early in the overall process.

Affinity Chromatography
Affinity chromatography involves the use of packing which has been chemically modified by
attaching a compound with a specific affinity for the desired molecules, primarily biological
compounds. The packing material used, called the affinity matrix, must be inert and easily
modified. Agarose is the most common substance used, in spite of its cost. The ligands, or
"affinity tails", that are inserted into the matrix can be genetically engineered to possess a
specific affinity. In a process similar to ion exchange chromatography, the desired molecules
adsorb to the ligands on the matrix until a solution of high salt concentration is passed through
the column. This causes desorption of the molecules from the ligands, and they elute from the
column. Fouling of the matrix can occur when a large number of impurities are present,
therefore, this type of chromatography is usually implemented late in the process.
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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.
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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.
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Fig:5.5 Principle of chromatography

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