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Unit 3. Chromatography-I
Chromatography is an old technique derived from the Greek word “chrome” means color and
“graph” means development and it was defined as

“the separation technique used for the separation of different color pigments”.

But later on, this technique was so much developed that even colorless substances can
also be separated.

The new definition is

The separation technique which is capable to separate, identify and quantify the
component of a mixture. Separation is achieved by the difference in migration rates of
different components on the stationary phase under the influence of the mobile phase.

Tswett definition
In 1906 A.D Tswett stated that, a chromatography is a method in which a component of a
mixture is separated on an absorbing column, or a flowing system.
IUPAC Definition
The international Union of Pure and Applied Chemistry (IUPAC) have drafted a recommended
definition of chromatography.
“Chromatography is a physical method of separation in which the components to be separated
are distributed between two phases, one of which is stationary phase while the other (the mobile
phase) moves in a definite direction.”
The stationary phase may be solid, or liquid supported on a solid.
“Chromatography is a technique in which the components of a mixture are separated based on
differences in the rate at which they are carried through a fixed or stationary phase by a gaseous
or liquid mobile phase”.

3.1 Classification of Chromatographic Techniques

Chromatographic processes can be classified according to the type of equilibration process
involved, which is governed by the type of stationary phase. Various bases of equilibration are:
(1) adsorption, (2) partition, (3) ion exchange, and (4) pore penetration.
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Adsorption Chromatography

The chromatography in which stationary phase is a solid on which the sample components are
adsorbed is called adsorption chromatography. The adsorption chromatography in which the
mobile phase may be a liquid is called liquid-solid chromatography and when the mobile phase
is a gas called gas-solid chromatography.
In adsorption chromatography, the components distribute between the two phases through a
combination of sorption and desorption processes. Thin-layer chromatography (TLC) is a special
example of adsorption chromatography in which the stationary phase is a plane, in the form of a
solid supported on an inert plate, and the mobile phase is a liquid.

Partition Chromatography

The chromatography in which stationary phase is a liquid supported on an inert solid is called
partition chromatography. The partition chromatography in which the mobile phase may be a
liquid is called liquid-liquid partition chromatography and when the mobile phase is a gas
called gas-liquid chromatography, GLC).
The liquid-liquid partition chromatography in which a polar stationary phase (e.g., methanol on
silica) is used with a nonpolar mobile phase (e.g., hexane) is call normal-phase
chromatography. This favors retention of polar compounds and elution of nonpolar compounds.
The liquid-liquid partition chromatography, in which a nonpolar stationary phase is used with a
polar mobile phase, is call reversed-phase chromatography. This favors retention of nonpolar
solutes and more polar solutes more readily eluted.

Ion Exchange Chromatography

Ion exchange chromatography uses an ion exchange resin as the stationary phase. The
mechanism of separation is based on ion exchange equilibria. Solutes ions of the opposite charge
are attracted to the stationary phase. The mobile phase is a liquid.

Size Exclusion Chromatograph

In Size Exclusion Chromatography, solvated molecules are separated according to their size
by their ability to penetrate a sievelike structure (the stationary phase).
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(adsorption chromatography) or are coated with a thin layer of liquid phase (partition
chromatography). In gas chromatography, the more common form today is a capillary column in
which microparticles or a liquid are coated on the wall of the capillary tube.

3.2 Principles of Chromatographic Separations

Chromatography operates on the same principle as extraction, but one phase is held in place
while the other moves past it. Figure 3.1 shows a solution containing solutes A and B placed on
top of a column packed with solid particles and filled with solvent. When the outlet is opened,
solutes A and B flow down into the column. Fresh solvent is then applied to the top of the
column and the mixture is washed down the column by continuous solvent flow. If solute A is
more strongly adsorbed than solute B on the solid particles, then solute A spends a smaller
fraction of the time free in solution. Solute A moves down the column more slowly than solute B
and emerges at the bottom after solute B. We have just separated a mixture into its components
by chromatography.

The mobile phase (the solvent moving through the column) in chromatography is either
a liquid or a gas. The stationary phase (the one that stays in place inside the column) is most
commonly a viscous liquid chemically bonded to the inside of a capillary tube or onto the surface
of solid particles packed in the column. Alternatively, as in Figure 3.1, the solid particles
themselves may be the stationary phase. In any case, the partitioning of solutes between mobile
and stationary phases gives rise to separation.

Fluid entering the column is called eluent. Fluid emerging from the end of the column is
called eluate:

The process of passing liquid or gas through a chromatography column is called elution.
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Figure 3.1 The idea behind chromatography: solute A, with a greater affinity than solute B for
the stationary phase, remains on the column longer.

Columns are either packed or open tubular. A packed column is filled with particles of stationary
phase, as in Figure 3.1. An open tubular column is a narrow, hollow capillary with stationary
phase coated on the inside walls.

3.3 Thin Layer Chromatography

Thin layer chromatography (TLC) is a planar form of chromatography useful for wide-scale
qualitative analysis screening and can also be used for quantitative analysis. The separation is
based upon the differences in the relative adsorption rates of the components on the stationary
phase. The stationary phase is a thin layer of finely divided adsorbent supported on a glass or
aluminum plate, or plastic strip.

Stationary phases for TLC

The stationary phase consists of a finely divided powder (particle size 10 to 50 µm). it can be
adsorbent, an ion exchange, or a molecular sieve, or it can serve as the support for a liquid film.
An aqueous slurry of the powder is prepared, usually a binder such as plaster of paris, gypsum,
or poly(vinyl alcohol) is added to help it to adhere to the glass plate. The slurry is spread on the
plate in a thin film, typically 0.1 to 0.3 mm, using a spreading adaptor to assure uniform
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thickness. The solvent is evaporated off and adsorbent are activated by placing in an oven at 110
o
C for several hours. Commercially prepared plates and strips on plastic are available.

Adsorbent

The most commonly used stationary phases are adsorbents. Silica gel, alumina, and powdered
cellulose are the most popular. Silica gel particles contain hydroxyl groups on their surface
which will hydrogen bond with polar molecules. Adsorbed water prevents other polar molecules
from reaching the surface, so the gel is activated by heating to remove the adsorbed water.
Alumina also contains hydroxyl group or oxygen atoms. Alumina is preferred for the separation
of weakly polar compounds, but silica gel is preferred for polar compounds such as amino acid
and sugars. Magnesium silicates, calcium silicates, and activated charcoal may also be used as
adsorbents.

Mobile phases for TLC

The choice of solvent depends upon the nature of sample. In adsorption chromatography, the
eluting power of solvents increases in the order of their polarities. Usually the solvent with
greater polarity as compared with sample is selected. A single solvent or at most two or three
solvent should be used whenever possible.
The developing solvent must be of high purity. The presence of small amounts of water
or other impurities can produce irreproducible chromatograms.
Some common solvent used as a mobile phase are; hexane, acetone, alcohol, water.
Methanol + water, benzene + cyclohexane, benzene + methanol,
Developing the chromatogram

A typical setup for thin layer chromatography is shown in figure 3.2. A thin pencil line is drawn
across the plate a few centimeters from the bottom, and the sample is spotted on this for future
reference in Rf measurements. The spot must be made as small as possible for maximum
separation and minimum tailing. The plate is placed in a chamber with its end dipping in the
developing solvent. A closed chamber must be used to saturate the atmosphere with the solvent
and prevent it from evaporating from the surface of the plate as it moves up. The developing may
take 10 minutes to 1 hour, but it require no operator time.
Usually ascending chromatography is performed in TLC, but other techniques like
descending chromatography and two dimensional are also in use.
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Figure 3.2 Thin layer chromatography setup

Detection of the Spots

If the solutes fluoresce (aromatic compounds), they can be detected by shining an ultraviolet
light on the plate. A pencil line is drawn around the spot for permanent identification. Color-
developing reagents are often used. For example, amino acid and amines are detected by
spraying the plate with a solution of ninhydrin, which is converted to a blue or purple color.
After the spots are identified, they may be scraped off and the solutes washed off (eluted) and
determined quantitatively by a micromethod.
Frequently, colorless or nonfluorescent spot can be visualized by exposing the developed
plate to iodine vapor.

3.4 Ion Exchange Chromatography

Ion exchange chromatography is particularly well suited for the separation of inorganic ions,
both cations and anions, because the separation is based on exchange of ions in the stationary
phase. It has also proved to be extremely useful for the separation of amino acids.
The stationary phase in ion exchange chromatography consists of beads made of
polystyrene polymer cross-linked with divinylbenzene. The cross-linked polymer (resin) has
free phenyl groups attached to the chain, which can easily be treated to add ionic functional
groups. There are two types of the ion exchangers.
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3.4.1 Cation Exchange Resins

These resins contain acidic functional; groups added to the aromatic ring of the resin. The
__
strong-acid cation exchangers have sulfonic acid groups, SO3H, which are strong acid,
__
much like sulfuric acid. The weak-acid cation exchangers have carboxylic acid groups,
CO2H, which are only partially ionized. The protons on these groups can exchange with
other cations:

Where Rz represents the resin. The equilibrium can be shifted to the left or right by
increasing [H+] or [Mn+] or decreasing one with respect to the amount of resin present.

3.4.2 Anion Exchange Resins

Basic groups on the resin in which the hydroxyl anion can be exchanged with other anions
make up the anion exchange resins. There are strong-base groups (quaternary ammonium
groups) and weak-base groups (amine groups). The exchange reaction can be represented by
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Where R represent organic groups, usually methyl.

The strong-base exchangers can be used over the pH range 0 to 12, but the weak base
exchangers only over the range of 0 to 9.

3.4.3 Crosslinkage

The greater the cross linkage of the resin, the greater the difference in selectivities.
Increasing the crosslinkage is expressed by manufacturers as percent of divinylbenzene.
Generally, crosslinkage also increases the rigidity of the resin, reduces swelling, reduces
porosity, and reduces the solubility of the resin. The degree of crosslinkage is expressed
by the manufacturers as percent of divinylbenzene. Generally, crosslinkage of 8 to 10%
is used.

zwitterion (isoelectric point) will pass through both columns, while the positively and
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negatively charged amino acids will each be retained by one of the columns. The groups can
be further subdivided by changing the pH.

3.5 High Performance Liquid Chromatography (HPLC)

High performance liquid chromatography (HPLC) is the most versatile and widely used
type of elution chromatography. The technique is used by chemist to separate and
determine species in a variety of organic, inorganic, and biological materials. In liquid
chromatography, the mobile phase is a liquid solvent containing the sample as a mixture
of solutes.

Principle of HPLC

HPLC uses a tightly packed column containing small particles of the stationary phase. As a
greater surface area of stationary phase is exposed to sample components so the efficiency of
column is increased. The column of HPLC is tightly packed so to obtain a useful flow rate of
liquid mobile phase greater pressure is required through the column. This pressure is
achieved with the help of pumps.
After passing through the pump the mobile phase reaches the injection system where the
sample which is in most cases is the mixture of different components is injected into the
mobile phase. The injection system is positioned just before the start of column. Then sample
enters the column where separation occurs. The column is maintained at constant
temperature. After leaving the column, the components enter the detector. Different kinds of
detectors are used.
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Figure 3.3 Basic components of high performance liquid chromatograph

INSTRUMENTATION
Pumping pressure of 1000 to 3000 psi are required to provide flow rate of 1 to 2 mL/min
in columns of 3 to 5 mm diameters and 10 t0 30 cm long, although in certain instances
pressures up to 6000 psi may be required. Probably 80 to 90% of HPLC separations are
performed with pressure of less than 1200 psi, and even some polyurethane column
materials require very low pressures near atmospheric pressures.

High performance liquid chromatography apparatus consists of following parts:

1. Mobile-Phase Reservoirs and Solvent Treatment System

A modern HPLC apparatus is equipped with one or more glass reservoirs, each of which
contains 500 mL or more of a solvent. The solvent reservoirs can be filled with a range of
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solvents of different polarities, provided they are miscible, or they can be filled with
solutions of different pH and are mixed in the buffer volume. The solvents must be free of
dissolved gases and dust. The gas bubble and dust interfere with the performance of most
detectors. Degassers may consist of a vacuum pumping system, a distillation system, a
device for heating and stirring, or a system for sparging, in which the dissolved gases are
swept out of solution by fine bubbles of an inert gas that is not soluble in the mobile phase.

An elution with a single solvent or a solvent mixture of constant composition is

isocratic elution.
In gradient elution, two (and sometimes more) solvent system that differs significantly in
polarity is used. The ratio of the two solvents is varied in a pre-programmed way during the
separation, sometimes continuously and sometimes in a series of step.
Modern HPLC instruments are often equipped with proportioning valves that introduce
liquid from two or more reservoirs at ratios that can be varied continuously.

2. Pumping System

Due to great resistance offered by small particles size of the column to the flow of mobile
phase pumps are used to get a considerable pressure and flow rate through column. The
pump must fulfill the following requirements
i. Ability to generate pressures of up to 6000 psi.
ii. Capable to deliver a pulse- less flow.
iii. Flow rates ranging from 0.1 to 10 mL/min.
iv. They should be constructed from such material that is inert to solvent.
The most commonly used pump for HPLC is the reciprocating pump. This has a small
cylindrical piston chamber that is alternately filled with mobile phase and emptied via back-
and-forth movement of the piston. This produces a pulsed flow that must be damped.
Reciprocating pumps have a number of advantages;
i. They have a small internal volume, are capable of high output pressures.
ii. They can readily be used for gradient elution.
iii. They provide constant flow rates, independent of solvent viscosity or column
backpressure.
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Other pumps used are motor-driven syringe pumps and pneumatic (constant pressure)
pumps.

3. Sample Injection System

A typical injection system is shown in Figure 3.4. This consists of a stainless steel ring with
six different ports, one to the column. A movable Teflon cone within the ring has three open
segments, each of which connects a pair of external ports. Two of the ports are connected by
an external sample loop of known or fixed volume. In one configuration, the cone permits
direct flow of effluent into the column, and the loop can be filled with the sample. The cone
can then be rotated 30o to make the sample loop part of the moving stream, which sweeps the
sample into the column. Sample of a few microliters can be injected at a pressure up to 6000
psi.
Sample can be introduced manually into the valve with a syringe to fill the sample loop.

Fig.3.4. Sample loop injector

4. Column for HPLC

Liquid chromatographic columns are usually constructed from stainless steel tubing. Most
column range in length from 10 to 30 cm and have inside diameter of 2 to 5 mm. Column
packings typically have particle sizes of 3 to 10 µm. Columns of this type provide 40,000 to
60,000 plates/m. Recently microcolumns have become available with inside diameter of 1 to
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4.6 mm and lengths of 3 to 7.5 cm. These columns, which are packed with 3- or 5-µm
particles, contain as many as 100,000 plates /m and have the advantage of speed and
minimal solvent consumption.

Packing materials for column

The most common packing for liquid chromatography is prepared from silica particles,
alumina particles, porous polymer particles, and ion-exchange resins.

Guard Columns
Often, a short guard column is positioned ahead of the analytical column to increase the life
of the analytical column by removing particulate matter and contaminants from the solvents.
In addition, in liquid-liquid chromatography, the guard column serves to saturate the mobile
phase with the stationary phase so that losses of the stationary phase from the analytical
column are minimized. The composition of the guard-column packing should be similar to
that of the analytical column.

5. Detectors
The detector should be able to recognize when a substance is eluted from the column.
Therefore it has to monitor the change in the mobile phase composition convert this into an
electric signal and then convey it to the recorder.
The ideal detector should be
i. Small and compatible with liquid flow
ii. able to monitor small amounts
Many detectors are used in HPLC but some of the commonly used are:
a. Ultraviolet detector
b. Fluorescence detector
c. Differential Refractive index detector

a. Ultraviolet detector

UV-visible detectors are the most commonly used type of detector due to their sensitivity,
wide linear response rang and ability to monitor many different analytes as they elute
from a HPLC column. Most UV-visible detectors employ a UV-visible light source in
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conjunction with one or more photodiodes and a flow cell through which the eluent
passes, as shown in Figure 3.5.
Optical (photon) absorption detectors may be used in the wavelength ranges of 200-900
nm provided that the eluent possesses chromophores that absorb in the UV, visible, or
near-infrared ranges. It is sensitive to a large number of organic compounds.
Many different monochromatic UV-visible light sources including low pressure mercury
vapor cadmium and zinc lamps can be used for specific application; however, the most
widely sued detectors employ a tungsten lamp in conjunction with a deuterium lamp to
provide a continuous emission spectrum over a 200-900 nm wavelength range. A
monochromatic filter or diffraction grating is then used so that the flow cell and hence the
eluent are illuminated within a narrow wavelength range.

Fig. 3.5. A UV-visible detector for HPL

b. Fluorescence Detector

Some compounds absorb radiation of shorter wavelength and emit the radiation of longer
wavelength upon de-excitation, this phenomena is called fluorescence. Fluorescence
detectors can obviously only be used to monitor fluorescent compounds, yet may in some
instances offer sensitivities 1000 times greater than those obtainable via UV-visible
detection. These detectors measure the intensity and wavelength of the emitted radiations.
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The light of suitable wavelength is passed through cell and emitted radiation is detected
at right angle.
Care must be taken to avoid the unsuitable solvent or dissolved O2 in the solvent because
they quench the fluorescence or show decrease in fluorescence intensity.

c. Differential Refractive index detector

The differential refractive index is often called a “universal detector”. This measure
changes in refractive index of the eluent that result from the presence of solutes as they
emerge from the column. It cannot be used effectively with gradient elution due to a
change in the baseline (a change in the solvent index of refraction as the gradient is
changed) nor when the solvent has an index of refraction close to that of the solutes. This
detector is rugged and will detect concentration of about 10-5 to 10-6 g/mL (10 to 1 ppm).

3.6 High-Performance Partition Chromatography

The most widely used type of HPLC is partition chromatography, in which the
stationary phase is a second liquid that is immiscible with the liquid mobile phase.
Partition chromatography can be subdivided into liquid-liquid and liquid-bonded-phase
chromatography. The difference between the two lies in the way that the stationary phase
is held on the support particles of the packing.
In liquid-liquid partition chromatography, the stationary phase is a solvent that is held
in place by adsorption on the surface of packing particles.
In liquid-bonded-phase partition chromatography, the stationary phase is an organic
species that is attached to the surface of the packing particles by chemical bond.

3.7 High-Performance Adsorption Chromatography

The pioneering work in chromatography was based on adsorption of analyte species on a

solid surface. In adsorption chromatography, the stationary phase is the surface of a
finely divided polar solid. The analyte species are adsorbed onto the surface of a polar
packing.
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Finely divided silica and alumina are the only stationary phase that finds extensive use
in adsorption chromatography.

3.8 Analysis by HPLC

HPLC is widely used for both qualitative as well as quantitative analysis. In case of
qualitative analysis the retention time of component is compared with the reference
standard. In case of quantitative analysis the peak height is measured. The height of the
peak is the measure of the concentration i.e. greater the concentration greater will be the
peak height.

Applications of HPLC
The HPPLC is a versatile separation technique and has developed extensive applications in
nearly every field of analysis. It can be used for organic and inorganic analysis.

The bonded phase partition chromatography is used for

i. Separating soft drink addititives and organophosphate insecticides.
ii. Analysis of pharmaceutical compounds
iii. The separation of amino acids, proteins, carbohydrates, and lipids.
iv. The separation of condensed aromatic, surfactants and dyes.
v. The separation of pesticides, herbicides, phenol, and polychlorinated biphenyls.

Currently, liquid-solid HPLC is extensively used for

i. The separations of relatively nonpolar, water-insoluble organic compounds with
molecular masses that is less than about 5000.
ii. Separation of isomeric mixtures such as meta and para substituted benzene
derivatives.

Dr. Muhammad Naeem Khan

Assistant Professor
Department of Chemistry
Allama Iqbal Open University
Islamabad