Chromatography Technique under CBPBU UG syllabus

1. Dr. Santanu Chakravorty
CHEMISTRY-DSE: ANALYTICAL METHODS IN
CHEMISTRY
Dr. Santanu Chakravorty
Assistant Professor
Mathabhanga College
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2. Dr. Santanu Chakravorty
Syllabus
Chromatography: Classification, principle and efficiency of the
technique. Mechanism of separation: adsorption, partition & ion
exchange. Development of chromatograms: frontal, elution and
displacement methods. Qualitative and quantitative aspects of
chromatographic methods of analysis: IC, GLC, GPC, TLC and HPLC
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3. Dr. Santanu Chakravorty
Principles of Chromatography
Chromatography now refers to any of a diverse group of techniques that effect a
separation through a distribution of sample between two immiscible phases. The
requirement to distinguish chromatography from other separation technique is
that one phase be stationary while the second phase be mobile and percolating
through the first phase resulting in selective retention of the components of a
mixture by the stationary phase. The stationary phase which may be solid or liquid
or may consists of a mixture of a solid and liquid , is finely divided and is fixed in
place. The mobile phase, which may be liquid or gaseous, fills the interstices of the
stationary phase and is able to flow through the stationary phase. The physical
states of the mobile and stationary phases give rise to four basic type of
chromatography
i. liquid-solid chromatography, (LSC) ii. Liquid-liquid chromatography, (LLC) iii. Gas-
liquid chromatography, (GLC) iv. Gas-solid chromatography (GSC)
Solid stationary phase also gives rise to low exchange chromatography. Separation
of the components, or solutes of a sample results from differences in their rate of
adsorption.
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4. Dr. Santanu Chakravorty
Classification
• The purpose of applying chromatography which is used as a method of quantitative analysis
apart from its separation, is to achieve a satisfactory separation within a suitable time
interval. Various chromatography methods have been developed to that end. Some of them
include column chromatography, thin-layer chromatography (TLC), paper chromatography,
gas chromatography, ion exchange chromatography, gel permeation chromatography, high-
pressure liquid chromatography, and affinity chromatography.
Types of chromatography
• Column chromatography
• Ion-exchange chromatography
• Gel-permeation (molecular sieve) chromatography
• Affinity chromatography
• Paper chromatography
• Thin-layer chromatography
• Gas chromatography
• Dye-ligand chromatography
• Hydrophobic interaction chromatography
• Pseudoaffinity chromatography
• High-pressure liquid chromatography (HPLC)
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5. Dr. Santanu Chakravorty
Efficiency of Chromatography techniques
Chromatographic methods will separate ionic species, inorganic or organic, and molecular
species ranging in size from the lightest and smallest, helium and hydrogen, to particulate
matter such as single cells . No single configuration will accomplish this, however. Little
preknowledge of the constituent of a mixture is required. At its best, chromatography will
separate several hundreds of components of unknown identity and unknown concentrations,
leaving the components unchanged. Amounts in the parts per billion range can be detected
with some detectors. The solute can range from polar to nonpolar—i.e., water-soluble to
hydrocarbon-soluble.
• Substances of low critical temperature or low molecular weight, such as the gases at
laboratory conditions showing dispersive or London intermolecular forces only, are separated
with molecular sieves or gas-solid techniques. Gas-liquid chromatography is applicable to
species with high critical temperatures and normal boiling points as high as 400 °C.
Substances that are solids at normal laboratory conditions with molecular weights below
1,000 are best separated with liquid-solid or liquid-liquid systems. Lower members of the
molecular weight scale range are amenable to supercritical fluid separations. Size-
exclusion methods are involved at molecular weights above 1,000. Field-flow fractionation
extends the size range to colloids and microscopic particles.
• Separations are fast, ranging from analysis times of a few minutes to several hours. The
prechromatographic world would have considered a time of several hours to separate
multicomponent mixtures to be miraculously fast. Now several hours is considered excessive,
and there is much emphasis on increasing speed.
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6. Dr. Santanu Chakravorty
Adsorption Chromatography
Adsorption chromatography is a type of LC in which chemicals are retained based on their adsorption and
desorption at the surface of the support, which also acts as the stationary phase. This method is also
sometimes referred to as liquid-solid chromatography. Retention in this method is based on the
competition of the analyte with molecules of the mobile phase as both bind to the surface of the support.
The degree of a chemical's retention in adsorption chromatography will depend on (1) the binding
strength of this chemical to the support, (2) the surface area of the support, (3) the amount of mobile
phase displaced from the support by the chemical, and (4) the binding strength of the mobile phase to the
support. Electrostatic interactions, hydrogen bonding, dipole-dipole interactions, and dispersive
interactions (ie, van der Waals forces) all may affect retention in this type of chromatography. The binding
strength of the mobile phase with the support in adsorption chromatography is described by the mobile
phase's elutropic strength. A liquid or solution that has a large elutropic strength for a given support will
act as a strong mobile phase for that material because this mobile phase will tend to bind tightly to the
support and cause the analyte to elute more quickly as it spends more time in the mobile phase. As an
example, a relatively polar solvent such as methanol will have higher elutropic strength for a polar support
such as silica than a nonpolar solvent such as carbon tetrachloride. In the same manner, a liquid or
solution that has a low elutropic strength for a support would represent a weak mobile phase for that
support in adsorption chromatography (eg, carbon tetrachloride on silica). Three types of adsorbents are
generally used in adsorption chromatography: (1) polar acidic supports, (2) polar basic supports, and (3)
nonpolar supports. The most common polar and acidic support used in adsorption chromatography is
silica. The surface silanol groups on this support tend to adsorb polar compounds and work particularly
well for basic substances. Alumina is the main type of polar and basic adsorbent that is used in adsorption
chromatography. Like silica, alumina retains polar compounds, but alumina works especially well for polar
acidic substances. Other types of supports that can be used in adsorption chromatography are nonpolar
adsorbents such as charcoal and polystyrene.
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7. Dr. Santanu Chakravorty
Column Chromatography
Column chromatography in chemistry is a chromatography method used to isolate
a single chemical compound from a mixture. Chromatography is able to separate
substances based on differential adsorption of compounds to the adsorbent.
Compounds move through the column at different rates, allowing them to be
separated in fractions. The technique is widely applicable, as many different
adsorbents can be used with a wide range of solvents. The technique can be used
on scales from micrograms up to kilograms. The main advantage of column
chromatography is the relatively low cost and disposability of the stationary phase
used in the process. The latter prevents cross contamination and stationary phase
degradation due to recycling column chromatography can be done using gravity to
move solvent, or using compressed gas to push the solvent through the column.
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•In column chromatography the stationary phase is packed into a glass or metal column.
•The mixture of analytes is then applied and the mobile phase, commonly referred to as the
eluent, is passed through the column either by use of a pumping system or applied gas
pressure.
•The stationary phase is either coated onto discrete small particles (the matrix) and packed
into the column or applied as a thin film to the inside wall of the column.
•As the eluent flows through the column the analytes separate on the basis of their
distribution coefficients and emerge individually in the eluate as it leaves the column.

8. Dr. Santanu Chakravorty
Column Chromatography Technique
• Column chromatography is a technique in which the substances to be separated
are introduced onto the top of a column packed with an adsorbent, passed
through the column at different rates that depend on the affinity of each
substance for the adsorbent and for the solvent or solvent mixture, and are usually
collected in solution as they pass from the column at different times.
• It is a solid-liquid technique in which the stationary phase is a solid & the mobile
phase is a liquid or gas.
• It was developed by the American chemist D.T Day in 1900 while M.S. Tswett, the
Polish botanist, 1906 used adsorption columns in his investigations of plant
pigments.
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9. Dr. Santanu Chakravorty
Elution, Eluent and Rf value
In a liquid chromatography experiment for example an analyte is generally
adsorbed, or bound to an adsorbent in a liquid chromatography column. The
adsorbent, a solid phase, is a powder which is coated onto a solid support. Based
on an adsorbents composition, it can have varying affinities to hold onto other
molecules-forming a thin film on its surface. Elution then is the process of
removing analytes from the adsorbent by running a solvents, called an eluent pass
the adsorbent/ analyte complex.
The eluent is the carrier portion of the mobile phase. It moves the analytes
through the chromatograph. In liquid chromatography, the eluent is the liquid
solvent, in gas chromatography, it is carrier gas.
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10. Dr. Santanu Chakravorty
Elution, Eluent and Rf value
Rf is the retardation factor, which is the ratio of the distance traveled by a
compound in a mobile phase compared with the distance traveled by the front of
the mobile phase itself. It is always greater than or equal to zero, and less than
equal to 1.
• A chromatogram is first to be developed with a suitable solvent (mobile phase),
depending upon the nature of analytes, and the stationary phases. The developed
chromatogram is then dried and the positions (migration values) are measured for
analytes and the solvent front. The Rf (retardation/retention factor) values can be
calculated by using the given procedure using the above experiment.
• A prepared sample solution (A+B) is applied on the chromatogram paper and run
through a mobile phase. Analyte (A) and (B) separate out because of different
affinities with mobile phase (solvent). The relative measurements are taken for the
analytes, the solvent front, and the point where the mixture (A+B) was applied.
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11. Dr. Santanu Chakravorty
Rf value calculation
• For the analyte (A)
• Rf = Distance moved by analyte (A) / Distance moved by solvent front
Rf = 2.9 / 4.0 = 0.725
• For the analyte (B)
• Rf = Distance moved by analyte (B) / Distance moved by solvent front
Rf = 1.3 / 4.0 = 0.325
• So, the Rf values for the analytes (A) and (B) are 0.725 and 0.325
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12. Dr. Santanu Chakravorty
Thin layer Chromatography
Different compounds in the sample mixture travel at different rates due to the
differences in their attraction to the stationary phase and because of differences in
solubility in the solvent. By changing the solvents, or perhaps using a mixture, the
separation of compounds can be adjusted. Chemists often use TLC to develop a
protocol for separation by chromatography and use TLC to determine which
fraction contain the desired compounds. Separation of compound is based on the
competition of the solute and the mobile phase for binding sites on the stationary
phase. For instances, if normal phase silica gel is used as the stationary phase, it
can be considered polar. Given two compounds that differ polarity, the more polar
compound has a stronger interaction with the silica and is, therefore, better able
to displace the mobile phase from the available binding sites. As a consequence,
the less polar compound moves higher up the plate.
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13. Dr. Santanu Chakravorty
TLC Technique
• Thin layer chromatography technique used to separate non-volatile mixtures. This-
layer chromatography is performed on a sheet of glass, plastic or aluminum foil,
which is coated with a thin layer of adsorbent material, usually silica gel, aluminum
oxide. This layer of adsorbent is known as the stationary phase.
• After the sample has been applied on the plate, a solvent or solvent mixture is
drawn up the plate via capillary action. Because different analytes ascend the TLC
plate at different rates, separation is achieved. For example, with silica gel, a very
polar substances, non-polar mobile phase such as heptane is used.
• After the experiment, the spots are visualized. Often this can be done simply by
projecting ultraviolet light onto the sheet; the sheets are treated with a phosphor,
and dark spots appear on the sheets were compounds absorb light impinging on a
certain area.
• To quantify the results, the distance traveled by the substance being considered is
divided by the total distance traveled by the mobile phase. This ratio is called
retardation factor. In general, a substance whose structure resembles the
stationary phase will have low Rf, while one that has a similar structure to the
mobile phase will have high retardation factor.
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14. Dr. Santanu Chakravorty
Difference between column chromatography
and thin layer chromatography
The main differences are:
• TLC has a stationary phase of alumina or silica gel. Column Chromatography is
packed uses its stationary phase with an appropriate matrix material, such as silica.
• TLC is carried for the against gravity. Column Chromatography is run under gravity.
• TLC uses for the analytical purpose. Column Chromatography uses for the
preparative purpose.
• Column Chromatography take more time to separate than TLC.
• TLC needs less quantity of solvent to separate the analytes . Column
chromatography required more amount of solvent.
• TLC needs more polar solvent compared to the column chromatography.
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15. Dr. Santanu Chakravorty
Partition Chromatography
Partition Chromatography Principle
• In partition chromatography, the separation of the components from the sample
takes place through the process of partition the components between two phases,
where both the phases are present in liquid form. In this procedure, the
immiscible solid surface that is covered with the liquid surface on the stationary
phase is in the mobile phase.
• The stationary phase immobilizes the liquid surface which ultimately changes into
a stationary phase. The components are separated just after the mobile phase
shifts from the stationary phase. The separation is because of the differences in
partition coefficients.
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16. Dr. Santanu Chakravorty
Technique
• For a simple comprehension of the partition chromatography, here we are explaining the procedure
for conducting paper chromatography.
Apparatus required:
• Chromatography jar, Capillary tube, Stationary phase
• Mobile phase – Chloroform, methanol, acetone, or ethanol
Steps:
• Selecting an appropriate type of development – Based on solvent, mixture, paper complexity, etc.
the type of development is selected. Usually ascending paper chromatography is utilized as it is a
simple technique to perform and also the chromatograms are quickly acquired.
• Choosing a stationary phase – Based on pores size and quality of the analyte, filter paper
(stationary phase) is chosen.
• Preparing the sample – The sample is prepared by dissolving the component in a proper solvent
that is employed during the procedure of producing mobile phases.
• Spotting the sample on paper – The sample mixture is applied on the filter paper at an appropriate
position.
• Developing the chromatogram – With the help of a chromatographic jar, the chromatogram
development is determined by drenching the paper into the solvent or mobile phase. Through the
capillary action, the mobile phase is allowed to run over the test sample.
• Paper drying and identifying the compounds – After the development of the chromatograms, the
filter paper is dried with the aid of an air dryer. Paper possessing different bands of molecules can
be studied in the UV cabinet. Rf values are also figured out.
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17. Dr. Santanu Chakravorty
Ion Exchange Chromatography
• IEC is a type of liquid chromatography in which ions are separated by their adsorption onto a
support that contains fixed charges on its surface. This method relies on the interaction (or
exchange) of ions in the sample or mobile phase with fixed ionic groups of the opposite
charge that are bound to the support and act as the stationary phase. Depending on the
charge of the groups that make up the stationary phase, the types of ions that bind to the
column may be either cations (ie, positively charged ions) or anions (ie, negatively charged
ions). These two methods are referred to as cation-exchange chromatography and anion-
exchange chromatography, respectively. Supports for cation-exchange chromatography
contain negatively charged functional groups. These groups may be the conjugate bases of
strong acids, or the conjugate bases of weak acids. The supports used in anion-exchange
chromatography are usually the conjugate acids of strongly basic quaternary amines, or the
conjugate acids of weak bases.
• A strong mobile phase in IEC is usually a mobile phase that contains a high concentration of
competing ions. The presence of these competing ions will make it more difficult for a
charged analyte to bind to the fixed charges that act as the stationary phase. A weak mobile
phase in IEC is one that contains few or no competing ions or that otherwise promotes
binding by charged analytes to the column. Changing the competing ion concentration is the
most common approach for adjusting the retention of analyte ions in IEC. The retention of
ions in this method also may be affected by (1) pH, (2) the type of competing ion used, (3)
the type of fixed charges used as the stationary phase, and (4) the density of these fixed
charges on the support.
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18. Dr. Santanu Chakravorty
Ion Exchange Chromatography Technique
Key steps in the ion exchange chromatography procedure are listed below:
• An impure protein sample is loaded into the ion exchange chromatography column
at a particular pH.
• Charged proteins will bind to the oppositely charged functional groups in the resin
• A salt gradient is used to elute separated proteins. At low salt concentrations,
proteins having few charged groups are eluted and at higher salt concentrations,
proteins with several charged groups are eluted.
• Unwanted proteins and impurities are removed by washing the column.
• A pH gradient can also be applied to elute individual proteins on the basis of their
isoelectric point (pI) i.e. the point at which the amino acids in a protein carry
neutral charge and hence do not migrate in an electric field. As amino acids are
zwitter ionic compounds they contain groups having both positive and negative
charges. Based on the pH of the environment, proteins carry a positive, negative,
or nil charge. At their isoelectric point, they will not interact with the charged
moieties in the column resin and hence are eluted. A decreasing pH gradient can
be used to elute proteins using an anion exchange resin and an increasing pH
gradient can be used to elute proteins from cation exchange resins.
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19. Dr. Santanu Chakravorty
Gas-Liquid Chromatography
• GLC is a process by which a mixture is separated into its constituents by a moving
gas passing over a liquid stationary phase. Separation of the mixture into its
constituents occur by partitioning the sample between a mobile gas phase (known
as carrier gas) and a thin layer of non-volatile liquid coated on an inert support.
• A gas-liquid chromatograph consists of the following parts: A supply of carrier gas
from a high pressure cylinder, sample injection system and flow controller, the
column and the detector
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The sample is introduced as a gas at the head of the column. As a result,
components having finite solubility in the stationary liquid phase distributed
themselves between this phase and the mobile gas phase, in accordance with

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Gas-Liquid Chromatography
equilibrium law. Elution is the carried out by forcing an inert gas (eg He, N2, H2 or
Ar) through the column. The rate of movement of the various component along
the column depends upon their tending to dissolve in the stationary liquid phase.
Components having negligible solubility in the stationary phase move more rapidly
through the column, while the components whose distribution coefficient favors
the solvent liquid phase move slowly through the column.
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Typical chart record peaks corresponds to individual components detected

21. Dr. Santanu Chakravorty
Difference between GLC and Column
Chromatography
Gas chromatography is in principle similar to column chromatography, but has
several notable differences. First, the process of separating the compounds in a
mixture is carried out between a liquid-stationary phase and a gas mobile phase,
whereas in column chromatography the stationary phase is solid and mobile phase
is a liquid. Second, the column through which the gas phase passes is located in an
oven where the temperature of the gas can be controlled, whereas column
chromatography has no such temperature control. Finally the concentration of a
compound in the gas phase is solely function of the vapor pressure of the gas.
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22. Dr. Santanu Chakravorty
High Performance liquid Chromatography
(HPLC)
The separation principle of HPLC is based on the distribution of the analyte
(sample) between a mobile phase (eluent) and a stationary phase (packing
material of the column). Depending on the chemical structure of the analyte, the
molecules are retarded while passing the stationary phase. The specific
intermolecular interactions between the molecules of a sample and the packing
material define their time “on-column”. Hence, different constituents of a sample
are eluted at different times. Thereby, the separation of the sample ingredients is
achieved. A detection unit (e.g. UV detector) recognizes the analytes after leaving
the column. The signals are converted and recorded by a data management system
(computer software) and then shown in a chromatogram. After passing the
detector unit, the mobile phase can be subjected to additional detector units, a
fraction collection unit or to the waste. In general, a HPLC system contains the
following modules: a solvent reservoir, a pump, an injection valve, a column, a
detector unit and a data processing unit. The solvent (eluent) is delivered by the
pump at high pressure and constant speed through the system. To keep the drift
and noise of the detector signal as low as possible, a constant and pulseless flow
from the pump is crucial. The analyte (sample) is provided to the eluent by the
injection valve.
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