UNIT IV SEPARATION METHODS

General description of chromatography – Band broadening and optimization of column

performance- Liquid chromatography – Partition chromatography – Adsorption chromatography
– Ion exchange chromatography -size exclusion chromatography- Affinity chromatography-
principles of GC and applications – HPLC- Capillary electrophoresis – Applications.

4.1 General description of chromatography

• Chromatography comprises a group of powerful separation methods that find applications in
all branches of science.
• Chromatography was invented and named by the Russian botanist Mikhail Tswett.
• He used the technique to separate various plant pigments such as chlorophylls and
xanthophylls by passing solutions of these compounds through a glass column packed with
finely divided calcium carbonate.
• The separated species appeared as colored bands on the column, which accounts for the name
he chose for the method (Greek chroma meaning “color” and graphein meaning “writing”).
• Chromatography allows the separation, identification, and determination of closely related
components of complex mixtures;
• In all chromatographic separations the sample is dissolved in a mobile phase, which may be a
gas, a liquid, or a supercritical fluid.
• This mobile phase is then forced through an immiscible stationary phase, which is fixed in
place in a column or on a solid surface.
• The two phases are chosen so that the components of the sample distribute themselves
between the mobile and stationary phases to varying degrees.
• Those components strongly retained by the stationary phase move only slowly with the flow
of mobile phase.
• In contrast, components that are weakly held by the stationary phase travel rapidly.
• Because of these differences in migration rates, sample components separate into discrete
bands, or zones, that can be analyzed qualitatively and quantitatively.
Classification of Chromatographic Methods:
Chromatographic methods can be categorized in two ways:
 Column chromatography
 Planar chromatography
• In column chromatography, the stationary phase is held in a narrow tube through which
the mobile phase is forced under pressure.
• In planar chromatography, the stationary phase is supported on a flat plate or in the
interstices of paper. In this arrangement, the mobile phase moves through the stationary
phase by capillary action or under the influence of gravity.
• Another classification of chromatographic methods is one based on the types of mobile
and stationary phases and the kinds of equilibria involved in the transfer of solutes
between phases.
 • Three general categories of chromatography are:
• gas chromatography (GC),
• liquid chromatography (LC), and
• supercritical fluid chromatography (SFC).
• As the names imply, the mobile phases in the three techniques are gases, liquids, and
supercritical fluids, respectively.
• Only LC can be performed either in columns or on planar surfaces;
• GC and SFC, on the other hand, are restricted to column procedures so that the column
walls enclose the mobile phase.
Elution in Column Chromatography:
• Figure below shows schematically how two substances A and B are separated on a
packed column by elution.
• The column consists of a narrow-bore tubing packed with a finely divided inert solid that
holds the stationary phase on its surface.
• The mobile phase occupies the open spaces between the particles of the packing.
• Initially, a solution of the sample containing a mixture of A and B in the mobile phase is
introduced at the head of the column as a narrow plug, as shown in Figure at time t0.
• The two components distribute themselves between the mobile and the stationary phases
as fresh mobile phase is added continuously and A and B are washed through the column.
• This process is called elution.

Figure (a) Diagram showing the separation of a mixture of components A and B by column
elution chromatography. (b) The output of the signal detector at the various stages of elution
shown in (a).
 • In modern chromatography, continuous addition is accomplished by means of a pump in
LC and SFC or by the application of pressure in GC.
Working:
• With the first introduction of fresh mobile phase, the eluent— the portion of the sample
contained in the mobile phase—moves down the column, where it further distributes
between the mobile phase and the stationary phase (time t1).
• Interaction of the solute with the fresh mobile phase and the stationary phase takes place
as the mobile phase washes the sample through the column.
• As fresh mobile phase flows through the column, it carries solute molecules down the
column in a continuous series of interactions with the two phases
• Because solute movement can occur only in the mobile phase, the average rate at which a
solute zone migrates down the column depends on the fraction of time it spends in the
mobile phase.
• This fraction is small for solutes strongly retained by the stationary phase (component B)
and large when the solute resides mostly in the mobile phase (component A).
• Ideally, the resulting differences in rates cause the components in a mixture to separate
into bands, or zones, along the length of the column (t2).
• Isolation of the separated species is then accomplished by passing a sufficient quantity of
mobile phase through the column to cause the individual zones to pass out the end, where
they can be detected or collected (times t3 and t4).
Analyte Dilution:
• Figure above illustrates an important general characteristic of the chromatographic
process—namely, dilution of analytes always accompanies chromatographic separation.
• Thus, the size of the original zone containing the analytes in the figure is noticeably
smaller than either of the two zones that reach the detector, meaning that significant
dilution of the analytes and band spreading have occurred while they were being
separated.
• As a result, the detectors employed for separated analytes must often be more sensitive
than would be required if the separation process were unnecessary.
Chromatograms
• If a detector that responds to solute concentration is placed at the end of the column and
its signal is plotted as function of time (or of volume of the added mobile phase), a series
of peaks is obtained
• Such a plot, called a chromatogram, is useful for both qualitative and quantitative
analysis.
• The positions of peaks on the time axis can be used to identify the components of the
sample; the areas under the peaks provide a quantitative measure of the amount of each
component.
 4.2 Band broadening and optimization of column performance
• A chromatographic separation is optimized by varying experimental conditions until the
components of a mixture are separated cleanly in a minimum amount of time.
• Optimization experiments are aimed at either
(1) reducing zone broadening (the component moves through the column as a zone which
is detected by the detector and translated into a peak). or
(2) altering relative migration rates of the components.
• Zone broadening is increased by those kinetic variables that increase the plate height of a
column.
• Migration rates, on the other hand, are varied by changing those variables that affect
retention and selectivity factors.

A poor separation of components A and B

An improvement in resolution leads to a partial separation of

the peaks.
 A further improvement in resolution has led to separation
of the peaks.

• Resolution refers to the extent of separation of the peaks of interest on a chromatogram.

Column resolution
• Resolution RS is a quantitative measure of the ability of the column under specific
operating conditions to separate (or resolve) two analyte peaks.
• The resolution for a given stationary phase can be improved by lengthening of the
column thus increasing the number of plates, but a negative effect of this approach is the
increased time required for separation and the band broadening that will occur.
Optimising column performance - Reduction of peak broadening :
• Variables that may be adjusted to reduce peak broadening are those that lead to an
improvement in column efficiency.
• Making adjustments to increase the number of theoretical plates (N) or decreasing plate
height (H) will therefore reduce the tendency of peaks to broaden.
The variables to be adjusted include:
• linear velocity of the mobile phase (too low and too high reduces efficiency)
• diameter of packing particle (smaller is generally better)
• thickness of the liquid coating of the stationary phase (HPLC)
• column temperature (GC)
• diameter of the column (narrower is better).
Optimising column performance – Altering migration rates
• Migration rates are altered with the aim of ensuring that the components of interest all
elute from the column within a reasonable time and are sufficiently separated from each
other.
• The extent of separation in this regard indicates how effective the chromatographic
column is.
• The variables to be adjusted that influence migration rates include:
• column temperature (GC)
• mobile phase composition (HPLC) eg 70% methanol/30% water  50%/50%,
 • gradient elution chemical composition of the stationary phase – use a different type of
column special chemical effects – incorporating a species in the stationary phase that
interacts with one or more of the sample components to enhance resolution.
• The specific parameters relating to the study of migration rates:
• Capacity factor
• Selectivity factor.
4.3 Liquid chromatography
• High Performance Liquid Chromatography (HPLC) is the most versatile and widely used
type of elution chromatography.
• The technique is used by scientists for separating and determining 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.
• The types of HPLC are often classified by separation mechanism or by the type of
stationary phase.
• The varieties include
(1) partition, or liquid- liquid, chromatography;
(2) adsorption, or liquid-solid chromatography;
(3) ion-exchange, or ion, chromatography;
(4) size-exclusion chromatography;
(5) affinity chromatography, and
(6) chiral chromatography
• Early liquid chromatography (LC) was carried out in glass columns with diameters of
10–50 mm.
• The columns were packed with 50- to 500-mm lengths of solid particles coated with an
adsorbed liquid that formed the stationary phase.
• To ensure reasonable flow rates through this type of stationary phase, the particle size of
the solid was kept larger than 150–200 mm; even then, flow rates were at best a few
tenths of a milliliter per minute. Thus, separation times were long—often several hours.
• To overcome this, the particles of stationary phase packings with diameters, 3–10 μm
was developed.
• Sophisticated instruments were developed which operated at high pressures , which
distinguished from original gravity-flow methods. These methods are called high-
performance liquid chromatography (HPLC).
The terms HPLC and LC can be used interchangeably
Scope of HPLC :
• LC is the most widely used of all of the analytical separation techniques.
• The reasons for the popularity of HPLC are:
 Its sensitivity,
  its ready adaptability to accurate quantitative determinations,
 its ease of automation,
 its suitability for separating nonvolatile species or thermally fragile ones, and
 above all, its widespread applicability to substances that are important to industry,
to many fields of science, and to the public.
• Examples of such materials include amino acids, proteins, nucleic acids, hydrocarbons,
carbohydrates, drugs, terpenoids, pesticides, antibiotics, steroids, metal-organic species,
and a variety of inorganic substances.
• Figure below reveals that the various liquid chromatographic procedures are
complementary in their application.

FIGURE: : Applications of liquid chromatography.

• For solutes having molecular masses greater than 10,000, size-exclusion chromatography
is often used.
• For lower-molecular-mass ionic species, ion-exchange chromatography is widely used.
• Small polar but nonionic species are best handled by reversed-phase methods.
Effects of Particle Size of Packings:
 • The efficiency of an LC column should improve dramatically as the particle size
decreases.
• A reduction of particle size from 45 to 6 μm results in a tenfold or more decrease in plate
height.
Extracolumn Band Broadening in LC :
• In LC, significant band broadening sometimes occurs outside the column packing itself.
This extracolumn band broadening occurs as the solute is carried through open tubes such as
those found in the injection system, the detector region, and the piping connecting the various
components of the system
• Here, broadening arises from differences in flow rates between layers of liquid adjacent
to the wall and the center of the tube.
• As a result, the center part of a solute band moves more rapidly than the peripheral part.
• It has been shown that the contribution of extracolumn effects Hex to the total plate
height is given by

where
u is the linear-flow velocity (cm/s),
r is the radius of the tube (cm), and
DM is the diffusion coefficient of the solute in the mobile phase (cm2/s).
 Extracolumn broadening can become quite serious when small-bore columns are used.
 Here, the radius of the extracolumn components should be reduced to 0.010 inch or less,
and the length of extracolumn tubing made as small as feasible to minimize this source of
broadening.
4.3 Liquid chromatography - Instrumentation
• Pumping pressures of several hundred atmospheres are required to achieve reasonable
flow rates with packings of 3–10 mm, which are common in modern LC.
• Because of these high pressures, the equipment for HPLC tends to be more elaborate and
expensive than equipment for other types of chromatography.
• Figure below is a diagram showing the important components of a typical LC instrument.
Mobile-Phase Reservoirs and Solvent Treatment Systems :
• A modern LC apparatus is equipped with one or more glass reservoirs, each of which
contains 500 mL or more of a solvent.
• Provisions are often included to remove dissolved gases and dust from the liquids.
 FIGURE Block diagram showing components of a typical apparatus for HPLC.
• Since, dissolved gases can lead to irreproducible flow rates and band spreading; in
addition, both bubbles and dust interfere with the performance of most detectors.
• To avoid this , Sparger is used.
• In Sparging method , the dissolved gases are swept out of the solution by fine bubbles
of an inert gas that is not soluble in the mobile phase.
• Another convenient way of treating solvents before introduction into the reservoir is to
filter them through a millipore filter under vacuum.
• This treatment removes gases as well as suspended matter.
• An elution with a single solvent or solvent mixture of constant composition is termed an
isocratic elution.
• In gradient elution, two (and sometimes more) solvent systems that differ significantly in
polarity are used and varied in composition during the separation.
• The ratio of the two solvents is varied in a preprogrammed way, sometimes continuously
and sometimes in a series of steps.
• Modern HPLC instruments are often equipped with proportioning valves that introduce
liquids from two or more reservoirs at ratios that can be varied continuously.
Pumping Systems :
• The requirements for liquid chromatographic pumps include
1) the generation of pressures of up to 6000 psi (lb/in.) or 414 bar
2) pulse-free output,
 3) flow rates ranging from 0.1 to 10 mL/min,
4) flow reproducibilities of 0.5% relative or better, and
5) resistance to corrosion by a variety of solvents.
• The high pressures generated by liquid chromatographic pumps are not an explosion
hazard because liquids are not very compressible.
• Thus, rupture of a component results only in solvent leakage.
• However, such leakage may constitute a fire or environmental hazard with some solvents.
• Two major types of pumps are used in LC:
1. the screw-driven syringe type and
2. the reciprocating pump.
• Reciprocating pumps are used in almost all modern commercial chromatographs.
Reciprocating Pumps :
• Reciprocating pumps usually consist of a small chamber in which the solvent is pumped
by the back and forth motion of a motor driven piston

FIGURE : A reciprocating pump for HPLC.

• Two ball check valves, which open and close alternately, control the flow of solvent into
and out of a cylinder.
• The solvent is in direct contact with the piston.
• As an alternative, pressure may be transmitted to the solvent via
a flexible diaphragm, which in turn is hydraulically pumped by a reciprocating piston.
• Reciprocating pumps have the disadvantage of producing a pulsed flow, which must be
damped because the pulses appear as baseline noise on the chromatogram.
Sample-Injection Systems :
• The, sample volumes injected into the system must be very small—a few tenths of a
microliter to perhaps 500 μL.
• Furthermore, it is convenient to be able to introduce the sample without depressurizing
the system.
• The most widely used method of sample introduction in LC is based on sampling loops,
such as that shown in Figure below.

Columns for HPLC :

• Liquid-chromatographic columns are usually constructed from smooth-bore stainless
steel tubing.
• HPLC columns are sometimes made from heavy-walled glass tubing and polymer tubing,
such as polyetheretherketone (PEEK).
• In addition, stainless steel columns lined with glass or PEEK are also available.
• Hundreds of packed columns differing in size and packing are available.
• The cost of standard-sized, non speciality columns ranges from $200 to more than $500.
• Specialized columns, such as chiral columns, can cost more than $1000.
Column Temperature Control:
• For some applications, close control of column temperature is not necessary and columns
are operated at room temperature.
• Often, however, better, more reproducible chromatograms are obtained by maintaining
constant column temperature.
• Most modern commercial instruments are now equipped with heaters that control column
temperatures to a few tenths of a degree from near ambient to 150°C.
Detectors :
• LC detectors are analytical instruments adapted with flow cells to measure low
concentrations of solutes in liquid streams.
Types of Detectors :
Liquid chromatographic detectors are of two basic types:
 Bulk-property detectors
 Solute-property detectors
 Bulk-property detectors respond to a mobile-phase bulk property, such as refractive
index, dielectric constant, or density, that is modulated by the presence of solutes.
 Solute-property detectors respond to some property of solutes, such as UV absorbance,
fluorescence, or diffusion current, that is not possessed by the mobile phase.
 The most widely used detectors for LC are based on absorption of UV or visible
radiation.
 Fluorescence, refractive-index, and electrochemical detectors are also widely used.
 Mass spectrometry (MS) detectors are currently quite popular. Such LC/MS systems can
greatly aid in identifying the analytes exiting from the HPLC column
 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.

4.4 Partition chromatography

• Partition chromatography is a type of chromatography.
• It is a method of separation in which the components present in the mixture get
distributed more likely into two liquid phases because of differences in partition
coefficients.
• It is based on differences in retention factor K as well as distribution coefficient Kd of the
analytes using liquid for both stationary as well as the mobile phase.
• The retention factor or capacity factor (k) is defined as the ratio of time an analyte is
retained in the stationary phase to the time it is retained in the mobile phase

• Distribution coefficient Kd  is a ratio of solid phase to solute concentrations.

• Partition chromatography can be divided into
1) liquid-liquid chromatography and
2) bonded-phase liquid chromatography.
• The partition chromatography is the basic principle involved in many separation
techniques like high performance liquid chromatography and gas chromatography.
• It is a method of separation in which the components present in the mixture get
distributed more likely into two liquid phases because of differences in partition
coefficients.
Bonded-Phase Packings:
• The supports for most bonded-phase packings for partition chromatography are prepared
from rigid silica, or silica-based, compositions.
• These solids are formed as uniform, porous, mechanically sturdy particles commonly
having diameters of 1.5–10 μm, with 3- and 5-μm particles being most common.
• The surface of fully hydrolyzed silica (hydrolyzed by heating with 0.1 M HCl for a day
or two) is made up of chemically reactive silanol groups.
• That is,

• Typical silica surfaces contain about 8 μmol/m2 of OH groups and have surface areas of
100–300 m2/g.
Normal- and Reversed-Phase Packings:
• Two types of partition chromatography are distinguishable based on
the relative polarities of the mobile and stationary phases.
 Normal-phase chromatography
 Reversed-phase chromatography
 In normal-phase chromatography , highly polar stationary phases is triethylene glycol or
water; a relatively mobile phase. nonpolar solvent is hexane or i-propyl.
 In reversed-phase chromatography, the stationary phase is nonpolar, often a
hydrocarbon, and the mobile phase is a relatively polar solvent (such as water, methanol)
 In normal-phase chromatography, the least polar component is eluted first; increasing the
polarity of the mobile phase then decreases the elution time.
 With reversed-phase chromatography, however, the most polar component elutes first,
and increasing the mobile-phase polarity increases the elution time.
 Bonded-phase packings are classified as reversed phase when the bonded coating is
nonpolar in character and as normal phase when the coating contains polar functional
groups.
 The major advantage of reversed-phase separations is that water can be used as the
mobile phase. Water is an inexpensive, nontoxic, UV-transparent solvent compatible with
biological solutes. Also, mass transfer is rapid with nonpolar stationary phases
Method Development in Partition Chromatography:
• Method development tends to be more complex in LC because in a liquid mobile phase
the sample components interact with both the stationary phase and the mobile phase .
• The success of a partition chromatographic separation is often critically dependent on
whether the mobile phase is, say, acetonitrile, hexane, or dioxane.
Column Selection in Partition Chromatographic Separations:
 • Successful chromatography with interactive mobile phases requires a proper balance of
intermolecular forces among the three active participants in the separation process—the
solute, the mobile phase, and the stationary phase.
• These intermolecular forces are described qualitatively in terms of the relative polarity of
each of the three reactants.
• The polarities of the various analyte functional groups increase in the following order:
• Hydrocarbons < ethers < esters < ketones < aldehydes < amides < amines < alcohols.
• Water is more polar than compounds containing any of the preceding functional groups.
• Often, in choosing a column for a partition chromatographic separation, the polarity of
the stationary phase is matched roughly with that of the analytes; a mobile phase of
considerably different polarity is then used for elution.
• This procedure is generally more successful than one in which the polarities of the solute
and mobile phase are matched but different from that of the stationary phase.
• Here, the stationary phase often cannot compete successfully for the sample components;
retention times then become too short for practical application.
• At the other extreme, of course, is the situation where the polarity of the solute and
stationary phases are too much alike and totally different from that of the mobile phase.
• Here, retention times become inordinately long.
• In summary, then, polarities for solute, mobile phase, and stationary phase must be
carefully blended if good partition chromatographic separations are to be realized in a
reasonable time.
Effect of Solvent Strength on Retention Factors :
• Solvents that interact strongly with solutes are often termed “strong” solvents.
• Strong solvents are often, but not always, polar solvents.
• Solvent strength depends on the nature of the analyte and stationary phase. Several
indexes have been developed for quantitatively describing the polarity of solvents.
• The most useful of these for partition chromatography is the polarity
index P’, which was developed by Snyder.
• The polarity index is a numerical measure of the relative polarity of various solvents.
Applications of Partition Chromatography :
• The table below lists the few typical examples of the multitude of uses of partition
chromatography in various fields
 4.5 Adsorption chromatography
• Adsorption, or liquid-solid, chromatography is the classic form of LC first introduced by
Tswett at the beginning of the last century.
• Because of the strong overlap between normal- phase partition chromatography and
adsorption chromatography, many of the principles and techniques used for the former
apply to adsorption chromatography.
• In fact, in many normal-phase separations, adsorption-displacement processes govern
retention.
• Finely divided silica and alumina are the only stationary phases that find use for
adsorption chromatography.
• Silica is preferred for most applications because of its higher sample capacity.
• The adsorption characteristics of the two substances parallel one another. For both,
retention times become longer as the polarity of the analyte increases.
• For both, retention times become longer as the polarity of the analyte increases.
• The polarity index P’, which gives idea to the strengths of solvents for adsorption
chromatography.
• Eluent strength Ɛ0 is a much better index, which is the adsorption energy per unit area of
solvent.
• The eluent strength depends on the adsorbent, with Ɛ0 values for silica being about 0.8 .
• Because of the versatility and ready availability of bonded stationary phases, use of
traditional adsorption chromatography with solid stationary phases has decreased in
recent years in favor of normal-phase chromatography.
 4.6 Ion exchange chromatography
• Ion exchange chromatography definition (or ion chromatography) is a process that
allows the separation of ions and polar molecules based on their affinity to the ion
exchanger.
• It can be used for almost any kind of charged molecule including large proteins, small
nucleotides, and amino acids.
• The principle of separation is thus by reversible exchange of ions between the target ions
present in the sample solution to the ions present on ion exchangers.
• In this process two types of exchangers i.e., cationic and anionic exchangers can be used.
• Cationic exchangers possess negatively charged group, and these will attract positively
charged cations. These exchangers are also called “Acidic ion exchange” materials,
because their negative charges result from the ionization of acidic group.
• Anionic exchangers have positively charged groups that will attract negatively charged
anions. These are also called “Basic ion exchange” materials.
• Ion exchange chromatography is most often performed in the form of column
chromatography.
Principle of ion exchange chromatography:
• This form of chromatography relies on the attraction between oppositely charged
stationary phase, known as an ion exchanger, and analyte.
• The ion exchangers basically contain charged groups covalently linked to the surface of
an insoluble matrix.
• The charged groups of the matrix can be positively or negatively charged.
• When suspended in an aqueous solution, the charged groups of the matrix will be
surrounded by ions of the opposite charge.
• In this “ion cloud”, ions can be reversibly exchanged without changing the nature and the
properties of the matrix.
• Ion-exclusion chromatography finds numerous applications for identification and
determination of acidic species in milk, coffee, wine, and many other products of
commerce.
• Salts of weak acids can be analyzed because they are converted to the corresponding acid
by the hydrogen ions in the exchanger. Weak bases and their salts can also be determined
by ion-exclusion chromatography.
Working:-
• Consider a column having E- Y+ cation exchanger in which E - is negative charged
exchanger and Y+ is the mobile counter ion.
• Let X+ be the cation in the sample having charge greater than Y+ .
The X+ ion can exchange sites with the counter ion Y+ with satisfying the following relationship;
E - Y+ X+ → E - X+ Y+ + The remaining neutral and negatively charged particles
Desired bounded cation (X+ ) can now be eluted by either of the two ways;
1. By adding a component M+ having magnitude of charge more than that of X+ so that M+
will replace X+ and X+ will be eluting out.
 2. By changing pH of the solvent (mobile phase so that X+ have no charge and is then
unbounded from the matrix and can be eluted out.

The 4 basic steps of ion exchange chromatography:-

1. Equilibration
2. Sample application and wash
3. Elution
4. Regeneration
Requirement:-
1.Column :-
glass, stainless steel or polymers
2.Packing the column :-
Wet packing method: A slurry is prepared of the eluent with the stationary phase powder and
then carefully poured into the column. Care must be taken to avoid air bubbles.
3.Application of the sample:-
After packing, sample is added to the top of the stationary phase, use syringe or pipette.
This layer is usually topped with a small layer of sand or with cotton or glass wool to protect the
shape of the organic layer from the velocity of newly added eluent.
4.Mobile phase:-
Acids, alkalis, buffers.
5.Stationary phase:-
The ionic compound consisting of the cationic species (M+) and the anionic species (B-)
6.Elution:-
Components of mixture separate & move down the column at different rates depending upon the
affinity of the ion for ion exchanger.The eluates are collected at different stages
7. Analysis of the eluate:-
Spectrophotometric, flame photometry polarographic, conductometri.
Factors affecting Ion –Exchangr Separation
A . Ion Exchange Resin:
The swelling factor and cross linking is important for the effective separation. The cross linking
should be controlled as its affects the exchanger’s capacity. Swelling helps in proper exposure of
charged functional groups for exchange of ions.
swells less → separation of ions of different sizes is difficult.
B. Nature of exchanging ions:
1. valency of ions.
2.Size of ions
3.Polarizability
4.Concentration of solution.
5.Concentration & charge of ions
C . pH of the mobile phase
D . Ionic strength
E . Mobile phase modifiers
F . Temperature
G. Buffer: The pH of the buffer should impart the same charge to the sample ions as present in
the Column. Anionic Exchange Chromatography should be carried out with cationic buffers and
vice versa because buffer ion will indulge in ion exchange, which will be of no use.
Ion Exchangers:-
There are three classes of ion exchangers
1. Resins:- Ion exchange resins are used for the separation of small molecules
2. Gels:- Ion exchange gels are used for the separation of large molecules like proteins ,nucleic
acids
3. Inorganic exchangers:- Separations involving harsh chemical conditions (high temperature ,
high radiation levels, strongly basic solutions or powerful oxidizing agents) employ inorganic
ion exchangers

Classification of ion exchange resins:-

According to the chemical nature they classified as-
Strongly acidic cation exchanger:-
sulphonic acid groups attached to styrene and divinyl-benzene copolymer.
Weakly acidic cation exchanger:-
carboxylic acid groups attached to acrylic and divinyl-benzene co-polymer.
Strongly basic anion exchanger:-
quaternary ammonium groups attached to styrene and divinyl-benzene co-polymer.
Weakly basic anion exchanger:-
poly alkyl amine groups attached to styrene and divinyl benzene co- polymer.
According to the source:-
Natural resins :
Cation - Zeolytes, Clay
Anion - Dolomite
Synthetic resins: Inorganic & Organic resins
Organic resins:- are polymeric resin matrix.
Exchange Medium
• The choice of ion exchangers depends upon the stability, molecular weight,and ionic strength
of the sample components.
• The volume of exchanger used for separation is usually 2.5 fold greater than to exchange with
the ion in the sample.
• The ion exchanger are packed in column having suitable buffer.
• The ion exchangers are of two types;
Cation Exchangers
Anion Exchangers
 4.7 Size Exclusion Chromatography
 Size-exculsion chromatography (SEC), also called gel filtration or gel-permeation
chromatography (GPC), uses porous particles to separate molecules of different sizes.
 It is generally used to separate biological molecules, and to determine molecular weights
and molecular weight distributions of polymers
 It is usually applied to large molecules or macromolecular complexes such as proteins
and industrial polymers.
 When an aqueous solution is used to transport the sample through the column, the
technique is known as Gel filtration chromatography .
 When an organic solvent is used as a mobile phase,the technique is known as Gel-
permeation chromatography.
 The separation of molecules is called fractionation.
 Size of pores in beads determines the exclusion limit (what goes through the beads and
what goes around the beads)
 PRINCIPLE
 A mixture of molecules dissolved in liquid (the mobile phase) is applied chromatography
column which contains a solid support in the form of microscopic spheres, or “beads”
(the stationary phase).
 The mass of beads within the column is often referred to as the column bed.
 The beads act as “traps” or “sieves” and function to filter small molecules which become
temporarily trapped within the pores.
 Larger molecules pass around or are “excluded” from the beads . Large sample molecules
cannot or can only partially penetrate the pores, whereas smaller molecules can access
most or all pores.
 Thus, large molecules elute first, smaller molecules elute later, while molecules that can
access all the pores elute last from the column. Particles of different sizes will elute
(filter) through a stationary phase at different rates.

Total column volume (Vt)

Vt = Vg + Vi + V0
where
Vg --is the volume occupied by the packing
Vi --is the volume of solvent in the pores
V0 --is the free solvent volume (similar to injection volume)

COMPONENTS OF A SEC
1. Stationary Phase
2. The Mobile Phase
3. The Columns
4. The Pump
5. Detectors
STATIONARY PHASE:
• Stationary Phase Semi-permeable, porous beads with welldefined range of pore sizes .
• Beads are crosslinked polymers
• Degree of crosslinking is controlled carefully to yield different pore sizes.
• Smaller pore sizes are used for rapid desalting of proteins or for protein purification.
• Intermediate pore sizes are used to separate relatively small proteins.
• Very large pore sizes are used for purification of biological complexes.
• Stationary phase used for gel exclusion chromatography include dextran (Sephadex™),
polyacrylamide and dextranpolyacrylamide (Sephacryl™).
• Each is available with a variety of different ranges of pore size in the beads, permitting
separation of macromolecules of different size.

A good stationary phase should have

following properties:
• It be chemically inert.
• It should be inexpensive.
• It should not react with component to be separated.
• It should not react with eluent.
• It should be colorless, uniform in size and shape.
• It should be mechanically stable.
Soft gel e.g.- dextran(Sephadex), Polyacrylamide gels
Separation of proteins.
Semi-rigid gel e.g.- bio beads
Separation of non-polar polymers in non-polar solvents.
Highly rigid gels and glasses
Separation of polar systems.
Dextran
• A homopolysaccharide of glucose residues.
• It’s prepared with various degrees of cross-linking to control pore size.
• It’s bought as dry beads, the beads swell when water is added.
• The trade name is sephadex.
• It’s mainly used for separation of small peptides and globular proteins with small to
average molecular mass.
Polyacrylamide
• these gels are prepared by cross linking acrylamide with N,N-methylene bis acrylamide.
• The pore size is determined by the degree of cross-linking.
• The separation properties of polyacrylamide gels are mainly the same as those of
dextrans.
• They are sold as bio-gel P. They are available in wide range of pore sizes.
Agarose
• Linear polymers of D-galactose and 3,6 anhydro-1-galactose.
• It forms a gel that’s held together with H bonds. It’s dissolved in boiling water and forms
a gel when it’s cold.
• The concentration of the material in the gel determines the pore size.
• The pores of agarose gel are much larger than those of sephadex or bio-gel p.
 • It’s useful for analysis or separation of large globular proteins or long linear molecules
such as DNA
Mobile phase
• The liquid used to dissolve the biomolecules to make the mobile phase is usually called a
buffer.
• The mixture of biomolecules dissolved in the buffer is called the sample.
• The choice of mobile phase to be used in any separation will depend on the type of
separation to be achieved and component to be separated.
• The most common eluents in for polymers that dissolve at room temperature.e.g.-
Tetrahydrofuran , Chloroform , Dimethyl formamide.
SOLVENT SELECTION
• The solvents used for mobile phase of SEC are limited to those follows following criteria:
• The solvent must dissolve the sample completely.
• The solvent has different properties with solute in the eluent: typically with solvent
refractive index (RI) .
• solvent must not degrade the sample during use Otherwise, the viscosity of eluent will
gradually increase over times.
• The solvent is not corrosive to any components of the Equipments.
Mobile Phase Preparation
• high purity of solvent is recommended.
• Filter mobile phase solvents using 0.5 micron filter to remove any particular impurities
such as dusts, insoluble salts.
• Antioxidant is added to trichlorobenzene to keep solvent stable in high temperature.
• Other additives eliminate adsorption or interaction of solutes with column packing
materials
Sample preparation
• The sample solutions are supposed to be prepared in dilute concentration (less than 2
mg/mL)
• A good solvent can dissolve a sample in any proportion in a range of temperatures.
• Samples with broad molecular weight distribution may require higher concentrations.
• It is recommended to filter the sample solutions before injecting into columns in order to
get rid of clogging and excessively high pressure problems.
• Agitation and filtration Generally filtration is required to remove insoluble impurities.
• Do not agitate and filter samples that contain very high MW (>1 million).
COLUMNS
Commercially Available Columns
• analytical column- 7.5–8mm diameters.
• Preparative columns-22–25mm for.
• Usual column lengths-25, 30, 50, and 60 cm.
• Recently, narrow bore columns- 2–3mm diameter have been introduced, which save time
and solute
Selecting SEC column
• Shorter columns save time and solvent.
• Small particles (typically 5 mm) provide a better resolution.
• On the other hand, 5 mm (or even 3 mm) packings are more sensitive towards
contamination by samples containing impurities.
 • Particles as large as 20 mm have been recommended for very high-molecular-weight
polymers.
Selecting SEC column
• Shorter columns save time and solvent.
• Small particles (typically 5 mm) provide a better resolution.
• On the other hand, 5 mm (or even 3 mm) packings are more sensitive towards
contamination by samples containing impurities.
• Particles as large as 20 mm have been recommended for very high-molecular-weight
polymers.
• Small particle size packings can sometimes result in shear degradation of large polymer
molecules because the space between particles is very narrow.
• Columns with different porosity or mixed-bed columns, provide a better separation.
Handling SEC Columns
• A column set in SEC should be always run in the same mobile phase.(isocratic)
• SEC columns should never be operated in a backward direction.
• Care should also be taken in connecting columns or in sample injection.
• Replacing a clogged inlet frit is a dangerous operation which can reduce column
performance.
• A damaged or dirty check valve of pump, can also reduce column life.

PUMP
 A highly constant flow rate has to be maintained during the entire chromatogram. This is
very important in SEC.
 A change of the flow rate of only 0.1% can cause an error in molar mass of up to 10%.
 Most pumps can only reproduce the flow rate to 0.2–0.3%.
 In-line filters in the solvent reservoir may prevent particles from coming into the pump
heads, which might damage the check valves or the pump seals.
 Types-)Syringe pumps, Reciprocating pumps
DETECTOR
Concentration sensitive detectors
 Bulk Property Detectors- Refractive Index (RI) Detector
 Solute Property Detectors- Ultraviolet (UV) Absorption Detector
 Evaporative Detectors- Evaporative Light Scattering Detector (ELSD)
Molar mass sensitive detectors
 Light Scattering Detectors
 Low Angle Light Scattering (LALS) Detectors
 Multiangle Light Scattering (MALS) detectors
Viscosity Detectors- Differential Vscometers
 Other :- Flame Ionization Detector (FID), A Mass
 Spectrometer or A Fourier Transform Infrared (FTIR) Spectrometer
ADVANTAGES
 Short analysis time.
 Well defined separation.
 Narrow bands and good sensitivity.
 There is no sample loss.
 Small amount of mobile phase required.
 The flow rate can be set.
DISADVANTAGES
 Limited number of peaks that can be resolved within the short time scale of the GPC run.
 Filtrations must be performed before using the instrument to prevent dust and other
particulates from ruining the columns and interfering with the detectors.
 The molecular masses of most of the chains will be too close for the GPC separation to
show anything more than broad peaks
APPLICATION
 Proteins fractionation
 Purification
 Molecular weight determination.
 Separation of sugar, proteins, peptides, rubbers and others on the basis of their size.
 This technique can be determine the quaternary structure of purified proteins.
 SEC is a widely used technique for the purification and analysis of synthetic and
biological polymers, such as protein, polysaccharides and nucleic acid.
 Various species of RNA and viruses have been purified using agarose gels.
 For Desalting
 For copolymerisation studies

4.8 Affinity Chromatography

 Affinity chromatography involves covalently bonding a reagent, called an affinity ligand,
to a solid support.
 Typical affinity ligands are antibodies, enzyme inhibitors, or other molecules that
reversibly and selectively bind to analyte molecules in the sample.
 When the sample passes through the column, only the molecules that selectively bind to
the affinity ligand are retained.
 Molecules that do not bind pass through the column with the mobile phase.
 After the undesired molecules are removed, the retained analytes can be eluted by
changing the mobile-phase conditions.
 The stationary phase for affinity chromatography is a solid such as agarose or a porous
glass bead to which the affinity ligand is immobilized.
 The mobile phase in affinity chromatography has two distinct roles to play.
 First, it must support the strong binding of the analyte molecules to the ligand.
 Second, once the undesired species are removed, the mobile phase must weaken or
eliminate the analyte-ligand interaction so that the analyte can be eluted.
 Often, changes in pH or ionic strength are used to change the elution conditions during
the two stages of the process.
 Affinity chromatography has the major advantage of extraordinary specificity.
 The primary use is in the rapid isolation of biomolecules during preparative work.

4.9 Principles of GC and applications

GC – Gas Chromatography :
• Separation in GC is based on different distributions of the molecules of the components
being separated between the mobile gas phase and the stationary phase.
• Gas chromatography differs from other forms of chromatography in that the mobile
phase is a gas and the stationery phase is a solid, the sample is sent as vaporized form
and the components are separated as vapors.
• In performing a gas chromatographic separation, the sample is vaporized and injected
onto the head of a chromatographic column.
• Elution is brought about by the flow of an inert gaseous mobile phase. In contrast to most
other types of chromatography, the mobile phase does not interact with molecules of the
analyte; its only function is to transport the analyte through the column.
• There are two types of gas chromatography:
• gas-liquid chromatography (GLC) and
• gas-solid chromatography (GSC).
• GLC is used throughout all fields of science; its name is usually shortened to gas
chromatography (GC).
• GSC is based on a solid stationary phase in which retention of analytes occurs because of
physical adsorption.
• The application of GSC is limited because of semipermanent retention of active or polar
molecules and severe tailing of elution peaks.
• Tailing is a result of the nonlinear nature of the adsorption process.
 • Thus, this technique is not widely used except for the separation of certain low
molecular- mass gaseous species;
• In GLC the analyte is partitioned between a gaseous mobile phase and a liquid phase
immobilized on the surface of an inert solid packing or on the walls of a capillary tubing.
• The concept of GLC was first suggested in 1941 by Martin and Synge, who were also
responsible for the development of liquid-liquid partition chromatography.
• More than a decade was to elapse, however, before the value of GLC was demonstrated
experimentally and this technique began to be used as a routine laboratory tool.
• In 1955 the first commercial apparatus for GLC appeared on the market.
• Since that time, the growth in applications of this technique has been phenomenal.
• Currently, nearly a million gas chromatographs are in use throughout the world.
Retention Volumes :
• Liquid Chromatography concentrates on retention times and Gas chromatography
concentrates on retention volumes.
• The relationship between the two is given by

where
 F is the average volumetric flow rate within the column;
 V and t are retention volumes and times, respectively; and
 The subscripts R and M refer to species that are retained and not retained on the
column.
 The flow rate within the column is not directly measurable. Instead, the rate of gas flow
as it exits the column is determined experimentally with a flow meter
 For popular soap-bubble-type flow meters, where the gas is saturated with water, the
average flow rate F is related to the measured flow rate Fm by

Where
Tc is the column temperature in kelvins,
T is the temperature at the flow meter, and
P is the gas pressure at the end of the column.
Usually P and T are the ambient pressure and temperature.
The term involving the vapor pressure of water, PH2O, is a correction for the pressure used when
the gas is saturated with water.
• Both VR and VM depend on the average pressure within the column—a quantity that lies
intermediate between the inlet pressure Pi and the outlet pressure P (atmospheric pressure).
• The pressure drop correction factor j, also known as the compressibility factor, accounts for
the pressure within the column being a nonlinear function of the Pi/P ratio.
• Corrected retention volumes and , which correspond to volumes at the average
column pressure, are obtained from the relationships
-------------(1)
where j can be calculated from the relationship

----------------------------------------------------(2)
The specific retention volume Vg is then defined as

-------------------------(3)
where mS is the mass of the stationary phase, a quantity determined at the time of column
preparation.
Relationship between Vg and K:
• The specific retention volume Vg can be related to the distribution constant Kc by

------------------------------------------------------------(4)
Combining this with Equation (2) we, get

------------------------------------------------------------------(5)

--------------------------------------------------------------------(6)
The density of the liquid on the stationary phase ρS is given by

------------------------------------------------------------------------(7)

------(8)
Applications of GC :
• GC has two roles
• First, GC is a tool for performing separations.
 • In this role, GC methods are unsurpassed when applied to complex organic, metal-
organic, and biochemical systems made up of volatile species or species that can be
derivatized to yield volatile substances.
• The second role that GC plays is in the completion of an analysis.
• In this role, retention times or volumes are used for qualitative identification, and peak
heights or peak areas provide quantitative information.
• For qualitative purposes, GC is much more limited than most of the spectroscopic
methods.
• Thus, an important trend in the field has been in the direction of combining the
remarkable separation capabilities of GC with the superior identification properties of
such instruments as mass, infrared, and nuclear magnetic resonance spectrometers

4.10 Capillary Electrophoresis

 Electrophoresis is a separation method based on the differenti al rate of migration of
charged species in an applied dc electric field.
 Electrophoresis on a macro scale has been applied to a variety of difficult analytical
separation problems: inorganic anions and cations, amino acids, catecholamines, drugs,
vitamins, carbohydrates, peptides, proteins, nucleic acids, nucleotides, polynucleotides,
and numerous other species.
 An electrophoretic separation is performed by injecting a small band of the sample into
an aqueous buffer solution contained in a narrow tube or on a flat porous support medium
such as paper or a semisolid gel.

Figure Schematic of a capillary electrophoresis system.

 A high voltage is applied across the length of the buffer by means of a pair of electrodes
located
 at each end of the buffer.
 This field causes ions of the sample to migrate toward one of the electrodes. The rate of
migration of a given species depends on its charge and its size.
 Separations are then based on differences in charge-to-size ratios for the various analytes
in a sample. The larger this ratio, the faster an ion migrates in the electric field.
Types of Electrophoresis
Electrophoretic separations are currently performed in two different formats:
 i. slab electrophoresis and
ii. capillary electrophoresis.
Slab electrophoresis:
 Slab electrophoresis is a classical method
 Slab separations are carried out on a thin flat layer or slab of a porous semisolid gel
containing an aqueous buffer solution within its pores.
 This slab has dimensions of a few centimeters on a side and, like a chromatographic thin-
layer plate, is capable of separating several samples simultaneously.
 Samples are introduced as spots or bands on the slab, and a dc electric field is applied
across the slab for a fixed period.
 When the separations are complete, the field is discontinued and the separated species are
visualized by staining in much the same way as was described for thin-layer
chromatography
 Slab electrophoresis is now the most widely used separation tool in biochemistry and
biology.

The Basis for Electrophoretic Separations:

The migration rate v of an ion (cm/s) in an electric field is equal to the product of the field
strength E (V cm-1) and the electrophoretic mobility µe (cm2 V-1 s-1). That is,
v =µeE---------------------------------(1)
 The electrophoretic mobility is in turn proportional to the ionic charge on the analyte and
inversely proportional to frictional retarding factors.
 The electric field acts on only ions. If two species differ either in charge or in the
frictional forces they experience while moving through the buffer, they will be separated
from each other.
 Neutral species are not affected by the field and are thus not separated.
 The frictional retarding force on an analyte ion is determined by the size and shape of the
ion and the viscosity of the migration medium.
 For ions of the same size, the greater the charge, the greater the driving force, and the
faster the rate of migration.
 For ions of the same charge, the smaller the ion, the smaller the frictional forces, and the
faster the rate of migration.
 The ion’s charge-to-size ratio combines these two effects.

Capillary electrophoresis
 Capillary electrophoresis (CE) yields high-speed, high-resolution separations on
exceptionally small sample volumes (0.1–10 nL in contrast to slab electrophoresis, which
requires samples in the μL range).
 Additionally, the separated species are eluted from one end of the capillary, so
quantitative detectors, similar to those found in high-performance liquid chromatography
(HPLC)
Migration Rates in CE
As Equation -1 shows, the migration rate of an ion v depends on the electric field strength. The
electric field in turn is proportional to the magnitude of the applied voltage V and inversely
proportional to the length L over which it is applied.
Thus

-----------------------------------------(2)
 This relationship indicates that high applied voltages are desirable to achieve rapid ion
migration and fast separations.
 It is desirable to have rapid separations, but it is even more important to achieve high-
resolution separations.