Introduction to Chromatography

Definition
Chromatography is a separation technique based on the different interactions of
compounds with two phases, a mobile phase and a stationary phase, as the
compounds travel through a supporting medium.

Components:
mobile phase: a solvent that flows through the supporting medium

stationary phase: a layer or coating on the supporting medium that interacts

with the analytes

supporting medium: a solid surface on which the stationary phase is bound or

coated
 The analytes interacting
most strongly with the
stationary phase will take
longer to pass through
the system than those
with weaker interactions.

 These interactions are

usually chemical in
nature, but in some cases
physical interactions can
also be used.
Types of Chromatography:- chromatography can be classified based on the type of mobile
phase, stationary phase and support material
Types of Chromatography
1) The primary division of chromatographic techniques is based on the type of mobile
phase used in the system:

Type of Chromatography Type of Mobile Phase

Gas chromatography (GC) gas
Liquid chromatograph (LC) liquid

2) Further divisions can be made based on the type of stationary phase used in the
system:

Gas Chromatography
Name of GC Method Type of Stationary Phase
Gas-solid chromatography solid, underivatized support
Gas-liquid chromatography liquid-coated support
Bonded-phase gas chromatography chemically-derivatized support
Liquid Chromatography
Name of LC Method Type of Stationary Phase
Adsorption chromatography solid, underivatized support
Partition chromatography liquid-coated or derivatized support
Ion-exchange chromatography support containing fixed charges
Size exclusion chromatography porous support
Affinity chromatography support with immobilized ligand

3) Chromatographic techniques may also be classified based on the type of

support material used in the system:

 Packed bed (column) chromatography

 Open tubular (capillary) chromatography

 Open bed (planar) chromatography

Theory of Chromatography

1) Typical response obtained by chromatography

Chromatogram - concentration versus elution time

Wh

Wb

Inject
Where:
tR = retention time tM = void time
Wb = baseline width of the peak in time units
Wh = half-height width of the peak in time units
The separation of solutes in chromatography depends on two factors:

 a difference in the retention of solutes (i.e., a difference in their

time or volume of elution
 a sufficiently narrow width of the solute peaks (i.e, good
efficiency for the separation system)

Peak width & peak position

determine separation of peaks
2) Solute Retention:
A solute’s retention time or retention volume in chromatography is directly
related to the strength of the solute’s interaction with the mobile and stationary
phases.

Retention on a given column pertain to the particulars of that system depends

on the size of the column and the flow rate of the mobile phase

Capacity factor (k) more universal measure of retention, determined from tR.

k = (tR –tM)/tM

capacity factor is useful for comparing results obtained on different systems

since it is independent on column length and flow-rate.
Capacity factor (k)

The value of the capacity factor is useful in understanding the retention

mechanisms for a solute, since the fundamental definition of k is:
n Astationary phase
k =
n Amobile phase

k is directly related to the strength of the interaction between a solute with the
stationary and mobile phases.

nAstationary phase and nAmobile phase represents the amount of solute present in each
phase at equilibrium.

Equilibrium is achieved or approached at the center of a chromatographic peak.

When k' is < 1.0, separation is poor
When k' is > 30, separation is slow
When k' is = 2-10, separation is optimum

Load Eluent
(sample) (solvent)

k’ > 1000 k’ < 0.001

Compounds of
Compounds of
interest released
interest stick to
from sorbent
sorbent

Waste Extract
 Mobile phase Mobile phase
Load
Sample
(injection)
1 < k’ < 10
Compounds of Differential
interest in a interaction with
plug stationary phase
separates
compounds in
time and space.

Detector Detector
A simple example relating k to the interactions of a solute in a column is
illustrated for partition chromatography:

A (mobile phase) KD A (stationary phase)

where: KD = equilibrium constant for the distribution of A between the

mobile phase and stationary phase

Assuming local equilibrium at the center of the chromatographic peak:

As KD increases, interaction of the solute with the stationary phase becomes

more favorable and the solute’s retention (k) increases
 Separation between two solutes requires different KD’s for their interactions
with the mobile and stationary phases

since DG = -RT ln KD

peak separation also represents different changes in free energy

3) Efficiency
Efficiency is experimentally related to a solute’s peak width.
 an efficient system will produce narrow peaks
 narrow peaks  smaller difference in interactions in order to separate two
solutes

Efficiency is theoretically related to the various kinetic processes that are

involved in solute retention and transport in the column
 determine the width or standard deviation (s) of peaks

Estimate s from peak widths, assuming

Gaussian shaped peak:
Wh
Wb = 4s

Wh = 2.354s

Dependent on the amount of time that a solute spends in the column (k’ or tR)
Number of theoretical plates (N)
compare efficiencies of a system for solutes that have different retention times

N = (tR/s)2

or for a Gaussian shaped peak

N = 16 (tR/Wb)2

N = 5.54 (tR/Wh)2

The larger the value of N is for a column, the better the column will be able to
separate two compounds.
 the better the ability to resolve solutes that have small differences in retention
 N is independent of solute retention
 N is dependent on the length of the column
Plate height or height equivalent of a theoretical plate (H or HETP):
compare efficiencies of columns with different lengths:

H = L/N
where: L = column length
N = number of theoretical plates for the column

Note: H simply gives the length of the column that corresponds to one theoretical
plate

H can be also used to relate various chromatographic parameters (e.g., flow rate,
particle size, etc.) to the kinetic processes that give rise to peak broadening:

Why Do Bands Spread?

 Eddy diffusion
 Mobile phase mass transfer
 Stagnant mobile phase mass transfer
 Stationary phase mass transfer
 Longitudinal diffusion
Eddy diffusion (A-term) – a process that leads to peak (band)
broadening due to the presence of multiple flow paths through a
packed column.

As solute molecules travel through the column,

some arrive at the end sooner then others simply
due to the different path traveled around the
support particles in the column that result in
different travel distances.

Longer path arrives at end of column after (1).

Longitudinal diffusion (B-term) – band-broadening due to the
diffusion of the solute along the length of the column in the flowing
mobile phase.

The degree of band-broadening due to longitudinal diffusion depends

on the:
 diffusion of the solute
 flow-rate of the solute through the column
Mobile phase mass transfer (c-term) – a process of peak broadening
caused by the presence of different flow profile within channels or
between particles of the support in the column.

A solute in the center of the channel moves more

quickly than solute at the edges, it will tend to
reach the end of the channel first leading to band-
broadening

The degree of band-broadening due to eddy diffusion and mobile

phase mass transfer depends mainly on the:

 size of the packing material

 diffusion rate of the solute

Stagnant mobile phase mass transfer (C-term) – band-broadening
due to differences in the rate of diffusion of the solute molecules
between the mobile phase outside the pores of the support (flowing
mobile phase) to the mobile phase within the pores of the support
(stagnant mobile phase).

Since a solute does not travel down

the column when it is in the stagnant
mobile phase, it spends a longer time
in the column than solute that
remains in the flowing mobile phase.

The degree of band-broadening due to stagnant mobile phase mass

transfer depends on the:
 size, shape and pore structure of the packing material
 diffusion and retention of the solute
 flow-rate of the solute through the column
Stationary phase mass transfer (C-term) – band-broadening due to the
movement of solute between the stagnant phase and the stationary
phase.

Since different solute molecules

spend different lengths of time in the
stationary phase, they also spend
different amounts of time on the
column, giving rise to band-
broadening.

The degree of band-broadening due to stationary phase mass transfer

depends on the:
 retention and diffusion of the solute
 flow-rate of the solute through the column
 kinetics of interaction between the solute and the stationary phase
Van Deemter equation: relates flow-rate or linear velocity to H:

H = A + B/µ + Cµ
where:

m = linear velocity (flow-rate x Vm/L)

H = total plate height of the column
A = constant representing eddy diffusion &
mobile phase mass transfer
B = constant representing longitudinal diffusion
C = constant representing stagnant mobile phase
& stationary phase mass transfer

 One use of plate height (H) is to relate these kinetic process to band
broadening to a parameter of the chromatographic system (e.g.,
flow-rate).

 This relationship is used to predict what the resulting effect would

be of varying this parameter on the overall efficiency of the
chromatographic system.
Plot of van Deemter equation shows how H changes with the linear
velocity (flow-rate) of the mobile phase

µ optimum

Optimum linear velocity (µopt) - where H has a minimum value

and the point of maximum column efficiency:

µopt is easy to achieve for gas chromatography, but is usually too small for liquid
chromatography requiring flow-rates higher than optimal to separate compounds
4) Measures of Solute Separation:
separation factor (a) – parameter used to describe how well two solutes are
separated by a chromatographic system:

 = k′2/k′1 k′ = (tR –tM)/tM

where: k′1 = the capacity factor of the first solute
k′2 = the capacity factor of the second solute, with k′2 ≥ k′1

A value of  ≈ 1.1 is usually indicative of a good separation

Does not consider the effect of column efficiency or peak widths, only retention.
Rs is preferred over a since both
retention (tr) and column efficiency
(Wb) are considered in defining peak
separation.

Rs ≥ 1.5 represents baseline

resolution, or complete separation
of two neighboring solutes  ideal
case.

Rs ≥ 1.0 considered adequate for

most separations.
Resolution
Describes how well 2 compounds are separated

selectivity
efficiency
retention

Example: Given the following data and a 24.7 cm column

Compound Retention Time (min) Peak Width(WB, min)
Non-retained 3.1 -
A 5.4 0.41
B 13.3 1.07
C 14.1 1.16
D 21.6 1.72

Calculate the resolution between species C and D. What column

length is required for a resolution of 1.5?
Gas chromatography (GC)

 Is a technique used for separation of volatile substances, or

substances that can be made volatile

 A mobile phase only pushes the sample (has no interaction

with the sample components)
 called carrier gas

 The components of the sample come in contact with the SP.

 The different components of the sample have different

affinities for the SP which results in differential migration of
solutes, thus leading to separation

27
GC: classified into two

1. Gas-solid chromatography (GSC)

 The stationary phase: is a solid like silica alumina.

 Separation of analytes is based on the difference in
their adsorption on the solid material

 adsorption chromatography (the SP is also called

adsorbent

 This GC technique: most useful for the separation

and analysis of gases like CH4, CO2, CO, ... etc.
28
2. Gas - Liquid Chromatography (GLC)
 The SP is a liquid immobilized (supported) on the surface of an
inert solid.
 Analytes are separated based on the difference in their solubility
in the liquid SP
 Partition chromatography
 Possible types of interactions between the stationary liquid and
sample components in GLC
 Dispersion (London) force
 typical for alkanes and benzene
 Orientation force
 between permanent dipoles (e.g. H-bonding)
 Induction force
 between permanent and induced dipoles
 Formation of charge transfer complexes
 between aromatic hydrocarbon and metal ion

29
GLC is a commonly used format of GC
because:

 K of solutes (CS/CM) is much larger in GSC

because molecules are adsorbed on the SP
 tR is inconveniently large

 GSC needs the elution of adsorbed solute

molecules with another solvent
Increases cost

30
Instrumentation of GC

31
Fig.1. Block diagram of a typical gas chromatography
 a) The Carrier Gas reservoir

A high pressure gas cylinder in which the carrier

(mobile) gas is filled

A carrier gas should have the following properties:

 Highly pure (> 99.9%)

 Inert so that no reaction with SP or instrumental
components
 A higher density (larger viscosity) carrier gas
 Compatible with the detector
 A cheap and available carrier gas with low
molecular weight (e.g. H2, He, Ne, Ar, N2, CO2)
32
 Longitudinal diffusion (B-term)

 An important factor contributing to band

broadening

HL = K DM /u

Where DM is the diffusion coefficient of

solute in the carrier gas.

 This term can be minimized:

 Lower diffusion (that is, high density) of
MP,
 higher flow rates.

33
b) Flow control

 Controls the flow rate of the carrier gas

 Controlled MP flow rate:

 Provides reproducible retention times of
analytes
 Minimizes signal drift in the detector
c) Injector

 The sample is introduced into the injector through a self-

sealing silicone rubber septum.

 The carrier gas flows through the injector carrying

vaporized solutes (small amount in mL range).

 The temperature of the injector should be adjusted so

that flash vaporization of all solutes occurs.

 If the temperature of the injector is not high enough (at

least 50 degrees above highest boiling component),
band broadening will take place.
35
 Syringe

Septum

Carrier
Gas

Vaporization
Chamber

To
Column
36
 Sample
 Gas, liquid or dissolved solid
 non-gaseous samples should be volatized upon
introduction
The injection unit is kept in an oven
The applied temperature should not decompose the
sample/analyte
 Non-volatile and unstable substances can also be
chemically modified to volatile and stable forms via
chemical reaction
 derivatization)
 E.g. silylation

Introduction of silyl group (R3Si-) to a molecule

Volatile and
thermally stable
 d) Column
Types of GC column

Packed column

Capillary (open)
column
Column type vs. separation

 Lower resolution
 Fewer peaks

 Better resolution
 More peaks
 Faster analysis
Packed GC column
 Easy to make and use
 Limited resolution (N < 8,000)
 Outside: solid tubing made from stainless steel
 Because of strength
 Glass when more inert substrate is needed
 Inside: tightly packed with inert support
 Solid supports should be inert and have high surface area
 Typically diatomaceous earth or fluorocarbon polymer

 Stationary liquid phase is coated on the solid support

 3-10% by weight of the solid support
Capillary (open) column
 Most common and efficient
 High resolution (N > 100, 000)
 Outside: solid tubing made from fused silica
 Inert, flexible, strong and easy to use

 Inside: column is an open tube

 Very low resistance to flow
 Long lengths possible (L > 100 m)
 SP is a thin, uniform liquid film coated on the wall of the
tubing (thus, A-term in van Deemter equation is zero )

H = B/u +
Cu
Column temperature
 Kept in an oven to enable analytes stay in their
vaporized form
 Oven temperature: should not be higher than the
boiling point of the analyte(s)
 Two modes of column temperature adjustment
 Isothermal
 Temperature of oven remains constant
 Applied for simple mixtures containing substances of similar
volatilities
 Temperature programming
 Achieved by continuously raising the column
temperature during the elution process
 applied for analytes with wide range of volatilities
 Accelerates the elution rate of analytes that, otherwise,
would take a very long time to elute
42
Fig. 2. Effect of temperature in gas chromatogram. a) Isothermal at
45oC. b) Isothermal at 145oC. C) Programmed 30oC to 180oC
e) Detector
 Produces signal(s) characteristic to the substance(s)
eluted from the column
 The sample components, after detection, exit the GC
through the exhaust (vent)

 Requirements:
 High sensitivity (should produce signal for traces of
analytes)
 Wide dynamic linear range
 Operation temperatures up to 400 oC
 Fast response time
 Good stability and reproducibility
 Nondestructive
44
Thermal Conductivity Detector (TCD)
 The heart of the detector is a
heated filament which is cooled by
helium carrier gas.
 Any solute passes across the
filament will not cool it as much as
He and H2 because He and H2 has
the highest thermal conductivity.
 This results in an increase in the
temperature of the filament which
is related to concentration.
 The detector is simple,
nondestructive, and universal but is
not very sensitive and is flow rate
sensitive.
45
46
Flame Ionization Detector (FID)
 This is one of the most sensitive
and reliable, but destructive
detectors.
 Separate two gas cylinders, one
for fuel (H2) and the other for O2
or air are used in the ignition of
the flame of the FID.

 Analytes are ionized

 Ions are collected on a charged
electrode
 The resulting currents is
measured

47
 Sensitive to compounds containing C-C or C-H
bonds

 Weakly sensitive to carbonyl, amine, alcohol, amine

groups

 The FID detector is not responsive to air, water,

carbon disulfide.

 This is an extremely important advantage where

volatile solutes present in water matrix can be easily
analyzed without any pretreatment.
48
 Electron Capture Detector (ECD)

 The mechanism of sensing relies on electronegative

atoms, like halogens, will capture electrons from a b-
emitter (usually 63Ni).
 In absence of halogenated compounds, a high current
signal will be recorded due to high ionization of the
carrier gas (N2)
 while in presence of halogenated compounds the signal
will decrease due to lower nitrogen ionization. 49
 Sensitive to halogen containing compounds and thus has
applications in the detection of pesticides and polychlorinated
biphenyls (Sensitive: 10-15 g/s).

 to electronegative groups (halogens)

 Largely non-destructive

 Insensitive to amines, alcohols and hydrocarbons

 Limited dynamic range (102)

 Mass sensitive detector

50
 Problem
Consider the following compounds: If the compounds in the table
below are run on an non-polar SP GC column, which compound will
have the lowest retention time? Highest?
Compound Boiling point (°C)
Methyl cyclohexane 101
Pentane 36
Octane 126
2,3-Dimethyl octane 165
Heptane 98
Consider the following compounds: If they are run on a polar SP GC
column, which column will have the lowest RT? Highest?
Compound Boiling point (°C)
Propionic acid 141
2-Hexanol 140
Isoamyl acetate 142
3,4-Dimethylheptane 140
3. High Performance of Liquid Chromatography
(HPLC)

Classical LC

 SP is held in a narrow
tube

 The solvent passes

though SP (carrying the
sample) by the action of
gravity under atmospheric
pressure

52
Limitation of classical LC

 The columns are used once

 makes the column packing tedious and expensive
 Packing materials have large particle size (150-250
mm)
 low column efficiency
 MP flow rate cannot be adjusted (gravity action)
 Lengthy operation time
 Manual analysis solutes
 Labor intensive, time consuming

53
 High performance liquid chromatography
(HPLC)
 Developed in the mid 1970’s
 LC with increased efficiency
 Uses small size (3-10 μm) stationary material uniformly
packed in a column of very small diameters (3-5 mm)

 A high pressure is applied to force the MP through the

tightly-packed column
Samples moves smoothly (non-pulsed)

 Columns are reusable

 Flow rate adjustment is possible

54
 55
Block diagram showing components of a typical apparatus for HPLC
1. Reservoirs

 Reservoir is a glass (or stainless steel) bottle which contains

the MP (solvent)
 Mobile phase should be:
 Inert towards the sample components and the SP
 Less viscous (reduces pressure)
 Highly pure (free from impurities and dissolved gases
 Impurities: causes interference in the separation and
detection
 Dissolved gases: form bubbles which interfere in the
pumping of the MP and in the detection of analytes
 Removal of dissolved gases = degassing
 are removed from the solution by bubbling of an
inert gas of low solubility like Helium
56
 MP composition: Isocratic versus gradient elution
 Isocratic elution: performed with a single solvent (or constant
solvent mixture)
Gradient elution: continuous change of solvent composition to
increase MP strength

Improvement in separation effectiveness

of Gradient elution

57
2. Pump
 The quality of a pump is measured by
how steady and reproducible a flow it
can produce. A fluctuating flow
creates detector noise that obscures
weak signals.
 Requirements of pumping system:
 Create non-pulsing solvent flow.
 Flow rates from 0.1-10 mL/min.
 Flow control and flow
reproducibility of a maximum Reciprocating pump (in
error of 0.5%. 90% of systems)
 Corrosion resistant (stainless steel
and Teflon).
 Generation of high pressure up to
58
6000 psi (400 Bar)
 3. Injector
When the valve is rotated 60° counterclockwise, the content of the
sample loop is injected into the column at high pressure.

59
Sample preparation

 In general, the “dilute and shoot” approach can be used for

most liquid samples

 A common process of “grind → extract → dilute → filter” is

used for most solid sample

 More complex samples, such as lotions, creams, and

physiological samples (serum or plasma) might require
additional sample clean-up and extraction such as liquid-liquid
extraction or solid-phase extraction (SPE).

60
4. The Column
 It is made of stainless steel tubing

 Column length is: 10 – 30 cm

 Column Internal diameter is: 4 – 10 mm
 Column packing typically have particle sizes 5
or 10 µm. Column of this type often contain
40000-60000 plates/m
 It is expensive and easily damaged by dust or
particles in the sample or solvent.
Protection:
1. by periodically renewed guard columns
containing the same SP like main column.
2. by passing solvents and samples through
filters (0.5 mm). 61
Van Deempter for HPLC

H ∞ A + Cm
5. Detection
 Requirements of a detector:
 Sensitive to low concentrations of every analyte.
 Small volume to avoid peak broadening.
 Linear response.
 Insensitive to changes in temperature or solvent composition.

63
5.1 UV detector

Use ulteraviolet radiation source in a

flow cell
 Employ deuterium, tungsten or xenon
lamps with monochromators to choose
the optimum λ.

The flow cell

64
5.2. Fluorescence detector

 Fluorescence is measured after

excitation of the eluate with a laser.
 Advantage: very sensitive
 Disadvantage:
response to few analytes.
Derivatization of originally non-
fluorescing analytes by covalent
attachment of fluorophors to analytes
either prior to chromatographic
separation, or by adding derivatization
reagents to the eluate between column
and detector.

65
 Partition Chromatography
 The separation can be performed into two ways based on stationary
phase (normal phase or reverse phase)
 In normal-phase chromatography, the least polar component is eluted
first; increasing the polarity of the mobile phase then decreases the
elution time. In contrast, with reversed-phase chromatography, the most
polar component elutes first, and increasing the mobile phase polarity
increases the elution time.

Bonded-phase packing 66
Problem
Consider the following three compounds
HOCH2CH2OH CH3CH2CH2CH2CH2CH3 C6H5CH3
(ethylene glycol) (hexane) (toluene)
where toluene has a polarity that is in between the two other
compounds.

i) In what order would the compounds elute in normal-phase

HPLC? For a given mobile phase composition, the retention time
of ethylene glycol is 5.6 minutes. Will the retention time increase or
decrease by increasing the polarity of the mobile phase?

ii) In what order would the compounds elute in reversed-phase

HPLC? For a given mobile phase composition, the retention time
of hexane is 5.6 minutes. Will the retention time increase or
decrease by increasing the polarity of the mobile phase? 67
 Adsorption Chromatography

Stationary phase (adsorbents):

 The most common are alumina and silica gel in which the
interactions with solute molecules is due to OH groups present on
their surface.

 More polar molecules are adsorbed more strongly and will elute
more slowly

 Strength of adsorption of polar groups (solutes) on polar support is

in the following order:
-C=C- < O-CH3 < -COOR < >C = O < -CHO < -NH2 < -OH < -COOH
Olefins < Ethers < Esters < Lactones < Aldehydes < Amines < Phenols <
Acids.

68
Applications in separation of natural products
 Alumina: sterols, dyestuffs, vitamins, esters, alkaloids and
inorganic compounds.
 Not used for compounds containing phenolic or carboxylic
groups

 Silica gel: sterols and amino acids

 Carbon: peptides, carbohydrates and amino acids

 Calcium carbonate: carotenoids and xanthophylls

69
Selection of the solid support

The support material should:

 adsorb and retain the stationary phase.
 expose as large surface as possible to the mobile
phase
 be mechanically stable.
 be easy to pack.
 not retard the solvent flow

Examples of solid supports:

Silica gel, diatomaceous earth and cellulose

70
Size-exclusion or gel permeation
chromatography
 Porous polymeric matrix: formed of spongy
particles, with pores completely filled with the
liquid mobile phase (gel).

 The gels (polymers) consist of open, three-

dimensional networks formed by cross-linking of
long polymeric chains.

 The pore size varies with the degree of cross-

linking.

 The diameter of the pores is critical as separation

is based on that molecules above certain size are
totally excluded from the pores because they can
not enter the gel.

 The interior of the pores is reached, partially or 71

wholly, by smaller molecules.
Mobile phase

Mobile phase is a liquid as water or dilute acohol

Separation mechanism

 Based on difference between the solutes molecular

weights.

 Molecules will distribute themselves outside and inside the

pores according to their size.

 Larger are excluded, medium sized enter half-way and

smallest permeate all the way.

72
Applications of GPC to natural products

 Separation of mixture of mono- and polysaccharides.

 Separation of amino acids from peptides and proteins.

 Separation of proteins of different molecular weights.

 Separation of polysaccharides and soluble RNA.

 Separation of myoglobin and haemoglobin.

73
 Ion Exchange Chromatography
 Process by which ions of an electrolyte solution are brought into
contact with an ion exchange resin.

 The ion exchange resin is an insoluble polymer consisting of a

"matrix“ that carries
 fixed charges (not exchangeable)
 mobile active ions "counter ions" which are loosely attached to
the matrix.

 In water, the counter-ions move more or less freely in the framework

and can be replaced by ions of the same sign present in the
surrounding solution.

74
Cation Exchangers
 Active ions (counter ions) are cations.
The polar groups attached to the matrix are acidic
(sulphonic acids, carboxylic acids, phenols, phosphoric acids)

Example: a cation exchanger in the free carboxylic acid

form:

Example: X-COO- H+
 X = Frame work (matrix)
 -COO- = fixed charge (anionic), non-exchangeable
 H+ = counter ion (cation), Exchangeable

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 Regeneration of the resin
 Ion exchange process is generally reversible e.g in the following:

X-2Na+ + Ca2+ 2Cl - → X-Ca2+ + 2Na+ Cl –

 The cation exchanger could be exhausted after exchanging all Na+

for Ca2+

 the exchanger could be regenerated (loaded again with Na+) by

contacting it with excess Na+ ions e.g. a solution of NaCl.

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 Anion Exchangers

 Active ions (counter ions) are anions.

 The polar groups attached to the matrix are tertiary
or quaternary ammonium groups (basic).
 Example: Anion exchanger in the quaternary
ammonium form:
 Example: X-NR3+OH –

 X = Framework (matrix)
 -NR3+ = Fixed charge (cationic), non-exchangeable
 -OH– = counter ion (anion), Exchangeable

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Applications of Ion Exchange Chromatography
 Water softening: Removal of Ca2+, Mg2+ and other multivalent
ions causing hardness of water by filtration through a layer of
strong cation resin.

 Water demineralization: Removal of cations and anions

dissolved in water. Usually carried by the two step technique in
which two columns of strongly acid cation exchanger in [H+] form
and strongly basic anion exchanger in [OH-] form are used in
sequence.

 Neutralization: Cationic exchanger in [H+] can be used to

neutralize alkali hydroxide and anionic exchanger in [OH-] form to
neutralize the acidity.

 Separation of electrolytes from non-electrolytes

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 Affinity Chromatography

 Analytes are separated based on

biochemical interactions (enzyme-
substrate interaction)

 The SP has a biochemical that has

affinity to a specific type of analyte

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