SR.NO TOPIC PAGE.

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1 INTRODUCTION 1

2 PRINCIPLE 2

3 CLASSIFICATION & PROPERTIES 3

4 TYPICAL ION EXCHANGE EXPERIMENT 12

5 EXPERIMENTAL TECHNIQUE 14

6 SEPARATION FACTOR 18

7 FACTORS AFFECTING SEPARATION 21

FACTOR

8 TECHNIQUES 25

9 DETECTION IN ION CHROMATOGRAPHY 30

10 APPLICATIONS 35

1
INTRODUCTION

Ion-exchange can be described as the process of the reversible,

stoichiometric exchange of ions of same charge between a mobile
liquid phase and an insoluble solid (stationary phase).An ion
exchanger is an insoluble material liberating the counter ions
(mobile ions) by electrolytic dissociation. Amongest all the
chromatographic techniques, ion exchange chromatography are
considered to be a very versatile method and is very useful in the
separation of ions of similar properties. It is treated as column
technique because the ion exchange material is normally packed in
the column.

Many natural substances like clays and zeolites (aluminosilicates)

have ion-exchange properties. They were used to purify water since
19th century. However, from the standard point of analytical
chemistry, it was not until 1935 that ion exchange became important
to analytical chemist. At that time, a series of polymeric ion
exchangers were first synthesized by Holmes. These exchangers had
the advantage of high exchange capacities .they were readily
penetrated by solvents, possessed sufficient stability and provided
reproducible response .Now a days, most of the ion-exchange
materials used are synthetic resins.

PRINCIPLE

2
Ion Exchange Chromatography relies on charge-charge interactions between the
protiens in your sample and the charges immobilized on the resin of your
choice.Ion exchange chromatography can be sub divided into cation exchange
chromatography,in which postively charged ions bind to a negatively charged
resin;and anion exchange chromatography,in which the binding ions are
negative,and the immobilized functional group is postive.Once the solutes are
bound,the column is washed to equilibriate it in your starting buffer,which
should of low ionic strength,and then the bound molecules are eluted off using a
gradient of a second buffer,which steadily increases the ionic strength of the
eluent solution.Alternatively,the PH of the eluent buffer can be modified as to
give your protien or the matrix a charge at which they will not interact and your
molecule of interest elutes from the resin.If you know the PH ,you want to run at
and need to decide what type of ion exchange to use paste your protien sequence
into the titration curve generator.If it is negatively charged at the PH you
wish,use an anion exchanger.Ofcourse this means that your protien will be
bindind under the condition you choose.In many cases,it may be more
advantageous to actually select conditions at which your protien will flow through
while the conatminates will bind.This mode of binding is referred to as “flow
through mode”.This is a particularly good mode to use this type of mode to bind
up endotoxins or other highly negatively charged substances well at the same
time relatively simply flowing your protien through the matrix.

Ion Exchange Resins : Classification and

Properties

3
Ion exchange resins are highly ionic, covalently cross linked,
insoluble polyelectrolytes supplied as beads. The beads have either
have a dense internal structure with no discrete pores or a porous
multichanneled structure. They are commonly prepared from styrene
a various level of cross linking agent divinyl benzene, which control
the porosity of the particles. Porous beads can also be made by
adding homopolystrene, which is soluble in precursor beads are post
functionalized to yield the finished resin Acrylic based; ion exchange
resins are also available. These ionic polymers contain two types of
ions, those which are bound within the structure and the oppositely
charged counter ions which are free. The property of ion exchange is
a consequence of Donnan exclusion-when the resins is immersed in
a solution in which it is soluble, the counter ions are mobile and can
be exchanged for other counter ions from the surrounding medium;
ions of the same type of charge as the bound ions do not have free
movement into and out of the polymer. Ion exchange resins have
been classified based on the charge on the exchangeable counter
ion and the ionic strength of the bound ion.

Thus there are four primary types of ion exchange resins:

1. Strong cation exchange resins: containing sulfonic acid group or

the corresponding salts.

2. Weak cation exchange resins: containing carbolic acid groups or

the corresponding salts.

3. Strong anion exchange resins: containing quaternary ammonium

groups. Of the se there are two types :

Type 1: resins contain triakylammmonium chloride or hydroxide

Type 2 : resin contains dialkyl 2-hydroxyethyl ammonium chloride

or hydroxide.

4. Weak anion exchange resins: containing ammonium chloride or

hydroxide.

Additional type of ion exchange resins include blends of cation and

anion exchange resins, called as MIXED BED RESIN.A resin which
contains both an anion and a cation as bound ion are called as
AMPHOLYTIC.

4
Some ion exchange resins are prepared from chelating properties
making them highly selective towards certain ions. In addition to
there use in ion exchange, organic polymer supports, many of which
are based on PS-DVB resins, are being used as polymer catalysts in
the expanding research area involving heterogenization of
homogenous catalysts and as a polymeric supports and reagents in
combinatorial chemistry. The internal structure of the resin beads,
i.e whether micro porous or macroporous,is important in the
selection of an ion exchanger.Macroporous resins with their high
effective surface area, facilitate the ion exchange process. Also they
give access to the exchange sites for larger ions, can be used with
almost any solvent, irrespective of whether it is a good solvent for
the uncrossed linked polymer, and take up the solvent with little or
no change in volume. They make more rigid beads, facilitating ease
of removal from reaction system. In the case of micro porous resins
since they have no discrete pores, solute ions diffuse through the
particle to interact with exchange sites. Despite diffusional
limitations on reaction rates, these resins offer certain advantages:
they are less fragile, requiring less care handling, react faster in
funtionalization and application reactions, and possess loading
capacities. In addition to being functions of bead morphology, the
kinetics of exchange depend on the particle size distribution of resin.
It is enhanced by monodisperse resin.

BASIC REQUIREMENTS OF USEFUL RESINS :

• The resin must be sufficiently crossed linked to have only

negligible solubility.

• In order to permit diffusion of ions through the structure at

constant and finite rate, the resin must be sufficiently
hydrophilie.

• The swollen resin must be denser than water.

• The must be chemically stable.

• It must contain sufficient number of accessible ionic exchange

groups.

CLASSIFICATION OF ION EXCHANGERS :-

5
According to their origin both, organic and inorganic ion exchangers
may be natural or sythetic.For chromatographic purpose organic
synthetic ion exchangers, ion exchangers and ion exchange
derivatives of cellulose, polydetran and agarose are of the greatest
importances.

There are four basic types of resins which are commonly used in the
ion exchange chromatography.

Strongly acidic cation exchange resins – Sulphonate polystyrene

resins belong to this class. They are useful between the ph ranges 1
– 1.4.These are used mainly in the fractionation of cations, inorganic
separation, lanthanides, vitamins, peptides and amino acids.

Weakly acidic cation exchange resins – Carboxylic

polymethacrylate is an example of such type of resins. They are
effective between ph 5 – 14.These is useful in functionation of
cations, biochemical separation, transistion elements, amino acids,
antibiotics and organic base.

Strongly basic anion exchange resins – Quaternary ammonium

polystrene belongs to this class and it is effective between ph 0 and
12.This type of resins are useful in fractionation of
anions,halogens,alkaloids,vitamin b complex, fatty acids etc.

Strongly basic anion exchange resins – phenol formaldehyde

and polyamine polystrene resins belong to this class. They are
effective in the ph range 0-9 and can be used for the fractionation of
the anionic complexes of metals, anions of different
valancies,vitamins and amino acids.

CATION EXCHANGE RESINS : -

Resins in which liable ions are cations are known as cation exchange
resin. Thus a cation exchange resin is a high molecular weight, cross
linked polymer having acid mobile ions or functional groups such as
sulphonic, carboxylic, phenolic etc.In this cation exchanger the
hydrogen ions are mobile and exchangeable with other cations.The
anions remain attached to the resin network a widely used cation
exchange resin is obtained by copolymerization of styrene with little
of divinyl benzene followed by sulphonation.

6
Cation exchange may be sub divided into:

• Strongly acidic.

• Weakly acidic cation exchangers.

A) Strongly acidic cation exchanger behaves like a strong acid

and is almost completely ionized over a wide range of pH;such
ion exchanger can exchange ion rapidly. These resins are
useful for the chromatography of amino acids, rare earths and
other substances. An example is the exchanger containing the
sulphonic solution of NaCl is passed through the resin; the H+
will be replaced by Na+ ion because the sulphonate group has
less attraction for the hydrogen ion than for the sodium ion.
The equilibrium can be represented as :

7
B) A weakly acidic cation exchanger mainly contain carboxylic
group or phenolic.Such as exchanger ionizes in alkaline media
only and should therefore be used at pH greater than 7.The
rate of exchange has found to increase with increase in pH.

ANION EXCHANGE RESINS : -

8
Resins in which ions are anions are known as anion exchange
resins. The anion exchange resin is a high molecular weight, cross
linked polymer containing basic functional group such as amino
groups, quaternary ammonium group, halides group etc.A widely
used anion exchange resin prepared by co-polymerization of
styrene and a little divinyl benzene followed by chromethylation
and interaction with base such as trimethyl amine.

Anion exchanger may be sub divided into:

A) Strongly basic anion exchanger: - With positively charged

quaternary ammonium groups attached to cross linked
polystrene framework belongs to this class. These resins ionize
over the wide range of pH.They are useful in fractionation of
anions, halogens, alkaloids, vitamin B complex etc.

B) Weakly basic anion exchanger: Ionize in acid media only and

should therefore be used at pH less than 7.

AMPHOTERIC:-

9
Amphoteric ion exchanger contains both cation exchanging and
anion exchanging groups in their matrix. These ion exchangers
are capable of forming internal salts which dissociates in contact
with electrolytes and bind both its components. However they can
be easily regenerated with water dipolar ion exchangers; are a
special kind of atmospheric ion exchangers. On the matrix are
bound amino acids which dipoles in aqueous solution. These types
of ion exchangers are suitable for the chromatography of
biopolymer with which the dipole interacts selectively.

10
Resin type Chemical Usual Common Selectivity
constitution form of trade name
purchase Rohm DOWEX
& chemical
Hass

Strongly acidic Sulphonic acid Aryl-SO Amberli- Dowex Ag+>Rb+>

cation exchangers groups attached to te 50w Cs+>R+
the styrene and IR-120 NH4+>H+>
divinylbenzene Li+>Zn2+>
copolymer >Cu+>Ni2
+>Co2+

Weakly acidic Carboxylic acid R-COO-Na- Amberli- --------- H+>>Ag+>

cation exchangers groups attached to te K+>Na+>>
acrylic & IR 50 Fe+>Ba2+
divinylbenzene Ca2+>
copolymer Mg2+

Strongly basic Quaternary Aryl- Amberli- Dowex- I>Phenol

anion exchanger ammonium groups CH2N(CH3)3 te 1 Ic>HSO4-
attached to styrene +Cl- IRA-400 >CIO3-
& divinylbenzene >NO3->
copolymer Ba->CN->
HSO3->NO2-
>Cl-
>HCO3->
HCOO->
Acetate>
OH->F-

Wealky basic Polyalkylamine Aryl-NH(R)2-Cl- Amberli- Dowex Aryl-SO

anion exchanger groups attached to te 3 3h>Citri
styrene & IR-45 c>CrO3>
divinylbenzene co HSO4>ta
polymer Taric>ox
Alic>H3P
O4>HNO**>
HBr>
HCL>H

11
PROPERTIES OF ION EXCHANGE RESINS:-
Ion exchange resin of different origin, structure and components will
have different properties. The most important properties of ion
exchange resin are color, density, mechanical strength, particle
strength andcapacity,selectivity,cross
linking,swelling,porosity,surface area and chemical resistant.

• Cross linking is very important in ion exchange resin, because

it effects swelling and strength of the resin. As the cross linking
in the resin decreases, the resin swelling increases.Divinyl
benzene is the most common employed cross linking joining
the chains together at various position.

• Swelling increases as the cross in resin decreases. Almost all

the ion exchange resin swells when placed in the water. This
probably is due to the hydration of their ions. When the resin is
in the swollen condition, small ions can diffuse in and out. It is
very difficult to maintain electrical neutrality under these
conditions. Thus if Ca2 ion enter inside the resin, and then it is
most essential that the H+ ions must leave the place so that
electrical neutrality is maintained.

• Particle size is also the important factor on which the effective

separation is based Particle size ranges of 50-100 mesh or
100-200 mesh are mostly commonly employed for effective
separation since equilibrium constant.

• Since the equilibrium constant on which the separation

depends is influenced by the temperature changes, the later
also affects the effective separation.

• The degree of ionization at a given pH and the nature and

concentration of the ion in solution are also important. The
effect that the pH of the medium has on the exchange capacity
of all material is significant, when pH increases the exchange
capacity of cation exchanger increases, and that of anion
exchangers decreases and vice-versa.

12
TYPICAL ION EXCHANGE EXPERIMENT
The simplest apparatus for ion exchange analysis consist of a
burette provided with a glass- wool plug or sintered glass disc at the
lower end .A glass- wool pad may be placed at the top of the bed of
resin and the eluting agent is added on top of the bed of resin should
be of small particle size so as to provide a large surface of contact.
In all cases, the diameter of the resin bed should be less than one-
tenth of that of the column. The resin should be stirred with water in
an open beaker for several minutes, any fine particle if present, is
removed by decantation. The resin slurry is then transferred portion-
wise to the column previously filled with water. To ensure the
removal of trapped air bubbles or any particles or an uneven
distribution of resin granules, the column is backwashed before use.
A stream of distilled water or deionised water is run up through the
bed from the bottom at a sufficient flow rate to loosen and suspend
the exchanger granules. The excess of water is drained off. However,
the water level must never fall below the surface of resin throughout
the operation.

Procedure:-

The solution containing a mixture of solutes is first passed

through a column packed with ion exchange resin. The cations in
solution undergo exchange with the hydrogen or any other cation
that may be present in the ion exchanger. The cation which has the
maximum capacity to undergo the exchange process is held further
down the column in the order of their decreasing capacities to
undergo the exchange reaction .Another solution containing a
stronger exchanger such as H+ ions’ is then used as an eluent .HCl
is a good eluent in many cases. It is allowed to percolate slowly
through the column. H+ ions displace the other cations held on the
resin. The eluate i.e. the liquid coming out from the bottom of the
column, contains first the components (cation) which is least firmly
held on the resin. It is followed by other components (cation) in the
order of increasing strength with which they are held by the

13
resin.Thus, the elute coming out last from the bottom of the column
is that which was most firmly held by the resin.

The process of removing absorbed ions is known as elution,

the solution employed for elution is term as eluent, and the solution
resulting from elution is called the eluate.

If the ion exchange column is loaded with several ions of similar

charge and if the concentration of components in successive
portions of the eluate is plotted against the volume of the eluate, an
elution curve is obtained as

14
EXPERIMENTAL TECHNIQUE
There are three ways of performing an ion exchange separation: by
column chromatography, by batch methods, and by expended bed
absorption. This section will mostly deal with column
chromatography.

Column chromatography:

COLUMN:-

Column should be so designed that there should be no hindrance in

the flow of liquid. The liquid is poured on the top of the column and
all operations are carried out in down flow direction. The liquid
moves down the column and comes in contact with the ion exchange
resin and as a result exchange takes place. As the liquid moves,
more the ions are exchanged and finally all the ions in the eluted
solvent are completely exchanged with the resin. It should be noted
that there should be no air bubble in column and the column is not
allowed to drain out.

The column geometry is dependent entirely on the separation factor.

The latter can be improved by increasing the length of the column. It
should be noted that the length of the column can be increased only
upto to certain height, called critical length, beyond which it cannot
be further increased. The diameter of the column depends upon the
material to be separated. Generally the ratio of 10:1 or 100:1
between height and diameter is maintained in most of the
experiments. Columns should b neither too wide nor too narrow in
sizes, otherwise uneven flow of liquid is expected to occur.

PACKING OF THE COLUMN :-

The column is held in a vertical position and the slurry of resin is

then poured into the column. It should be noted that their should be
no air bubble in the column. More over swelling effect are avoided;
otherwisethey will interfere with the flow rate and packing of the
column.The slurry is added in several parts and the reason is allowed

15
to settle down in between each addition. It is also advisable to bring
the resin in equilibrium with solvent before packing the column.

When the packing is complete, the eluent is allowed to pass through

the column, for certain time in order to ensure uniform rate flow over
the whole cross section of the column. Then the level of the liquid is
so adjusted that it remains over the top of the resin bed. Now the ion
exchange resin column is ready for the experiment and the sample
solution to be separated is now poured on top of the resin in the
column by making use of a micropipette or micro syringe.

It has been observed that a high range of exchange has been

affected with a resin of low cross linking, and small particle size. The
rate of exchange has also been the solution.

The column in ion exchange chromatography may be

operated, by elution, frontal analysis and displacement analysis.

COLUMN OPERATION WITH AN ION EXCHANGE RESIN

The column operation is quite better and efficient method than

batch operation .the resin is placed on top of the glass wool plug in a
vertical tube (for most analytical purpose, a burette has been
employed).By passing a sufficiently concentrated solution of the
desired cation through the resin column (or resin bed), a strong
acidic cation exchange resin can easily be converted completely into
the desired ionic form. It should be noted that the liquid level must
always be above the top of the resin bed, otherwise air bubbles
become entrapped and the active surface available for the ion
otherwise air bubbles become entrapped and the active surface
available for the ion exchange is reduced .Thus it is desirable to
place the column at a constant rate(e.g. 3-5 ml per minute per sq.cm
of the resin bed cross section).A complete conversion of sodium
chloride to hydrochloric acid can therefore be achieved by column
operation method, because the solution as it passes through the
column will counter fresh, uniequilibrated layers of resin and
conversion to HCl will be complete.

It should however be noted that the exchange capacity of the

bed must be greater than the amount of sodium ion to be exchanged
.Moreover, the solution of sodium chloride must be sufficiently

16
dilute, otherwise a concentrated solution of HCl will obtained and
equilibrium would not favor the complete exchange of sodium for
hydrogen.

The column becomes exhausted, when it is used for a

prolonged time. The column can be regenerated by passing a
concentrated solution of an appropriate ion.

SAMPLE PREPARATION

Sample concentration:-

The amount of sample which can be applied to column

depends on the dynamic capacity of the ion exchanger and the
degree of the resolution required .For the best resolution it is not
usually advisable to use more than 10-20% of this capacity
(23).Information on the available capacities for the different
exchangers is given in the relevant product sections. Methods for
determining available and dynamic capacities are given later in this
chapter.

Sample composition:-

The ionic composition should be the same as that of the

starting buffer. If it is not, it can be changed by gel filtration on
sephadex G-25 using e.g.Amersham Biosciences Disposable PD-10,
fast Desalting Column hr 10/10 or Hitrap Desalting Columns, dialysis,
diafiltration or possibly by addition of concentrated start buffer.

Sample volume:-

If the ion exchanger is to be developed with the starting buffer

(isocratic elution), the sample volume is important and should be
limited to between 1 and 5%of the bed volume. If however, the ion
exchanger is to be developed with a gradient, starting conditions are

17
normally chosen so that all important substances are adsorbed at
the top of the bed. In this case, the sample mass applied is of far
greater importance than the sample volume. This means that large
volumes of dilute solutions, such as pooled fractions from a
preceding gel filtration step or a cell culture supernatant can be
applied directly to the ion exchanger without prior concentration.

Ion exchange thus serves as a useful means of concentration a

sample in addition to fractionating it. If contaminants are to be
adsorbed, and the component of interest is allowed to pass straight
through, then the sample volume is less important than the amount
of contaminant which is present .Under these conditions there will be
no concentration of the purified component, rather some degree of
dilution due to diffusion.

Sample viscosity :-

The viscosity may limit the quantity of sample that can be

applied to column .a high sample viscosity causes instability of the
zone and an irregular flow pattern. The critical variable is the
viscosity of the sample relative to the eluent. A rule of thumb is to
use 4 cp approximately 5% .Approximate relative viscosities can be
quickly estimated by comparing emptying times from a pipette .if
the sample is too viscous, due to high solute concentration, it can be
diluted with start buffer. High viscosity due to nucleic acid
contaminants can be alleviated by precipitation with a poly-cationic
macromolecule such as polyethylene mine or protamine
sulphate.Nucleic acid viscosity can also be reduced by digestion with
endo nuclease. Such additives may however be less attractive in an
industrial process since they will have to be proven absent from the
final product.

Sample preparation:-

In all forms of chromatography, good resolution and long

column life time depend on the sample being free from particulate
matter. It is important that “dirty” samples are cleaned by filtration

18
or centrifugation before being applied to the column. This
requirement is particularly crucial when working with small particle
matrices, such as Minibeads(3µm), MonoBeads(10µm),SOURCE(15
and 30µm) and sepharose high performance (34µm).The “grade” of
filter required for sample preparation depends on the particle size of
the ion exchange matrix which will be used. Samples which are to be
separated on a 90 µm medium can be filtered using a 1µm filter. For
3,10,15,30 and 34µm media, samples should be filtered through a
0.45µm filter. When sterile filtration or extract clean samples are
required, a 0.22µm filter is appropriate. Samples should be clear
after filtration and free from visible contamination by lipids. If turbid
solutions are injected onto the column, the column lifetime,
resolution and capacity can be reduced. Centrifugation at 10000g for
15 minutes can also be used to prepare samples. This is not the
ideal method of sample preparation but may be appropriate if
samples are of very small volume or adsorb nonspecifically to filters.

SEPARATION FACTOR:-
Ion exchange chromatography is based on the exchange of ions
between a solid ion exchanger and ions present in the solution. The
exchange of ions obey the law of mass action. For example, the
equilibrium between the ion exchanger and the solution can be
represented as,

19
The conditional ion exchange equilibrium constant in the first
approximation can be expressed as

[B-] / [A-] = KAB [B+] / [A+]

Where [B-] and [A-] are the concentrations of the ions in the solid
phase and [B+] and [A+] are the concentrations of ions in the liquid
phase. KAB is conditional ion exchange equilibrium constant. It should
be noted that the sorption of ions depend on the following factors:

1) Nature and structure of the ion exchanger.

2) Nature of analyzed substances.
3) Experimental conditions such as pH, temperature, etc.

It should also be noted that sorption is physiochemical process

resulting in the absorption of gases, vapors, radiations and solutes
by a substance form the surrounding medium.

Sorption also includes, i.e., sorption on the interface, absorption i.e.

sorption by the bulk of the sorbent and other process.

For dilute solutions, the activities may be replaced by the

concentration of exchanging ions in solution, because activity
coefficients are equal to unity. The activities of ions in an ion
exchanger may also be replaced by the concentrations if the ratio of
activities of ions to powers corresponding to the reciprocal of the
ionic charge is assumed to be a constant quantity. Thus if ion
exchange proceeds according to the equation,

Z2M1AZ1 + Z1M2Z2+ Z1M2AZ1 + Z2M1Z1+

Where Z1 and Z2 = Charges of exchanging ions.

20
 M1 and M2 = First and second exchanging ions of metals

A = Anion of resin

Applying law of mass action

K = [M2AZ2] Z1
[M1Z1+] Z2

[M1AZ1]Z2 [M2Z2+] Z1

OR

[M2AZ2]1/Z2 = Kc C21/ Z2

[M1AZ1]1/Z1 C11/ Z1 .…(5)

Where C1 and C2 are the concentrations of exchanging ion in

solution and KC is concentration exchange constant which depends
on temperature and the chemical nature (the charge, and size of
ions and their hydration) of exchanging ions and ions exchanger. It
characterizes the strength of incorporation of a given ion in the
lattice of an ion is the change of the cation, and it increases with
atomic number of the element involved.

Equations (5) is known as Nikolsky equation and is valid if the

volume of the ion exchanger does not change in the course of an
experiment, the ions in the solution do not react with the ion
exchange radical, molecular and ionic sorption is absent, and the
ions to be separated freely permeate into an exchanger.

Static exchange capacity may be defined as the number of mg

equivalent of the ion adsorbed per a certain time interval by 1g of
the dry ion exchanger. The thermodynamic exchange capacity may
be defined as the number of ions adsorbed by a layer of ion
exchanger 20cm high and 1 sq. cm in the cross section at the rate of
migration of 0.5 dm2 per hour.

21
The adsorptive effect of an ion is characterized by the DISTRIBUTION
COEFFICIENTS.

D = Cr.m

Cs.V

Where Cr and Cs are the equilibrium concentrations of ions in the

respective phases and m is the mass of the exchanger in grams. V is
the volume of aqueous phase in cm3.

The SEPARATION FACTOR α is the ratio of the distribution

coefficients of the two components (for ions of like charge).Thus

α = DA

DB

Where DA and DB are the distribution coefficients of two components

respectively.

FACTORS AFFECTING SEPARATION FACTOR :-

Ion exchange is a physio-chemical process. Hence both chemical and
purely physical variable influence the separation factor.

Chemical variable includes:

• pH

• Nature of ions being separated

• Concentration of ions in solution

22
 • Tendency of ion to hydrate

• Chemical composition of the exchanger etc.

The pH effect on ion exchanging process depends upon the chemical

composition of the exchanger as well as on the type of exchange.
The exchange capacity of a cation exchanger generally increases in
pH of the solution, while that of an anion exchanger is decreased. In
case an ion exchanger contains weak acid groups such as – COOH,-
OH etc, an increase in pH cause an increase in the sorption capacity.
In the case of an ion exchanger who contains strong acidic groups
such as –SO3 H the sorption capacity remains constant over a wide
range of pH.Sorbability of ions increase in their charges, atomic
masses and ionic radii.

The physical variables include:

• Rate flow of solution in the column

• Size of exchanger grains

• The height of the column

• Temperature of the solution etc.

Optimum separation is effected by selecting an appropriate amount

of an ion exchanger. It is possible to calculate the value of the ratio
of the exchanger mass to the volume of the analyzed solution in cm3
i.e. m/V provided the distribution coefficient D and the capacity
alpha of the ion exchanger is known.

Ion exchange constant vary with the cations involved and are
markedly different from unity. The rates of the movements of the
ions through a layer of an ion exchanger are inversely proportional
to the values of ion exchange constant. In this way, a number of
mixtures of a complex composition can be separated into their
components.

In displacement elution, the extraction of the ions by an ion

exchange is affected with the help of a solution containing ions of a
higher ion exchange constant. These ions displace the ions absorbed
by the resin which pass into the solution. Instead of ions with a
higher exchange constant, it is better to use ions with a lower

23
constant but present in a high concentration. Example of such ion is
H+ ions. For this purpose HCL solution are commonly employed. It
should be noted that efficient separation of ion can be achieved only
when their is a considerable difference in ion exchange constants.

Hydrated ions with a similar radius have a greater charge have

higher distribution coefficient, and therefore similar elements are
eluted from the column in the following sequence.

Li Na k Rb Cs

Mg Ca Sr Ba

Lu Yb Tu Er Ho Dy Tb Gb Eu Sm
Pm

A considerable more complete separation can be achieved in

complex forming elution. In this case,desorption can be
accomplished with the help of a solution of a compound which
together with the cation absorbed by the resin, form a complex ion
bearing a charge of opposite sign. For example in the desorption of
the cations of rare earth elements by a citrate solution, the cation
pass onto solution as the complex ion.

Better separation takes place if there is equilibrium between the ions

in the solution and on the ion exchange resin. An increase in the
surface area of the ion exchanger also improves the condition for
attainment of an equilibrium and separation. For good separation to
be ensured, the solution must be passed slowly through the ion
exchange resin. The rate of attainment of equilibrium is also affected
by the temperature. A rise in temperature in many cases improves
the result of separation. The temperature, however, changes also
the equilibrium constant of the exchange, which may either improve
of the impair the separation condition.

The separation is also influenced by the composition of cation

exchange resin. Resins with high content of divinyl benzene have a
high swelling ability and ion exchange capacity.

The length of column also plays an important role in separation

process. For the separation to proceed more rapidly, the columns are
expected to be short. Short columns are used for separation of

24
anions and cations.Columns of greater length is needed for
separation of cations.

CHOICE OF BUFFER : -
As with the choice of an ion exchanger, there are a number of
variables which have to be considered. These include:

1. The choice of buffer pH and ionic strength.

2. The choice of buffering substances.

3. The price of the buffer if it is to be used in production process.

BUFFERS : -

Choice of buffer pH and ionic strength

The choice of the buffer pH has been discussed in the previous

section. It should be pointed out, however that in many application
the optimum separation may be achieved by choosing conditions so
that major and troublesome contaminants are bound to be
exchanger while the substance of the interest is eluted during the
wash phase. This procedure is sometimes referred to as “starting
state elution”.

The highest ionic strength which permits binding of the selected

substances and the lowest ionic strength that causes their elution
should normally be used as starting and final ionic strengths in
subsequent column experiments. A third and higher ionic strength
buffer ionic strength buffer is frequently employed as a wash step
before column regeneration and re use.

The required concentration of the start buffer will vary depending on

the nature of the buffering substances. In the majority of cases a

25
starting ionic strength of at least 10mM is required to ensure
adequate buffering capacity. Salt also play a role in stabilizing
protein structures in solution and so it is important that the ionic
strength should not be so low that protein denaturation or
precipitation occur. A major advantage of using Amersham
Bioscience ion exchangers is that they have excellent capacities and
so the initial ionic strength of the buffer can be quiet high with out
significant affecting capacity for sample 77

In case of prepacked ion packed ion exchangers and columns, which

can be run conveniently quickly, trial experiments using salt
gradients with allow the determination of an optimal starting ionic
strength

In the case of sephadex based exchanges for batch application or

where column running times are prohibitively long a simple test tube
technique is recommendation as a test for a suitable ionic strength.

Choice of buffer substances

If the buffering ions carry a charge opposite to that of the functional

groups of the ion exchanger they will take part in the ion exchange
process and cause local disturbances in pH, it is preferable,
therefore to use buffering ions with the same charge sign as the
substituent groups on the ion exchanger. There are of course
exceptions to this rule as illustrated by the frequency with which
phosphate buffers are cited in the literature in connection with anion
exchangers. In those instances when a buffering ion which interacts
with the ionic groups on the matrix is used, extra care must be taken
to ensure that the system has come to equilibrium before application
of sample.

TECHNIQUES
Recent advances in ion chromatography are mostly related to highly
selective separation and to extremely low detection limits. Through
the choice of stationary phase and the variation of eluent
composition, higher separation selectively can be obatined.Analyzing
the samples in which analytes are presented at low concentration in
the presence of high ionic strength or extreme pH matrix, the major

26
matrix ions would displace the adsorbed analyte ions on the
stationary phase. The result would be band broadening and poor
separation because the extreme pH of sample causes disturbance to
the equilibria in the eluent.Therefore, technique relating to sample
preparation and separation need to make progress.

Both suppressed and non-suppressed detection modes have been

applied to the analysis of samples.However, the most common mode
of detection for ion chromatography is suppressed conductivity in
which a suppressor is used to reduce the background conductivity of
the eluent and to increase the detection signal of analyte ions. The
suppressor is a cation or anion- exchanger, installed after the
separation column to replace the counter ions with H+ and OH-,to
change the highly ionized eluent ions into weakly ionized species.
Progress in the area of continous electrochemically regenerated
devices associated with suppressed conductivity Recent detection
has also been made.

ION CHROMATOGRAPHY BASED ON SUPPRESSORS :-

The first used suppressor was an ion exchange column in the form of
H+ or OH-,2 but this technique is restricted by the limited capacity
of the suppressor, which must be regenerated periodically off-line.
This problem has been overcome with the introduction of the hollow-
fiber membrane suppressor.However; a problem of this kind of
suppressor is the low surface area of the fiber, resulting in low ion
exchange and suppression capacity. The appearance of the micro
membrane suppressor, in which two flats of membrane were
inserted into three ion exchange screens as a sandwich, solved this
problem.

Electrolytic membrane suppressors

The suppressor mentioned above employs diffusion alone for ion

transfer. More recently, an electric membrane based
suppressor.This,a “Self Regenerating Suppressor” (SRS) has a
design similar to that of the micro membrane supressor

27
were used to allow the generation of H+ or OH- necessary from
water. In this device, no external regenerate is needed. In analysis,
an anion SRS is used and H+ ions produced from anode migrate
across the cation-exchange membrane to transfer OH- ions into
water and meanwhile counter-cations in the eluent are driven by
electric field to cathode. Gas waste from electrolysis reaction and
liquid waste will move out of the regenerant chamber.Cation analysis
will operate in the same way except that OH- instead of H+ will
migrate across the anion exchange membrane.

SRS can be performed in a couple of ways: “recycle mode” and

“external water mode ”.The “recycle mode” is operated in the
manner in which effluent from the conductivity detector servers as
the source of the water required for electrolysis. While an external
source of water is supplied to the regenerant chamber of electrolysis
in an “external water mode”. The performance difference in between
them comes from the fact that higher flow rate of external water
employed in “external water mode” would result in a lower noise and
better sensitivity.Upto now the “recycle mode” is the most
commonly used operated mode for most samples because of its ease
to operate.Recently,it was found that the problem of low sensitivity

28
could be circumvented if an inert gas was used to supplement the
effluent flow from detector. This mode called “gas assisted recycle
mode” speeds up the removal of eluent counter-ions and electrolysis
products, hence leads to a performance comparable to “external
water mode”.

“Packed column mini suppressor ”

“Solid phase chemical suppression” employs a valve configuration

and two suppressor catridges.The devices offers no significant
improvements because the cat ridge needs to be regenerated off-
line from time to time. A modified device,” Electrochemically
Regenerated Ion Suppression”(ERIS) was developed in which two
solid-phase electrochemical suppressor cells replaced the
catridges.Suppressor cells are composed of cation exchange resins
or anion exchange resins with electrodes being applied. At the start,
eluent from the separation column enter into one cell, and normal
suppression reaction occurs in this cell, and the effluent from the
detector cell will flow into the second cell, where regeneration of ion
exchange resins take place as water is electrolyzed. The
performance of this device is equivalent to that of self regenerated
suppressor. It has an advantage over membrane-based suppressors
in that it can withstand high back-pressures.

“Continuously regenerated packed-column suppressor”

A newer design of packed column suppressors has been developed,

which combines the benefits of both electrolytic SRS suppressor and
packed column mini-suppressors. The mechanism of this device is
based on the principles of “ion reflux”, in which electrically polarized
resins are used with water as an eluent to regenerate the required
H+ ions. In anion analysis. the effluent from the separation column is
introduced in the opposite flow direction of H+ ions generated at the
anode, hence the counter cation in the eluent are displaced by the
continous H+ ions and driven towards the cathode, where they
combine with OH- ions generated at the cathode. Because the H+
ions would not be depleted as long as the electrolysis reaction

29
occurs, the suppression capacity of this device is higher and
background.

SINGLE COLUMN ION CHROMATOGRAPHY :

Recently, commercial ion chromatography instrumentation that

requires no suppressor column has become available. This approach
depends on the small difference in conductivity between sample ions
and the prevailing eluent ions. To amplify these differences, low
capacity exchangers are used that permit elution with solution with
low electrolyte concentration. The typical eluent used in
nonsuppressed IC are phthalic acid and p-hydroxybenzoic acid for
the determination of anions and the methanesulphonic acid for the
determination of cations.The equivalent conductance values of
chloride, sulphate and other common anions significantly greater
than that of eluent and their for a positive peak is detected as the

30
anions are carried through the detector. The equivalent conductive
value of sodium, potassium, calcium, magnesium and other common
cations are significantly lower than that of the cations (H-) in the
eluent.In this instance a negative peak is detected as the cations are
carried through the detected.

Non suppressed IC is easier to perform, and it is a technique for

determining ions of weak acids such as cyanide and sulfide, which
are non conducting after chemical suppression but show a higher
baseline noise pharmaceutical analyses can be performed in the non
suppressed mode because the quantification limits are usually in the
upper mg per L to low percentage levels.

DETECTION IN ION CHROMATOGRAPHY

The following detection methods are available with ion-exchange
chromatography:

1. Conductivity detection.

2. Electrochemical detection

3. Potentiometric detection.

4. Spectroscopic detection.

5. Post column reaction detection.

 Conductivity detection :-

Conductivity detection has two major advantages for inorganic

ion analysis. First all the ions are electrically conducting, so
that the detector should be universal in response, and second,
the detectors are relatively simple to construct and operate.
Conductivity detection will be discussed here in terms of the

31
principle of operation and performance characteristics, modes
of detection, cell design post column signal enhancement i.e.
suppression and applications.

Principle of operation:

The mobile phase eluting through the detector is in fact a

conducting electrolyte. It flows through two electrodes across
which potential is applied. The more current conducted by the
solution, the higher is the electrical conductivity. The
conductance of a solution is determined by several factors,
including the ionic strength and the type of species in the
solution, as well as the temperature. The specific conductance
depends on the cross sectional area (cm2) of the electrodes
inserted into the solution, and L(cm) is the distance between
them, and will vary with concentration. The conductance is
increased for cells in which the electrodes are large in surface
area and are close together. The equivalent conductance is
subject to activity effects such as ion-ion interactions,
therefore the relationship between G and C becomes non-
linear at high ionic strength. Since the conductance of the
solution results from both the anions and cations of the
electrolyte, conductance is calculated for individual anions and
cations in the solution. Most of the common anions and cations
have limiting equivalent ionic conductance of 30-100.The most
conducting cation is the hydronium ion and the most
conducting anion is the hydroxyl ions; their values are 350 and
198 respectively. The conductance of an ion increases with its
charge density and decreases with its viscosity. Therefore
when stroelutropic multiply charged ions are needed in the
mobile phase they can exert high background, therefore, large
ions such as phthalate, citrate, or trimesate are used in such
cases.

Sensitivity detection can result as long as there is a

considerable difference in the ionic conductance of the solute
and the mobile phase ion’s. This difference can be positive or
negative, depending on whether the eluent ions are strongly
or weakly conducting. If the ionic conductance of the eluent

32
ions is low, then an increase in conductance occurs when the
solute enters the detections cell, due to higher conductance.
In general this detection mode is referred to as direct. On the
other hand, when the mobile phase ions are highly conducting,
a decrease in conductance occurs when the solute enters the
detection cell, due to lower conductance. This mode of
detection is referred to as indirect.

Direct conductivity detection is used for most IC methods

involving the separation of anions.Eluent for non-suppressed
IC formed from the salts such as potassium hydrogen
phthalate or sodium benzoate contain competing anions with
moderately low conductance.Similarly,direct conductivity
detection is possible with eluents containing with organic
bases. Indirect conductivity detection can be applied to anions
using hydroxide eluents and to cation using mineral acid
eluents.

Electrochemical detection :-

The term “electrochemical detection” is applied loosely to

describe a range of detection technique involving the
application of electric oxidation-reduction potential via
suitable electrodes to sample solution, containing oxidizable or
reducible solutes. The resulting current is measured as
function of time. Electrochemical detection has been applied
in situation where extreme sensitivity is required. Most
commonly the electrochemical detector has been operated in
tandem with a conductivity detector, which acts as a universal
detector that gives a more general sample analysis.

Voltametery:-

33
 Voltametery is a well-established technique in which a
changing potential is applied to a working electrode with
respect to a reference electrode. The current resulting from
the reaction of analyzed species at the working electrode is
measured. The key factor is that the applied potential is varied
over the course of the measurement.

Amperometry and coulometry :

The term amperometry describes the technique in which a

fixed potential is applied to a working electrode with respect
to a reference electrode. The working electrode is located in
the flow-cell through which the mobile phase passes and the
current resulting from the oxidation –reduction reactions
occurring at the working electrode is measured. The analyte to
be detected undergoes a Faradaic reaction if the applied
potential has appropriate polarity and magnitude.When the
reaction is incomplete, causing only a fraction of the total
analyte to react, the detection mode is termed
amperometry,while when the working electrode has larger
surface area and the reaction is complete the mod is called
coulometry.

Spectroscopic Methods :-

Spectroscopic methods of detection are very common in ion

chromatography and are second in their abundance. This
mode of detection can be divided to two major categories:
molecular and atomic spectroscopy. Molecular spectroscopy
includes methods such as UV-IS absorption, refractive index,
fluorescence and phosphorescence. Atomic spectroscopy
includes flame atomic absorption, flame atomic emission and
plasma atomic emission.

Molecular Spectroscopy :-

34
a) UV-VIS Absorption :

Many inorganic cations and anions do not have significant

absorption in the UV-VIS range of the spectrum, therefore the
direct detection cannot be used typically. However their are
areas where ions can be detected directly by their UV or visible
detection in the 185-220 nm range.

Detection of non absorbing ion can be achieved using indirect

photometric mode, similarly to the indirect conductivity mode
of detection. In the indirect mode highly absorbing ionic
species are used as the eluents with high background, and the
detector response is zeroed on them. The non absorbing
solutes are detected as negative peaks, since the detector
measures the difference of absorption between the high
background and the nonabsorbing species. The polarity of the
detector can be then reversed and the peak appear positive
.Benzenepolycarboxylic acid salts, such as
phthalate,benzoate,phenylphosphonate,p-toluenesulfonate or
trimellitate are used as chromophoric eluent anions that
enable the sensitive detection of union UV-VIS absorbing ion.

b) Fluorescence:

Fluorescence detection is well known for its sensitivity. Since

most of the ionic species analyzed by the ion chromatography
do not exhibit fluorescence, direct mode of detection has only
a limited scope. Usually the mobile phase includes a chelate or
an ion pair reagent that forms a species with the ions that
produces a signal in the fluorescence detector. It is more likely
to find works that utilize the indirect mode of fluorescence
detection.

c) Refractive index(RI):

Most of the solutes for ion chromatography is used normally

are not detectable directly by refractive index detectors. The
general exceptions are carboxylic acids, large species such as
polyphosphonates or sulphonium ions and some inorganic ions.
In cases where the ions cannot be detected directly by the RI
detector, an indirect mode of detection was used.

35
Atomic spectroscopy :- The combination of HPLC separation with
various forms of atomic spectrometry gives a method of great
sensitivity as well as a time resolved detection of species.

a. Flame atomic absorption (AA) and atomic emission (AE).

Direct coupling of atomic absorption spectrometer to an HLPC

system requires means to match the flow rates of the two
techniques. The output of the IC system needs to be relatively high
to accommodate the atomic absorption instrument; therefore, pure
water is added some times as a “make up” solvent.

b. Inductively coupled plasma(ICP)

ICP with emission spectroscopy or with mass spectroscopy have

emerged as a replacement to emission spectrometers and act as
detectors for ion chromatography in recent years. The
introduction of HPLC coupled directly to ICP MS led to the used of
these properties in speciation analysis.

The coupling of ion chromatography(IC) with ICP MS made

possible the elimination of gram amounts of matrix in cases
where it could be converted into an anion form, so that ultra-trace
amounts of the cationic impurities could be determined. In the
semiconductor field, such analyses have been carried out on
matrices of Mo, W, Re, As and P.

Post column reaction

Detection by post-column reaction (PCR) involves the chemical

reaction of the solutes as they elute from the column on the fly,
prior to their introduction to the detector. The main goal of such a
procedure is to enhance selectivity and specificity to solutes of
small quantities in the present of large quantities of interferences
in the sample matrix.

Some of the post column reagents are ammonium molybdate,4-

(2-pyridylazo) resorcinol, pyridine-2, 6-dicarboxylic
acid,phenylfluorone,2-(5-bromo-2-pyridylazo)-5-(diethyl
amino)phenol.

36
APPLICATIONS OF ION EXCHANGE CHROMATOGRAPHY :-

Ion exchange chromatography has proved to be excellent tool for

solving many complicated problems in the field of biology, organic
and inorganic chemistry. In biological field, it has especially been
used in the separation of hydrolyze products of nucleic acid. In
organic chemistry, a number of separations are based on ion
exchange. Among these are the separations of acids, amino acids
peptides and nucleotides etc.In organic chemistry, it used in the
separation of rare earth etc.Some important applications of ion
exchange chromatography are briefly described below:

1) Demineralization of water:-

Ion exchange resins have been used for the complete

demineralization of water for chemical use. Water is first allowed
to pass through a cation exchanger, which removes the anion
replacing them with the exact amount of OH- ions necessary to
neutralize the acid from the first operation.

Thus if water is passed through two column, the first containing

cation exchange resin in the hydrogen form and the second anion
exchange resin in the hydroxide form (M+) and anions(X-),the
reactions are :

The resulting water is as pure as distilled water and is far les

expensive. Colloidal material of course not recovered. Water
purification by this method in comparison to distillation has two main
advantages,

The ion exchange procedure as cheaper as well as faster than

distillation in the laboratory.

37
 2) Determination of sodium and potassium in the mixture :-
According to beulankamp and Reimen,the separation of potassium
and sodium ions in a mixture can be done by introducing the sample
at the top of a containing a sulphonic acid resin saturated with H+
ions. The column is then eluted with a solution of 0.7M HCl at a flow
rate of 0.6mL/sq.min.Sodium ions, being the less strongly held, move
down the column more rapidly and can then be collected before
potassium ions appear in the effluent. The solutions are evaporated
to dryness and the alkali chlorides weighed. Divalent ions, if present
do not come out of the cation exchange resin until all the potassium
ions have appeared in the effluent.Partion of the sodium ions in each
theoretical plate column involves,

The potassium ion behaves in a similar way but is partition

coefficient is numerically larger. The alkali chlorides are then
redissolved and analyzed by titration of the Cl- ion by the Mohr
method.

3) Separation of transition metals: - Kraus and Moore have

reported the separation of transition metals by introducing the
sample at the top of the column containing a strongly basic anion
exchange resin in the chloride form. In this process stepwise rather
than continuous change in the eluent was effected. The column was
initially filed with 12M HCl and the sample was introduced at the

38
top.Elutionwas carried out by successively more dilute solution of
HCl.The Ni was not retained at all even in the presence of
concentrated HCl.When the HCl was diluted to 6M,the acid caused
the elution of Mn;Co came out at 4M,Cu at 2.5M,and Fe at 0.5M and
Zn at 0.0005M HCl.This type of elution indicates the relative
stabilities of the complex chloride anions and varying affinity of the
resin and of water for these ions as chloride ions.

4) Separation of interfering ions of opposite charge: - Ion

exchange resin have also been used for the removal of interfering
ions, particularly where the ions have a charge opposite to the
species being determined. For example,Fe,AI and other cations
cause interference in the determination of sulphate,because of their
tendency to co precipitate with the barium sulphate.The solution to
be analyzed is passed through a column containing a cation
exchange resin as a result of which all cation are retained and
corresponding number of protons(H+) are liberated. The sulphate
ion passes unhindered through the column and its analysis can be
performed on the effluent. Similarly phosphate ion, which interferes
in the analysis of Ba++ or Ca++,can be removed by passing the
sample solution through a column containing an anion exchange
resin. Ion exchange chromatography has also beer used to remove
PO4 from the solution in which cations are being determined by the
acid-alkali scheme of analysis or M3+ is being determined
quantitatively in the form of hydroxide; to remove trivalent metal
from a solution where sulphate sulphur is determined by gravimetric
technique technique, and so on. In the first case, when the solution
is present through the cation exchanger, the cations are
sorbed,while the phosphate ion remains in the solution. For the
subsequent removal of ion exchange to the left, the cation
exchanger again passes over into H+ form and the cations enter the
solution containing no PO4 ions. While determining SO4 ions of
trivalent metals are separated by passing the solution through
strongly acidic cation exchanger H2SO4 remains in the solution,
where it is determined.

(5) Total conten5t of cation in a solution: - T he stoichiometric

nature of ion exchange resin allows us to find the total content of
cations in a solution by titrating an amount of protons equivalent to
them and obtained by reaction of the cation exchanger and the
solution being analyzed.

39
(6)Concentration of traces of an electrolyte:- Concentration of
traces of an ion form a very dilute solution can also be carried by
making use of ion exchanger resins. For example, traces of metallic
elements can be collected from larger volumes of natural waters by
making use of cation exchange resins. The resin is first treated with
HCl to liberate the ions .As a result the solution becomes more
concentrated for further analysis.

(7)Conversion of salts to acids or bases:- The total salt content

of a sample can also be determined by using ion exchanger resins.
When the sample solution is passed through a cation exchange resin
in the acid form, the cations present in the sample are absorbed or
retained by the exchanger and an equivalent quantity of H+ ions is
released .These can be collected in the washing from the column
and then titrated.

Similarly, a standard solution of an acid can be prepared

from a salt. For example, acid form of a cation exchange resin can
be titrated against a weighed quantity of solution of chloride. The
salt liberates an equivalent quantity of HCl, which is collected in
washings and diluted to a known volume .In an analogous manner;
the OH- ions are liberated when the salt is treated with an anion
exchange resin. In addition to above applications, ion exchange
chromatography is an extremely valuable tool in the separation of
complex mixture of compounds of biological interest .For example,
Scott and his coworkers have resolved peaks corresponding to more
than 100 constituents of urine. They used gradient elution with
parallel column of anion and cation exchange resins. Forty six amino
acids have been separated using ion exchange chromatography. The
peaks have been observed with a photometric detector.

(8) Separation of amphoteric metals from non-amphoteric

metals:- Ion exchange chromatography has successfully been used
in the separation of constituents of a mixture, separation of cations
from anions ,isolation of cations ,isolation of anions etc.In these
cases amphoterism, complex formation and regulation of acidity of
solutions are frequently used. For example, when non-amphoteric
metals such as Fe, Cu etc are separated from amphoteric metals
such as Zn,Sn,Al etc,the mixture is passed through a column packed
with a cation exchanger and then washed with an alkali solution. The
non-amphoteric metals are adsorbed by the cation exchanger as

40
hydroxides while the amphoteric metals are eluted as AlO 2- ,Zn2- ions
etc.

(9) Separation of metals, alloys and high alloy steels :-Pure

metals, alloys and high alloy steels can be analyzed using
complexation processes. The method is based on different stability
of the complexes formed at a definite pH value. For example,
addition of HCl to a mixture of Cu2+,Zn2+,Cd2+,PB2+and Bi3+ ions
produces the chloride complexes [CuCl4], [CdCl4], [PbCl3] - and
[BiCl4]-.Their stability increases from copper to bismuth. The
solutions obtained are percolated through a column packed with an
anion exchanger, which adsorbs all the complexes. The metals are
then eluted successively with dilute HCl, water and nitric acid,2N HCl
elutes copper,6NHCl elutes zinc,0.3N HCl elutes cadmium, lead is
eluted with water and bismuth id eluted with HNO3.

(10) Separation of substances processing related properties:-

Ion exchange chromatography has also been used for the separation
of substances processing related properties such as cations of alkali
and alkaline earth metals, rare earth and transuranium elements,
twin elements such as zirconium and hafnium, cis-trans isomeric
complexes of cobalt and platinum. It is only this method that is
applied to the quantitative separation of copper from Cu-Fe alloys
with the iron content below 50%.

(11)Analysis of natural and industrial waters:- Ion exchange

chromatography is an efficient concentration technique often used in
the analysis of natural and industrial waters for their content of
heavy metals.

(12) Separation of complex mixtures of biochemical

compounds:- Ion exchange chromatography is an extremely
valuable tool in the separation of complex mixtures of compounds of
biochemical interest. Scott and coworkers have resolved peaks
corresponding to over 100 constituents of urine using parallel
columns of anion and cation exchanger, with the gradient elution.

Peaks in an ion exchange chromatogram of mixed amino acids,

observed with a photometric detector. Complete special purpose

41
chromatographs for the analysis of amino acid mixtures are also
available commercially.

(13) Production of analytical concentrates:- The concentrations

of substances can be increased 200-500 fold by percolating large
volumes of dilute solutions through an ion exchanger layer and
subsequent extraction of the adsorbed substances with a small
amount of solvent .This method is generally used in the separation
of non –ferrous metals in the production of rare
elements,uranium,radioactive isotopes etc .

(14) Identification of ions: - The selectivity of colorless ion

exchangers can be used to detect colored ions in a mixture.
Depending on their sorpitivity, the ions are distributed in the cation
exchanger by zones, namely, the lower the sorptivity of a cation, the
lower in the column will its zone be. The separation of a mixture into
different colored zones.

REFERENCES
Instrumental methods of chemical analysis : By B.K.Sharma.

Pathways of analytical chemistry :- By Kelkar,Mishra.

Principles of instrumental analysis :- By Skoog.

Basic concepts of analytical chemistry :- By Khopkar

42