Paper No: 2Analytical Chemistry

Module: 38 Ion exchange chromatography

Development Team
Principal Investigator
Prof. R.K. Kohli
&
Prof. V.K. Garg &Prof.Ashok Dhawan
Co- Principal Investigator
Central University of Punjab, Bathinda
Dr. J. N. Babu,
Paper Coordinator
Central University of Punjab, Bathinda
Dr. Varinder Kaur
Content Writer Department of Chemistry,
Panjab University, Chandigarh
Content Reviewer Prof. Ashok Kumar
Punjabi University, Patiala

Anchor Institute Central University of Punjab

Analytical Chemistry
Environmental
Sciences Ion exchange chromatography
 Description of Module

Subject Name Environmental Sciences

Paper Name Analytical Chemistry

Module
Ion exchange chromatography
Name/Title
Module Id EVS/AC-II/09

Pre-requisites

Objectives

Keywords

Analytical Chemistry
Environmental
Sciences Ion exchange chromatography
Module 38: Ion exchange chromatography
Objectives: To study the basics of ion exchange chromatography and know the following
questions.
1. What is ion exchange chromatography?
2. How it works?
3. What are the ion exchangers?
4. Where ion exchange chromatography is applied?

Analytical Chemistry
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 MODULE 38 ION EXCHANGE CHROMATOGRAPHY

1. Description

It is the chromatographic techniques, which is used to separate the charged molecules such as ions,
large proteins, nucleotides and amino acids, etc. It was discovered by an agricultural chemist who
worked on the adsoption of ammonium salts to soils and followed by several scientists for the
respective separation of rare earth elements, anionic species, and proteins. With the more
advancement, anion exchange and cation exchange chromatography came into existence. It is also
known as ion chromatography. Nowadays, ion exchange chromatography is popular for potential
applications in pharmaceutics, biotechnology, environment science, agriculture science and industries.

2. Principle

Ion exchange chromatography, the separation of charged analytes takes place on the basis of ionic (or
electrostatic) interactions between analytes and the stationary phase consisting of ionic functional
groups (ionic exchangers). The driving force for the separation of ions is competitive ionic binding
and repulsion between the similarly charged analyte ions/stationary phase ions.

3. Theory of ion exchange

The basic steps and components of ion exchange chromatography are same as the liquid column
chromatography i.e. mobile phase, stationary phase and eluent. However, the properties of stationary
phase are specific in ion exchange chromatography. The stationary phases used in ion exchange
chromatography consist of insoluble surfaces with exchangeable ions and are called as ion exchangers.
The ionic functional groups fixed on the surface of the stationary phase are referred as fixed ions while
the exchangeable ions with opposite charge are referred as counter ions. The counter ions have
tendency to move under the influence of electric field, diffusion or via exchange with similarly
charged external ions. The counter ions may include protons (H+), hydroxide groups (OH-), uni-
positive/negative ions (Na+, K+, Cl-), double charged ions (Ca2+, Mg2+), and polyatomic ions (SO42-,
PO43-), organic bases (NR2H+) and acids (COO-), etc. Therefore, the ion exchangers are classified into
two categories; 1) Cation exchangers when fixed ion is negatively charged 2) Anion exchangers when
fixed charge is positively charged.
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 To understand the mechanism of ion exchange, an illustration is given below. Suppose S+ is
stationary material carrying a positively charged fixed ion and negatively charged counter ion E -.
When it is exposed to mobile phase having negatively charged analyte A-, the counter ion E- moves
into the mobile phase and analyte ion A- interacts with the stationary phase. This exchange between
counter ion and analyte ion establishes equilibrium and at equilibrium both the A- and E- are
distributed between S+ and mobile phase. However, co-ion of the analyte does not take part in the
exchange process and electroneutrality of the solution is maintained by the flow of equal charges
between the two phases.

S+E- + A-  S+A- + E-

The tendency of ions/charged molecules to interact with the charged stationary phase vary
considerably due to their overall charge, charge density and surface charge distribution, which results
in the variable retention time of each components and helps in their separation.

4. Configuration of ion exchange system

The ion exchange systems are of two types; classical system and modern system.

4.1 Classical ion exchange chromatographic system

The classical ion exchange system consisted of and open tubular column and mobile phase or eluent.
The glass column (1-2 cm diameter) is filled with loosely bound ion exchanger material. The mobile
phase consisting of a competing ion is passed through the column from the top to bottom to run under
gravity. After the saturation of column with the mobile phase, sample solution containing components
is poured into the column from the top and flow of eluent (mobile phase) is stopped for some time to
allow the equilibration. Then, the flow of eluent is resumed and eluent is collected at the bottom at

Analytical Chemistry
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 regular intervals of time. Each fraction of eluent is analyzed for the eluted components and parameters
are evaluated.

(Reproduced from http://www.biochemden.com/ion-exchange-chromatography/)

The classical ion exchange chromatography is not much preferred because it involves flow of solvent
under gravity which makes the separation process slow, and leads to poor resolution and poor
efficiency.

4.2 Modern ion exchange chromatography

The modern ion chromatography is able to solve the problems associated with classical ion exchange
chromatography. Like high performance liquid chromatographic technique, it involves use of pump to
push the mobile phase at a high flow rate, rigid column filled with ion exchanger of high quality, and a
detector to analyze the components continuously eluted with the eluent. The working and
instrumentation of ion chromatography is same as HPLC, however the specific characteristics of the
eluent (mobile phase), and stationary phase (ion exchanger) are discussed below.
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 4.2.1 Eluent (mobile phase)

The main component required for the ion exchange between the stationary phase and the mobile phase
is competing ion. This ion helps in the elution of the analyte ion bound with the ion exchange material
within suitable time period. Therefore, eluent in ion exchange chromatography consists of aqueous
solution of a salt (or mixture of salts) with small percent fraction of organic solvent. An additional
buffer is added to the eluent (if required) otherwise the salt mixture itself acts as a buffer in most of the
cases. The separation process is governed by various factors associated with the eluent like nature and
concentration of competing ions, pH and flow rate of the eluent and temperature of the eluent.

The separation of components is highly influenced by the nature and concentration of

competing ions. The affinity of a competing ion to replace analyte ion from the stationary phase makes
is determined in terms of selectivity coefficient of that particular competing ion. This selectivity
coefficient varies with the nature of competing ion therefore separation is affected by the type of salt
solution selected as an eluent. Secondly, the higher is the concentration of the competing ion in the
eluent, higher is the displacement of the analyte ion from the stationary phase. This shifts the direction
of equilibrium and elution of analyte increases.

The time spent by the analyte on the stationary phase and displacement of the analyte by
competing ion directly depends upon the flow rate. Therefore, flow rate of the eluent should be
appropriate as high flow rates give rise to less resolution and low flow rates make the process time
consuming. In conrast, temperature has less effect on the separation process. Sometimes, increase in
temperature increases the diffusion within matrix and therefore, increases the elution volumes.

4.2.2 Ion exchangers (stationary phase)

A stationary phase in chromatographic techniques remains fixed. In case of ion exchange

chromatography, the stationary phase is termed as ion exchanger due to its ability to change the ion
with mobile phase. The ion exchangers possess two main structural components; 1) a polymeric
substrate possessing a fixed ion, 2) exchangeable ion. The fixed substrate is insoluble in the solvents,
however, the its exchangeable ion is mobile in nature and can be exchanged with other ions having
similar charge. The fixed polymeric network may be of organic or inorganic origin and can be exist

Analytical Chemistry
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 naturally or may be synthesized. However, on the basis of exchangeable ions attached to the fixed
substrate, ion exchangers can be classified into cation exchangers or anion exchangers.

Ion exchanger

cation anion
exchnagers exchangers

inorganic inorganic
organic cation organic anion
cation anion
exchanger exchanger
exchanger exchanger

natural synthetic natural synthetic natural synthetic

strongly basic weakly basic

strongly acidic weakly acidic anion anion
exchanger exchanger

Cation exchangers Anion exchangers

1. Cation exchangers are the materials 1. Anion exchangers are the materials which
which possess negatively charged fixed possess positively charged fixed substrate and
substrate and exchangeable positive ions. exchangeable negative ions.
2. These are further classified into strongly 2. These are classified into strongly basic and
acidic and weakly acidic cation exchangers. weakly basic anion exchangers.
3. The strongly acidic cation exchangers 3. The strongly basic anionic exchangers work
exist in ionized form in a wide pH range. under wide pH range.
4. The weakly acidic exchangers can ionize 4. The weakly basic anionic resins work under
only a narrow pH range. Therefore, weakly low pH conditions only.
acidic cation exchangers require sufficiently
high pH working conditions.
S-COOH + NaOH S-COO-Na+ + H2O S-NH2 + HCl S-NH3+Cl-
Examples of strongly acidic cation Examples of strongly basic anion
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 exchangers exchangers
Sulphonic acid Quaternary amine
Examples of weakly acidic cation Examples of weakly basic anion exchangers
exchangers Tertiary amine, secondary amine, primary
carboxylic acid, phosphonic acid, amine
phosphinic acid, phenolic, arsenic acid,
selenonic acid

5. Characteristics of ion exchangers

5.1 Ion exchange capacity

The ion exchange capacity of an ion exchanger determines the number of ions displaced on the ion the
exchanger. It depends upon the number of functional groups present per unit weight of the substrate. It
is determined both for the dry state as well as wet state of the exchanger. The determination of ion
exchange capacity involves saturation of known amount of ion exchanger with a particular ion
followed by the washing and quantification of the displacement of exchangeable ion from the material.
The ion exchange capacity is reported in terms of milliequivalent per gram or milliequivalents per
litre.

5.2 Swelling characteristics

The ion exchangers of organic origin show swelling when come in contact with the mobile phase.
These materials are highly cross-linked and have folded structures. In the presence of mobile phase,
the polymeric networks unfold and tend to accommodate the solvated ions. As a result the water
molecules enter into the spaces and the polymeric substrate shows swelling. The swelling property
decides the mechanical strength of the exchanger.

5.3 Ion exchange selectivity

It determines the affinity of an ion exchanger for a particular ion. It depends upon the nature of
polymer.

The main factors which affect the ion exchange selectivity are as follows:

1). Charge on the analyte ion

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 2). Size of the analyte ion

3). Degree of cross-linking of the exchanger

4). polarizability of the analyte ion

5). Ion exchange capacity of the exchanger

6). Interaction between the exchanger and the analyte ion

7). functional group on the exchanger

For example; ion exchange selectivity of strong acid cation exchanger decreases with the decrease in
the charge on the cation.

Pu4+>La3+>Ce3+>Pr3+>Eu3+>Y3+>Sc3+>Al3+>Ba2+>Pb2+>Sr2+>Ca2+>Ni2+>Cu2+>Co2+>Zn2+>Mg2+>Tl+
>Ag+> Cs+>Rb+>K+>NH4+>Na+>H+>Li+

6. Buffer

The pH of the eluent is very important parameter for the separation of charged components on the ion
exchange chromatography. Therefore, buffers are added to the mobile phase or salts/mixture of salts
are selected which can act as buffer to maintain a constant pH throughout the experiment. The pH
variation can occur due to the co-ions released from the analyte or the exchangeable ions released
from the stationary phase during equilibration. The buffers having high buffering capacity, wide
working pH, good solubility, high purity and low cost are suitable for ion-exchange chromatography.
The buffer should be inert towards the functional groups of the exchanger and should have negligible
conductivity. Some shortcomings of using buffer solutions include the interactions of buffer with
stationary phase or mobile phase, precipitation of the mobile phase, etc. The best conditions for ion
exchange chromatography are slightly acidic to slightly alkaline conditions i.e. pH range 6.0-8.5.
Some common buffers include phosphate buffer (anionic buffer) and ethanolamine, Tris and Tricine
(as cationic buffers).

7. Detection

The detection process is common as used in HPLC. However, conductivity detector is the best detector
in ion exchange chromatography. It offers excellent sensitivity when the conductance of the eluted
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 solute ion is measured in an eluent of low background conductance. In addition, UV-Vis detectors are
also used with ion exchange chromatography.

8. Various stages in exchange chromatography

In brief, the system is saturated with the mobile phase and sample is introduced into the system
manually or using an autosampler. The mobile phase is selected according to the nature of analytes
present in the sample. Usually it is a buffered aqueous solution and it carries the sample from the loop
to stationary phase. The stationary phase is also selected on the basis of the nature of analytes. Then,
the retained analytes (anions or cations) are eluted by increasing the concentration of a similarly
charged species. These ions displace the analyte ions from the stationary phase, which are delivered to
the detector for detection. The main stages for the interaction between analyte and stationary phase are
summarized below.

STAGE 1 EXCHANGER SATURATION The ion exchanger is saturated with the

mobile phase having desired pH and
ionic strength. In this stage, exchanger
consists of exchangeable ions such as
sodium ion, chloride ion etc.

STAGE 2 SAMPLE INTRODUCTION The sample is introduced in the

system. The charged analyte ions
displace the counter-ions of the
stationary phase and bind reversibly
with the exchanger. The extra
components are washed out with the
mobile phase.

STAGE 3 ELUTION The properties of the mobile phase are

changed to make it unsuitable for the

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 binding of analyte with the exchanger.
Usually, the ionic strength of the
mobile phase is increased or pH is
changed as per the requirement. This
results in the elution of weakly bound
analytes followed by the strongly
bound moieties.

STAGE 4 Washing The column is washed properly to

remove the impurities present in the
system if any

STAGE 5 Re-equilibration The ion exchanger is again

equilibriated to regenerate the
stationary phase.

9. Disadvantages of ion exchange chromatography

Apart from the various advantages, ion exchange chromatography has some disadvantages.

1. It requires buffers in the mobile phase, which require proper washing making the process lengthy.
2. The efficiency of column decrease because buffers left in the column may contaminate the
column.
3. Sometimes, it is difficult to achieve control over selectivity and resolution by ion exchange
chromatography.
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 10. Applications of Ion Exchange Chromatography

1. It is used for the separation and analysis of amino acids, lanthaides, nucleic acids, proteins,
vitamins, organic acids and bases etc.
2. It is used as preconcentration techniques for trace analysis.
3. It is used to remove interferences due to radicals.
4. This is most effective method for water purification and softening of drinking water.

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  A. Braithwaite, F. J. Smith, Chromatographic Methods, 4th Edition
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