PRINCIPLES OF ION EXCHANGE

CHROMATOGRAPHY

Instrumentation
PRINIPLES OF ION EXCHANGE
CHRAOMATOGRAPHY
Learning objectives
Upon completion of this module you should be able to :
Explain the basic principles, operation and application of IC and explain the
basis for the common IC detection methods.

Table of contents

1-definition

2-types of Ic

3-history

4-principle

5-basic process of IC

6-stationary phase or ion exchange materials used in IC

7-detection methods

8-chromatogram

9-basic instrumentation

10-conditions to be remembered in IC

11-applicaions of IC
Principles of Ion exchange
chromatography
The most popular method for the purification of proteins and other charged
molecules is ion exchange chromatography.

1-Definition
It is a type of liquid solid chromatography that is specially applicable
to ionic species.

Or

Ion exchange chromatography is an exchange of ions between two

electrolytes or an electrolyte solution and complex.

2-Types
Two types of ion exchange technique include

 CATION_EXCHANGE CHROMATOGRAPHY
 ANION EXCHANGE CHROMATOGRAPHY

a-CATION EXCHANGE CHROMATOGRAPHY

In cation exchange chromatography positively charged molecules are
attracted to a negatively charged solid support.

 Commonly used cation exchange resins are S-resin, sulfate

derivatives; and CM resins, carboxylate derived ions.
b-ANION EXCHANGE CHROMATOGRAPHY
In anion exchange chromatography, negatively charged molecules are
attracted to a positively charged solid support.

 Commonly used anion exchange resins are Q-resin, a Quaternary amine;

and DEAE resin, DiEthylAminoEthane.

3-History
Ion Chromatography (IC) methods were first reported around 1850 when
H.Thomson and J.T. Way used various clays as an ion exchange and
extracted labile calcium, magnesium, and ammonium ions.

 In 1927, the first zeolite column was used to remove Mg2+ and
Ca2+ from water.
 Cation exchange using a sulfonated polystyrene/divinylbenzene
column was developed in the 1940s as part of the Manhattan project.
Very large columns were used to concentrate and purify the
radioactive nucleotides required for the atom bomb.
 In the late 1940s anion exchange was performed with the attachment
of a quaternary ammonia on the polystyrene/divinylbenzene support.
 The industrialization of the technique occurred in the 1970s
when a suppressor column was developed that enabled
conductivity detection.
4-Principle
In ion exchange chromatography a reversible exchange of ions is possible
between ions in a liquid phase and solid , insoluble substances containing
ionic sites.

5-Basic process of IC

The basic process of chromatography using ion exchange can be

represented in 5 steps:

 eluent loading
 sample injection
 separation of sample
 elution of analyte A
 elution of analyte B

These steps are elaborated below:

Step 1: The eluent loaded onto the column displaces any anions bonded to
the resin and saturates the resin surface with the eluent anion.

(key: Eluent ion = , Ion A= , Ion B = )

Step 2: A sample containing anion A and anion B are injected onto the
column. This sample could contain many different ions, but for simplicity
this example uses just two different ions ready to be injected onto the
column.

Step 3: After the sample has been injected, the continued addition
of eluent causes a flow through the column. As the sample elutes (or moves
through the column), anion A and anion B adhere to the column surface
differently. The sample zones move through the column as eluent gradually
displaces the analytes.

In reality not every eluent ion is removed from the surface of the column.
It depends on the amount of analyte loaded. A better representation of the
column can be seen by just looking at a slice of the column where separation
is occuring, as shown in the figure below.
Step 4: As the eluent continues to be added, the anion A moves through
the column in a band and ultimately is eluted first.

Step 5: The eluent displaces anion B, and anion B is eluted off the column.

The overall 5 step process can be represented pictorally:

6-Stationary phase or ion exchange
materials
There are a number of different resins or stationary phases that have been
developed for use in IC. The main classes of substances used are:

 modified organic polymer resins,

 modified silica gels,
 inorganic salts,
 glasses,
 zeolites,
 metal oxides and
 cellulose derivatives.

Note: The most commonly used resins are the silica gels and polymer
resins.Some of the commonly used resins are explained here . . .

a-Ion Exchange Resins

The first resins were prepared by condensation of phenol,sulphuric
acid with formaldehyde.The product contained –SO3H,-OH and –COOH
reactive groups.
 However most of the modern polystyrene based resins use only
one type of grouping.
 Extremely difficult separations of radioactive isotopes, rare earth
elements and amino-acids are achievable by these resins.
 The most widely used type of resin is prepared by a
polymerization of styrene and divenyl benzene.
 They are further of two types.

Cation exchangers :They are subdivided into strong acid

types containing _SO3H groups and weak acid types containing
_COOH groups.

Anion exchangers: If basic functional groups are

introduced ,the resin can exchange anions rather than cations.
b-Chelating resins
These are produced bychelating functional groups which show selective
affinities for certain metals e.g thiophenol and 8-hydroxy quinoline.

c-Ion exchange gels

By introducing the appropriate exchanging groups into the matrix of the gels
used for gel filteration ,it is possible to produce media which is useful for
separation of polyelectrolytes.

 Common gels used are sephadex,agarose,bio-gel,porous glass and

styra gel.

d-Ion exchange cellulose

This is derived from carbohydrate polymers. These are used for fractionation
of poly-electrolytes.

 They may be fibrous or microgranular.

e-Liquid ion exchangers

The are used in column form by coating them onto a solid support.

 e.g tri-n-octylamine(TNOA).

f-Inorganic ion exchangers

These are used at higher temperatures or under conditions of high energy
radiation.

 Hydrated zirconium oxide and micro-crystalline heteropolyacid salts.

Selection of resins
A wide variety of resins are available for commercial use.however, the choice
of a suitable resin is necessary for IC as efficiency of the process is directly
affected by resin .Following properties should be taken into account.

 They should have appropriate mesh size.

  High rate of cross linking that increases the rigidity and reduces
swelling of its polymeric structure.
 Resin chosen should have a good quality.
 Analytical grade resins are preffered as they are carefully sized and
washed.
 A resin of known weight should be used .It must be dried or brought to
a known moisture content in hydrostate.

7-Detection Methods

In order to have useful information, you need to be able to detect what

comes out of the column. The analytes are ions that come out in separate
sample bands, which means there is a small part of the solution coming out
with a higher concentration of ions.
NOTE: There are many different possibilities of detecting ions. Due to its
simplicity, most instruments use conductivity.

 Conductivity
Conductivity is the measure of a material’s ability to conduct electricity.
Since conductivity is proportional to the number of ions in solution, it is the
primary method of detection for ion chromatography.

Other methods

Other detection methods have been coupled with IC, including

 mass spectrometry
 atomic spectroscopy
 fluorescence
 luminescence
 UV-Vis, and
 potentiometric.

NOTE :Most require post-column reactions to generate the signal or are so

selective they are not useful in detecting multiple analytes simultaneously.
8-Chromatograms

The time to elute an analyte is a function of how long the analyte is retained
on the column, therefore the output of IC is a graph of conductivity as a
function of time, called a chromatogram.

Consider for example if the two ions are traveling at the same speed, set by
the flow of the eluent then from the figure, you should be able to see that
the path in red is much shorter than the path in blue.

Since the path is shorter, and the ions are traveling at the same speed, the
ion following the red path will emerge first. Thus a normal chromatogram
peak will have a gaussian distribution, symmetric around the mean, as seen
in the figure .
Example of chromatogram of Poland Springs bottled water. Each separate
peak is due to a different cation.

NOTE :The area under each peak is used to calculate the concentration of
each ion.

9-Basic Instrumentation
The loading of the sample onto the column varies with the instrument. The
sample is eluted off of the column, through the detector. The signal from
the detector is converted into the chromatograph.

 The pressures required for most IC instruments is at least 600 psi. In

order to achieve this pressure a double piston high pressure pump is
used, one such example is shown in the picture.
  The injection of the sample onto the column is performed using a
multiport valve that is inline with the eluent tubing. Different
instruments have slightly different styles, with the most common a
direct port for injection.
 Another example is where an autosampler is used. In the photo to the
left, an autosampler is shown(fig a), where a peristaltic pump (fig b)
pulls the sample up the autosampler tube and into the sample loop on
a six-port valve.

(A) autosamper peristaltic pump B)

 Software controls the six-port valve and at the appropriate time the
valve switches to have the sample flow onto the column.

 The six port valve works by first having the flow of the sample go
through the sample loop (figure below) and then to the waste. This
fills the sample loop with the sample. At a set period, the valve turns
connecting different lines coming in. The eluent now forces the
sample from the sample loop onto the column.
  One instrument, a Lachat 8500 QuickChem with IC, is shown below
illustrating a guard column, analytical column, and the suppressor
cartridge. The guard column is used to protect the analytical ion
column from contamination.

10-Important conditions to be followed in IC

Buffer pH

As a rule, the pH of the mobile phase buffer must be between the pI

(isoelectric point) or pKa (acid dissociation constant) of the charged
molecule and the pKa of the charged group on the solid support.
Salt Gradients
As in most other modes of chromatography (SEC being the exception) a
protein sample is injected onto the column under conditions where it will be
strongly retained. A gradient of linearly increasing salt concentration is then
applied to elute the sample components from the column. An alternative to
using a linear gradient is to use a step gradient.

Varying pH
Many chromatographers also use changes in pH to affect a separation.For
example in cation exchange chromatography, raising the pH of the mobile phase
buffer will cause the molecule to become less protonated and the result is that the
protein no longer can form a ionic interaction with the negatively charged solid
support, which ultimately results in the molecule to elute from the column.

11-Applications of ion exchange

chromatography

IC is utilized in numerous industrial and research settings including a test for

authentic tequila and for environmental analyses such as the determination
of anions (PO43-, Cl-, NO3-, etc) in surface waters.
Following are various applications that are commonly associated with ion
exchange chromatography.(continued on next page>>>)

 The treatment of water for drinking, use (commercial, industrial, and

residential), and wastewater treatment. Ion exchangers can soften the
water, deionize it, and even be used in desalination.

 Preparation of various acids, bases, salts, and solutions is also aided

by ion exchange.

 The recovery of valuable metals is also possible with resins.

 Industrial drying of treatment of gases is accomplished often with ion

exchange.
 The food industry uses ion exchange in a variety of ways, ranging from
wine-making to sugar manufacture.

 In the medical world,it is used for development and preparation of key

drugs and antibiotics, such as streptomycin and quinine, to treatments
for ulcers, TB, kidneys, and much more.

 Ion exchange is used to prevent coagulation in blood stores and in

dextrose, as well.

 An ion exchange is also useful in death, as it plays a role in the

treatment of formaldehyde.

 In soil science, cation exchange capacity is the ion exchange capacity

of soil . Soils can be considered as natural weak cation exchangers.

 A very important case is the PUREX process (plutonium-uranium

extraction process), which is used to separate the plutonium and the
uranium from the spent fuel products from a nuclear reactor.

 In planar waveguide manufacturing, ion exchange is used to create the

guiding layer of higher index of refraction.