ION EXCHANGE

CHROMATOGRAPHY
Bio separation presentation
Group 4
CONTENT
What is ion exchange chromatography ?

Working Principle

Instrumentation and steps

Types and examples

Parameters

Strengths and limitation

Applications
 ION EXCHANGE CHROMATOGRAPHY (IEX)

• Ion exchange chromatography, or IEX, is a class

of liquid chromatography (LC) used to separate
organic and inorganic molecules.
• Separate charged Biomolecules based on their
interaction with the oppositely charged particles
of the stationary phase and subsequent release
from the column by modifying the pH or the
ionic strength of the mobile phase.

Fig. interactions in IEX column

2022 Bio separation presentation 3


provide a continuous constant
flow of the eluent through the IC
injector, column, and detector
The suppressor reduces the
background conductivity of the
chemicals used to elute samples
from the ion-exchange column
Generally uses changes in
electrical conductivity to
analyze extractant
serves as a protective factor for ion
exchange column
• Sample introduction
• Liquid samples may be injected
directly
• solid samples need only to be
dissolved in an appropriate
solvent INSTRUMENTATION
 MECHANISM AND PRINCIPLE

This form of chromatography relies on the

attraction between oppositely charged
stationary phase, known as an ion exchanger,
and analyte.
• The ion exchangers basically contain
charged groups covalently linked to the
surface of an insoluble matrix.
• The charged groups of the matrix can be
positively or negatively charged.
• When suspended in an aqueous solution,
the charged groups of the matrix will be
surrounded by ions of the opposite
charge.
• In this “ion cloud”, ions can be reversibly
exchanged without changing the nature
and the properties of the matrix.

Fig: 5 main steps to the process of IEX 

 ANION EXCHANGE CHROMATOGRAPHY
VS
CATION EXCHANGE CHROMATOGRAPHY
Anion exchange
• Columns have positively charged functional groups
covalently bonded to the stationary phase
• Used for the separation of anions such as deprotonated
or “acidic” protein molecules that have more carboxylic
acid groups on their surface
• Sample functional groups : Quaternary ammonium salts
(Q) (CH 3 ) 3  N + CH 2 O, Diethylamino ethyl (DEAE)
(C 2 H 5 ) 2 N CH2 CH 2 O -
Cation exchange
• Columns have negatively charged functional
groups bonded to stationary phase particles.
• Used for the separation of cations such as
protonated “basic” proteins that have more amino
groups on their surface.
• Sample functional groups: Sulfopropyl (SP) -
OCH 2 CH 2 CH 2 SO 3 -

The ion exchange columns are further classified as strong and weak anion and cation exchangers depending on their
ion exchange capacity.
• Strong exchangers retain their ion exchange capacity over a wide • Weak exchangers are effective over a narrow pH range where they
pH range as they do not take up or lose protons with changes in the are ionized.
mobile phase pH.
 PARAMETERS AFFECTING IEX
• porosity and size of the polymeric resin impacts the resolution.
Column 
• Smaller particle sizes provide improved resolution but increase the system backpressure

• pka values should be borne in mind when selecting buffers for IEX. To maintain the desired pH, the
Buffers concentrations of buffers should typically be in the range of 20 – 50 mM
• For cation exchange chromatography, the elution buffer pH is maintained in the range 4-7 and for
Buffer pH anion exchange in the range 7-11

Ionic • Buffers containing as much as 300 to 500 mM of salts such as sodium chloride, potassium chloride,
strength sodium bromide, sodium sulfate or sodium acetate are typically used for elution.
• Additives such as urea, sugars or detergents are added to increase the solubility of the analytes, prevent
Additives their precipitation or to ensure analyte stability by inhibiting enzyme activity.

• As the pH of the buffer determines the ionization state of a molecule, the pH of the sample buffer is
Sample maintained at 0.5 to 1.5 pH unit above or below the pI value of analyte of interest. This ensures that
preparation  the molecules are charged appropriately for either anion exchange or cation exchange chromatography

Flow rate  • Flow rate at which the resolution is not impacted isused to improve throughput

7
 STRENGTHS AND LIMITATIONS OF IEX
CHROMATOGRAPHY
Strengths
High loading capacity
High resolution group separation of molecules of interest from
other molecules and impurities
 
Can separate proteins differing by only one charged amino acid
or molecules with only small differences
 
Fast separations as IEX can be carried out at high flow rates
High recoveries of target molecules  
Limitations
Expensive buffers and instrumentation
Sample injection solvent must have low ionic strength and
controlled pH which may require an additional buffer exchange
step
 
Weak ion exchange columns are sensitive to pH changes and
must be used within the prescribed range to maintain capacity
and resolution
Coupling with mass spectrometry (MS) is limited due to high
salt concentration in the eluent
 APPLICATIONS OF IEX

Extensively applied for the separation and purification of monoclonal antibodies (mAbs) and
proteins. 

Fractionation of crude protein mixtures and separation of seventeen proteins have been carried out
using anion exchange chromatography with a linear pH gradient.

IEX is used to remove contaminants such as viruses during downstream purification process.

sugars and nucleic acids have also been separated using IEX.
THANK YOU
Group Members

Nathan Jude Serpes

Pushti Verma

Tushima sharma

Kajal

Jeneeta