Ion exclusion chromatography separates ionic compounds from non-ionic compounds using an ion exchange stationary phase. Ionic substances pass quickly through the column while non-ionic substances are retained. The technique was first introduced in 1953 and uses either cation or anion exchange resins. Key applications include determining neutral molecules, strong inorganic acids and bases, carboxylic acids, and amino acids.

1. Presented By
Chintu Lahkar
M.pharm, Roll no- 02
Pharmaceutical chemistry
NETES Institute Of Pharmaceutical Science
Mirza, Kamrup-781125,Assam
ION
EXCLUSION
CHROMATOGRAPHY

2. CONTENTS
 Introduction
 Principle
 Instrumentation
 Applications
 Reference

3. INTRODUCTION
 First introduced by Wheaton and Bauman in 1953.
 Useful technique for separation of ionic and non-ionic substances.
 IEC is opposite to ion-exchange chromatography.
 Ion exclusion chromatography (IEC) separates ionic from non-ionic compounds
using an ion exchange stationary phase.
 Negatively charged ions are separated on cation-exchange resins, while positively
charged species are separated on anion-exchange resins.
 The same column can be used for both ion exchange and ion exclusion
chromatography.
 IEC is also referred to by several other names, including ion exclusion partition
chromatography, ion chromatography- exclusion mode, and Donnan exclusion
chromatography.

4. PRINCIPLE
 Ionic substances pass quickly through the column. Non-ionic (molecular) or
partially ionized substances are held up and are eluted more slowly. Functions by
Donnan exclusion mechanism.
 Mobile phase- liquid (water or aqueous acid or base)
 Stationary phase- ion exchange resins
 Cationic
 Anionic
 Chromatographic system consists of 3 phases.
 Mobile phase
 Resin phase
 Occluded liquid phase

5. Contd.
 The mobile phase passes through the interstitial volume existing between the beads of
the ion exchange resin.
 The resin phase is the solid resin network and functionalized groups, which can be
considered to be a semipermeable ion exchange membrane separating the flowing
mobile phase from the stationary occluded liquid inside the resin.
 An occluded liquid phase is formed by mobile phase that becomes trapped within the
pores of the resin phase. This trapped liquid acts as the stationary phase of the system

6.  When the column is filled with water, pumped through as a mobile phase, the
water molecules accumulate as hydration spheres around the dissociated functional
groups of the support.
 Water contained in the pores of the support and in the hydration, spheres is
immobilized, thus forming the stationary phase.
Contd.

7. Contd.
 The commonly accepted retention mechanism of IEC involves the formation of a
pseudo semipermeable membrane around the resin stationary phase
 Ionic solutes of similar charge to the stationary phase (generally sulfonated cation
exchangers are used for weak acid analytes), experience repulsion from the resin
surface, whereas neutral species can penetrate the resins pores and stationary
occluded phase, thus experiencing retention
Due to the high density of charged
groups in the stationary phase a
potential difference is generated
which leads to the formation of a
virtual membrane - the Donnan
membrane. This charged membrane
only permits neutral molecules to
reach the stationary phase. Charged
molecules can not pass the Donnan
membrane

8.  Stationary phase
 Column
• Glass, stainless steel or polymer
• Length: Diameter ratio 20:100 to
100:1
 Sacking the column
Wet packing method
 Application of the sample
After packing sample is added to the top of
the column, use syringe or pipette
 Mobile phase
Acid, Alkali, Buffers
INSTRUMENTATION

9.  ELUENTS: 3 types of eluents are used in ion exclusion chromatography.
• Water eluent
• Acid eluent
• Complexing eluent
 DEGASSING UNIT: Removing dissolved gases in mobile phases is an important
step for ensuring proper function of pump check valves, and to prevent outgassing
in the detector flow cell.
 ANALYTICAL COLUMN:
• The analytical column consists of stationary phase (wet packing) with reactive
layer bonded to inert polymeric particles.
• Stationary phases must satisfy implicitly a number of requirements as narrow
granulometric distribution (monodisperse), large specific surface area,
mechanical resistance, stability under acid and basic pH and rapid ion transfer.
Contd.

10. Contd.
 The most common resins used in ion exclusion chromatography are high-capacity
PS-DVB-based strongly acidic cation exchange resins of 5 μ particle size.
 Styrene/divinylbenzene copolymers are the most widely used substrate materials.
Since they are stable in the pH range between 0 to 14, eluents with extreme pH
values may be used.
 The co-polymerization of styrene with divinylbenzene is necessary in order to
obtain the required stability of the resin.

11. Contd.
 SUPPRESSOR: Suppression plays a key role in the analysis of anions and organic
acids using ion chromatography and conductivity detection. Suppressor is a device
placed between the column and the detector, and acts to reduce the background
conductivity of the eluent and enhance the conductivity of the analytes. For anion
analysis, the suppressor is a high-capacity cation exchange membrane or resin in the
acid form. It removes cations from the eluent and replaces them with H+.
• decreases the background conductivity of the eluent
• minimizes baseline noise
• transforms analytes in free anions with protons as counter ions (which involves a
remarkable increase in the conductivity signal)
• optimizes the signal-to-noise ratio
• increases the detection sensitivity of the measurement system

12. Contd.
 DETECTOR:
 Conductivity Detector:
• In ion chromatographic practice, conductivity detectors are most commonly used.
• The conductivity of the solutions is an additive property, depending on the quality of
the ions (mobility) and the number of ions (concentration).
• In principle, the conductivity detector can be used for some non-aqueous eluents.
The sensitivity of these detectors depends on the temperature during the separation
and detection the temperature must be kept strictly constant.
 Detectors with UV- Visible spectrophotometry:
• Detectors with UV-Visible spectrophotometry are often used for detection.
• This is used in cases where the component is absorbed in the UV-Visible range.

13. Contd.
• Examples include iodide, nitrite, nitrate, iodate or chromate ions. The detector is
photodiode and the cell is a quartz cuvette.
• Deuterium and tungsten lamps are used as a source of light. In addition, a diode
array detector can be used, if the purpose is to simultaneously detect light
absorption at different wavelengths.
 Fluorescent based Detectors:
• We can detect fluorescent materials with fluorescence-based detectors.
• The principle of detection is that the components of the sample are excited by a
given wavelength light and the components emit light and we can detect this light.
• For biological samples, this type of detection method is common

14. APPLICATIONS
 Determination of neutral molecules: Neutral compounds such as sugars and
alcohols can be separated by IEC.
 Determination of strong inorganic acids: Sulphate, nitrate and chloride ions, and
strong base cations such as sodium, ammonium, potassium. Magnesium and
calcium ions commonly found in acid rainwater.
 Separation of carboxylic acids: - The separation of carboxylic acids is the most
common application of ion-exclusion chromatography
 Determination of Weak inorganic acids and bases: - IEC has found increasing use
for the determination of weakly ionized inorganic anions such as fluoride, nitrite,
phosphate, sulphite, arsenite, arsenate, bicarbonate, borate and cyanide.
 Determination of water in some organic solvents.
 Determination of amino acids and its derivatives.

15. REFERENCE
 D. A. Skoog, F J. Holler, S. R. Crouch, “Principles of Instrumental Analysis”, 6th
edition, Thomson Brooks/Cole, page no: 839-847.
 S. Ahuja, M. W. Dong, “Handbook of Pharmaceutical Analysis by HPLC”,
Volume 6, ELSEVIER Academic Press, page no: 219- 253.
 G. D. Christian, P. K. Dasgupta, K. A. Schug, “Analytical Chemistry”, 7th edition,
John Wiley & Sons, page no: 653-665.
 M. Mori, K. Tanaka, Q. Xu, M. Ikedu, H. Taoda, W. Hu, "Highly sensitive
determination of hydrazine ion by ion-exclusion chromatography with ion-
exchange enhancement of conductivity detection." Journal of Chromatography
A 1039, no. 1-2 (2004): 135-139.
 B. K. Glód, "Ion exclusion chromatography: parameters influencing
retention." Neurochemical research 22 (1997), pageno:1237-1248.

16. OPEN TO DISCUSSION

17. THANK YOU