This document discusses ion exclusion chromatography, which uses an ion exchange stationary phase to separate ionic and nonionic substances. Ionic substances pass quickly through the column while nonionic substances are retained longer. Separation depends on whether substances are ionized and repelled by the resin or able to enter the resin network if nonpolar or partially ionized. Detection methods include conductivity detectors and UV-visible or fluorescence detectors. Applications include separation of carboxylic acids, inorganic anions, amino acids, and determination of water in organic solvents.

1. ION- EXCLUSION
CHROMATOGRAPHY

2. INTRODUCTION…
 Useful technique for the separation of ionic and nonionic
substances.
 Ion exchange stationary phase.
 Separation by partition.
 Ionic substances - pass quickly through the column.
 Nonionic (molecular) or partially ionized substances are held
up and are eluted more slowly.

3. Alternative names…
 Ion Exclusion Partition Chromatography
 Ion Chromatography-Exclusion Mode
 Donnan Exclusion Chromatography

4. Principle
 A poly(styrene}divinylbenzene) (PS-DVB) based strongly acidic
cation exchange resin in the hydrogen form.
 3 distinct components:
1. A solid resin network with charged functional groups (the
membrane).
2. Occluded liquid within the resin beads (the stationary phase).
3. The mobile liquid between the resin beads (the mobile phase or
eluent).

6.  Molecules that are partially dissociated will show retention between
that of fully ionized and non-polar solutes.
 Analytes are separated by
 (i) exclusion or repulsion if they are ionized and have the same
charge as the resin, or
 (ii) adsorption if they are non-polar or partially ionized.

7.  The ratio of the concentrations of ionized to neutral forms
of an analysed compound is determined by its dissociation
constant and is equivalent to the solute effective charge.
 Strong acids that are completely dissociated are
electrostatically repulsed.
 Eluted unseparated in the column dead volume (Vm),
which corresponds to the volume of the mobile phase in
the column.

8.  Undissociated molecules are able to enter the resin
network.
 Eluted together with a retention volume equal to the sum
of the inner and dead column volumes.
 The inner column volume means the volume of its
stationary phase.

9.  Kd - Distribution coefficient.
 Ka
S and Ka
M - dissociation constants in the
stationary and mobile phases,respectively.
 a - functional group activity
 - the activity coefficient of hydrogen ions in the
stationary phase,
 Kp - neutral molecule partition coefficient
 f - functional group dissociation constant,
 cb - the buffer concentration
 Kb - dissociation constant.

10. INSTRUMENTATION

11. Stationary Phases
•High capacity PS-DVB-based strongly acidic cation
exchange resins of 5 µm particle size.

12.  Polymethacrylate based weakly acidic cation exchange resin.
 Unfunctionalized silica gel.
Mobile phases:
 Methanol
 Ethanol
 Butanol
 Glycerol
 Acetonitrile.

13. SUPPRESSOR
Reduce the background conductivity of the eluent and enhance
the conductivity of the analytes.
Minimizes baseline noise
Increases the detection sensitivity of the measurement system
Optimizes the signal-to-noise ratio

14. Detection
 Conductivity detectors
 Most popular and universal detection method

15. Ions in solution help to transport current.

16.  Detectors with UV-Visible spectrophotometry
 Used in cases where the component is absorbed in the UV-
Visible range.
 Eg: iodide, nitrite, nitrate, iodate or chromate ions.
 Detector is photodiode and the cell is a quartz cuvette.
 Deuterium and tungsten lamps are used as a source of
light.

17. Fluorescence-based detectors
 Components of the sample are excited by a given
wavelength light and the components emit light.
 Detection of biological samples.

18. APPLICATIONS

19. V0 - nitric acid, 1- formic acid, 2-
acetic acid, 3- propionic acid, 4-
butyric acid, 5- valeric acid, 6-
caproic acid, 7- heptanoic acid, 8-
caprylic acid, 9- pelargonic acid,
10- capric acid.
SEPARATION OF CARBOXYLIC ACIDS

20. Determination of weakly ionized inorganic anions.
 Fluoride, nitrite, phosphate, sulfite,
arsenite, arsenate, bicarbonate, borate
and cyanide from seawater and waste
waters.
 Separation of bicarbonate in tap waters
by IEC with conductimetric detection by
elution with water.
 (A) Raw tap water) (10-fold dilution);
(B) tap water after softening treatment;
(C) tap water (10-fold dilution). Peaks:
1, strong acid anions; 2, bicarbonate ion.

21. Strong Inorganic Acids
 Sulfate, nitrate and chloride ions, and strong base cations such
as sodium, ammonium potassium, magnesium and calcium ions
commonly found in acid rainwater.

22. Neutral Compounds
 Neutral compounds such as sugars and alcohols can be
separated by IEC.

23. Determination of water in some organic solvents.
 PS-DVB based cation exchange resin in the hydrogen
form.
 Eluent - methanol containing a small amount of strong
acid.
 Spectrophotometric detection.
 Peak at at 310nm.

24. Amino acids and amino acid derivatives.
 Hippuric and orotic acid were determined using
photodiode array detection.
 The two peaks of hippuric and orotic acids were
monitored at 210 and 280 nm.

25. REFERENCE
 Instrumental analysis – Doglas A skoog, F. James Holler, Timothy A. Nieman, 5th
edition, page no:
 Instrumental methods of chemical analysis- H.Kaur, page no: 1139
 R.E. Smith, ‘Ion Chromatography Applications’, CRC Press, Boca Raton, 1988.
 http://www.chromacademy.com/lms/sco111/theory_of_hplc_ion-
chromatography.pdf.
 https://www.researchgate.net/publication/233910695_Principles_and_Applications_
of_Ion_Exclusion_Chromatography.
 https://academic.oup.com/chromsci/article/51/7/655/472405.