The slides covers brief description of ion exclusion chromatography. i hope the slides will be helpful for any further details you can contact me through email. mail id - sobhigaba@gmail.com

1. PREPARED BY- SOBHI
GABA

2. Name: SOBHI GABA
Designation: STUDENT
Department: M. PHARMA
(PHARMACEUTICAL
CHEMISTRY)
E-mail: sobhigaba@gmail.com

3. 
 Ion-exclusion chromatography was first introduced
by Wheaton and Bauman in 1953.
 It involves the use of strong anion- or cation-
exchange resins for the separation of ionic solutes
from weakly ionized or neutral solutes.
INTRODUCTION

4. 
Principle of ion exclusion
chromatography

5. 
 In this mode of chromatography , the charge sign on
the ion-exchange resin used is the same as that of the
weakly ionized solutes.
 That is, solutes with a partial negative charge (such
as carboxylic acids) are separated on a cation -
exchange resin having anionic sulfonate functional
groups, whereas solutes with a partial positive
charge (such as weak bases) are separated on an
anion exchange resin having cationic quaternary
ammonium functional groups.
 This is the opposite situation to that occuring in ion-
exchange chromatography.
Principle continued…

6.  The principle of this type of chromtography is based on
Donnan Exclusion Mechanism
 Non ionic or partially ionized molecules are held up the
stationery phase whereas the strongly ionised molecules
experiences repulsion from the stationery phase due to which
they are eluted first.
 The principles of ion-exclusion chromatography can be
illustrated in a schematic manner by considering the
chromatographic system to be comprised of three distinct
phases.
 The first of these is the flowing eluent, which passes between
the beads of ion exchange resin (i.e. through the interstitial
volume).
 The second zone is the polymeric network of the resin material
itself, together with its bound ionic functionalities,
 the third zone is liquid occluded inside the pores of the resin
bead.
 The polymeric resin can be considered as a semi-permeable,
ion-exchange membrane which separates the flowing eluent
from the stationary occluded liquid inside the resin

7. Figure presenting the 3 phases of the chromatographic system in ion
exclusion chromatography
RESIN – CATION EXCHANGE RESIN
(a) acidic solutes, such as acetic acid and HCI, and
(b) basic solutes, such as NH3 and NaOH.

8. 
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, where as neutral species can penetrate the
resins pores and stationary occluded phase, thus
experiencing retention.

9. 
 When the column is filled with water, which is pumped
through as mobile phase, the water soluble molecules
build up 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
 The basic mechanism is that the neutral and uncharged
molecules can penetrate the resin, whereas similarly
charged co-ions are repelled owing to the presence of
dissociated functional group immobilized in the
stationary phase.
RETENTION MECHANISM

10.  Thus, analytes are
separated by :
i. Exclusion or
repulsion if they are
ionized and have the
same charge as the
resin.
ii. Adsorption if they
are non-polar or
partially ionized.

11. 
 The eluent is water or aqueous acid or base with little or
no organic modifier, which offers environmental and
economic benefits in addition to compatibility with
aqueous sample matrices.
 Difficult separations such as simple aliphatic carboxylic
acids (e.g., formate, acetate, propionate and butyrate) are
possible.
 It is compatible with a wide range of detection methods.
 The columns can be ion exchange columns, reversed-
phase or normal-phase columns, or dynamically modified
reversed phase with an ion pairing agent.
 It is stable for long-term analysis of complex samples such
as wine or mustard.
ADVANTAGES

12. 
I. STATIONERY PHASE
1. COLUMN
 Glass, stainless steel or polymer
 Length: Diameter ratio 20:100 to 100:1
2. PACKING THE COLUMN
 Wet packing method
II. APPLICATION OF THE SAMPLE
 After packing sample is added to the top of the column, use syringe or
pipette
III. MOBILE PHASE
 Acid, Alkali, Buffers
IV. ELUTION
 Components of mixture separated & move down the column at different
rates depending upon the affinity of the ion for ion exchanger.
V. ANALYSIS OF THE ELUATE
 spectrophotometric.,Conductometric
PRACTICAL REQUIREMENTS

13. 
INSTRUMENTATION

14. 
 3 types of eluents are used in ion exclusion chromatography.
1) Water eluent
2) Acid eluent (dilute solutions of strong mineral acids to be
employed for the elution of anionic solutes, or dilute solutions
of strong bases to be employed for the elution of cationic
solutes).
3) Complexing eluent (The retention and detection properties of
some solutes can be enhanced if a complexing agent is added
to the eluent. An example of this approach is the use of a
mannitol eluent for the determination of boric acid, in which
the mannitol serves to complex the boric acid to form a species
which is more easily detectable by conductivity measurements
than is boric acid alone).
I. ELUENTS

15. 
 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.
II. Degassing unit

16. 
 The analytical column consists of stationery phase (wet
packing)
 The column packing consist of a reactive layer bonded to inert
polymeric particles.
 Stationary phases must satisfy implicitly a number of
requirements as narrow granulometric distribution (mono-
disperse), large specific surface area, mechanical resistance,
stability under acid and basic pH and rapid ion transfer.
 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 copolymerization of styrene with divinylbenzene is
necessary in order to obtain the required stability of the resin.
III. ANALYTICAL COLUMN

18. 
 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+ . Suppression
• 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
IV. SUPPRESSOR

19. Analyte:
Na+
Cl–
+ RSO3
–
H +
H +
Cl–
+ RSO3
–
Na+
This example of an anion analysis includes a sodium analyte counter
ion. This ion is replaced with a proton with an equivalent conductivity
that is five times higher. This significantly increases the conductivity of
the sample solution and therefore also the signal strength. Salts from
weakly dissociated acids (e.g., sodium carbonate/sodium hydrogen
carbonate) are used as eluent.
Eluent
Na+
HCO3
–
+ RSO3
–
H +
H2CO3 + RSO3
–
Na+
The eluent counter ions are also replaced with protons. The carbonic
acid that is produced in this way is unstable and only weakly
dissociated, meaning that lower background conductivity is measured.
Depending on the eluent composition, background conductivity
values of 10 to 20 µS/cm are typical for chemical suppression.

20. 
 Detectors used in this type of chromatography are as
follows:-
1) Conductivity Detector
2) Detectors with UV- Visible spectrophotometery
3) Fluorescent based Detectors
DETECTORS

21. 
 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.
CONDUCTIVEITY DETECTORS

22. 
 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.
 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.
UV- VISIBLE DETECTORS

23. 
 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.
FLUORESCENT BASED
DETECTORS

24. 
I. Separation of carboxylic acids
II. Determination of Weak inorganic acids and bases
III. Determination of strong inorganic acids.
IV. Determination of neutral molecules.
V. Determination of water in some organic solvents
VI. Determination of amino acids and its derivatives
APPLICATIONS

25. 
 The separation of carboxylic acids is the most
common application of ion-exclusion
chromatography
Separation of carboxylic acids

26. 
 Solutes such as fluoride ,carbonate , cyanide, borate,
sulfite, phosphates, nitrite, arsenite, arsenate and
ammonium have been determined using this
approach.
 Interference from strongly ionized species is
minimal because these solutes are unretained and
appear at the column void volume.
 Ion-exclusion chromatography can therefore readily
separate weakly ionized solutes in samples
containing high concentrations of ionic species, e.g.
seawater and wastewater.
Determination of Weak
inorganic acids and bases

27. 
Determination of strong
inorganic acids

28. 
Determination of neutral
molecules.

29. 
Determination of water in some
organic solvents

30. 
Determination of amino acids
and its derivatives

31. THANKS
For Further Detail/SUGGESTIONS Please
Contact
ISF COLLEGE OF PHARMACY, MOGA
(An Autonomous College)
Ghal Kalan, GT Road, Moga- 142001, (Pb.)
E- mail: director@isfcp.org
Website: www.isfcp.org