This document summarizes a seminar on ion exchange chromatography. It introduces the topic, covering the basic principles of how ion exchange chromatography separates ions and polar molecules using stationary phases with positively or negatively charged groups. It then discusses the types of ion exchange resins, including classifications based on chemical nature and source. Examples of applications are given, such as separating similar ions, water softening, demineralization, separating sugars and amino acids. The document concludes by discussing some advantages and disadvantages of ion exchange chromatography.

1. Seminar on ion exchange chromatography
PRESENTED BY
MANOJ KUMAR . M
H.T.NO.636217885003
M.Pharmacy 1st year
UNDER GUIDANCE OF
DR.S.Y. MANJUNATH
DEPARTMENT OF PHARMACEUTICAL ANALYSIS
SRIKRUPA INSTITUTE OF PHARMACEUTICAL SCIENCES
[AFFILIATED TO OSMANIA UNIVERSITY]
[Approved by PCI;AICTE]

2. INTRODUCTION
 Ion exchange chromatography is a chromatographic process
that separates ions & polar molecules based on their affinity
to the ion exchangers i.e. cationic or anionic exchange resins.
 Ion exchange chromatography retains analyte molecule on
the column based on ionic interactions.
 Essentially the molecule undergo electrostatic interactions
with opposite charge on the stationary phase matrix.
 Columns used for ion exchange are characterized by the
presence of charged groups covalently attached to the
stationary phase.
 Anion exchangers contain bound positive groups, where as
cation exchangers contain bound negative groups

3. Principle…..
 Reversible exchange of ions b/w ions present in the
solution & ion exchange resin
 Cation exchange chromatography retains positively
charged cations because the stationary phase displays a
negatively charged functional group.
 Anion exchange chromatography retains anions using
positively charged functional group.

 BCMXRBMCXR

 AMBXRBMAXR

4. CLASSIFICATION OF RESINS
According to the chemical nature they classified as-
1. Strong cation exchange resin
2. Weak cation exchange resin
3. Strong anion exchange resin
4. Weak anion exchange resin
According to the Source they can -
Natural : Cation - Zeolytes, Clay
Anion - Dolomite
Synthetic : Inorganic & Organic resins
Organic resins are polymeric resin matrix The resin
composed of – polystyrene ( sites for exchangeable functional
groups ), divinyl benzene ( cross linking agent ) – offers
stability

5. Structural types of ion exchange resins
a) Pellicular type with ion exchange resins:
 »30 - 40μ with 1-2μ film thickness
 »Very low exchange capacity
b) Porous resin coated with exchanger beads
 » Size 5 - 10μ
 » Porous & highly efficient
c) Macroreticular resin bead
 » Not highly efficient & low exchange capacity
d) Surface sulfonated & bonded electrostatically with anion
exchanger
 ≫ less efficient & low exchange capacity

6. Physical properties of ion exchange resins
Cross linking:
 It affects swelling & strength & solubility
Swelling:
 When resin swells, polymer chain spreads apart
 Polar solvents → swelling
 Non-polar solvents → contraction
 Swelling also affected electrolyte conc.
Particle size & Porosity
 ↑surface area & ↓particle size will ↑rate of ion exchange
 Particle size 50-100 mesh / 100-200 mesh
Regeneration
 Cation exchange resin are regenerated by treatment
 Anion exchange resin are regenerated by treatment with NaOH,

7. Ion exchange resin should have following
requirements:
 It must be chemically stable
 It should be insoluble in common solvents
 It should have a sufficient degree of cross linking
 The swollen resin must be denser than water
 It must contain sufficient no. of ion exchange groups
Practical requirements:
 column
 Packing of column
 Application of sample
 Mobile phase
 elution
 Analysis of eluate

8. Schematic Diagram of Ion exchange
chromatography

9. Column:
 Column used in the laboratories are made up of glass but those
used in industries are made up of either high quality stainless
steel or polymer, which are resistant to strong acids and alkalis.
The separation is improved by increasing the length of the
column but the length cannot be increased beyond a critical
length. Uneven flow of liquid is possible in case of column too
wide or too narrow in size . A Dimension of column is 20:1 to
100:1 for the higher efficiency can be used.
Packing of the column:
 In this wet packing method is used. The resins is mixed with the
mobile phases and packed in the column uniformly. The sample
to be separated is dissolved in the mobile phases and introduced
all at once into the column.
Application of the sample:
 After packing the column, the solution to be analyzed is added to
the top of the column and allowed to pass through the bed of ion
exchanger. For this purpose the syringe or pipette is utilized

10. Mobile phase:
 The organic solvents are less useful so they are not used these
days. Only different strength of acids, alkalis and buffer are used
as eluting solvent . E.g.: 0.1 N HCl, 1N NaOH, phosphate buffers,
acetate buffers, borate buffers, phthalate buffers, etc.
Developments of the chromatogram and elution:
 After introduction of the sample, development of the
chromatogram is done by using different mobile phases. The
aqueous salt solution is adjusted to a constant ionic strength. The
choice of the mobile phase depends on the selectivity of the resin
for the solute ions. Two types of elution techniques are used:
a. Isocratic elution
b. Gradient elution
 In Isocratic elution technique, the same solvent composition is
used i.e., same strength of acids or alkalis or buffers are used.

11.  In gradients elution techniques, initially less acidic or basic
mobile phase is used. Then, acidity or basicity is increased at
regular intervals. The different fraction of the elution is collected
volume wise or time wise and analyzed
 Analysis of the elute or Detection:
Different fractions are collected with respect to the volume or
time is analyzed for their contents. Several methods of analysis
can be used which depends upon the nature and quantity of the
ionic species are:
* Conductometric method
* Amperometric methods
* Flame photometric method
* Polarography
* UV. Spectroscopy
* Radiochemical methods using Geiger Muller counter,
ionization chamber method .

12.  Conductivity detector is the most common and useful detector in
ion exchange chromatography. Conductivity detection gives
excellent sensitivity when the conductance of the eluted solute
ion is measured in an eluent of low background conductance.
Therefore when conductivity detection is used dilute eluents
should be preferred and in order for such eluents, to act as
effective competing ions, the ion exchange capacity of the
column should be low. The results obtained are stored in
computer.

13. Advantages :
 Detectability: useful for the detection of many inorganic salts
and also for the detection of organic ions with poor uv
absorptivity like alkyl amines or sulfonates.
 Preparative separations: usually preferred because of the
availability of volatile buffers . volatile buffers makes the
removal of mobile phase easier.
 Useful to resolve very complex samples, i.e. in the case of multi
step separation
 Useful for separation of mixtures of biological origin, in organic
salts and some organo- metallics
Disadvantages:
 Column efficiency is less
 It is difficult to achieve control over selectivity and resolution
 Stability and reproducibility of the columns become
questionable after repeated use

14. APPLICATIONS:
1.Separation of similar ions
 A mixture of sodium, hydrogen and potassium can be
separated using cation exchanger resin.
 A mixture of Chloride, bromide, and iodide can be
separated using basic anion exchange resin.
Method: Mixture of chloride, bromide & iodide is passed
through basic anion exchanger using 0.5M sodium nitrate as
eluant. Chloride will first elute. Raise the concentration of
Sodium Nitrate, Bromide will elute, raise the concentration of
Sodium Nitrate further, iodide ion will elute.
.

15. 2.Softening of hard water:
 Hardness of water due to ca, mg and other divalent ions.
This water is passed through cation exchanger charged with
the sodium ions. Ca & Mg ions retained in the column while
sodium is exchanged.
3.Complete demineralization of water:
 Removal of both cations & anions.
 Step A) Hard water is first passed through an acidic cation
exchanger- Ca, Mg & Na are exchanged by H+ions.
 Step B) This water is then passed thro a basic anion
exchanger – Cl, NO2, SO4- are exchanged by OH- ions of
the exchanger.
4.Separation of sugars:
 sugars-borate complexes. This complex is separated on
Dewax. In this disaccharides separated from mono.

16. 5.Separation of Amino Acids:
 protein after hydrolysis is introduced to a short column on
special polystyrene sulphonic acid resin at pH 2 and eluted
with 0.35N sodium citrate buffer of pH 5.25. acidic &
neutral Amino acids first leave the column as unseparated
then others.
6.Medicinal importance:
 Anionic resins are introduced in the treatment of ulcer
while cation exchangers have been used to remove Na+
from body during the treatment of hypertension and
edema. The resins are also used as a diagnostic aid in
gastric acidity tests. The resins have been successfully
used with other medicinal agents to achieve delayed action
dosages

17. 7.Removal of interfering radicals:
Phosphate ion is the interfering with the calcium & barium ions.
Phosphate is removed using sulphonic acid cation exchanger.
Calcium & barium ions exchanged with H+ ions while phosphate
ion pass through the column. The process has to be repeated so
that the phosphate ions are completely removed. Now, the
calcium and Ba+ ions held by resin will be removed by using
suitable eluent. Finally, these ions are estimated by the usual
methods

18. 8.Other applications
 For the measurement of various active ingredients in medicinal
formulations,
 For the measurement of drugs and their metabolites in serum and
urine, for residue analysis in food raw materials,
 For the measurement of additives such as vitamins and
preservatives in foods and beverages.

19. Analysis of Carbohydrates by High-Performance
Anion-Exchange Chromatography with Pulsed
Amperometric Detection (HPAE-PAD)
 Methods for the liquid chromatographic analysis of
carbohydrates have often employed silica-based amino bonded
or polymer-based, metal-loaded, cation-exchange columns,
with refractive index (RI) or low-wavelength ultraviolet (UV)
detection.
 These analytical methods require attention to sample solubility,
sample concentration and, in the case of the metal loaded
cation-exchange columns, also require column heating. In
addition, RI and low-wavelength UV detection methods are
sensitive to eluent and sample matrix components. This usually
precludes the use of gradients and often requires stringent
sample cleanup prior to injection.
 As a result, an improved chromatographic technique known as
high-performance anion exchange (HPAE) was developed to
separate carbohydrates.

20.  Coupled with pulsed Amperometric detection (PAD), it
permits direct quantification of nonderivatized carbohydrates
at low - picomole levels with minimal sample preparation and
cleanup.
 HPAE chromatography takes advantage of the weakly acidic
nature of carbohydrates to give highly selective separations at
high pH using a strong anion-exchange stationary phase.
HPAE-PAD is extremely selective and specific for
carbohydrates because:
 1. Pulsed amperometry detects only those compounds that
contain functional groups that are oxidizable at the detection
voltage employed
 2. Neutral or cationic sample components in the matrix elute
in, or close to, the void volume of the column. Therefore,
even if such species are oxidizable, they do not usually
interfere with analysis of the carbohydrate components of
interest.

21. Anion-Exchange Chromatography
Mechanism of Separation
 Although anion-exchange chromatography has been used
extensively to analyze acidic carbohydrates and
glycopeptides.
 It has not been commonly used for analysis of neutral
sugars.
 However, examination of the pKa values of the neutral
monosaccharaides listed in below Table shows that
carbohydrates are in fact weak acids. At high pH, they are
at least partially ionized, and thus can be separated by
anion-exchange mechanisms.
 This approach cannot be used with classical silica-based
columns because these matrices dissolve at high pH.
Anion exchange at high pH is, however, ideally suited

22. Table1: Dissociation constants of some common carbohydrates
(in water at 25 °C)
Sugar pka
Fructose 12.03
Mannose 12.08
xylose 12.15
Glucose 12.28
Galactose 12.39
Ducitiol 13.43
Sorbitol 13.60
α-methyl glucoside 13.71

23. Thermo Scientific™ Dionex™ CarboPac™
Columns
1.Dionex CarboPac PA1 and PA-100 Columns
 Dionex, now part of Thermo Fisher Scientific, designed the
Dionex CarboPac series of columns specifically for carbohydrate
anion-exchange chromatography.
 These columns permit the separation and analysis of mono-,
oligo-and polysaccharides.
 The Dionex CarboPac PA1 and Dionex CarboPac PA100 are
packed with a unique polymeric, nonporous, Thermo
Scientific™ Dionex™ MicroBead™ Pellicular resin. Dionex
MicroBead resins exhibit rapid mass transfer, high pH stability
(pH 0–14), and excellent mechanical stability that permits back
pressures of more than 4000 psi (28 MPa). Column
reequilibration after gradient analysis is fast, generally taking 10
min or less.

24.  The Dionex CarboPac PA1 is particularly well-suited to the
analysis of monosaccharides and the separation of linear
homopolymers, while the Dionex CarboPac PA100 is optimized
for oligosaccharide resolution and separation.
2. Dionex CarboPac MA1 Column
 Reduced carbohydrates (also called sugar alcohols) have
traditionally been a difficult class of carbohydrates to separate by
liquid chromatography.
 They are weaker acids than their nonreduced counterparts
(compare the pKas of glucose and sorbitol or galactose and
dulcitol in Table 1), and are therefore poorly retained on the
Dionex CarboPac PA1 and PA100 columns.
 The Dionex CarboPac MA1 was developed to address the
challenge of retaining and separating extremely weak acids.
 This column is packed with a macroporous polymeric resin which
has an ion-exchange capacity 45 times that of the Dionex
CarboPac PA1

25.  . As a result, weak anions bind more strongly to the column,
requiring higher sodium hydroxide concentrations for elution.
The increase in hydroxide ion concentration leads to greater
ionization of the sugar alcohols, with greatly improved
retention and resolution on the column.
 Nonreduced neutral oligosaccharides can also be analyzed on
the Dionex CarboPac MA1 column, although their analysis
times are longer than on the Dionex CarboPac PA1 and PA100
columns. Retention of carbohydrates on the Dionex CarboPac
MA1 can be manipulated by altering the sodium hydroxide
concentration of the eluent (Table 2). Note that the elution
order of several of the compounds changes with the sodium
hydroxide concentration.

26. Table 2. k´ Values of selected analytes on the
Dionex CarboPac MA1 Column
carbohydrate 0.05 0.14 0.25 0.38 0.50
Glucose 15.70 7.19 4.31 2.91
Mannose 13.55 6.15 3.72 2.53
Galactose 17.82 8.25 4.99 3.43
Glycerol 1.13 0.99 0.89 0.80 0.72
M-inositol 1.32 1.08 0.86 0.69 0.56
S-inositol 1.63 1.30 1.02 0.81 0.64
Glcnol 1.81 1.40 1.09 0.89 0.75
Flucitol 1.94 1.63 1.40 1.18 1.05
Sorbitol 6.43 4.72 3.33 2.55 2.06
Dulcitol 6.52 5.04 3.73 2.87 2.26
Mannitol 8.98 6.38 4.37 3.28 2.63
Fucose 10.34 4.72 2.52 1.69 1.25
lactitol 14.97 9.61 5.49 3.57 2.43
Eluent concentration (M Naoh)

27. Comparison of the Dionex CarboPac
MA1, PA1, and PA100
characteristic Dionex CarboPac
MA1
Dionex carbo PA1 Dionex CarboPac
PA100
Recommended
applications
Mono- and disaccharide
alcohol
analysis in food
products,
Monosaccharide
compositional
analysis, linear
homopolymer
separations,
saccharide
purification
Oligosaccharide
mapping and
analysis
Resin composition 8.5-μm-diameter
vinylbenzylchloride
divinylbenzene
macroporous
substrate fully
functionalized with an
alkyl quaternary
ammonium group
10-μm-diameter
polystyrene/
divinylbenzene
substrate
agglomerated with
350-nm
Dionex MicroBead
quaternary
amine
functionalized latex
10-μm-diameter
ethylvinylbenzene/
agglomerated with
350-nm
Dionex MicroBead
quaternary
amine functionalized
latex

28. Dionex MicroBead
latex
cross-linking
N/A, no latex 5% cross-linked 6% cross-linked
Anion-exchange
capacity
4500 μeq per 4 ×
250-mm column
100 μeq per 4 × 250-
mm column
90 μeq per 4 × 250-
mm column
Recommended flow
rate
0.4 mL/min (4 ×
250-mm column)
1 mL/min (4 × 250-
mm column)
1 mL/min (4 × 250-
mm column
Organic solvent
compatibility
0% 0-2% 0-100%
pH compatibility pH 0–14 pH 0–14 pH 0–14
Maximum back
pressure
2000 psi (14 MPa) 4000 psi (28 MPa) 4000 psi (28 MPa)

29. Pulsed Amperometric Detection
I. Theory of Operation
Pulsed amperometry permits detection of carbohydrates with
excellent signal-to-noise ratios down to approximatel 10 picomoles
without requiring derivatization. Carbohydrates are detected by
measuring the electrical current generated by their oxidation at the
surface of a gold electrode. The products of this oxidation reaction
also poison the surface of the electrode, which means that it has to be
cleaned between measurements. This is accomplished by first raising
the potential to a level sufficient to oxidize the gold surface. This
causes desorption of the carbohydrate oxidation products. The
electrode potential is then lowered to reduce the electrode surface
back to gold.

30. A.Monosaccharides—Neutral and Amino Sugars
These sugars can be successfully separated on the Dionex
CarboPac PA1 column using isocratic conditions with 16 mM
sodium hydroxide as the eluent. Because the concentration of
sodium hydroxide used for the separation is only 16 mM, the
column should be regenerated after each run. Otherwise,
carbonate will start to contaminate the column, irrespective of
the care taken to eliminate it from eluents and samples.
Regenerate the column by washing it with 200 mM sodium
hydroxide for 10 min at a flow rate of 1.0 mL/min. This
procedure will also remove other strongly bound contaminants
such as peptides and amino acids.
Separation of neutral and amino monosaccharides derived from glycoproteins

31. B. Sugar Alcohols
Mono- and oligosaccharide sugar alcohols can be separated using
the Dionex CarboPac MA1 column with sodium hydroxide eluents.
Examples of isocratic separations are shown in Figures 1 and 2.
Gradients can be used to improve separations (Figure 3) or to
accelerate the elution of late-eluting components (Figure 4). Table 2
shows that the elution order of certain carbohydrates may be altered
by changing the sodium hydroxide concentration
Figure 1. Isocratic separation of a
group of alditols plus glucose and
fructose on the Dionex CarboPac
MA1 column
Figure 2. Separation of reducing and
nonreducing carbohydrates. Food alditols and
aldoses are separable under isocratic
conditions on the Dionex CarboPac MA1.

32. Figure 3. Separation of alditols found in biological fluids. The NaOH gradient
improves the separation of sorbitol and dulcitol, which are poorly resolved at
NaOH concentrations that permit resolution of glycerol from inositol
Figure 4. Separation of monosaccharide alditols released by direct s-elimination
from glycoproteins. The hydroxide gradient following the isocratic separation
of the first three components accelerates the elution of mannitol as well as any
oligosaccharide alcohols that may have been released during the s-elimination
process.

33. REFERENCE:
 B.K. Sharma; Instrumental Methods of Chemical
Analysis; page. No: 123-160.
 G. Vidya Sagar; A Text Book of Pharmaceutical
Analysis; volume-II; page. No: 13 – 18.
 Gurdeep R. Chatwal, Sham K. Anand; Instrumental
Methods of Chemical Analysis; page. No: 2.662-2.672.