ION-EXCHANGE

MODERN PHARMACEUTICAL ANALYSIS AND

TECHNIQUE

Seminar
On

ION EXCHANGE CHROMATOGRAPHY

Topic: Factors affecting resolution, chromatographic
parameters and Applications

Presented By: Govind Rashmi K.

Department of P
Department of
harmaceutical Pharmacognocy
Chemistry

Submitted To: Dr. Somashekar P L

Copy To: Mr/Ms_________________

Government College of Pharmacy, Bangalore

P. Kalingarao Road, Subbaiah Circle, Mission Road,
Bengaluru, Karnataka
560027

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ION-EXCHANGE

INTRODUCTION:

Ion exchange chromatography denotes mutual separations of exchangeable ions. Separations

of different cations from each other are based on different absorbances on cation
exchangers, which may be utilized to effect a quantitative separation if proper conditions are
chosen. Similarly, anion exchangers may be used for mutual separation of different anions.
Elution development is the most important method in chromatographic separations for
analytical purposes.

Definition of Ion exchange chromatography:

Ion exchange chromatography is the process by which a mixture of similar charged ions can be
separated by using an ion-exchange resin which exchanges ions according to their relative affinities.
Ion exchange chromatography that allows the separation of ions and polar molecules based on their
affinity to the ion exchanger.

Ion exchange resins:

Substances capable of sorption exchange of ions with the electrolyte solution are known as
ion exchangers. Ion exchangers are porous solids swelling in water without dissolving in it.
According to the , composition of the main skeleton which binds the ionogenic groups into
a whole system, the ion materials may be classified as (a) Mineral, and (b) Organic.

The ion exchange materials of mineral origin include numerous silicates, alumino silicates,
aluminium hydroxide, zirconium phosphate etc.
The ion exchange materials of organic origin are the products of chemical treatment of coal
or lignin, or manmade high molecular organic compounds containing the ionogenic groups.

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ION-EXCHANGE
Natural or artificial ion exchangers can be used for water treatment. Natural materials are
glauconites and humic coals. Manmade ion exchange materials are sulphonated coals
and synthetic ion exchange resins.
Ion exchange resins consists of beads of highly polymerised, cross-linked, organic
materials containing large number of acidic or basic groups. Although the resins are
insoluble in water, the active groups are hydrophilic and have varying degrees of affinity
for ionic solutes.
A cation exchange resin as a high-molecular- weight, cross-linked polymer containing
sulphonic, carboxyilic, phenolic, etc., groups as an integral part of the resin and an
equivalent number of cations.

(A) Styrene

(B) divinylbenzene

An anion exchange resin is a polymer containing amine (or quaternary ammonium) groups
as integral parts of the polymer lattice and an equivalent number of anions, such as
chloride, hydroxyl, or sulphate ions.

(D) Polystyrene

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The fundamental requirements of a useful resin are:
1. the resin must be sufficiently cross-linked to have only a negligible solubility;
2. the resin must be sufficiently hydrophilic to permit diffusion of ions through the
structure at a finite and usable rate;
3. the resin must contain a sufficient number of accessible ionic exchange groups and it
must be chemically stable;
4. The swollen resin must be denser than water.
There are four basic types of resins which are commonly used in ion exchange
chromatography-

 Strongly acidic cation exchangers (polystyrene sulphonic acid resins).

 Weakly acidic cation exchangers (a.g. polymethylacrylic acid resins).
 Strongly basic anion exchangers (polystyrene quaternary ammonium
resins).
 Weakly basic anion exchangers (polystyrene tertiary amine resins).

Selection of Suitable Systems

Specification of Ion Exchanges Ionic groups suitable chromatographic medium

Strong acid -S03- Acidic and alkaline

Weak acid -C00- Only alkaline

Strong base -N+R3 Acidic and alkaline

Weak base -NHR2 Only acidic

It is useful to classify ion exchangers according to the pK values of their ion groups.
However. Table 1 gives a simplified survey of their working ranges.

Table 1: Operating Range of Commonly used Ion Exchangers

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For practical use the dissolved material must be present also in the dissociated form if it is to
be retained by the resin. It is most convenient to work at a pH where one of the components will
be retained while the other does not interact with the ion exchanger.

The concentration of the participating ions of opposite charge also plays an important role.
The retained ions can be eluted with the aid of a high concentration of cations or anions
without changing the pH.

Ion exchange capacity:

The total ion exchange capacity of a resin is dependent upon the total number of ion- active
groups per unit weight of material, and the greater the number of ions, the greater will be the
capacity.

The total ion exchange capacity is defined as the number of milliequivalents of exchangeable
ions per gram of dry resin. usually expressed as millimoles per gram of exchanger. The
capacities of the weakly acidic and weakly basic ion exchangers are functions of pH, the
former reaching moderately constant values at pH above 9 and the latter at pH below 5.

V ×N
Capacity=
W

V ml of alkai solution

N in milliequivalents per litre

W weight of resin.

Chromatographic parameters:

1. Rate of ion exchange process: The rate of ion exchange process depends on the rate of
diffusion which is the slowest state on ion exchange process
2. Flow rate: Due to differences in rates of exchange and the fact that they may vary
significantly for different kinds of separation, flow rates are to be controlled (0.5-5
ml/min)
3. Mechanical strength: Polystyrene bead would have little mechanical strength and upon
adding functional group such as sulphonic acid to the polymer,solubility is greatly
enhanced. If the polymer is crosslinked by the incorporation of divinyl benzene,
mechanical strength is imparted to the resin.

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 ION-EXCHANGE
4. Swelling: Swelling is due to the tendency of particles to hydrate and electrostatic
repulsion of fixed ions with like charges Swelling is continued until an equilibrium is
reached between the osmotic pressure in the system and the opposing elastic force of the
flexible hydrocarbon chains.
5. Particle size: Large surface area and small particles will increase the rate of ion
exchange process.
6. Porosity: High porosity offers a large surface area covered by charged groups and so
provides a high binding capacity.
7. Selectivity: The ion exchange in solutions is a selective process. At the absolute
concentration of the solution decreases, polyvalent ions are adsorbed better than
monovalent ions, while at higher concentrations, monovalent ion is adsorbed.
8. Total capacity: Total capacity of a resin is determined by taking a weighed sample of
resin, placing it in a column and passing through a solution of Kcl through the column in
excess.
9. Exchange capacity: It depends on the quality of the ions extracted from water by one gram
of air dry ion exchanger.
10. Cross-linking: As the crosslinking in the resin decreases, the resin swelling increases.
Divinyl benzene is the most commonly employed crosslinking agent and imparts strength to
the polymer by joining the chains together at various positions.

Factors affecting resolution in ion exchange chromatography:

The technique used in ion exchange chromatography is generally the so-called liquid
chromatogram, which means that the eluate from the column is investigated either in small
fractions or continuously by observing some convenient physicochemical property related to the
concentration of the eluate.

The main parameters are- resin, particle size, eluent, temperature, flowrate, column
dimensions.

1. Resin:
Nature and properties of ion exchange resins -

Cross linking and swelling are important factors, when more cross linking agent is present, the resin
becomes more rigid and swells less (has small pore size). This makes separations of ions of
different sizes more difficult as they cannot pass through the pores present and it becomes selective
to ions of different sizes.

The nature of resin whether cationic or anionic exchanger, which determines strongly its
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 ION-EXCHANGE
selectivity. Cationic resin is selective for cations and vice versa.

Resin capacity (number of me-equivalents of replaceable ions per gram of dry resin) is
important.

2. Nature of exchanging ions:

Valence of ions:  At low concentrations, the extent of exchanges increases with increase
in valence; Ions with higher charge is more selective;
Th4+ > Al3+ > Ca2+ > Na+
Similarly in anions-
PO3-4 > SO2 > -4 I-

Size of ions:  For similarly charged ions; the exchange selectivity increases with decrease
in the size of hydrated ions;

Li+ < Na+ < NH4+ < K+ < Rb+ < Cs+ < H+

Polarizability: Highly polarizable ions are more selective. Exchange is preferred for
greater polarizable ions, as;
I- < Br- < Cl- < F-

Concentration of solutions: In dilute solutions poly-valent ions are generally absorbed

preferentially.

3. Eluent:
 The choice of eluent composition is in most separations more critical than the choice of
resin. Ions that carry different charges are more easily separated than ions of the same
charge.
 From dilute solutions containing ions of different charges those of higher charge are
preferentially retained by the resin.

 The differences in uptake, increase on dilution of the solution. Separations of ions of

different charges are therefore improved by diluting the eluent.
 Ions of equal charge, such as the alkali metals, can be effectively separated by the
elution development from a cation exchange column by means of hydrochloric acid.
 In most chromatographic works it is convenient to take advantage of the fact that the
uptake of a given material can be greatly affected by changes in solution conditions.
 In general the eluent composition should be chosen so that the separation factor, i.e.. the ratio
between the distribution coefficients attains a high value. It must however, be noted
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ION-EXCHANGE
that in practice the optimum conditions are not always obtained with an eluent composition
which corresponds to the highest possible separation factor.
 This is due to broadening of the elution curves, that occurs when the distribution
coefficients are high. For this reason it is desirable to find an eluent which gives high
separation factor but not too high distribution coefficients.

4. Temperature:
 High temperature increases the diffusion rate and therefore the number of theoretical plates
in a given column hence improved separations were observed when working at elevated
temperature.
 Other advantages which may be gained by working at elevated temperature, arc reduced
influence of non-ionic adsorption forces.
 Another advantage which is gained at elevated temperature is that the resistance to flow is
decreased.
 In this connection it must be stressed that independent of the temperature level it is necessary
that the temperature be constant over the whole cross-section of the resin bed.
 Care should therefore be taken that uneven heating effects from bunsen burners,
radiators, or sunlight are avoided.
 In certain separations, e.g., in the separation of amino acids according- to Stein and
Moore it has been observed that the separation can be improved when the temperature is
changed during the course of elution.

5. Flowrate
 The flow rate has a high influence on resolution. The height of a theoretical plate
increases with an increased flow rate. An increased flow rate means that the ions are
carried at a faster rate down the column giving them less time to diffuse into and through
the resin particles and thus, are prevented from reaching all exchanging groups.
 High flow rates will cause a broadening of the elution curves and thus an increased
overlap of the curves for the ions to he separated.
 The present trend in difficult separations is to work with extremely fine particles at not too
low flow rates, the solution being forced through the column with a pump and thus giving
a constant feed. In this way much time can be saved.
 A high flow rate is necessary in separations of short-lived radioactive products and with
such systems.
 In general, flow rates are in the range of 0.5 mL/minute to 5 mL/minute.
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ION-EXCHANGE

Fig: Effect of flow rate on IEX resolution. Separation of a 5 ml sample of myoglobin (peak 1),
ribonuclease A (peak 2), and cytochrome c (peak 3) on a 1 x 13 cm (8.7 ml)

6. Column Dimensions And Maximum Charge

 In many chromatographic separations of similar solutes where a number of theoretical
plates are required, an increase in length of a chromatographic column is made only to
improve the separation and not to increase the charge to a column because it actually
represents a reduced utilization of the resin.
 The column length is determined by the separation factor, it is the ratio between the
distribution coefficients of the solutes to be separated. This factor determines the number of
theoretical plates which is necessary for a quantitative separation.
 The diameter of column is determined by quantity of material to be separated.
 Generally a ratio of 10:1 or 100:1 between height and diameter is maintained in most of the
experiments.
 Column should neither be too wide or too narrow in sizes, otherwise uneven flow of
liquid is expected to occur.

7. Particle size:
 Partical size contribute directly to rate of exchange.
 Large surface area and small particles will increase the rate.
 The particle size that is used will depend on the length of column, diameter and whether gravity
or pumping is used to control the flow of the mobile phase.
 Most laboratory applications are successful with 100-200 mesh (0.147-0.074 mm).
 Smaller particles provide higher resolution but typically also require lower flow rates.
 Larger particles permit higher flow rates but yield lower resolution.
 Small-particle resin yields sharp, distinct peaks

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ION-EXCHANGE
 Larger-particle resin will yield broader, less defined peaks.
 Small-particle resins increase the backpressure on the column, which should not exceed the
column’s operating pressure unit.

Fig: Relationship between resin particle size, pressure, and resolution.

8. pH value:
 pH value is an important parameter for separation and can be controlled and adjusted
carefully by means of buffer substances.
 In order to prevent variation in matrix and protein net charge, maintenance of a constant
mobile phase pH during separation is essential to avoid pH changing which can occur when
both protein and exchanger ions are released into the mobile phase.
 By means of buffer substances pH value can be controlled and adjusted.
 Concentration of H+ and the buffering component influence the protein binding to the
stationary phase, chromatographic resolution and structural as well as functional integrity of
the protein to be separated.
 Thus a suitable pH range, in which the stability of sample is guaranteed, has to be identified.

APPLICATIONS:
Ion exchange chromatography can be applied for the separation and purification of many
charged or ionizable molecules such as proteins, peptides, enzymes, nucleotides, DNA,
antibiotics, vitamins and etc.

Ion exchangers are usually employed in a method for the separation of micro as well as
macro samples and are therefore, of practical use in quantitative analysis and purification.
Because of the macro application, ion exchangers can also be used on a pilot plant and
industrial scale. In general, the basic laboratory techniques common to adsorption and
partition column methods are also applied in ion-exchange column methods.
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Some of the important applications are discussed with the following examples:

1) Separation of complex mixtures

A wide variety of very complex mixtures have been separated using ion exchangers.
These include closely related elements such as Zr and Elf, rare earths, transuranium
elements, halides, alkali metals, and transition metals. Complex mixtures of organic
acids and bases, amino acids, peptides, and proteins have also been separated.

(a) Separation of lanthanides; During the second world war the separation of fission-
produces presented a major problem to analytical chemistry, To
separate these, synthetic resins were employed in combination with the technique
of chromatographic elution.

When the solution having a mixture of lanthanides is passed through a column

packed with particle of a suitable ion exchange resin, the cations present in
solution undergo exchange with the hydrogen or any other cation that may be

present in the ion exchange reaction gets held near the top while the other cations to
undergo exchange reaction.

(b) Purification of organic compounds extracted in water: Many natural products

extracted in water have been found to be contaminated with ions originally present
in water. Those ions can be removed by using ion-exchange processes.

(c) Separation of Sugars: This method was developed by Khym and Zill (1951). These
sugars are first of all converted into borate complexes. Separations of borate
complexes of sugars have been achieved on 11 x 0.9 cm columns of 200
—400 mesh Dowex 1 resin using a loading of 5-10 mg of borate complex and
flow rates 0.5-1 ml/min. Quantitative recovery of sugars is possible after separation of
the borate complexes. Similarly disaccharides can be separated from the
monosaccharides and the individual compounds of hexose and pentose mixtures
resolved.

2) Routine laboratory applications

Ion exchangers can be utilized in the laboratory in many other ways

a) Interfering ions can be removed.

b) Group separations are possible.

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c) Samples can be concentrated.
d) Charges on ions can be established.
e) Samples can be purified.
f) Standard solutions can be prepared.
g) Formation constants of complexes can be determined.

3) Water and waste water treatment

Ion exchange is used for water and wastewater treatment, primarily for removal of
"hardness ion" (Ca2+ and Mg2+) from water supplies, and for removal of iron and
from ground water supplies. It is also used for treatment of a variety of industrial
wastes to allow for recovery of valuable wastes materials or by products, particularly
ionic forms of precious metals such as silver, gold, and uranium. An

additional waste treatment application is for removal and recovery of radioactive

materials from nuclear reactors, hospital and laboratory wastes.

4) Water-softening

The removal of the hardness ions of Ca2+ and Mg2+ may be achieved with a strong acid
cation exchanger in the Na+ form. At low concentrations of most raw waters the resin
strongly prefers the divalent cation over sodium ion, so that the exchange reaction
proceeds to the right.
2RSO3-Na+ + Ca2+ (RSO3)2Ca2+ + 2Na+

5)De-alkalisation
In a water containing bicarbonates — most waters in Western and Central Europe do — calcium and
magnesium associated with bicarbonate ions are exchanged for hydrogen ions from a weakly acidic
cation exchange resin. This is called removal of temporary hardness.
The salinity of the treated water is lower than that of the feed water.

6)Routine analysis of amino acid mixtures, the 20 principal amino from blood serum or from the
hydrolysis of proteins are separated by ion exchange chromatography.

PRACTICAL APPROACH
Source: Coprinus comatus

Extraction: Stipe powder of C. comatus (100 g) was extracted three times with 1 L 95% ethanol

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 ION-EXCHANGE
under reflux for 2 h to remove lipid, and the residue was extracted three times with 2 L distilled
water for 2 h at 80 °C with intermediate centrifugation (2000 × g, 15 min). After concentrating the
collected aqueous supernatants to 400 mL (reduced pressure at 40 °C), a precipitation was performed
with 3 volumes of 95% ethanol. The precipitate was washed with ethanol and acetone, and then dried
at 40 °C, yielding crude polysaccharide material. Crude polysaccharide material was dissolved in 100
mL 0.2 M sodium phosphate buffer (pH 6.0), and after centrifugation the solution was applied to a
DEAE-Sepharose CL-6B column.

Stationary Phase: DEAE-Sepharose CL-6B column (3.5 cm × 30 cm).

Eluent: 0.2 M sodium phosphate buffer (pH 6.0), and linear gradient of 0.3–1.5 M NaCl in 0.2 M
sodium phosphate buffer (pH 6.0).

Detection: UV Detector, 490 nm (phenol–H2SO4 ) and 500 nm (Folin–phenol)

Analyte(s): Polysaccharides; disaccharide α,α-trehalose, α-D-glucan, β-D-glucan, α-L-fuco-α-

Dgalactan.

Limitations:

I. Even though ion exchange liquid chromatography is considered one of the most
efficient purification methods available, it has short comings as well. Compared
to other purification methods, separation efficiency is low.
II. Secondly, synthesis of ion exchangers is very complex, which slows down the
development of the technique. .
III. Moreover, polystyrene or other aromatic matrices have denaturing effects, leading
to possible dissociation of protein-protein, protein-RNA, -DNA complexes.
However, other matrices preserve the native structure of proteins well.
IV. Sometimes the swollen matrix by volume change complicates the
chromatographic operation as well. But, these drawbacks are quite minor and the
numerous applications described earlier prove its practical efficiency.

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References:
1. Ion exchange separations in analytical chemistry by Olof Samuelson, Wiley
publications, page no: 189-201.
2. Ion exchange resins by Robert Kunin, Wiley publications, page no: 110-115.
3. Vogel’s Textbook of quantitative chemical analysis by G.H Jeffery, J. Bassett,
J.Mendham, R.C. Denney, Fifth edition, Page no: 186-199.
4. Instrumental methods of chemical analysis by B.K Sharma, Page no: 124-154.
5. Instrumental methods of chemical analysis by Gurdeep R Chatwal and Sham K Anand,
page no: 2.662-2.671.
6. A textbook of analytical chemistry by Y. Anjaneyulu, K. Chandrashekhar, Valli
Manickam.

Review articles:

 Factors affecting selectivity in ion exchange chromatography by James S. Fritz,

Elsevier.
 Ion-Exchange Chromatography and Its Applications By Özlem Bahadir Acikara
http://dx.doi.org/10.5772/55744
 Recent trends in ionexchange chromatography international journal of pharmaceutics & drug
analysis VOL. 3 ISSUE 12, 2015; 403 – 416 ; http://ijpda.com; ISSN: 2348-8948
 Studies on Sustained Release Tablet using Ion Exchange Resins Acta Scientific
Pharmaceutical Sciences (ISSN: 2581-5423) Volume 3 Issue 4 April 201

Govind and Rashmi K. (2022-23), Govt College of Pharmacy, Bangalore 1