Ion Exchange Chromatography

Ion exchange chromatography (or ion chromatography) is a process that allows the separation
of ions and polar molecules based on their affinity to ion exchangers.

The principle of separation is thus by reversible exchange of ions between the target ions present
in the sample solution to the ions present on ion exchangers.

Types of Exchangers

In this process, two types of exchangers i.e., cationic and anionic exchangers can be used.

1. Cationic exchangers possess negatively charged group, and these will attract positively
charged cations. These exchangers are also called “Acidic ion exchange” materials, because
their negative charges result from the ionization of acidic group.

2. Anionic exchangers have positively charged groups that will attract negatively charged
anions. These are also called “Basic ion exchange” materials.

 Ion exchange chromatography is most often performed in the form of column chromatography.
However, there are also thin-layer chromatographic methods that work basically based on
the principle of ion exchange.

Working Principle of ion exchange chromatography

This form of chromatography relies on the attraction between oppositely charged stationary phase,
known as an ion exchanger, and analyte.
  The ion exchangers basically contain charged groups covalently linked to the surface of an
insoluble matrix.
 The charged groups of the matrix can be positively or negatively charged.
 When suspended in an aqueous solution, the charged groups of the matrix will be surrounded
by ions of the opposite charge.
 In this “ion cloud”, ions can be reversibly exchanged without changing the nature and the
properties of the matrix.

Instrumentation of ion exchange chromatography

Typical IC instrumentation includes: pump, injector, column, suppressor, detector and recorder or data
system.

1. Pump

The IC pump is considered to be one of the most important components in the system which has to
provide a continuous constant flow of the eluent through the IC injector, column, and detector.

2. Injector

Sample introduction can be accomplished in various ways. The simplest method is to use an injection
valve. Liquid samples may be injected directly and solid samples need only to be dissolved in an
appropriate solvent. Injectors should provide the possibility of injecting the liquid sample within the
range of 0.1 to 100 ml of volume with high reproducibility and under high pressure (up to the 4000
psi).

3. Columns

Depending on its ultimate use and area of application, the column material may be stainless steel,
titanium, glass or an inert plastic such as PEEK. The column can vary in diameter from about 2mm to
5 cm and in length from 3 cm to 50 cm depending on whether it is to be used for normal analytical
purposes, microanalysis, high speed analyses or preparative work.

Guard column is placed anterior to the separating column. This serves as a protective factor that
prolongs the life and usefulness of the separation column. They are dependable columns designed to
filter or remove particles that clog the separation column

4. Suppressor

The suppressor reduces the background conductivity of the chemicals used to elute samples from the
ion-exchange column which improves the conductivity measurement of the ions being tested. IC
suppressors are membrane-based devices which are designed to convert the ionic eluent to water as a
means of enhancing the sensitivity.

5. Detectors

Electrical conductivity detector is commonly use.

6. Data system

In routine analysis, where no automation is needed, a pre-programmed computing integrator may be

sufficient. For higher control levels, a more intelligent device is necessary, such as a data station or
minicomputer.

Second sub-category of liquid chromatography is known as ion-exchange chromatography. This

technique is used to analyze ionic substances. It is often used for inorganic anions (e.g., chloride,
nitrate, and sulfate) and inorganic cations (e.g., lithium, sodium, and potassium). It can also be used
for organic ions, although this is less common with the advent of reversed phase liquid
chromatographic methods that will be described later. Another significant application of ion-exchange
chromatography is as a step in the purification of proteins. Some of the substituent groups of amino
acids are charged (the total charge for a particular protein is a function of the pH of the solution), which
makes ion exchange a suitable method for protein purification.

The approach is to attach fixed ionic groups to the surface of a solid support. One common support
used in the formation of ion exchange resins is polystyrene-divinylbenzene copolymers. The fixed ions
are attached through a derivatization of the phenyl rings of the polystyrene. Common fixed ions involve
either sulfonate groups or quaternary amines as shown in Figure 56. The aromatic sulfonate groups are
strong enough acids that they are deprotonated at all but highly acidic pH values (pH < 1).

There must also be an exchangeable counterion associated with each of these fixed groups. In the case
of the sulfonate group, the counterion is a cation and this is a cation exchange resin. With the
quarternary amine phases, the exchangeable counterion is an anion and this is an anion exchange
resin. These counterions can be exchanged with each other. For example, this would enable you to

have a cation exchange resin in the sodium form or the hydrogen form .

An anion exchange resin could be in the hydroxide form (OH–) or chloride form (Cl–). One common
use of ion exchange resins is in the deionization of water. It is useful to consider a scheme using ion
exchange resins that would enable you to deionize water. If for example, we had water with sodiu m
chloride in it (Na+Cl–), we would need a way of removing both cations and anions. If we first passed
the water through a cation exchange resin in the hydrogen form, the sodium ions would exchange with
the hydrogen ions as shown in the top picture of Figure 58. If we then passed the water through an
anion exchange resin in the hydroxide form, the chloride ions would exchange with the hydroxide ions

The

H+ and OH– given off by the two resins would combine to form water. Eventually the resin will fill up
with impurity ions (Na+ and Cl– in this case), and we would either need to replace the resin or reactivate
it. We can reactivate the cation exchange resin by passing relatively concentrated hydrochloric acid
through it to remove all the Na+ and replace it with H+. We can reactivate the anion exchange resin by
passing a relatively concentrated solution of sodium hydroxide through it to remove all the Cl– and
replace it with OH–. To minimize how often we need to replace these resins or how frequently we need
to recharge them, it’s best to have resins with as high an ion exchange capacity as possible (the capacity
is determined by the number of phenyl rings that have been derivatized with fixed ions).

The capacity of ion exchange resins used for deionizing water is too high for analytical
purposes. Presumably, the concentrations of ions in samples that we want to analyze are relatively low.
If we use high capacity resins, the retention times will be much too long. One of the early impediments
to the use of ion exchange as an analytical method was the lack of methods to reproducibly synthesize
resins with low capacities. One of the first groups of people to figure out a way to do this was chemists
at Dow Chemical. They were motivated by a need to measure inorganic anions and cations at low
levels and realized that ion-exchange chromatography would be an ideal method for doing so.

With any chromatographic method, it helps to know some basic rules for predicting retention order. An
interesting example to consider in ion-exchange chromatography is the retention order for the
ions Li+, Na+, and K+ on a cation exchange resin. One thing we can consider is whether these ions
have different strength interactions with the fixed anion in the resin. All three ions have the same
charge. What should also be apparent is that lithium is the smallest ion with the densest charge,
potassium the largest with the most diffuse or fluffiest charge. We therefore might predict that the
attraction of the lithium ion for the sulfonate groups in the resin is the strongest of the three ions. It
turns out that the equation for ionic attraction has a distance term in it, and the closer the ions, the
stronger the attraction. Lithium being the smallest is effectively closer to the sulfonate and has the
strongest attractive force of the three for the resin. This would suggest a retention order with potassium
eluting first, sodium second, and lithium last as shown in Figure 59.

Figure 59. Retention order of Li+, Na+ and K+ on a cation-exchange column based on stationary phase
effects.

But there is also a mobile phase in chromatographic separations, and it is useful to examine whether
these ions have different relative stabilities in the mobile phase. If one of the three is most stable in the
mobile phase, it might be expected to stay in the mobile phase more than the others and elute first.
What might affect the stability of these ions in the mobile phase? The mobile phase in this separation
would be an aqueous phase. If we consider water, we know that it is unusual in having a very elaborate
network of hydrogen bonds. Any ion that dissolves in water will cause some disruption of this network.
The question to consider is which ion would be the most disruptive of the three to the hydrogen bond
network? Since the charges of these three ions are identical, this decision will be based on the size.
The larger potassium ion will be more disruptive of the hydrogen bonds, and more difficult for water
to accommodate. You could then say that Li+ is more stable in the water, stays in the mobile phase
more, and elutes first. You could phrase this another way by saying that the K+ is least stable in the
aqueous mobile phase, and so is “forced” into the resin by the mobile phase and elutes last. Sometimes
the chromatographic literature refers to this as the solvophobic effect. The K+ fears the solvent and so
spends more time in the resin. The relative retention order based on mobile phase effects would be
lithium first and potassium last, as shown in Figure 60, the exact opposite of what was predicted above
based on stationary phase effects

Figure 60. Retention order of Li+, Na+ and K+ on a cation-exchange column based on mobile phase
effects.

With conflicting predictions, the only way to know which one is more important is to inject the ions
and determine the retention order. In this case, the measurements show that the Li+ elutes first and
the K+ elutes last. The mobile phase effects are more significant in determining the retention order.

Now suppose we had two ions with exactly the same size, but one had a charge of 1+ and the other a
charge of 2+. Examining stationary phase effects, the equation that describes the attraction between
two ions has the charges of both ions in it. The greater the charge of the ions, the greater the attraction.
We would therefore expect the 2+ ion to show greater attraction for the fixed sulfonate anions and
elute later than the 1+ ion as shown in Figure 61.
 Figure 61. Retention order on a cation exchange column for ions of the same size but different
charge based on stationary phase effects.

Examining mobile phase effects, both ions would have a similar extent of disruption of the hydrogen
bond network since both have the same size. What we then need to consider is the strength of the
attraction between the positive ion and the sphere of water molecules that surround it (remember, these
cations would be surrounded by the slightly negative oxygen atoms of water molecules). This
represents an electrostatic attraction, and again is dependent on charge. Therefore the 2+ ion is more
stable in the water and ought to elute first as shown in Figure 62, the exact opposite of what was
predicted based on stationary phase effects.

Figure 62. Retention order on a cation exchange column for ions of the same size but different
charge based on mobile phase effects.

Faced with conflicting predictions, it’s necessary to perform the experiment and see which comes out
first. In this case, the measurements show that the 1+ ion elutes first, the 2+ ion elutes second. The
stationary phase effects are more important in this case in determining the retention order. The likely
reason why the stationary phase effects are more significant is that the 2+ ion can actually bind
simultaneously at two adjacent sulfonate sites as shown in Figure 63.
 Figure 63. Representation of a +2 ion associating at two anionic sites on the resin.

The likelihood that this occurs can be observed if a lanthanide(III) ion is added to these resins. In this
case, the resin particles actually shrink in size as the lanthanide ion is added. The reason for the
compression is that the binding of three sulfonate groups to the lanthanide causes the polymer to
collapse in a bit to fit these groups around the lanthanide.

The separation of Li+, Na+, and K+ described in the prior problem would often be done on a polystyrene
resin using a fairly dilute solution of hydrochloric acid (perhaps 0.1 M) as the mobile phase. The bound
ions would be sulfonate groups and the mobile counter ion would be the H+ ion. An important issue is
how to detect these ions. They do not absorb ultraviolet or visible light in the accessible portion of the
spectrum. They do not absorb infrared light. Conductivity is one possibility for performing the
measurement. The conductivity of a solution is a measure of the extent to which the solution conducts
electricity. Dissolved ions are needed for a solution to conduct electricity, and the higher the
concentration of ions, the higher the conductivity. We can measure the conductivity of a solution quite
sensitively. In fact, this is the reading that is performed on water that has been purified by passage
through a MilliQ water purification device to see just how well the water has been deionized. The only
problem with trying to apply a direct conductimetric measurement is that the hydrochloric acid in the
mobile phase produces too high a background signal. The chemists at Dow who had developed low
capacity ion exchange resins recognized this problem as well and devised an ingeneous way to remove
the conductivity of the eluent ions (HCl) but retain the conductivity of the alkali ions they wanted to
detect.

What they did was use a device called a suppressor column. If we imagine measuring Na+ in a
solution containing sodium chloride (Na+Cl–), we first start with a cation exchange resin in the H+ form
and would have Na+Cl– and H+Cl– eluting out the end of the column when the sodium band comes off
as shown in Figure 64.
 Figure 64. Effluent from the analytical column when analyzing a sample containing sodium.
H+Cl– is the eluent ion

Note that there is still a high concentration of HCl mixed with the NaCl as it elutes from the column.
This HCl will interfere with the measurement of conductivity. Suppose we took the column eluent and
then passed it through an anion exchange resin in the hydroxide form as shown in Figure 65. The
Cl– ion of the Na+Cl– would exchange with the hydroxide, converting this into sodium hydroxide
(Na+OH–), a conducting electrolyte because it stays ionized. The Cl– ion of the HCl would exchange
with the hydroxide, converting this into H+ and OH–, which is non-conducting water. We can therefore
measure a conductivity that only relates to the amount of sodium ion in the original sample. An
analogous scheme, which had the columns reversed, could be used to measure the conductivity of
anions that were separated on an anion exchange column in the hydroxide form.

Figure 65. Effect of the suppressor column on the eluent from the column in Figure 64.

Eventually, the hydroxide counterions in the suppressor column will all become replaced with chloride
ions and the device would not work anymore. The suppressor column must be periodically regenerated
in the hydroxide form.

Today, instead of using suppressor columns to remove the conductivity of the eluent ions, membrane-
based electrolytic neutralization devices are employed. The electrolysis of water can be used to
generate hydronium and hydroxide ions, and by proper design, the desired ion can be generated in such
a way to pass through a membrane and suppress the conductivity of the eluent ions. In some
instruments, similar electrolytic strategies are used prior to the analytical column to generate the eluent
ions as well. The use of this eluent generation technology leads to less background conductivity and
better sensitivity, making it especially useful for the analysis of low levels of ions.

Procedure of ion exchange chromatography

 Ion exchange separations are carried out mainly in columns packed with an ion-exchanger.

 These ionic exchangers are commercially available. They are made up of styrene and divinyl
benzene. Example. DEAE-cellulose is an anionic exchanger, CM-cellulose is a cationic
exchanger.

 The choice of the exchanger depends upon the charge of particle to be separated. To separate
anions “Anionic exchanger” is used, to separate cations “Cationic exchanger” is used.

 First the column is filled with ion exchanger then the sample is applied followed by the buffer.
The tris-buffer, pyridine buffer, acetate buffer, citrate and phosphate buffers are widely used.

 The particles which have high affinity for ion exchanger will come down the column along
with buffers.

 In next step using corresponding buffer separates the tightly bound particles.

 Then these particles are analyzed spectroscopically.

Applications of ion exchange chromatography

 An important use of ion-exchange chromatography is in the routine analysis of amino

acid mixtures.
 The 20 principal amino acids from blood serum or from the hydrolysis of proteins are separated
and used in clinical diagnosis.
 This is most effective method for water purification. Complete deionization of water (or) a
non-electrolyte solution is performed by exchanging solute cations for hydrogen ions and
solute anions for hydroxyl ions. This is usually achieved by method is used for softening of
drinking water.
 In the analysis of products of hydrolysis of nucleic acids. In this way, information is gained
about the structure of these molecules and how it relates to their biological function as carriers
of hereditary information.
 Chelating resins are used to collect trace metals from seawater.
 To analyze lunar rocks and rare trace elements on Earth.

Advantages of ion exchange chromatography

1. It is one of the most efficient methods for the separation of charged particles.

2. It can be used for almost any kind of charged molecule including large proteins, small
nucleotides and amino acids.

3. Ion exchange is used for both analytical and preparative purposes in the laboratory, the
analytical uses being the more common.

4. Inorganic ions also can be separated by ion-exchange chromatography

Limitations of ion exchange chromatography

 Only charged molecules can be separated.

 Buffer Requirement