Ion Pair Chromatography - Differentiating

When to Use Alkyl Sulfonates and

Perchlorates
HPLC

Ion pair chromatography (IPC) is one technique used to separate charged substances. It is widely
used to selectively analyze acids and bases, particularly with reverse phase chromatography.
However, customers often complain that setting analytical conditions for IPC can be troublesome
or good reproducibility is difficult to obtain. Such problems probably occur due to insufficient
consideration given to selecting pair ions (ions with the opposite charge of target components)
for adding to mobile phases, and to their use conditions. Therefore, the following explanation
uses alkyl sulfonates and perchlorates as an example, which are commonly used in IPC of bases
and cations, to provide an overview on how to determine which to use based on their respective
characteristics.

Alkyl Sulfonate
Fig. 1 Influence of Pair Ion Concentration on Component Retention Column: STR ODS-M,
Mobile phase: 10 mM citrate (sodium) buffer solution containing sodium 1-octanesulfonate (pH
4.6) / acetonitrile solution = 5/1 (v/v), Flowrate: 1.0 mL/min, Column temperature: 40 C, and
Detection: 280 nm. Components: (Gray) - Dopamine, - Epinephrine, - Norepinephrine

Alkyl sulfonate is a typical pair ion used in IPC of substances with a positive charge. Normally,
alkyl sulfonate with 5 to 12 carbons is used as a sodium salt. In general, the separation
mechanism used in reverse phase ion pair chromatography is explained as consisting of two
processes - an ion pair distribution process, which pairs the target components with pair ions and
captures them in a solid phase, and an ion exchange process, which retains the target components
by ionic interaction with the pair ions hydrophobically adsorbed to the solid phase. However, in
the case of alkyl sulfonate, the main mechanism is an ion exchange process. Therefore, the
higher the number of carbons in the alkyl sulfonate, the stronger the retention effect on the
component. In addition, for the same type of alkyl sulfonate, the lower the concentration of
organic solvent in the mobile phase, the stronger the retention. (If an alkyl sulfonate with a large
number carbons is used with an extremely low concentration of organic solvent, a pseudo-ion
exchange mode occurs, where once equilibrium is reached, target components can be retained
without adding pair ions to the mobile phase.) The pair ion concentration affects component
retention as well, but surfactants like alkyl sulfonates exhibit a unique relationship between
concentration and retention behavior. This relationship is shown in Figure 1. In areas where the
concentration is relatively low, component retention increases in a linear manner, but once it
becomes saturated at a given concentration (referred to as the "fold over point"), the retention
level reverses direction and begins decreasing. This is explained by the alkyl sulfonate forming
micelles, resulting in a secondary hydrophobic phase within the mobile phase. Therefore, there is
a limited range of pair ion concentration that can be used for IPC.

Perchlorate

Unlike alkyl sulfonates, perchlorates (normally used as a sodium salt) are not themselves
hydrophobic. Therefore, they cannot be used for ionic exchange purposes. Nevertheless, since
perchlorates have a large ion radius, they readily form ion pairs and, consequently, can be
considered completely in terms of the ion pair distribution process. Therefore, they have no fold
over point in the relationship between concentration and component retention, so retention keeps
gradually increasing as the concentration increases. Furthermore, the component retention effect
is uniform, regardless of organic solvent concentration.
However, since the retention of components by perchlorates is based on the hydrophobic
properties of the components themselves that appear when their charge is canceled by the
formation of ion pairs, it may not be applicable for some components.

Differentiating the Use of Pair Ions

Fig. 2 Ion Concentrations and Organic Solvent (acetonitrile) Concentration Range Effective for
Ion Pair Chromatography Red: Hexanesulfonate, Blue: Octanesulfonate, Yellow:
Decanesulfonate, Green: Perchlorate

Based on the characteristics of respective pair ions, the following describes practical
considerations for differentiating when to use different pair ions. The fundamental objective of
using IPC and these pair ions is to increase the retention strength of bases and cations, but they
are also used to suppress peak tailing (phenomenon common in ODS and other silica gel
columns). For the first of these objectives, alkyl sulfonate is more useful, because it can control
component retention over a wide range. For the second objective, perchlorates are preferable,
because they are easier to use and can be used for any component. Therefore, in general, alkyl
sulfonates are preferred for component retention, while perchlorates are preferred for suppressing
tailing. However, if target components are hydrophobic ions, there is no significant difference
between alkyl sulfonates and perchlorates. If an alkyl sulfonate is used, the concentration of the
organic solvent in the mobile phase is extremely important when determining the number of
carbons for the alkyl sulfonate. Eluting components at fixed positions using fixed organic solvent
concentrations requires that the fewer the number of carbons, the higher the concentration.
However, as shown in Figure 1, there is a limit to pair ion concentrations, so if the organic
solvent concentration is high, alkyl sulfonates with few carbons cannot be used. However, that
does not mean that higher carbon counts are necessarily preferable. Rather, fewer carbons is an
advantage in terms of column equilibrium. The lower the organic solvent concentration and the
lower the pair ion concentration, the longer it takes for the column to reach equilibrium, but if an
alkyl sulfonate with high carbon count is used with a low organic solvent concentration, it will
take a very long time for the column to stabilize because a low concentration is being used, of
course. Consequently, it is important to decide the organic solvent concentration before selecting
the number of carbons. Figure 2 shows general guidelines for determining use conditions (pair
ion concentrations and organic solvent concentrations) effective for respective pair ions. Use it as
a reference for selecting pair ions.

Setting Organic Solvent Concentrations

Decide the organic solvent concentration, by first considering the original hydrophobicity of the
target components. In other words, consider how well the mobile phase retains the target
components without including any pair ions. For example, if IPC is to be used to elute a
component in 10 minutes, mobile phase conditions must allow eluting the component within 10
minutes without including any pair ions. Consequently, the lower limit for organic solvent
concentration is the concentration that results in eluting the component in 10 minutes, so it is
necessary to set the concentration equal to or higher than that. If IPC is being used to simply
increase the retention strength of a component, any appropriate concentration equal to or higher
than that could be selected, but if components with similar hydrophobicity must be separated, the
concentration should be kept as close to the lower limit as possible, because the higher the
organic solvent concentration, the smaller the relative retention. If target components include not
only bases and cations, but also neutral substances or acids, the concentration that can adequately
retain such components is considered the upper limit.

Analysis of Ions Using High Performance Liquid Chromatography:

Ion-Chromatography

What Is Ion Chromatography?

Ion chromatography (IC) is an analytical technique for the separation and determination of ionic
solutes in water in general especially environmental in industrial processes, metal industry and
industrial waste water in biological systems in pharmaceutical samples in food etc. IC can be
classified as a liquid chromatographic method, in which a liquid permeates through a porous
solid stationary phase and elutes the solutes into a flow-through detector. The stationary phase is
usually in the form of small-diameter (5-10 mm) uniform particles, packed into a cylindrical
column. The column is constructed from a rigid material (such as stainless steel or plastic) and is
generally 5-30 cm long and the internal diameter is in the range of 4-9 mm. A high pressure
pump is required to force the mobile phase through the column at typical flow rates of 1-2
ml/min. The sample to be separated is introduced into the mobile phase by injection device,
manual or automatic, prior to the column. The detector usually contains low volume cell through
which the mobile phase passes carrying the sample components. A chromatographic system is
shown in Figure 1.
Any chromatographic system of the type shown in Figure 1 can be divided into instrumentation
and chemistry components. The instrumentation components are the pump, injector, detector
and data station, whereas the chemical components are the mobile phases and the stationary
phases. The instrumentation is typical to high performance liquid chromatography (HPLC) and
the chemistry components are the ones that determine that this mode of HPLC is dedicated to
analysis of ions. In any chromatographic mode the composition of the mobile phase provides the
chemical environment for the interaction of the solutes with the stationary phase. Separation can
be achieve by controlling and manipulating these interactions, which effect the relative retention
times of the various sample components. The types of solutes that can be determined using Ion
Chromatographic techniques are the following:
Inorganic ions such as Cl-, Br-, SO42- etc.
Inorganic cations, including alkali metal, alkaline earth, transition metal and rare earth ions, but
not neutral metal complexes.
Organic acids, including carboxylic, sulfonic, phosphonic acids etc.
Organic bases, including amines.
Ionic organo-metallic compounds.

The liquid chromatographic techniques applicable to the separations described above are termed
as the following:

Ion-exchange chromatography
Ion-exclusion chromatography
Ion-pair (Ion-interaction) chromatography
Capillary electrophoresis

The discussion here will include only ion-exchange chromatography.

Principles of the Separation

The mechanism of interaction of the solutes with the stationary phase determines the
classification of the mode of liquid chromatography. In ion chromatography the basic interaction
is ionic. The stationary phase is charged due to fixed anions or cations, which are neutralized by
counter ions of the corresponding opposite charge. The counter ions can be exchanged by other
ions either from the mobile phase or from the sample, hence the name ion-exchange
chromatography.

Figure 2 illustrates the principle of retention by exchange of anions in anion-exchange

chromatography, and Eqn 8 describes it as an equilibrium.
The functional groups on the stationary phase's surface are fixed positively charged species
(M+). At equilibrium these positively charged functional groups are neutralized by the counter
ions from the running mobile phase (C-). In the second and the third steps, the anionic sample
components (A-) enter the column and distribute between the stationary and the mobile phases
by displacing the counter ions, and being displaced by the mobile phase ions back and forth. The
distribution equilibrium is determined by the competition between the sample components and
the anions of the mobile phase on the charged sites of the stationary phase. The process can also
be described as shown in Eqn 8:

8) M+C- + A- ---> M+A- + C-

The electroneutrality of the solution must be maintained during the ion-exchange process,
therefore, the exchange is stoichiometric so that a single monovalent anion A- displaces a single
monovalent counter ion C-. The process of cation retention is similar, however, the stationary
phase is negatively charged and the counter ions are positively charged.
Types of Stationary Phases
Ion-exchangers are characterized both by the nature of the matrix used as a
support and the nature of the ionic functional groups on the surface. Table 1 shows
the types of functional groups commonly encountered in ion chromatography.

CATION EXCHANGERS ANION EXCHANGERS

Sulfonic acid -SO3- H+ Quaternary amine -N(CH3)3+ OH-

Carboxylic acid -COO- H+ Quaternary amine -N(CH3)2 (EtOH)+ OH-

Phosphonic acid PO3- H+ Tertiary amine -NH(CH3)2+ OH-

Phosphinic acid HPO2- H+ Secondary amine -NH2(CH3)2+ OH-

Phenolic -O- H+ Primary amine -NH3+ OH-

Arsonic -HAsO3- H+

Selenonic -SeO3- H+

Cation-exchangers functional groups can function as such only when they are ionized, therefore
they are classified into strong acid and weak acid types accordingly. The strong acidic functional
groups are ionized over a wide pH range, in contrast to the weak acidic functional groups, which
are ionized over a limited pH range. Sulfonic acid exchangers are strong acid types, whilst the
remaining cation-exchangers functional groups in Table 1 are weak. The weak acidic functional
group requires the use of pH higher than its pKa. For example, a carboxylic functional group
such as Resin-COOH will be able to retain cation only in its Resin-COO- form, which exists
mainly at pHs above its pKa.
Similarly, anion-exchangers are classified as strong base and weak base exchangers. Quaternary
amine functional groups form strong anion-exchangers, whilst less substituted amines form weak
base exchangers. The strong base will be positively charged over a wide pH range, therefore will
be able to function as an anion-exchanger, in contrast to the weak anion-exchangers. A weak
anion-exchangers such as Resin-NH2 for example, requires pH sufficiently low enough to
protonate the amine group into Resin-NH3+. Most of the ion-chromatography separations, using
silica or polymeric ion exchangers perform on strong anion-exchanger (SAX) or strong cation
exchangers (SCX).
Matrixes
The types of matrixes used as support for stationary phases in ion chromatography can be
divided to three: silica-based, synthetic organic polymers and hydrous oxides.
Silica-Based
There are two distinct groups of silica-based materials. One group includes the functionalized
silica, where a functional group is chemically bonded directly to the silica particle. The second
group is the polymer-coated silica, in which the silica particles are first coated with a layer of
polymer, such as polystyrene, silicone or fluorocarbon, and this layer is then functionalized. The
main advantage of such particles over the total polymeric ones are the faster diffusion of the
solutes throughout the thin layer of the polymer, which leads to better mass transfer between the
two phases, the stationary and the mobile. Improved mass transfer leads to better efficiency of
the separation.
The functionalized silica-based ion-exchangers are produced by chemically bonding quaternary
amines to form strong anion exchangers and alkylsulfonates to form strong cation exchangers.
Their capacity is usually moderate to high, requiring either UV-VIS detection or conductivity
with suppression. The polymeric coated silica have low capacities, therefore, they are suitable
for non-suppressed ion-chromatography.
The most important advantage of silica-based stationary phases is the better chromatographic
efficiency, stability and durability in high pressures. A serious drawback of the silica-based
stationary phases is the limited pH range over which the columns can be operated: 2 > pH < 7.
Another drawback of the silica based particles is the affinity of the exposed silica and the free
silanols on the surface to metal ions with high charge density, such as transition metals. Those
are irreversibly adsorbed on the surface, causing interference with the analysis.

Polymer-based ion-exchangers
Polymeric supports for ion-exchange chromatography are called also resins. These materials are
produced by chemically derivatization of synthetic organic polymers, and they are the most
widely used types of ion-exchangers. These resins are manufactured by first synthesizing a
polymer with suitable physical and chemical properties, and then they are further reacted to
introduce the ionic functional groups. Most ion-exchange resins are made from copolymers
consisted of styrene and divinylbenzene (PS-DVB), and some are consisted of copolymers of
divinylbenzene and acrylic or methacrylic acid (PMMA).
The fact that a low degree of functionalization required for ion chromatography implies that a
significant proportion of the surface area of the resin exists as a neutral polymer, mainly aromatic
moieties. It can therefore be expected that some of the reversed-phase character of the original
polymer will be retained, and surface adsorption effects will contribute to the retention of organic
ions. These effects are the reason for differences in ion-exchange selectivity between resin- and
silica- based ion-exchangers and the need for eluents containing polar organic solvents to control
the selectivity.

Comparison
The prime advantage of resin-based ion-exchangers is their tolerance towards eluents and
samples with extreme pH values, between 0-14, in contrast to the silica-based stationary phases,
whose pH limits are 2-7. This wide range of pH values enables the exploitation of selectivity
effects of multi-charged or weakly ionizable solutes.
The polymeric resins are subject to pressure limitations, because they are relatively soft
materials, as a result, the column lengths and flow rates are limited. Macroporous (uto 1000 A)
resins are relatively more rigid and stable, therefore, they can be used in long columns and
higher flow rates.

Hydrous Oxide
Minerals, such as aluminosilicates, alumina, silica or zirconia can act as ion-exchangers because
the skeleton or matrix material carries an excess charge which is neutralized by mobile counter
ions. Anion - cation separations on a mixed-bed alumina - silica column. The metal oxide can
act as both an acid or base and indicate the possibilities of cation- and anion-exchange behavior
respectively:

9.1 =M-O-H =M-O- + H+

9.2 =M-O-H =M + + OH-

The ion-exchange properties of hydrous metal oxides show strong pH dependence. The pH
values at which the reactions in Eqn 9 occur are dependent on the type of hydrous oxide under
consideration. The matrix is a cation exchanger at low pH values and an anion exchanger at high
pH values and it has an isoelectric pH range, depending on the surface chemistry and the type of
buffer that is used to maintain the pH. For example, the isoelectric point for silica is 2 and for
alumina it can be 3.5 in citrate buffer and 9.2 in carbonate buffer. The pH is therefore a powerful
selectivity controlling parameter in the hydrous oxide stationary phases.

Characteristics - Ion capacity

The ion capacity of the ion-exchanger is determined by the number of functional groups per unit
weight of the stationary phase. The most commonly used unit is milliequivalents of charge per
gram of dry packing, or milliequivalents per ml of wet packing. In the second case it is
customary to state the type of counter-ion present in the stationary phase, since it affects the
degree of swelling of the packing and hence its volume. The ion-exchange capacity of a
stationary phase plays a significant role in determining the concentrations of competing ions
used in the mobile phase for elution. Higher capacity stationary phases generally require the use
of more concentrated mobile phases, which are problematic when high performance ion-
chromatography is concerned, due to the use of conductometric detectors, which cannot function
well with high salts concentrations. Typical ion-exchange capacity in IC is 10-100 mequiv/g.

Characteristics - Swelling
Organic stationary phases consist of cross linked polymer chains containing ionic functional
groups. When such materials come into contact with water they tend to swell, with swelling
pressures up to 300 atmospheres with high ion-exchange capacities. The higher the ionic
capacity and lower cross linking the more sensitive the polymer is to swelling. The content of
the mobile phase is very significant to the effect of swelling. Macroporous resins with high cross
linking and small ion-exchange capacities are commonly used as stationary phases for high
performance ion chromatography.

Characteristics - Selectivity
The relative affinities of different counter ions to the stationary phase show considerable
variation with the type of ion-exchanger and the conditions under which it is used. There are
cases where simple ion-exchange mechanism may not be the sole retention mechanism, such as
cases where there are ion-exclusion effects exist or adsorption to the stationary phase matrix
rather than to the functional groups. However, it is still possible to provide approximate
guidelines for the relative affinities of the ion-exchangers for different ions. The properties of
the solute ions, the mobile phase ion and the counter ions that affect the extent of the ionic
interactions are the following:
The charge on the solute ion
The size of the solvated ion
The degree of cross-linking of the ion-exchange polymers
The polarizability of the solute ion
The ion exchange capacity of the stationary phase
The type of functional group on the stationary phase
The extent of interactions with the stationary phase matrix of the support.
As a rule, an increase of the charge-density (charge / solvated size) of the solute ion increases,
its affinity for the stationary phase. Higher charge with smaller solvated ion radius result in
higher retention due to higher coulombic interactions. This trend becomes more pronounced in
more diluted mobile phases.
The order of relative affinities of cations to strong acid cation exchange stationary phases are
generally in the following order:
Pu4+ >>
La3+ > Ce3+ > Pr3+ > Eu3+ > Y3+ > Sc3+ > Al3+ >>
Ba2+ > Pb2+ > Sr2+ > Ca2+ > Ni2+ > Cd2+ > Cu2+ > Co2+ > Zn2+ > Mg2+ >
UO22+ > >
Tl+ > Ag+ > Cs+ > Rb+ > K+ > NH4+ > Na+ > H+ > Li+ >

From this series it can be concluded that cation-exchange mobile phases of 0.1 M KCl are
stronger then those containing 0.1 M NaCl, provided that all other parameters are identical.
The order of relative affinity of anions on strong base anion exchangers follow the general order
of:
citrate> salicylate> ClO4 > SCN- > I- > S2O32- > WO42- > MoO42- > CrO42- > SO42-
>SO32- > HPO42- > NO3- > Br- > NO2- > CN- > Cl- > HCO3- > H2PO4- > CH3COO- > IO3-
> HCOO- > BrO3- > ClO3- > F- > OH-
Higher degree of cross linking result in ion-exclusion effects, i.e., exclusion of ions with higher
solvated radii from the stationary phases pores. Since these ions are also less retained, they
elute faster than the smaller, more charged ones, which can enter the small pores. Ions with high
charges and small radius are polarizable are therefore retained longer. The effects of the last two
properties in the above list are hard to predict. Therefore, it is not possible to provide clear-cut
guidelines, regarding the control of the separation, based on these two properties..

Properties of Mobile phases

Elution strength of the mobile phase is controlled by changing ionic strength, pH or type of
anions. The mobile phases used in IC are typically aqueous salt solutions which can be
classified into groups of similar characteristics as the following:
5.1 Compatibility with the detection mode - Suppressed or Non-suppressed.
5.2 Nature of the competing ion
5.3 Concentration of the competing ion
5.4 Mobile phase's pH
5.5 Buffering capacity of the mobile phase
5.6 Ability to complex the ionic sample components
5.7 Organic modifiers

Compatibility with the detection mode - Suppressed or Non-suppressed.

The detection mode that is used is the major factor that determines the types of mobile phases
suitable for the desired separation. The detector signal obtained by the background, i.e., the
mobile phase itself, must not be too high, otherwise it would be difficult to obtain linearity, wide
dynamic range and stability of the baseline. When high sensitivity is needed, highly responding
mobile phase (highly conducting in conductivity detector and highly absorbing in UV-VIS
detector) will render it impossible to be used. If a highly conducting mobile phase is the only
option for a particular separation, or high sensitivity is a must, the mobile phase should be
selected so that its conductivity will be suppressed, using a suppressor between the column outlet
and the detector.

Nature of the competing ion

In qualitative terms, the mobile phase characteristics which influence solute retention are the
relative affinities of the sample ions and the mobile phase's competing ions. The affinity of the
mobile phase ions to the stationary phase is governed by the same factors that effect the affinity
of the solute ions, i.e., charge density, degree of hydration, polarizability etc. Mobile phase ions
of higher affinity to the stationary phase are stronger, and will result in lower interactions of the
sample ions with the stationary phase, hence lower retention times.

Concentration of the competing ion

The concentration of the counter ion in the mobile phase effects the retention of the sample ions
as well, higher concentrations result in stronger competition, and displacement of the sample
ions from the stationary phase, hence lower retention. The effect of concentration on the
competition between the solute and the phase's ions is much more pronounce for singly charged
ions than for doubly charged ions, although the latter is a stronger eluent.
It is therefore most convenient to choose the type of mobile phase by initially selecting the
appropriate charge. The next step will be considering additional effects on selectivity such as
size, polarizability etc. within the group of mobile phase salts having the desired charge. The last
consideration will be manipulating mobile phase salts concentration to produce the required
separation.

Mobile phase's pH

The Mobile phase's pH is a key parameter in determining its characteristics, as it influences the
charges on both the mobile phase's ions and the solute ions. The effect of pH is particularly
important in the separations of anions, where it may effect their ionization. The charge on the
acid anion increases with pH, so the eluting power of weak acid eluents increases with pH until
the acid is completely dissociated. The opposite trend occurs for weak bases in the mobile
phase. With decreasing pH a higher degree of protonation occurs and the mobile phase becomes
a stronger eluent. Similarly, the degree of ionization of solute ions that derived from weak acids
or bases will be pH dependent. In this case, increased solute charge will increase its affinity to
the functional groups on the stationary phase, hence increase their retention. Examples of solutes
showing these effects are F-, CO32-, PO43-, SiO32-, CN- and amines. When these ions are
present in mixtures with other ions that show no pH dependency, the control of mobile phase's
pH becomes an important variable to be manipulated in the optimization of the separation.

Buffering capacity of the mobile phase

Since both mobile phase's and solutes' ions can be effected by the pH, the buffering capacity of
the mobile phase is very important, and should be maintained high. Polyprotic solute ions'
retention can be significantly changed with pH, as their charge can increase from singly to
doubly and triply charged. In such cases it is very important to make sure that the mobile phase
pH is kept constant, using high capacity buffers.

Ability to complex the ionic sample components

When metallic ions separations are considered, the ability of the mobile phase's salts to complex
them is a very important variable. The complexing agent forms complexes with the metal ions
that may change its original charge and degree of ionization. The new species have now
different retention times, therefore, separation is effected. The degree of complexation depends
on the concentration of the complexing agent as well as on the pH of the mobile phase.

Organic modifiers
Water miscible organic solvents , such as methanol, ethanol, glycerol, acetonitrile and acetone
are used sometimes as additives to the mobile phase for the ion-exchange separations. Ion-
chromatographic separation of alkali metals in organic solvents. These solvents can effect
variety of parameters related to the separation process, such as alter affinity of organic ions to the
stationary phase, alter the degree of complexation when such process occurs, change the degree
of ionization of weak acidic and basic ions either in the mobile or the stationary phases or in the
samples.

Ion-Suppression in Ion-Chromatography

Suppression in ion chromatography is needed when conductivity detectors are used and the
mobile phase is intensively conducting, saturating the detector's response. A device, called the
suppressor, is inserted between the ion-exchange separator column and the detector. The device
releases hydronium ions or hydroxyl ions dependent on the characteristics of the mobile phase,
to convert it to the corresponding non-ionized species hence reduce their conductance. The
suppressor modifies in fact both the mobile phase and the separated solutes coming out of the
separator column, so that the mobile phase's conductance is reduced and that of the solutes is
enhanced, hence detectability of the solutes is improved. The suppressor requires a regenerant
(or scavenger) solution to enable it to operate for extended periods.
The most simple means to accomplish suppression of an acidic mobile phase is to pass it through
a cation-exchange column in the hydrogen form. The most simple example for the function of a
suppressor is the case of Cl- ion as a solute eluted by an eluent that composes of NaHCO3. The
eluent reaction in the suppressor is given in Eqn 9 I and the reaction of the solute with the
suppressor is given in Eqn 9II.

9 I ) Resin-H+ + Na+ HCO32- --> Resin-Na+ + H2CO3

9 II) Resin-H+ + Na+ Cl- --> Resin-Na+ + HCl

The combined result of these two processes is that the mobile phase's conductance is reduced
greatly whilst the conductance of the sample ions is enhanced by the replacement of sodium ions
(50 S. Cm2/equiv.) with hydronium ions (350 S.cm2/equiv.). The detectability of the solute is
therefore enhanced.
A similar procedure can be applied to cation-exchange chromatography, when the suppressor is
an anion-exchange column in the OH- form, which provides hydroxyl ions to the stream. A
simple example would be the separation of Na+ ions using HCl in the mobile phase. The
processes of suppression are shown in Eqns 10 I and 10 II.

10 I) Resin-OH- + H+ Cl- --> Resin-Cl- + H2O

10 II) Resin-OH- + Na+ Cl- --> Resin-Cl- + Na+ OH-

The eluent is converted into water whilst the conductance of the sample band is increased due to
replacement of the Cl- ions (76 S.cm2/equiv) by OH- ions (198 S.cm2/equiv.).
Mobile Phases for Non-Suppressed Ion-Exchange Chromatography

Eluents for Anions

a. Aromatic carboxylic acids and their salts .

Salts of aromatic carboxylic acids, such as those shown in Fig (4.6 p85 chemdraw) are the most
widely employed eluent species in the separation of anions by non-suppressed IC. They have
low conductances, therefore, when used in dilute solutions they provide eluents with low
background conductance. The aromatic moiety is an intense UV chromophor, so aromatic acid
salts are also ideal for indirect spectrophotometric detection. All of these acids are relatively
weak, therefore the have buffering action, and since many of them are polyprotic, they can
provide the buffering action over a relatively wide range of pHs.
Mobile phases prepared from aromatic carboxylate salts are prepared very simply by mixing the
acids with the appropriate amounts of lithium hydroxide which is less conducting than Na+ or
K+. When high pH is needed to increase the retention of weak acidic solutes, a borate buffer is
used to raise the pH instead of the LiOH.

b. Aliphatic carboxylic acids

Mobile phases prepared from salts of aliphatic carboxylic acids have been employed widely in
non-suppressed. Citric, tartaric, succinic, fumaric, malic, fumaric, Acetic and formic have all
been used as eluent species.
With the exception of citrate, these are weak eluents, highly conducting, with weak to moderate
UV absorption and low ion-exchange selectivity coefficient. They are appropriate for the
separation of mixtures of weakly retained anions.

c. Aromatic and aliphatic sulfonic acids

Sulfonic acids are usually fully ionized in aqueous solution over the eluent pH range employed
in non-suppressed IC. Eluent pH is therefore not a critical factor in determretention times of the
solutes. Aromatic sulfonic acids have most of the advantages of aromatic carboxylic acids, i.e.,
low conductance, strong UV absorbance and large ion-exchange selectivity coefficient. They are
strong eluents, suitable for conductivity and for indirect spectrophotometric detection. Their
major drawback is their lack of buffering capacity, so if pH is important for the separation,
additional buffer must be separately added to the mobile phase.
Aliphatic sulfonic acids have higher conductance, which decrease with their chain length. The
have weak UV absorption and moderate ion-exchange selectivities. They are suitable for direct
UV detection.

d. Potassium Hydroxide
The hydroxide ion is the weakest ion-exchange competing anion and has a very high
conductance. It is suitable for weakly retained anions such as F-, ClO3-, BrO3-, Cl-, NO2-, Br-
and NO3-, or anions of weak acids that need high pH values to be retained such as phenols,
silicate, cyano sulfide and arsenite. The detection mode is usually indirect conductivity.
e. Polyol-borate complexes
It is well known that both boric acid or borate form neutral or anionic complexes with
polyhydroxy compounds such as mannitol, glucose, fructose, xylose, glycerol, sorbitol, sucrose
or maltose or acidic compounds such as gluconic, tartaric, glucoronic, and galactoronic. The
complex with the gluconic acid is the most widely used.

f. Ethylenediaminetetraacetic acid - EDTA

EDTA can be used as an aliphatic polycarboxylic eluent for anions as well as a strong
complexing agent for polyvalent metallic cations. The majority of its applications involve the
second property, the complexation capability.

g. Inorganic Salts
Inorganic anions such as Cl-, SO4= or PO43- can be used as strong eluents, but due to their high
conductance direct conductivity detection cannot be used. Other modes of detection can be UV
absorption, refractive index, electrochemical, and post column reaction.

Eluents for Cations

a. Inorganic acids
Dilute solutions of inorganic acids, such as nitric acid, are the most popular eluents for the
separation of alkali metal cations and amines by non-suppressed IC. The eluent strength is
determined solely by its pH. The hydronium ion is an effective competing cation for these
solutes and the very high conductance of the mobile phase enables a sensitive indirect
conductivity detection.
b. Organic bases
Organic bases become increasingly protonated with decreasing pH, hence they act as useful
cation-exchange eluents at low pH. Monovalent protonated bases are effective only to
monovalent amines while diprotonated bases are generally more suitable for the elution of
divalent cations.

Mobile Phases for Suppressed Ion Chromatography

As described above, the suppressor is a device inserted between the chromatographic column and
a conductivity detector. The goal is to reduce background conductance of the eluent and if
possible to enhance the conductance of the analytes ions. Suppressors operate through the
following mechanisms (see Table 2): 1. Exchange of eluent cations for hydronium ions, for
which mobile phases containing sodium salts of weak acids are suitable (carbonic, boric) 2.
Exchange of eluent anions for hydroxide ions, for which nitrate or chloride salts are suitable; 3.
Complete removal of the eluent ions by precipitation, such removal of Ba and Pb ions by
precipitation with SO42-. 4. Reduction of the ions charges in the mobile phase by complexing
them with Cu2+ or other complexant ions. Mobile phases suitable for these suppressors should
contain chelates.
Detection In Ion Chromatography
The following detection methods are available with ion-exchange chromatography:
Conductivity detection
Electrochemical (amperometric or coulometric) detection
Potentiometric detection
Spectroscopic detection
Post-column reaction detection.

Conductivity Detection

Conductivity detection has two major advantages for inorganic ion analysis. First, all the ions
are electrically conducting, so that the detector should be universal in response, and second, the
detectors are relatively simple to construct and operate. Conductivity detection will be discussed
here in terms of principle of operation and performance characteristics, modes of detection, cell
design, post column signal enhancement, i.e., suppression and applications.

Principle of Operation

The mobile phase eluting through the detector is in fact a conducting electrolyte. It flows
through two electrodes across which potential is applied. The more current conducted by the
solution, the higher is the electrical conductivity. The conductance of a solution is determined by
several factors, including the ionic strength and type of species in the solution, as well as the
temperature. The specific conductance depends on the cross sectional area (cm2) of the
electrodes inserted into the solution, and L (cm) is the distance between them, and will vary with
concentration. The conductance is increased for cells in which the electrodes are large in surface
area and are close together. The equivalent conductance is subject to activity effects such as ion-
ion interactions, therefore, the relationship between G and C becomes non-linear at high ionic
strength. Since the conductance of the solution results from both the anions and cations of the
electrolyte, conductance is calculated for the individual anions and cations in solution. Most of
the common cations and anions have limiting equivalent ionic conductance of 30-100 (S.cm2.eq-
1). The most conducting cation is the hydronium ion and the most conducting anion is the
hydroxyl ions; their values are 350 and 198 (S.cm2.eq-1) respectively. The conductance of an ion
increases with its charge density and decreases with its viscosity. Therefore, when stroelutropic
multiply charged ions are needed in the mobile phase they can exert high background, therefore,
large ions such as phthalate, citrate, or trimesate are used in such cases.

A sensitive detection can result as long as there is a considerable difference in the ionic
conductances of the solute and the mobile phases ions. This difference can be positive or
negative, depending whether the eluent ions are strongly or weakly conducting. If the ionic
conductance of the eluent ions is low, then an increase in conductance occurs when the solute
enters the detection cell, due to higher conductance. In general this detection mode is referred to
as direct. On the other hand, when the mobile phase ions are highly conducting, a decrease in
conductance occurs when the solutes enter the detection cell, due to lower conductance. This
mode of detection is referred to as indirect.

Direct conductivity detection is used for most IC methods involving the separation of anions.
Eluents for non-suppressed IC formed from salts such as potassium hydrogen phthalate or
sodium benzoate contain competing anions with moderately low conductance. Similarly, direct
conductivity detection is possible with eluents containing organic bases. Indirect conductivity
detection can be applied to anions using hydroxide eluents and to cations using mineral acid
eluents.

Electrochemical Detection

The term "electrochemical detection" is applied loosely to describe a range of detection

techniques involving the application of electric oxidation-reduction potential via suitable
electrodes to a sample solution, containing oxidizable or reducible solutes. The resulting current
is measured as function of time. Electrochemical detection has been applied in situations where
extreme sensitivity or selectivity is required. Most commonly the electrochemical detector has
been operated in tandem with a conductivity detector, which acts as a universal detector that
gives a more general sample analysis.

Voltammetry
Voltammetry is a well-established technique in which a changing potential is applied to a
working electrode with respect to a reference electrode. The current resulting from the reaction
of analyzed species at the working electrode is measured. The key factor is that the applied
potential is varied over the course of the measurement.

Amperometry and coulometry

The term amperometry describes the technique in which a fixed potential is applied to a working
electrode with respect to a reference electrode. The working electrode is located in the flow-cell
through which the mobile phase passes and the current resulting from the oxidation-reduction
reactions occurring at the working electrode is measured. The analyte to be detected undergoes a
Faradaic reaction if the applied potential has appropriate polarity and magnitude. Wen the
reaction is incomplete, causing only a fraction of the total analyte to react, the detection mode is
termed amperometry, while when the working electrode has larger surface area and the reaction
is complete the mode is called coulometry.

Spectroscopic Methods

Spectroscopic methods of detection are very common in ion chromatography and are second
only to conductivity detection in their abundance. This mode of detection can be divided to two
major categories: molecular and atomic spectroscopy. Molecular spectroscopy includes methods
such as UV-VIS absorption, refractive index, fluorescence and phosphorescence. Atomic
spectroscopy includes flame atomic absorption, flame atomic emission and plasma atomic
emission.

Molecular Spectroscopy:

a. UV-VIS Absorption
Many inorganic cations and anions do not have significant absorption in the UV-VIS range of the
spectrum, therefore, direct detection cannot be used typically. However there are cases where the
ions can be detected directly by their UV or visible detection in the 185-220 nm range.
Detection of non absorbing ions can be achieved using indirect photometric mode, similarly to
the indirect conductivity mode of detection. In the indirect mode highly absorbing ionic species
are used as the eluents with high background, and the detector response is zeroed on them. The
non absorbing solutes are detected as negative peaks, since the detector measures the difference
of absorbance between the high background and the nonabsorbing species. The polarity of the
detector can be then reversed and the peak appear positive. Benzenepolycarboxylic acid salts,
such as phthalate, benzoate, phenylphosphonate, p-toluenesulfonate or trimellitate are used as
chromophoric eluent anions that enable the sensitive detection of non UV-VIS absorbing ions.

b. Fluorescence
Fluorescence detection is well known for its sensitivity. Since most of the ionic species
analyzed by ion chromatography do not exhibit fluorescence, the direct mode of detection has
only a limited scope. Usually the mobile phase includes a chelate or an ion-pair reagent that
forms a species with the ions that produces a signal in the fluorescence detector. It is more likely
to find works that utilize the indirect mode of fluorescence detection.
c. Refractive Index (RI)
Most of the solutes for which ion chromatography is used normally are not detectable directly
by refractive index detectors. The general exception are carboxylic acids, large species such as
polyphosphonates or sulfonium ions and some inorganic ions. In cases where the ions cannot be
detected directly by the RI detector, an indirect mode of detection was used

Atomic spectroscopy

The combination of HPLC separation with various forms of atomic spectrometry gives a method
of great sensitivity as well as a time-resolved detection of species.

a. Flame atomic absorption (AA) and atomic emission (AE).

Direct coupling of atomic absorption spectrometer to an HPLC system requires means to match
the flow-rates of the two techniques. The output of the IC system needs to be relatively high to
accomodate the atomic absorption instrument, therefore, pure water is added some times as a
"make up" solvent.
b. Inductively coupled plasma (ICP)
ICP with emission spectroscopy or with mass spectroscopy have emerged as replacement to
flame emission spectrometers and act as detectors for ion chromatography in recent years. The
introduction of HPLC coupled directly to ICP MS led to the used of these properties in
speciation analysis. The coupling of ion chromatography (IC) with ICP MS made possible the
elimination of gram amounts of matrix in cases where it could be converted into an anionic form,
so that ultra-trace amounts of cationic impurities could be determined. In the semiconductor
field, such analyses have been carried out on matrices of Mo, W, Re, As and P.

Post column reaction

Detection by post-column reaction (PCR) involves the chemical reaction of the solutes as they
elute from the column on the fly, prior to their introduction to the detector. The main goal of
such a procedure it to enhance selectivity and specificity to solutes of small quantities in the
present of large quantities of interferences in the sample matrix. Some of the post column
reagents are amonium molybdate, 4-(2-pyridylazo)resorcinol, pyridine-2,6-dicarboxylic acid,
phenylfluorone, 2-(5-bromo-2-pyridylazo)-5-(diethylamino)phenol.