Analysis of Ions Using High Performance Liquid 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-pair (Ion-interaction) chromatography
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 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 pH’s 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).
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.
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.
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.
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.
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 à =M-O- + H+
9.2 =M-O-H à =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:
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.
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 pH’s.
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 analyte’s 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:
Electrochemical (amperometric or coulometric) detection
Post-column reaction 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 phase’s 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.
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 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 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.
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.
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
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.