Chapter 12

Ion Chromatography
John Statler
Dionex Corporation

Summary
General Uses
Separation and detection of ions and ionizable species Profiling, that is, determining the qualitative distribution of mixtures of oligomeric ions Simultaneous determination of several ions in mixtures Concentration of ionic species in samples of low concentration, often with simultaneous elimination of interfering matrix

Common Applications
Determination of inorganic anions or cations, organic acids, amines, amino acids, carbohydrates, or nucleic acids in a variety of samples Monitoring water quality Determination of the composition of industrial wastes Monitoring the quality of intermediates in industrial processes Determination of ionic composition of biological solutions Separation of components of mixtures before mass spectrometry or other spectroscopic techniques Identification of ionic impurities Purification of components from mixtures

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Samples
State
Liquids that are aqueous or water-miscible solvents can be analyzed directly. Water-immiscible liquids, solids, and gases must be extracted into or dissolved in aqueous solution before analysis.

Amount
Samples are usually introduced as 5- to 200-L volumes, although volumes as large as 100 mL can be introduced using chromatographic preconcentration techniques when additional sensitivity is needed.

Preparation
Dilution or dissolution and filtration are the most common sample preparation procedures. Extraction may be required for nonaqueous samples or preconcentration for dilute samples. The need for precolumn derivatization is rare.

Analysis Time
Excluding sample preparation, analysis times range from less than 3 min to more than 2 hr. Most commonly, however, analysis time is 10 to 15 min.

Limitations
General
Analyses are performed sequentially. Analytes can be misidentified or their quantities incorrectly determined if other components are not well separated. The analysis consumes eluent, which must be replenished regularly.

Accuracy
For routine analysis, accuracy is about 3%, but under carefully controlled conditions and with the use of an internal standard, relative standard deviation less than 1% is possible.

Sensitivity and Detection Limits

For a standard sample size of 25 L, detection limits are about 1 to 5 g/L (ppb) for most common inorganic ions. However, this can vary a great deal depending on the detector response of the analytes, the nature of the separation method, and interfering components in the sample.

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Complementary or Related Techniques

Atomic absorption, atomic emission, and inductively coupled plasma spectroscopies, used for determining the total amount of a metal rather than the amount of a certain ionic form of that metal Mass spectrometry, used to obtain information on chemical structure and molecular mass Nuclear magnetic resonance, used to obtain information on chemical structure Infrared spectroscopy, used to obtain information on chemical structure, particularly before ion chromatography to identify functional groups that can offer a key to separation or detection Capillary electrophoresis, used as a confirmatory technique because it relies on an unrelated separation mechanism

Introduction
Definition
In his comprehensive book on the subject, Small defines ion chromatography (IC) as the chromatographic separation and measurement of ionic species (1). Common applications of ion chromatography include the determination of simple anions, such as chloride and sulfate, simple cations, such as sodium and calcium, transition metals, lanthanide and actinide metals, organic acids, amines, amino acids, and carbohydrates.

Early Developments
Modern ion chromatography began with a report by Small, Stevens, and Bauman (2) wherein they described a way to combine an ion exchange chromatographic separation with simultaneous conductometric detection for the determination of anions including chloride, sulfate, nitrate, and phosphate, or of cations including sodium, ammonium, potassium, and calcium. The key element was their development of a device, later known as a suppressor, to lower the background conductometric signal resulting from the liquid mobile phase, or eluent, while enhancing the conductometric signal from the analyte ions. Originally, this pairing of ion exchange chromatography with suppressed conductometric detection was synonymous with IC. Eventually, however, the term expanded to include other detection methods and other chromatographic modes. The first of the alternative detection techniques, nonsuppressed conductometric detection, emerged in the late 1970s (3). Other routine techniques, including amperometry, optical absorbance, and fluorescence (4), as well as less common techniques such as inductively coupled plasma spectroscopy and mass spectroscopy, have been used in the years since then. Likewise, ion exclusion, ion pairing, and chelation chromatographies have been used in addition to ion exchange for the separation of ions (5). Nevertheless, even with all these methods of separation and detection falling within the modern definition of ion chromatography, IC as it is most often practiced today is still largely an ion exchange separation with suppressed conductometric detection.

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Improvements in Separation
Synthetic ion exchange resins have been available for many decades, but the first resins used in modern IC applications were surface-functionalized styrene divinylbenzene, in many ways very much like those in common use today. Common anions were separated in about 30 min. The alkali metal cations could be separated in about 25 min, but the alkaline earth cations required a separate analysis. These analyses, especially for the cations, seem slow by todays standards, but were vastly superior to the laborious, single ion, wet chemical options. On todays high-efficiency ion exchange resins, common anions can be resolved in less than 3 min, and the common group I and II cations can be determined together in about 10 min. Much of this improvement in speed has been a result of improvements in resin selectivity and uniformity. Most resins used for IC have a thin surface region of ion exchange sites, minimizing band broadening caused by diffusion of the ions into the resin interior. In some cases, analysis speed can also be increased with the use of gradient ion chromatography (6). This technique allows increasing eluent concentration to separate very weakly and very strongly retained ions in a short period of time.

Selectivity
The ideal ion exchange site is a permanent charge (that is, one whose charge does not change with pH) that does not have secondary (nonion-exchange) interactions. Since the beginning of IC, the most common functional group for anion exchange has been some sort of quaternary alkyl ammonium group. This group has a permanent positive charge, regardless of the pH of the surrounding eluent. The alkyl groups on the ammonium and the composition of the surrounding resin can be varied to create different anion exchange environments. This allows the manufacturing of columns to produce resins with different selectivities for specialized applications. Early cation exchangers usually had sulfonic acid groups as the cation exchange sites. The principal advantage of the sulfonic acid is that even at low pH it remains charged. However, these columns are most often used with column switching techniques or with eluents containing both monovalent and divalent ions to elute the monovalent and divalent analyte ions at similar times. In recent years, carboxylic acids have become popular because the affinities of mono- and divalent analyte cations for the functional group are more similar, so simple, unchanging isocratic eluents are capable of separating all cations in similar times.

Improvements in Detection
Conductivity, the most common detection method in IC, has advanced by reducing noise through improvements in electronics and better isolation and control of cell temperature, by improved response through detector cell design, and, in the case of suppressed conductometric detection, by improved suppressor design to decrease internal volume and dispersion. All of these have helped lower detection limits for many common ions to about 0.1 ng, or less than 10 g/L (ppb) for a 25-L injection. Electrochemical or amperometric detection as it was first used in IC was single-potential or DC amperometry, useful for certain electrochemically active ions such as cyanide, sulfite, and iodide. But the development of pulsed amperometric detection (PAD) for analytes that fouled electrode surfaces when detected eventually helped create a new category of IC for the determination of carbohydrates. Another advancement, known as integrated amperometry, has increased the sensitivity for other electrochemically active species, such as amines and many compounds containing reduced sulfur groups, that are sometimes weakly detected by PAD (7).

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Current Use
Types of Samples
IC is most often applied to aqueous samples or to solid samples that will dissolve in or can be extracted into aqueous solutions. For aqueous samples, sample preparation may be unnecessary or may consist of dilution or filtration before introducing the sample into the IC system. Insoluble solid samples, such as soil or air filters, are usually extracted into an aqueous solution for analysis. Analysis of gaseous samples presents some interesting challenges in sample preparation (8) and is currently an active area of research. Nonaqueous liquid samples, such as organic solvents, require either little sample preparation if the solvent is miscible with water, or a liquidliquid extraction to an aqueous solution if the solvent is immiscible.

Types of Analytes
What sets IC apart from most other techniques for the determination of ions is the ease with which it can determine several ionic analytes simultaneously. In principle, any species that can exist as an ion can be determined by IC. In addition to the common anions, such as chloride, bromide, sulfate, nitrate, and phosphate, and common cations, such as lithium, ammonium, magnesium, and calcium, a multitude of weak acids and bases can be ionized by adjusting pH. Amines are cationic below a pH of about 9. Carboxylic acids are anionic above a pH of about 3. Amino acids may be anionic at high pH or cationic at low pH. Sugars and similar carbohydrates, though not commonly considered ions, are actually very weak acids and can be chromatographed as anions above a pH of 12 or 13. Transition and lanthanide metals, though often considered cations, readily form complexes with chelating anions and are often chromatographed as anionic complexes.

How It Works
IC is the merging of a chromatographic technique for separating ions with a technique for detecting ions and determining concentrations. Although the separation and detection are closely linked in practice, the two processes can be conceptualized independently. Anion exchange and cation exchange are by far the two most common separation techniques, but the alternative chromatographic methods of ion exclusion, ion pairing, and chelation have some advantages in certain cases. Ions can be detected and measured using several methods depending on the sensitivity and specificity needed. Species that are ionic at or near neutrality can usually be detected using conductivity, the most common form of detection in IC. In many cases, however, amperometric, optical, ICP, or mass spectrometric methods may be preferred.

Separation: Ion Exchange

Ion exchange is a process in which a charged analyte (also called a solute or eluite) in a flowing solution competes with an eluent (mobile phase) ion of like charge for sites having the opposite charge on a stationary phase (Fig. 12.1). The sites of opposite charge are often called functional groups. Before introducing analyte ions into the systemthat is, before injectionthe functional group ions are paired with

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the eluent ions, maintaining electrical neutrality in the stationary phase. When a sample is injected, new ions compete with the eluent ions at the functional group site. Analyte ions that compete successfully (that is, those that have a high affinity for the ion-exchange site) are retained longer than ions that do not compete well with the eluent ions. As with all chromatographic processes, ion exchange can be thought of as a process involving the rapid movement of the analyte between two phases, in this case a liquid mobile phase and a solid stationary phase. In the stationary phase, the ions are immobilized. Ions travel through the column only in the mobile phase. The more time an ion spends in the stationary phase, the more slowly it moves through the column. The key to the separation of analyte ions is the differential affinities a functional group has for different analyte ions. For example, if analyte A has a higher affinity for the stationary phase site than does analyte B, A will compete with the eluent ions more successfully for those sites and be retained longer. A will therefore elute from the column after B. The relative affinities of analytes for the stationary phase are known as the selectivity. Selectivity is determined by many parameters of the separation, including type of functional group, stationary phase environment near the functional group, characteristics of eluent ions, eluent ion concentration, nonionic or oppositely charged eluent additives such as solvents or ionpairing agents, and temperature. The first two parameters are determined by the design of the ion exchange column and are usually optimized for a given class of analytes, such as common inorganic anions, organic acids, or inorganic cations. The other parameters can be adjusted by the analyst to tailor the sep-

Figure 12.1 Representation of the ion exchange process for an anion with hydroxide eluent.

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aration to specific requirements, but usually ion-exchange columns are designed with a specific set of chromatographic conditions in mind. The quantity of functional group sites in the stationary phase is known as the capacity. Capacity is usually expressed as the number of equivalents per column or equivalents per gram of resin. A higher capacity results in longer retention of the analyte ions. Capacity is independent of selectivity (that is, capacity can be increased or decreased without altering selectivity) and is determined by the resin manufacturer.

Separation: Ion Exclusion

Ion exclusion, in many respects, is complementary to ion exchange. Like ion exchange, the stationary phase is an ion exchange resin, although it has a very high ion exchange capacity and is the same charge as the analyte ions. Its greatest utility is the separation of weakly ionized species while eluting strongly ionized species in the void volume. The ion exclusion process (Fig. 12.2) relies on the establishment of an electrical potential between a dilute mobile phase and a stationary phase of high ion-exchange-site concentration. The relatively high concentration of ion exchange sites in the resin dictates a high concentration of counterions in the stationary phase to maintain electrical neutrality. However, diffusion forces tend toward equalizing counterion concentrations in the mobile and stationary phases, a situation that leaves the stationary phase charged the same as the analyte ions. This situation of high potential energy is often called the Donnan potential. The Donnan potential permits neutral molecules to enter the stationary phase, whereas analyte ions are repelled or excluded, hence the term ion exclusion. Weakly ionized species exhibit

Figure 12.2 Representation of the ion exclusion process for a carboxylic acid.

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intermediate behavior and are separated from one another based largely on their extent of ionization. The most common application of ion exclusion is for the separation of organic acids using a sulfonated macroporous cation exchange resin in the hydronium ion form. The separation of weak bases using a macroporous anion exchange resin is possible, but is much less common.

Separation: Ion Pairing

Ion-pair chromatography, also known as ion-interaction or dynamic ion-exchange chromatography, is a technique using a neutral hydrophobic stationary phase and a mobile phase containing a hydrophobic ion, sometimes called the ion-pairing agent, having a charge opposite to that of the analyte. A common explanation of the ion-pair mechanism is that the ion-pairing agent is associated with the stationary phase and functions as an ion-exchange functional group. For anionic analytes, quaternary ammonium ions, such as tetrabutylammonium, are most commonly used as ion-pairing agents. Alkylsulfonates, such as octanesulfonate, are most commonly used for cationic analytes. The most common stationary phases are alkyl-bonded porous silica resins, commonly used in reversed phase HPLC, and macroporous styrenedivinylbenzene polymers. Ion-pair chromatography, unlike ion-exchange chromatography, has some flexibility with respect to selectivity and capacity. Selectivity and capacity can be altered by simply changing the ion-pairing agent, and capacity can be increased by increasing the ion-pairing agents concentration. Historically, ion-pair chromatography has also had the advantage of compatibility with certain organic solvent modifiers, such as methanol or acetonitrile, added to the mobile phase to alter selectivity, but with the availability of solvent-compatible ion exchange resins today, organic modifiers can be used to alter selectivity in ion exchange as well. The presence of ion-pairing agents can complicate the use of conductometric and amperometric detections; nevertheless, both detection techniques are commonly used with ion-pair chromatography. The combination of ion-pair chromatography and suppressed conductivity detection is often called mobile phase ion chromatography (MPIC). Analytes most suited to ion-pair chromatography are large, hydrophobic ions because the slow mass transfer or secondary hydrophobic interactions that these types of ions exhibit with typical ionexchange stationary phases leads to poor efficiency, and often poor resolution.

Separation: Chelation
Certain organic groups, such as dicarboxylates, tend to form complexes with metal ions, but resins with such chelating functional groups have not found wide use as stationary phases. Although these resins have a high selectivity for certain metal ions, notably transition and lanthanide metals, the exchange process is too slow for efficient chromatographic separations. Instead, metals are usually chromatographed by anion exchange as anionic complexes of pyridine dicarboxylic acid or similar anionic chelating agents. Chelating resins are often used for metal concentration or matrix elimination. Many spectroscopic techniques experience interferences from main group metals, such as sodium and calcium, which can be present at concentrations that are several orders of magnitude higher than those of the metal analytes. IC, on the other hand, has the selectivity for interference-free determination of many transition or lanthanide metals.

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Detection: Suppressed Conductivity

In the mid-1970s, Small and coworkers (2) recognized that the very nature of ion exchange chromatography required an eluent ion to exchange with the analyte ion. Conductivity, a property shared by all ions, would be the ideal choice as a universal detection method, but the background conductance of the eluent would reduce the sensitivity of the technique. They solved this problem by using a second column, a suppressor column, having the opposite functionality as the analytical column and placing it between the analytical column and the detector. For determining anions, the suppressor column would be a cation exchange column in the hydronium ion form and for determining cations the suppressor would be an anion exchange column in the hydroxide ion form. When the salt of a weak acid or base is used as the eluent, the suppressor reduces the conductometric signal from the eluent. As an example, consider what would happen during suppression of an eluent containing sodium bicarbonate and sodium carbonate, a common eluent in IC (Fig. 12.3). The carbonate and bicarbonate anions exchange with the analyte anions (such as chloride) during the separation. The effluent then passes through a cation exchange column in the hydronium ion form. Sodium ion is exchanged for hydronium, forming carbonic acid, a weak acid having a very low conductometric signal. An analogous reaction occurs in the case of cations. Another benefit of the suppression reaction is that the response for the analyte ions is often increased. According to Kohlrauschs law, the measured conductivity of an ionic compound is the sum of the equivalent conductances of the anion and cation. The equivalent conductance of hydronium is the highest of all cations, so the measured conductance of each anion increases after suppression because the hydronium counterion contributes more to the total conductance. Similarly, hydroxide ion has the highest equivalent conductance of all anions, so the total conductance for cations increases after suppression. The principal disadvantages to this approach are that the suppressor column causes some dispersion of the analytes before detection and the suppressor must be discarded or regenerated as the hydronium ions are depleted and the column can no longer suppress the eluent. The suppressor device has changed through its evolution (9). Today it is commercially available in three versions: a packed column similar to the original one used about 20 years ago, a slurry of suspended ion exchange resin that is added to the effluent after the analytical column to accomplish the suppression reaction, and an ion-exchange membrane device that continuously supplies the suppressing ion from a chemical source or from the electrolysis of the water in recycled, suppressed eluent. The packed column suppressor is based on long-tested technology, but it must be regenerated or replaced periodically as it becomes exhausted. The postcolumn reagent approach has the lowest startup costs, but the suppressing resin is a consumable, discarded with the column effluent. The membrane devices have low dead volume, so they lose little efficiency during suppression, and the electrolytic version of the device has no consumable costs and almost no maintenance, but these types of suppressors have higher startup costs. Analytes detectable by suppressed conductance are those that, when suppressed, are ionic. Generally, this means that anions with pKas below about 5 and cations with pKbs below about 5 can usually be detected by this method. In other words, if the analyte is ionic at pH 7, the approximate pH of most eluents after suppression, suppressed conductance is a viable detection method.

Detection: Nonsuppressed Conductivity

Nonsuppressed IC, sometimes called single-column IC, operates with a higher background conductance. The higher background necessitates careful control of temperature, so conductivity detectors (Chap. 39) designed for nonsuppressed systems generally have thermostated cells and sometimes insulated chambers for the analytical column and the detector cell. Analytical columns designed for nonsuppressed IC are

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Figure 12.3 Suppression reaction in a column-style suppressor.

generally of lower capacity so that less concentrated eluents, which have lower background conductance, result in analysis times comparable to the suppressed systems. The detector must be able to detect a change in conductance as the analytes pass through it. This change may be an increase or a decrease. Most anion methods exhibit an increase because weakly conductive eluents, that is, eluent anions having equivalent conductances lower than the analyte anions, are used. Common nonsuppressed eluent systems include benzoate, phthalate, and borate/gluconate. Although they are much less common, highly conductive eluents such as sodium hydroxide have also been used for anion determination in nonsuppressed systems, producing a decrease in conductance as the analyte elutes. This decrease occurs because common anions have lower equivalent conductances that hydroxide. In these latter cases, the signal from the detector is commonly inverted because chromatographic convention displays analytes as positive peaks. Most nonsuppressed cation systems are analogous to the second anion system in that they use a highly conductive eluent, usually hydrochloric or nitric acid, and detect analyte cations as a decrease in the measured conductance. In this case, the decrease is due to the lower equivalent conductance of analyte cations compared to the very high equivalent conductance of hydronium. Small (1) compares nonsuppressed and suppressed conductometric detection on theoretical grounds. He calculates that detection limits for nonsuppressed IC are not as low as those for suppressed IC and the dynamic range is smaller than for suppressed methods. On the other hand, nonsuppressed

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methods have the advantage that weak acid anions and weak base cations can be detected more readily. For example, a cation with a pKb of 9 would be only about 1% ionic after suppression (pH of 7), but in 1 mM HCl (pH of 3) it would be about 99% ionic.

Detection: Single-Potential Amperometry

Any analyte that can be oxidized or reduced is a candidate for amperometric detection (Chap. 36). The simplest form of amperometric detection is single-potential, or direct current (DC), amperometry. A voltage (potential) is applied between two electrodes positioned in the column effluent. The measured current changes as an electroactive analyte is oxidized at the anode or reduced at the cathode. Singlepotential amperometry has been used to detect weak acid anions, such as cyanide and sulfide, which are problematic by conductometric methods. Another, possibly more important advantage of amperometry over other detection methods for these and other ions, such as iodide, sulfite, and hydrazine, is specificity. The applied potential can be adjusted to maximize the response for the analyte of interest while minimizing the response for interfering analytes.

Detection: Pulsed Amperometry

An extension of single-potential amperometry, (Chap. 36) is pulsed amperometry, most commonly used for analytes that tend to foul electrodes. Analytes that foul electrodes reduce the signal with each analysis and necessitate frequent cleaning of the electrode. In pulsed amperometric detection (PAD), a working potential is applied for a short time (usually a few hundred milliseconds), followed by higher or lower potentials that are used for cleaning the electrode. The current is measured only while the working potential is applied, then sequential current measurements are processed by the detector to produce a smooth output. PAD is most often used for detection of carbohydrates after an anion exchange separation, but further developments of related techniques (7) show promise for amines, reduced sulfur species, and other electroactive compounds.

Detection: Optical
The most commonly used optical detectors are absorbance detectors and fluorescence detectors (Chap. 25 and 26). Fluorescence detectors are rarely used in IC, but absorbance detectors are quite common. Absorbance is used for detection under three circumstances, direct photometric detection, indirect photometric detection, and photometric detection after a postcolumn derivatization. Some ions, most importantly certain anions, are chromophoric; that is they absorb light. Such is the case with the common anions nitrite and nitrate. These can be detected in the ultraviolet region, by monitoring absorbance at 215 nm, without detecting nonchromophoric anions such as chloride. Absorbance can also be used for detection of many organic ions, such as aryl amines and organic acids, following ion exchange chromatography. More commonly, however, ions are not chromophoric. Nevertheless, they can be detected indirectly using a chromophoric eluent ion of the same charge, a technique known as indirect photometric detection. During the ion-exchange process, ionic concentration remains constant at the stationary phase functional groups, so at the detector the presence of each equivalent of analyte requires the absence of an equivalent of chromophoric eluent ion. The use of indirect photometric detection is not very common today. By far, the most common use of absorbance detection in IC is with an ion-exchange separation fol-

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lowed by a derivatization reaction that renders the analytes chromophoric or, in some cases, more chromophoric. Ideally, the postcolumn reaction is fast (complete within seconds) and does not produce interferences (for example, by reaction with eluent components) in the absence of analytes. This technique is commonly used for the determination of transition metals and amino acids. As was mentioned earlier, transition metals are often chromatographed as anion complexes. By adding 4-(2-pyridylazo)resorcinol (PAR) to the column effluent, PAR complexes of the metals rapidly form and are detected by absorbance. Similarly, amino acids can be chromatographed by either anion or cation exchange, derivatized after separation with ninhydrin and detected by absorbance or, if lower detection limits are desired, derivatization with ortho-phthalaldehyde allows fluorescence detection.

Detection: Specialized Detectors

Recently, the scientific community has begun coupling ion-exchange separations with specialized techniques for detecting the analytes. The two most common are inductively coupled plasma (ICP) spectroscopy, useful for the determination of metals, and mass spectroscopy (MS). Coupled to ICP, IC (Chap. 21 and 22) is more a sample preparation step than a chromatographic separation. A column containing a chelating stationary phase is used to selectively concentrate metals from a sample matrix. An intermediate column wash step can selectively remove interfering metals if necessary. Then the column is washed with a strong eluent, delivering the metals of interest to the ICP. MS (Chap. 33) is not generally compatible with the highly ionic eluents commonly used in IC. However, just as with conductivity detection, eluent ions can be eliminated from the effluent by the use of a suppressor before it enters the MS (10). In these cases, the goal of IC-MS is not the quantification of the analytes, but rather their mass spectral identification and characterization.

What It Does
Instrumentation
Minimally, an IC system consists of an eluent reservoir, an analytical pump to deliver the eluent to the analytical column, an injection valve or other means of introducing the sample, an analytical chromatographic column, a detector, and a data processing device (Fig. 12.4). These components are the same components in an HPLC system. Here, however, we discuss each as it relates to IC. The eluent reservoir can be as simple as a bottle with a fluid line that leads to the analytical pump. Usually, however, the eluent is kept under a pressure of about 1/3 atm so that eluent is delivered to the analytical pump without fluid interruption. Eluents at high pH, such as sodium hydroxide, must be kept under an inert atmosphere to prevent carbon dioxide in the air from forming carbonate in the eluent and thereby altering the eluting ion. The pump used in IC is usually nonmetallic. This is because many IC methods use strong acids, strong bases, or high concentrations of salts that may corrode metallic systems. Materials such as polyetheretherketone (PEEK) are compatible with the vast majority of IC applications. Metallic pumps may be used if care is taken to maintain them properly. Many manufacturers of metallic pumps for IC recommend regular passivation of the system. High-sensitivity applications usually require a dual-piston pump for relatively pulse-free operation, although single-piston pumps with pulse dampers are usually sufficient for routine, mg/L-level

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Figure 12.4 The ion chromatographic system.

analysis. The most flexible systems have dual-piston gradient pumps, which allow the analyst to perform gradient methods or to mix eluents isocratically, aiding method development. The injection valve must introduce a reproducible sample volume into the IC system. The most common means of accomplishing this is by the use of a fixed-loop injection. A length of tubing of known volume is attached to the valve and switched into the eluent stream during injection. Many injection systems include an autosampler for unattended, high-volume work. The autosampler may have an injection valve built in, or it may deliver the sample to a remote injection valve. The former type is usually chosen if sample sizes are small because having an injection valve in the autosampler shortens the distance that the sample travels between sample vial and injection valve. Certain low-level IC methods use a concentration column in place of the injection loop on the injection valve. This concentration column may be a guard column of the type used for the analytical separation or it may be a column specifically designed for concentration in a particular application. If the concentration column has a high backpressure, an auxiliary pump is usually required to deliver the sample to that column. The chromatographic separation takes place on the analytical column. Usually, a guard column is placed before the analytical column to extend its lifetime. Most commonly, the guard column is simply a short, inexpensive version of the analytical column that is replaced as needed. Typically, the guard column also adds about 20% to the capacity of the analytical system. The detector ordinarily is one of the options discussed previously, namely conductometric, amperometric, optical, or some other method of measuring ions, along with any system used to facilitate detection. These systems include suppressors and postcolumn reaction devices. The simplest data processing device is a chart recorder that converts a detector output, usually a voltage within a predetermined range, into a timevoltage profile, called a chromatogram. However,

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this device relies on the analyst to convert the area or height of a peak drawn on the paper to a quantity of analyte, a labor-intensive step. A recording integrator does much the same thing as a chart recorder, but calculates the peak area automatically and may even calculate analyte quantities based on standards that were injected earlier. Some integrators can also send signals to the instruments, thereby controlling simple functions such as injections and detector range changes. Data processing through a computer offers the greatest flexibility and automation. As with the integrator, analyte quantities can be determined automatically, but the raw data can also be stored and reprocessed using different parameters at a later time. Computer-based systems usually have complete instrument control as well, so that complete operating conditions can be stored on computer and used later to reproduce the analytical method.

Analytical Information
Qualitative
Like other forms of liquid chromatography, IC can indicate sample composition. The components present elute at nearly unique retention times, determined either by separate injections of known standards of each ion, or more accurately by adding a small amount of a known standard to the sample and identifying the component that experiences an increase in peak size. An estimate of relative concentrations can also be made, but accurate concentrations can be determined only after calibrating the detector response. One can also obtain information about the distribution of components that differ in the number of repeating units. Examples are samples containing polyphosphates, such as polyphosphoric acid (Figure 12.5), linear oligosaccharides, such as hydrolyzed starch, oligonucleotides, and ionic surfactants, such as linear alkyl benzenesulfonic acid. Although each component may be completely resolved from other components, individual pure standards of each component are difficult if not impossible to obtain, so estimates of concentrations are only qualitative.

Quantitative
IC is first and foremost a quantitative technique. The purity of standards determines the accuracy of the technique, and because pure standards of most ions, usually in the form of salts, typically are easy to obtain, accuracy can be 1 to 2%. Analyte concentrations are determined by establishing a relationship between known concentrations of standards and their responses, in terms of either peak height or peak area. This is commonly known as the calibration curve. Such a calibration using independently chromatographed standards and samples, an external calibration, usually exhibits precision of less than 3% relative standard deviation or coefficient of variation (CV). The CV often can be reduced to below 1% by the use of an internal standard, a fully resolved component added to the injection of sample or standard. Peak responses are then normalized to the internal standard. Depending on the exact conditions used, and especially on how well the analyte and detection method are matched, detection limits can vary from below 1 g/L for a 25-L sample to above 1 mg/ L. Detection limits are sometimes lowered by the use of a concentrator column so that large volumes (as much as 100 mL) can be injected. For the most common applications, inorganic anions and cations using suppressed conductivity detection, detection limits are about 1 to 5 g/L for a 25-L sample.

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Figure 12.5 Anion profile of polyphosphoric acid.

It is usually desirable, though not necessary, to have a linear relationship between analyte concentration and response. The linearity is usually expressed by the coefficient of determination (r2) over the calibration range. Beers law for absorbance detection, and the high degree of dissociation of many species in dilute solutions for conductometric detection, are reasons why calibration curves are usually linear (r2 > 0.999) over three, and sometimes four, orders of magnitude. Similar linearities are common for amperometric and fluorimetric detection, but because exact conditions for detection can vary, so can linearity. For IC the dynamic rangethat is, the concentration range over which analytes can be determinedis a function not only of detection but also of separation. Although it is true that better detection limits can extend the dynamic range at low concentrations, it is column capacity that extends the range at high concentrations. The more analyte that can be injected into a system without overloading the column, the greater is the dynamic range. With most detection methods, there is little reason to use lowcapacity columns; however, nonsuppressed conductometric detection often uses a low-capacity column to allow the use of low eluent concentrations.

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Applications
Conductivity
The determination of most anions and cations, both inorganic and organic, is the mainstay of IC. It is used to analyze drinking water, rain water, soil, foods, chemicals, and countless other samples. Many municipal water facilities monitor ionic content of drinking water to ensure quality and safety (Fig. 12.6). The anion composition of rain and other forms of precipitation tells us a lot about our air quality (Fig. 12.7). Generally, high concentrations of anions in rain indicate high acid content. The ion composition of soil can help determine its suitability for agriculture. Proper reporting of the ionic content of foods and beverages is crucial to helping us maintain healthy diets. Several industries rely on high-purity reagents for their processes and IC is often used to monitor that purity. If ionic impurities are too high, poor-quality products may be produced. IC has also been used for more exotic applications such as determining the composition of moon rocks or the ionic content of ice found in Antarctica that is thousands of years old. Thousands of publications cover applications of IC with conductivity detection (Chap. 39). The analyst may want to consult one or more of the books listed in the References for a more complete discussion of the topic.

Amperometry
Although it is a relatively new technique, the determination of carbohydrates is one of the most common applications of IC with amperometric detection (Chap. 36). It is the pairing of the most selective separation technique with the most specific and sensitive detection technique for this class of comFigure 12.6 Cations in drinking water.

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Figure 12.7 Anions in rain water.

pounds. It has been applied to the analysis of foods (Fig. 12.8), oligosaccharide and polysaccharide profiling, and monosaccharide compositional analysis and oligosaccharide analysis of carbohydrates from glycoproteins. Amperometry is also used with more traditional ions, such as iodide and sulfite. Each of these ions can be detected by conductivity, but detection limits are much lower using single-potential or pulsed amperometry and, depending on the sample matrix, amperometry may be more specific for these ions than for other ions that are present. Examples of sulfite in beer and wine and iodide in foods have been reported. An interesting application of amperometric detection in IC is the detection of cyanide or sulfide using a silver working electrode. In these cases, it is not the analyte that undergoes oxidation but the working electrode. A very low potential is applied (0.05 or 0.00 V) and the current measured from the formation of silver complexes according to the reactions 2CN + Ag Ag(CN) 2 + e = 0 S + 2Ag Ag 2 S+2e Because these are weak anions, they can be chromatographed by either anion exchange or ion exclu 0

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Figure 12.8 Carbohydrate components of orange juice.

sion. In cases where high concentrations of chloride or other ions that react with silver may be present, ion exclusion is usually the better choice.

Optical
Certain inorganic ions, such as nitrate and nitrite, are chromophoric and can therefore be specifically detected using UV absorbance (Chap. 25) in complex samples such as waste water. Usually, however, the analyst is also interested in the nonchromophoric ions in the sample, so conductivity may be the better choice. Of course, there are a wide variety of applications for chromophoric amines and carboxylic acids. Aromatic amines and carboxylates can be detected by either UV absorbance or conductivity but, in general, as their molecular weights increase their conductometric responses decrease and their UV responses remain the same or increase. Metals, especially transition metals and lanthanide metals, are most commonly determined with IC by chromatographing them as anionic complexes, of oxalate or pyridinedicarboxylate for instance, and detecting them by absorbance after a postcolumn reaction (Fig. 12.9). In theory, this approach has the advantage over spectroscopic methods of being able to separately determine different oxidation states of a given metal. In practice, this works very well for Cr(III)/Cr(VI) and with some care can work for Fe(II)/Fe(III). However, because many eluents are prone to upset any redox equilibrium that may exist in the sample and because of the care needed in preventing standards from oxidizing or reducing, IC does not have wide application for oxidation state speciation. The determination of amino acids was perhaps the first widely used IC application. HPLC methods using precolumn derivatization and a reversed phase separation have replaced IC for amino acid anal-

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Figure 12.9 Transition metals concentrated from sea water using chelation concentration followed by IC.

ysis of purified proteins, but IC is still commonly used for physiological and other complex samples that are prone to interferences by reversed phase methods.

Hyphenated Techniques
IC Inductively Coupled Plasma Optical Emission Spectrometry
The coupling of IC, or more accurately chelation concentration, to ICP spectroscopy is a young technique and not widely used, but it has advantages over simple ICP for difficult samples. One of the most challenging types of samples for ICP is one with metals of interest at low concentration (5 to 10 g/L) and interfering metals (iron, aluminum, calcium) at much higher concentrations of about 50 to 100 mg/ L. Chelation concentration can be an automated sample preparation technique that concentrates the metals at low concentration, eliminates the interfering metal, and delivers the sample to the ICP in the column effluent. Standards are concentrated by the same process and delivered to the ICP in the same effluent. The technique has been used for mg/kg-level lanthanide metals in acid digested rock, g/Llevel transition metals in sea water, and a variety of other samples. See Chap. 21 and 22 for further discussion.

IC Mass Spectrometry
IC has been interfaced with MS not for quantitative analysis, but for analyte identification. An ion chromatographic separation, with either a volatile salt eluent or one that can be suppressed before entering the MS, is used to resolve the components. The technique has been applied to oligosaccharides and sulfonic acids (10). There are no commercial instruments dedicated to this technique, so the analyst wanting to work in this research area should be knowledgeable about both IC and MS. See also Chap. 33.

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Nuts and Bolts

Relative Costs
Complete system Components Single-piston pump Dual-piston pump Conductivity detector Amperometric detector Absorbance detector Fluorescence detector Autosamplers Data systems Recorders and integrators Computer-based Columns Suppressors Eluent reagents
$= 1 to 5K, $$= 5 to 15K, $$$= 15 to 50K

$$ to $$$ $ $$ $ to $$ $ to $$ $ to $$ $ to $$ $ to $$ $ to $$ $$ <$ <$ <$

The cost of an IC system is determined by required flexibility and, to a lesser extent, required sensitivity. A dedicated, low-cost system with a single-piston pump and a conductivity detector is reasonably priced and is capable of most routine anion and cation determinations. However, these systems do not allow the analyst to perform gradient separations or to mix eluents isocratically. Single-piston pumps are not as pulse-free as dual-piston pumps, so noise and detection limits are often higher. Complete systems are usually modular, so that the correct component (pump, detector, autosampler, data system) needed for an application can be configured within the system. A modular system is usually the best option if needs may change, requiring a different detector, or if several analyses are to be performed on the same system. If the system is to perform a new analysis, a new analytical column with optimum selectivity is usually needed. Periodically, columns may have to be replaced due to wear and tear, but the frequency of replacement depends largely on the types of samples and on how the samples are prepared before analysis. A multitude of sample preparation methods have been developed to eliminate sample components that may deteriorate analytical columns. However, many modern analytical columns are robust enough to allow injection of crude samples with regular cleaning of the columns, thus reducing the need for labor-intensive steps before analysis.

Vendors for Instruments and Accessories

I = instrument manufacturer; C = consumables supplier Alltech Associates Inc. (C) 2051 Waukegan Rd.

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Deerfield, IL 60015 phone: 708-948-8600, 800-255-8324 fax: 708-948-1078 email: 73554.3372@compuserve.com Internet: http://www.alltechweb.com/ Bio-Rad Laboratories, Life Science Group (C) 2000 Alfred Nobel Dr. Hercules, CA 94547 phone: 510-741-1000, 800-424-6723 fax: 800-879-2289 Internet: http://www.biorad.com Dionex Corp. (I & C) P.O. Box 3603, 1228 Titan Way Sunnyvale, CA 94088-3603 phone: 408-737-0700, 800-723-1161 fax: 408-730-9403 email: marcom@dionex.com Internet: http://www.dionex.com EM Separation Technology (I & C) 480 S. Democrat Rd. Gibbstown, NJ 08027-1297 phone: 609-224-0742, 800-922-1084 fax: 609-423-4389 Interaction Chromatography (I & C) 2032 Concourse Dr. San Jose, CA 95131 phone: 408-894-9200 fax: 408-894-0405 SaraSep, Inc. (I & C) 2032 Concourse Dr. San Jose, CA 95131 phone: 408-432-8536 fax: 408-432-8713 email: sarasep@sarasep.com Internet: http://www.sarasep.com Waters Corp. (I & C) 34 Maple Street Milford, MA 01757 phone: 508-478-2000, 800-254-4752 fax: 508-872-1990 email: info@waters.com Internet: http://www.waters.com

Required Level of Training

Operation of the IC system can be performed by anyone with a basic, high-school level understanding

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of chemistry. Instrument troubleshooting is a skill that is usually gained through experience. Many IC manufacturers offer basic courses on the operation and maintenance of their instruments for those who want some additional training. Integrators and computer-based data systems for IC are usually sufficient for routine quantitative analysis. Only minimal training is necessary to obtain accurate quantitative data, but a sound understanding of analytical chemistry and chromatography is needed to develop a method and to verify initially that the data system is programmed properly.

Service and Maintenance

Many modern IC components have internal diagnostics that can alert the analyst to the source of problems. Some systems even keep records of when maintenance tasks were last performed. The most common maintenance tasks are replacing the piston seals in the analytical pump, replacing lamps in the optical detectors, and replacing the reference electrode in the amperometric detector cell. Regular cleaning of the analytical column may also be necessary if sample matrices that may foul the column are injected. The operator manual for the column usually has instructions from the manufacturer on how best to clean the column.

Suggested Readings
SMALL, H., Ion Chromatography, New York: Plenum Press, 1990. WALTON, H. F., and R. D. ROCKLIN, Ion Exchange in Analytical Chemistry, Boca Raton, FL: CRC Press, 1990. WEISS, J., Ion Chromatography, 2nd ed. Weinheim, Germany: VCH. Verlagsgesellschaft mbH, 1995 (English translation).

References
1. H. Small, Ion Chromatography (New York: Plenum Press, 1989). 2. H. Small, T. S. Stevens, and W. C. Bauman, Analytical Chemistry, 47 (1975), 18019. 3. J. S. Fritz, Analytical Chemistry, 59 (1987), 335A44A. 4. R. D. Rocklin, Journal of Chromatography, 546 (1991), 17587. 5. H. F. Walton and R. D. Rocklin, Ion Exchange in Analytical Chemistry (Boca Raton, FL: CRC Press, 1990); J. T. Gjerde and J. S. Fritz, Ion Chromatography, 2nd ed. (New York: Heuthig, 1987). 6. R. D. Rocklin, C. A. Pohl, and J. A. Schibler, Journal of Chromatography, 411 (1987), 10719; W. R. Jones, P. Jandik, and A. L. Heckenberg, Analytical Chemistry, 60 (1988), 19779. 7. D. C. Johnson and W. R. LaCourse, Analytical Chemistry, 62 (1990), 589A97A. 8. P. K. Dasgupta, Analytical Chemistry, 64 (1992), 775A83A. 9. S. Rabin and others, Journal of Chromatography, 640 (1993), 97109. 10. R. A. M. van der Hoeven and others, Journal of Chromatography, 627 (1992), 6373; J. Hsu, Analytical Chemistry, 64 (1992), 43443.