Fifty Years of Ion Chromatography (IC): An Interview with IC Pioneer Joachim Weiss (Part 1)

Looking back over 50 years, what do you consider the most transformative milestone in the development of ion chromatography—and why?
Ion analysis before the introduction of ion chromatography (IC) included wet chemical methods such as photometry, titration, ion-selective electrodes, gravimetry, atomic absorption spectrometry (AAS), and polarography, to name just a few of the most important methods. Although most of these methods are used up to the present day, they are laborious, time-consuming, and—in many cases—prone to interferences. Based on the original concept developed by Small et al., the use of modern ion exchangers in combination with suppressed conductivity detection revolutionized ion analysis regarding simultaneousness, speed of analysis, sensitivity, selectivity, and cost (1).

However, in its original embodiments, IC was perceived to be an analytical method for only inorganic anions and, to a lesser extent, inorganic cations. The most important reason for this perception was the fact that anion or cation analyses were exclusively performed under isocratic conditions using predominantly carbonate–bicarbonate eluents in anion-exchange chromatography (AEX) and mineral acid eluents in cation-exchange chromatography (CEC). Thus, the peak capacities of these separations were limited. Hydroxide eluents, for instance, considered by Small et al. to be the eluent of choice in AEC (lower background conductivity), could not be used at that time due to carbonate impurities and the lack of high-capacity suppressor devices. The latter are necessary because hydroxide is a weaker eluent in comparison with carbonate–bicarbonate and thus must be used at much higher concentrations.

In the 50 years that encompass its development, IC has undergone enormous changes. In my opinion, the most transformative milestone in the development of IC is the introduction of reagent-free IC (RFIC) at the end of the 1990s, utilizing membrane technologies to generate, purify, and suppress eluents used in anion- or cation-exchange chromatography by means of continuous electrolysis (2).The most important advantages of this concept are the generation of high-purity eluents, the electric control of the eluent concentration being generated, less pump maintenance (only high-purity water is used as the carrier), and more consistent analytical results due to control by a chromatography data system (CDS). Since eluent concentration depends on the current applied to the electrodes, which can be kept constant or programmed over time, RFIC was the prerequisite for the application of gradient elution techniques in ion-exchange chromatography (IEC).

The introduction of RFIC was of particular importance for anion-exchange chromatography, because it allowed the generation of contaminant-free hydroxide eluents up to a concentration of 100 mmol/L (at a standard flow rate of 1 mL/min) for the first time. Knowing that the use of carbonate–bicarbonate eluents is impractical to perform gradient anion-exchange chromatography for several reasons, the significance of this development cannot be emphasized enough. As a result, the advantages of gradient elution (higher peak capacity and peak compression of late-eluting peaks) could be realized for anion-exchange chromatography in the RFIC mode to elute inorganic anions and organic acids with widely different retention behavior in the same chromatographic run. Analogously, RFIC allows the simultaneous elution of inorganic cations andorganic amines by cation-exchange chromatography using a methanesulfonic acid eluent.

How has IC evolved to meet the demands of increasingly complex sample types compared to other liquid chromatography (LC) techniques?
As with all liquid chromatography techniques, the samples to be injected for ion chromatography must be free of particulate matter to avoid clogging of the system (tubing and column frits) and to remove bacteria that can change the composition of the sample. Thus, sterile membrane filters with a pore diameter of 0.22 µm must be used for filtration offline at the point of sampling as a kind of sample preservation. In addition, inline filters or sample vials with filter caps can be used as a further precaution. In environmental water analysis, it is often necessary to dilute samples that exceed the working range of ion chromatography. This is particularly important when regulated methods are used to ensure that analyte concentrations fall within the calibrated range.

High analyte concentrations may also exceed column capacity, which, in turn, results in poor chromatography. In the past, sample dilutions were usually performed manually, which was labor-intensive and prone to errors. With the introduction of sophisticated autosamplers in combination with an inline sample conductivity and pH accessory, samples can be automatically diluted today if the measured conductivity exceeds a specified cutoff value. Alternatively, two injection loops with significantly different loop sizes can be mounted on an additional 10-port valve. With the respective chromatography software, sample concentrations are monitored. If the sample concentration is outside the specified range, the sample is automatically reinjected using a smaller injection loop.

While less contaminated samples, such as drinking water, do not require any other type of sample preparation, complex samples, including soil extracts, industrial waste, organic solvents, hydrogen peroxide, food, and many others, cannot be injected directly. Common IC sample preparation techniques include matrix elimination, sample neutralization, dialysis, and combustion techniques. Regarding matrix elimination, the most convenient way is the use of small disposable solid-phase extraction (SPE) cartridges containing a solid sorbent that allows the selective removal of interfering matrix components. SPE is currently the most widely used sample preparation technique in LC. If carried out offline, the SPE process includes activation, conditioning, sample loading, and elution, which is very laborious. SPE cartridges are available in a wide range of packing materials and sizes. Cartridges can be used singly or in series, depending on the matrix interferences to be removed.

A much more elegant and significantly less expensive way is to install inline cartridges between the autosampler and the IC injection valve, facilitating immediate, automated sample pretreatment. After loading a sample onto the injection loop, the sample is pushed through one or more cartridges onto a concentrator column, which, in turn, is switched in line with the analytical column to elute the ions of interest. In comparison with offline SPE, where a new cartridge must be used for every sample, inline cartridges can be used for a large number of samples due to the relatively small injection volume reaching the cartridge. Cross-contamination does not occur as inline cartridges are rinsed with eluent after every injection.

The best sample preparation technique for determining ionic impurities in concentrated acids or bases is AutoNeutralization, an automated neutralization of strongly acidic or strongly basic samples using special membrane suppressors. This replaced cumbersome manual dilution and neutralization procedures that would otherwise be required. An important example of this sample preparation technique is anion analysis in concentrated sodium hydroxide, as formed during chloralkaline electrolysis, for instance. The instrumental setup requires additional valves. The NaOH sample is transported with deionized water through a collection loop into the neutralization unit, which is connected to one of the valves, so that the sample can be passed through the neutralizer a second time if required. In the same way, purity control of amines in the semiconductor industry can be carried out. In the area of cation analysis, the determination of alkali and alkaline-earth metals in high-purity acids is the most important application.

During the past decades, inline dialysis techniques have been employed for analyzing a variety of samples (3). Passive dialysis is the most applied version of dialysis as a sample preparation technique in IC. It can be carried out in a relatively easy way and does not require sophisticated instrumentation. Passive dialysis is based on the selective transfer from a donor phase to an acceptor phase via a semipermeable membrane. The driving force for the migration of ions is the concentration gradient across the membrane, so that the dialysis process is completed when the concentrations in both liquid phases are balanced. Dialysis cells for continuous inline dialysis containing a cellulose acetate membrane with a pore size of 0.2 µm are available; the sample flow is countercurrent to the acceptor flow. A time-event program, controlled by the CDS, allows complete automation. Passive inline dialysis is almost completely applied in the areas of biomedical and food analysis. Application examples include inorganic anion analysis in engine coolants and untreated wastewater, as well as in processed milk and infant formula.

Finally, combustion IC (CIC), a sample preparation technique that is of utmost importance for the petrochemical industry, should be mentioned. It is predominantly used for the determination of the various halogens and sulfur in all kinds of combustible samples (4). Since petrochemicals are difficult to analyze by conventional IC, sample preparation is required to extract the analytes, which is costly and time-consuming. In automated combustion IC, samples are pyrolyzed in an oxidative atmosphere (Ar/O2) in a furnace at temperatures above 900 °C, and the resulting vapours are absorbed in an aqueous solution that can be directly introduced into an ion chromatograph. Typical application examples include anion analysis in coal and liquefied petroleum gas (LPG). All the sample preparation techniques mentioned above indicate that modern ion chromatography is well prepared to analyze even the most complex samples.

What were the key technological innovations that elevated IC from a niche technique to a mainstream analytical tool?
Many IC applications use conductivity detection. Since a conductivity detector is a bulk-property detector and ions cannot be eluted from ion exchangers without electrolytes in the mobile phase, a suppressor device is the key to turning a conductivity detector into a solute-specific one by converting the eluent into a less conductive form and the analytes into a more strongly conductive form. Thus, a suppressor device improves the sensitivity and the linear dynamic range of conductivity detection. In addition, the use of high-capacity separator columns that require higher ionic-strength eluents, as well as gradient elution techniques in combination with conductivity detection, would not be possible without a suppressor device. They are also a prerequisite for hyphenating ion-exchange chromatography with mass spectrometry.

While Small et al. used periodically-regenerated packed-bed suppressors in their original work, the introduction of continuously regenerated membrane suppressors in the mid-1980s was the most significant innovation in the development of suppressor devices (5). Membrane suppressors feature very low baseline drift and noise, high capacity, and overall improved system stability. However, they require a chemical regenerant (acid or base for anion or cation-exchange chromatography) that must be provided with, for instance, a peristaltic pump. To improve ease-of-use, displacement chemical regeneration (DCR) has been introduced. In the DCR mode, the analytical pump provides pulsation-free reagent delivery by replacing the regenerant with the eluent.

Thus, an additional pump is not required. The second most important step in the evolution of suppression technology was the introduction of self-regenerating suppressors in 1992. In those devices, regeneration is carried out via continuous electrolysis of the aqueous mobile phase after suppression. While the original self-regenerated suppressors were operated in the constant-current mode, the latest generation of electrolytically operated suppressors introduced in 2018 can be operated in the constant-voltage mode due to a unique resin formulation in the eluent channel inside. Such dynamically regenerated suppressors feature a remarkably low noise, especially under gradient elution conditions. Both chemical and electrolytic suppressors are used to the present day. While electrolytic suppressors are easier to use and do not require external regenerants, chemical suppression is preferred when the lowest noise, elevated column temperatures, or organic solvents in the mobile phase are required.

Electrolytic eluent generation as part of the RFIC concept has already been mentioned above. However, it was not only a key technological innovation to enable gradient elution of common inorganic and organic anions and cations. Small hardware modifications, resulting in more thorough eluent degassing, were the prerequisite to use electrolytic hydroxide generation for carbohydrate analysis by anion-exchange chromatography in combination with pulsed amperometric detection (PAD). The elution of monosaccharides, either derived from glycoproteins or present as free monosaccharides in plant extracts, fruit juices, coffee, and other beverages, requires very low hydroxide concentrations to achieve the desired resolution. If manually prepared, those eluents always contain residual carbonate impurities that require column rinsing with concentrated hydroxide solutions after every run to remove carbonate accumulated on the separator column. Although this procedure avoids retention time shifts, it is very time-consuming. In contrast, electrolytic hydroxide generation produces carbonate-free hydroxide eluents down to concentrations as low as 2 mmol/L. Further developments of the eluent generation technology resulted in the introduction of dual eluent generation to electrolytically generate potassium hydroxide–potassium methanesulfonate eluents for gradient elution of oligosaccharides, replacing the laborious manual preparation of NaOH–NaOAc with its purity problems for this type of analysis.

In terms of detection, a significant technological innovation was the introduction of the above-mentioned PAD in 1983, which opened up the field of carbohydrate analysis by anion-exchange chromatography (6). Over the years, it became the most sensitive and selective method for detecting carbohydrates (sugar alcohols, mono-, di-, and oligosaccharides) and their derivatives (sugar acids, sugar phosphates, sugar sulfates, and aminoglycoside antibiotics). Although it was known for a long time that all these compounds are electroactive, conventional DC amperometry does not work because of the oxidation products being formed that adsorb on the working electrode and thus change its characteristics. In pulsed amperometry, a series of positive and negative potentials is applied to the gold working electrode (pulse sequence or “waveform”) to oxidize the carbohydrates and subsequently remove the oxidation products electrochemically. The four-potential waveform used today is applied in 0.5 s, resulting in a data acquisition rate of 2 Hz, which is sufficient to characterize chromatographic signals. With this method, sensitive carbohydrate detection down to the femtomole range is achieved. With the introduction of integrated pulsed amperometry (IPAD), a variant of pulsed amperometry, at the end of the 1990s, electrochemical detection has expanded to include the analysis of amino acids and their derivatives (oxidized and phosphorylated amino acids), which can also be separated by anion-exchange chromatography (7). IPAD differs from PAD in that the oxidation potential is varied during data acquisition, exhibiting adsorption/desorption catalytic features at the gold working electrode. If this potential variation is carried out several times (multicyclic waveforms), even UV-transparent sulfur compounds with at least one pair of free electrons, such as modern sulfur-containing antibiotics (ampicillin, lincomycin), can be detected electrochemically, which is important for the pharmaceutical industry.

Hyphenated techniques such as the coupling of ion-exchange chromatography with inductively coupled plasma mass spectrometry (ICP-MS) or electrospray ionization (ESI)-MS have become increasingly important in recent years. The advantage of IC-ICP includes the ability to separate and detect metals with different oxidation states. The analytical interest in chemical speciation is because the oxidation state of an element determines toxicity, environmental behavior, and biological effects. Chromium(VI), for example, is highly toxic even in very small amounts, whereas chromium(III) is essential for lipid and carbohydrate metabolism. On the other hand, heavy and transition metals are often determined in complex samples such as body fluids, and considerable interferences may result. The current best method for chromium speciation is the coupling of a bifunctional ion exchanger with ICP-MS (8). This special separator column retains both the anionic chromium(VI) and the cationic chromium(III). Using a short column format (5 cm), both chromium species can be eluted in less than 150 s. Modern conventional high-capacity anion exchangers allow the separation of inorganic and organic arsenic species together with selenite and selenate in one chromatographic run. The hyphenation of IC with ICP is relatively straightforward; it only requires a capillary connection between the column end and the nebulizer of the ICP. The IC modules are typically controlled by the ICP software, which can process transient signals for quantitation. Hyphenation with ESI-MS (9). provides the analyst with mass-selective information. In principle, ion-exchange chromatography can be coupled to any kind of mass spectrometer. However, the eluents used to separate ionic species on an anion or cation exchanger are incompatible with mass spectrometry.

Therefore, either volatile eluents must be used, which can be pumped directly into the ion source, or, more commonly applied, a suppressor system must be placed between the ion exchanger and the electrospray interface. In these suppressor systems, the basic or acidic eluents are converted to water. In comparison with a conventional LC–MS system, the IC–MS setup often consists of an additional delivery system and a micro-tee to add an organic solvent to the column effluent before entering the electrospray interface for enhancing desolvation. IC–MS is usually applied when dealing with challenging applications. Single-quadrupole mass spectrometers, for instance, are often employed in series with suppressed conductivity detection as a complementary detection system if overlapping peaks of different nominal mass must be quantified. Typical examples are organic acids partly coeluting with standard anions as well as organic amines overlapping with inorganic cations. More demanding analyses, such as the determination of bromate, haloacetic acids, perchlorate, and other emerging contaminants in water at trace levels, are typically performed by IC‒ESI-MS/MS. High-resolution accurate mass (HRAM) MS is required, for instance, for the identification and quantification of metabolites by coupling anion-exchange chromatography to an orbital trap MS, or if samples with severe matrix problems have to be analyzed for contaminants at ultra-trace levels. All these examples clearly demonstrate the need for MS hyphenation to achieve the desired sensitivity and selectivity.

In what areas has IC had the most lasting and unique impact compared to other LC modalities?
Although the power-generating industry was the first one to adopt ion chromatography, today environmental and life science applications account for approximately 35% each. This is remarkable as many other industries, including the power-generating, semiconductor, food, electroplating, chemical and petrochemical, household products, agricultural, geological, and lately the pharmaceutical industries, are using IC, too. In environmental analysis, IC is the main analytical method for the determination of inorganic anions and cations, disinfection by-products, and other contaminants in all kinds of water samples, and has almost replaced traditional wet chemistry. However, IC is not limited to water samples; by utilizing the appropriate sample preparation methods, it can also be applied for the analysis of ionic constituents in soil and even in air. Gases are usually collected with liquid diffusion denuders that are integrated components of air sampling devices.

Life science applications include the analysis of organic acids in fermentation broths, carbohydrates derived from glycoproteins, oligonucleotides, and proteins. Not every analyst realizes that up to the present day, the highest-resolution separations of carbohydrates are obtained by anion-exchange chromatography. Monosaccharide and sialic acid analysis are critical ways to profile complex oligosaccharides derived from glycoproteins after acid hydrolysis. Anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) is the most convenient method for this type of analysis because it avoids fluorophore labelling that would be required for other LC techniques. In terms of intact protein characterization, the highest-resolution separations are also obtained by ion-exchange chromatography. For the separation of truncation variants of monoclonal antibodies (mAbs), anion-exchange chromatography became the “gold standard.”

Ion chromatography is indispensable for the analysis of ionic contaminants in high-purity water down to the single-digit part-per-trillion level, which is of fundamental importance for the power-generating and semiconductor industries. With the increase in integration density of semiconductor components in printed circuit boards, the demands on water purity rapidly increase. Large-volume injection techniques cannot be applied due to the lack of sensitivity. Therefore, the concept of reagent-free ion chromatography with electrolytic sample preparation (RFIC-ESP) has been developed to fully automate inline sample preconcentration and inline calibration. Many nuclear power plants and semiconductor production sites use this technology for online monitoring of high-purity water.

Ion chromatography has been adopted by many test and research laboratories in the food and beverage industry because it easily deals with complex matrices. The resistance to fouling of the stationary phases being used, as well as the sensitivity and selectivity of the employed detection methods, are the main reasons to apply IC methods for analyzing milk and meat products, beverages, canned food, infant formula, cereals, carbohydrates, and flavors. Not so long ago, the pharmaceutical industry—predominantly using RPLC techniques for the analysis of active pharmaceutical ingredients (APIs)—added IC to its analytical method portfolio for the analysis of counter ions, as APIs are usually administered in the form of tablets. Counter ion analysis not only includes inorganic anions and cations, but also organic acids and amines. If APIs are ionic—for example, bisphosphonates to treat osteoporosis—anion-exchange chromatography is by far superior to ion-pair chromatography, because the APIs can be separated together with excipients under gradient elution conditions and detected by suppressed conductivity. The analysis of sequestering agents in household products (washing powder, cleansing agents) is a perfect example to be performed on chromatography systems with metal-free fluidics; in this case, IC is superior to any other LC technique.

References
1. Small, H.; Stevens, T. S.; Bauman, W. C. Anal. Chem. 1975, 47, 1801.

2. Liu, Y.; Avdalovic, N.; Small, H.; Matt, R.; Dhillon, H. Presentation No. 1179, Pittcon, New Orleans, LA, USA, 1998.

3. van de Merbel, N. C. J. Chromatogr. A 1999, 856, 55.

4. ASTM International. ASTM D7359-08: Standard Test Method for Fluorine, Chlorine, and Sulfur in Aromatic Hydrocarbons and Their Mixtures by Oxidative Pyrolytic Combustion Followed by Ion Chromatography Detection (Combustion Ion Chromatography – CIC); ASTM International: Conshohocken, PA, USA, 2008.

5. Srinavasan, K. Sep. Sci. Technol. 2021, 13, 157–175.

 6. Hughes, S.; Johnson, D. C. Anal. Chim. Acta 1983, 149, 1.

7. Clarke, A. P.; Jandik, P.; Rocklin, R. D.; Liu, Y.; Avdalovic, N. Anal. Chem. 1999, 71, 2774.

8. Powell, M. J.; Boomer, D. W. Anal. Chem. 1995, 67, 2474.

9. Niessen, W. M. A. Liquid Chromatography–Mass Spectrometry, 2nd ed.; Marcel Dekker: New York, 1999.

Biography
Joachim Weiss graduated in chemistry in 1979 from the Technical University of Berlin (Germany). He worked in the field of liquid and gas chromatography at the Hahn-Meitner-Institute in Berlin and received his Ph.D. in analytical chemistry in 1982 from the Technical University of Berlin. In 2000, Guenther Bonn appointed him visiting professor at the Leopold-Franzens University in Innsbruck (Austria). Weiss habilitated in analytical chemistry at the Leopold-Franzens University (Austria) in 2002. In 2011, 2014, and 2016, Jacek Namiesnik appointed him visiting professor at the Technical University of Gdansk (Poland).

In 1982, Weiss started his professional career as an applications chemist at Dionex Corporation in Germany. From 1996 to 1997, he worked in the field of scanning probe microscopy at TopoMetrix GmbH in Germany as a European distributors manager and rejoined Dionex Corporation (now part of Thermo Fisher Scientific) in 1998. He is currently retired from the position of technical director for Dionex Products within the chromatography and mass spectrometry division (CMD) of Thermo Fisher Scientific in Germany.

Weiss is recognized as an international expert in analytical chemistry (especially in the field of liquid/ion chromatography). The 4^th edition of his Handbook of Ion Chromatography was published in 2016. In 2015, he was awarded the Maria Sklodowska Curie Medal of the Polish Chemical Society for his achievements in separation science.