Fifty Years Of Ion Chromatography (IC): An Interview with IC Pioneer Joachim Weiss (Part 2)

As someone deeply experienced in IC, what common misconceptions do you hear from reversed-phase LC users, and how would you correct them?
A common misconception I hear from reversed-phase LC users is the claim that hydroxide or methanesulfonic acid eluents typically used for anion or cation-exchange chromatography are incompatible with mass spectrometric detection. This would be true if ion-exchange chromatography is coupled directly with electrospray ionization mass spectrometry (ESI-MS). The truth is that an RFIC system incorporates an electrolytically regenerated suppressor, in which these eluents are converted to water that can be directed into the ion source of a mass spectrometer without any problems. It should be noted that older generations of mass spectrometers suffered from poor desolvation of this aqueous phase, affecting sensitivity, so that a small flow of an organic solvent had to be added to the suppressor effluent before entering the ion source. However, even this small modification of an IC-MS system is no longer required with the latest generation of mass spectrometers.

Some reversed-phase LC users believe that ion chromatography can be performed with a conventional, stainless steel-based or bio-inert HPLC system. This might be true if the determination of standard anions (or cations) is done only occasionally. Using an anion exchanger, for example, with an organic eluent such as potassium hydrogen phthalate (KHP) and indirect UV detection, the determination of standard anions is possible, but sensitivity and selectivity are compromised. Using such a nonspecific detection system, so-called system peaks are observed that can be negative or positive depending on the chromatographic conditions. Those system peaks appear somewhere in the chromatogram, potentially interfering with analyte peaks or prolonging the overall analysis time. Moreover, using a stainless-steel HPLC system, metal ions are leached out and accumulate on the guard and separator columns, regardless of whether the analytical pump is passivated with nitric acid or not (the black frits in the column fitting are proof of metal contamination). This leads to column fouling within a very short time. If ion chromatography is to be carried out on a routine basis, there is no alternative to a dedicated ion chromatograph with metal-free fluidics and suppressed conductivity detection.

Can you share a case where IC outperformed other LC techniques in a surprising or critical application?
Aside from the analysis of inorganic anions and cations under isocratic conditions with suppressed conductivity detection, where ion chromatography outperforms any other LC technique in terms of sensitivity, selectivity, and analysis time, I want to highlight a very topical subject: the analysis of ionic pesticides. Nonionic pesticides are commonly analyzed by either gas chromatography (GC)–MS or LC–MS, but anionic or cationic pesticides are not retained on reversed-phase columns.

To avoid pre- or postcolumn derivatization, it seems to be obvious to employ hydrophilic interaction liquid chromatography (HILIC) for the analysis of ionic pesticides using conventional HPLC instrumentation. However, in addition to column capacity issues, some HILIC columns suffer from metal contaminants leaching from conventional, metal-based HPLC systems. Indeed, HILIC in combination with tandem mass spectrometry works very well for a particular ionic pesticide such as glyphosate, but the European Food Safety Authority (EFSA) recently published a Reasoned Opinion indicating that the analysis of glyphosate for risk purposes should also include the metabolites: AMPA, N-acetyl AMPA, and N-acetyl glyphosate (1). These requirements represent an analytical challenge. Similarly, there is interest in glufosinate and its metabolites (N-acetyl glufosinate, 3-[methylphosphinico]-propionic acid), ethephon and its metabolite 2-hydroxyethane-phosphonic acid (HEPA), as well as fosetyl with its metabolite phosphonic acid. In addition, the generic extraction method for anionic pesticides used by many laboratories is based on quick polar pesticide extraction (QuPPe) developed by the European Reference Laboratory for single-residue methods.

One disadvantage of this approach is that several nonsuppressed IC and HILIC columns are required to cover all these anionic pesticides of interest, with limitations in sample capacity. Since the QuPPe method is based on the extraction with methanol–water, there is no effective clean-up for the removal of co-extractives. In contrast, all these analytes can be analyzed together at low mg/kg concentrations in a single analysis using IC-MS equipment. The higher column capacity provided by modern hyperbranched anion exchangers enables excellent resolution and stable retention times, even at higher sample/matrix loading. This results in improved reporting limits, especially for samples with complex matrices. Also, the system can be easily switched to the analysis of cationic pesticides and metabolites, such as chlormequat, mepiquat, paraquat, diquat, aminoglycosides, and others, which are also receiving a lot of interest. If using a dual-channel IC system configured for anions in one channel and cations in the other, the switch from one channel to the other can even be automated.

How has the role of IC in regulatory and compliance testing evolved, and what gives it an edge in trace-level ionic analysis?
Regulation is often considered to be an end in itself. The public sector requires accurate, litigable, and comparable analytical results for monitoring environmental quality. Analytics is expected to meet these requirements by applying standardized procedures. Regulatory work in ion chromatography in the early 1980s recognized the advantages of IC over established wet-chemical analytical methods, especially in the field of anion analysis

(gravimetry for sulfate). EPA Method 300.0 was the first standardized method developed in the mid-1980s for compliance monitoring of standard anions such as fluoride, chloride, nitrite, bromide, nitrate, orthophosphate, and sulfate in drinking water. In 1992, the method was also approved for standard anion analysis in wastewater. One year later, it was modified to include inorganic disinfection by-products such as bromate, chlorite, and chlorate as part B. After a lower level of bromate (10 ppb) was promulgated, EPA Method 300.0 was modified to EPA Method 300.1 to meet the new quantitation requirements. Today, EPA Method 300.1 and other equivalent methods are the most widely used IC applications in the US and in many other countries for environmental analysis (2).

Regulatory work for IC in Europe started in 1983 with the establishment of a working group by the German DIN organization (3). Since 1988, work has also been carried out by the International Organization for Standardization (ISO) if a project is of European or international interest. All ISO standards have been taken over by the European Committee for Standardization (CEN). There are currently six DIN EN ISO and two DIN standard methods for the determination of up to 29 different ions, including one for the simultaneous analysis of alkali and alkaline-earth metals (ISO 14911). While standard methods utilizing hyphenated techniques such as IC-ICP, IC-MS, and IC-MS/MS are not available in Europe, the US EPA published a method for bromate analysis by IC-ICP/MS (EPA Method 321.8) as well as a method for haloacetic acid analysis by IC-MS/MS (EPA Method 557). For perchlorate analysis, both IC-MS and IC-MS/MS methods are available (EPA Method 332.0). A speciality of European regulations is a standard method for polarizable anions such as iodide, thiocyanate, and thiosulfate (ISO 10304-03).

The US EPA and the European ISO are not the only organizations specifying IC. Other regulatory bodies, such as ASTM International and AOAC International, also published test methods for standard anions in drinking water (ASTM D4327-03 and AOAC 993.3) or hexavalent chromium (ASTM D5257-93). In China, the Ministry of Ecology and Environment published its own methods for the analysis of standard anions and cations in water and ambient air, as well as a method for adsorbable organic halogens. Several IC methods are approved in Japan for the analysis of anions in matrices such as industrial water (K0101), industrial wastewater (K0102), mine water, and wastewater (M0202). All these regulation examples indicate the versatile applicability and the widespread use of ion chromatography across the world. (4)

What do you see as the future for IC in an era increasingly dominated by mass spectrometry and multidimensional separations?
Since ion chromatography is part of liquid chromatography, it is not surprising that we observe similar trends in the development of IC. In analogy to the transition from HPLC to UHPLC, separator columns with smaller particle sizes are now also used in ion-exchange chromatography. In general, smaller particles produce higher-efficiency columns, resulting in higher-resolution separations of complex samples.

Alternatively, shorter columns of equivalent efficiency enable faster separations of ions in weakly contaminated samples without sacrificing resolution. However, smaller particles produce columns with a significantly higher backpressure, which could not be used in traditional IC instruments due to the pressure limitation of 20 MPa (3000 psi). Only with the introduction of high-pressure IC with a pressure rating of 34.5 MPa (5000 psi) did faster or higher-resolution separations become feasible. To increase the operating pressure of an IC system to that level, most of the hardware components had to be re-engineered. Since metal-free fluidics are required for an IC instrument to avoid corrosion by the eluents used for anion or cation-exchange chromatography, the pressure limit cannot be exceeded with the inert, polymeric materials (PEEK) currently available. Nevertheless, typical anion or cation profiles are obtained with modern 4-µm ion exchangers in approximately 5 min, cutting the analysis times in half in comparison with traditionally used 8-µm resins.

Another exciting area of development revolves around improving column capacity and selectivity. Researchers are constantly exploring new types of resin materials and surface modifications to enhance selectivity for the analysis of complex samples. On the one hand, high-capacity ion exchangers are the prerequisite to analyze neighbouring ions in the chromatogram at very disparate concentration levels. A typical example is the separation of sodium and ammonium in drinking water. Using the most modern high-capacity cation exchanger, both cations can be analyzed at a concentration ratio of up to 10,000:1. In general, high-capacity ion exchangers result in longer analysis times, which can be partly compensated for by utilizing higher ionic-strength mobile phases. However, this does not represent a problem as modern high-capacity membrane suppressors can easily handle eluent concentrations up to 100 mmol/L at standard flow rates.

For a long time, the dream of an ion chromatographer was the analysis of anions and cations in the same chromatographic run. This dream came true with the introduction of mixed-mode stationary phases supporting anion-exchange, cation-exchange, and hydrophobic interactions. Such trimodal columns are of vital importance for the pharmaceutical industry when analyzing APIs and their respective counter ions in the same chromatographic run. However, suppressed conductivity detection cannot be employed for this application as counter ions would be exchanged for hydronium or hydroxide ions in the respective suppressor system. If both the API and counter ion are chromophoric, a UV detector can be used. But in many cases, pharmaceutical counter ions, such as inorganic anions or cations, are nonchromophoric, so a nonspecific detection system, such as charged aerosol detection (CAD), is required. Bimodal stationary phases supporting AEX and RP interactions also offer improved resolution of highly polar, small-molecular-weight organic acids and are thus complementary to genuine anion exchangers. These two examples indicate a kind of dilemma, as it becomes increasingly difficult to distinguish between ion chromatography and conventional HPLC.

Multidimensional separations in ion chromatography do exist, but are not as significant as in HPLC, which is partly due to the huge variety of ion exchangers with different selectivities. However, if the concentration ratio between the matrix ion and the analyte ion becomes extreme, a single-dimensional separation will not work. A classic example is the determination of anionic impurities in ultrapure HF used in the semiconductor industry. Dilution of the sample is not an option because the limits of detection (LOD) for the analyte anions are compromised. This analytical challenge can only be solved by carrying out a preseparation of the fluoride matrix from other inorganic anions by ion-exclusion chromatography. In the second dimension, the analyte anions can then be separated by anion-exchange chromatography.

The use of mass spectrometric detection in ion chromatography is readily increasing, but not to the extent observed in HPLC, where approximately every third new liquid chromatograph is connected to any type of mass spectrometer. While common inorganic anion and cation analysis does not require mass spectrometric detection, lower allowable limits of ionic emerging contaminants cannot be analyzed without tandem or even HRAM mass spectrometry. Emerging contaminants are chemicals that have been detected in global drinking water supplies at trace levels and for which the risk to human health is not yet known. They not only include disinfection by-products, perchlorate, pesticides, and persistent per- and polyfluoroalkyl substances (PFAS), but also personal care products and endocrine disruptors.

The future of IC is not limited to advances in the separation and detection of ions; it also goes along with significant hardware and software improvements. Vendors are working hard to make an IC instrument more user-friendly, for example, through built-in plumbing diagrams, an intuitive fluidic layout, and an easy-to-use tablet control panel available in different languages. Conventional ferrule-type fittings, commonly employed in ion chromatography, are being replaced by finger-tight, zero dead volume connections (Viper fittings) that were originally developed for UHPLC. The recently introduced IC-compatible Viper fittings in the PEEK format are especially important when working with microbore columns. Automatic consumable tracking by radio-frequency identification (RFID) tags is another feature of the latest generations of ion chromatographs. Those tags allow the monitoring of various parameters associated with each consumable, such as the number of injections, eluent volume passed through the column, and pressure history. They also identify incompatible consumable combinations and monitor the performance of each component, indicating a potential need for its replacement. Instrument control and data acquisition without a CDS cannot be imagined today. While achieving, maintaining, and demonstrating compliance is something all commercial products have in common to keep up with ever-evolving standards and regulations, I just want to highlight here eWorkflows, which simplify the analysis with just a few mouse clicks without sacrificing flexibility. Fundamentally, all chromatography workflows are similar: samples are injected, chromatographic separations are performed, signals are captured, and results are generated.

However, workflows differ in the details such as the instrument conditions, injection sequence requirements, and the techniques by which results are calculated. These differences create complexity for operators, reducing their efficiency and increasing the risk of errors. An eWorkflow is a set of rules that captures all the unique aspects of a chromatography workflow and guides the operator through a minimal number of choices needed to run it. Using an eWorkflow, the operator simply selects the instrument, specifies the number of samples and the starting vial position in the autosampler, and starts the analysis. The software then runs the chromatography, processes the data, and produces the results. These benefits are important for routine analyses in quality control and compliance monitoring. eWorkflows can also help with method validation.

If you had to make a case for every LC lab to add IC to their toolbox, what would be your strongest argument today—especially in terms of ROI and versatility?
Ion chromatographs are usually not found in LC labs, as many analysts still perceive IC to be an analytical technique for inorganic ion analysis only. But no matter whether it is an inorganic or organic laboratory, an ion chromatograph should be added to the toolbox if ion analyses suitable for applying IC are carried out on a routine basis.

In this case, ROI is accomplished within a very short time as simple integrated ion chromatographs are not as expensive as they used to be years ago. If anion and cation analyses are required, the question often comes up whether this can be carried out using the same instrument and just adding a different guard column, analytical column, and suppressor. Even though a modern integrated ion chromatograph can accommodate two column sets and an additional valve to automatically switch from anion analysis to cation analysis and back, one should not forget that it takes at least two hours for system equilibration and calibration before the first sample can be injected. This approach only makes sense if the second type of analysis is carried out occasionally. For routine analysis of anions and cations, it is only slightly more expensive to invest in two separate integrated ion chromatographs and combine them with a single autosampler that enables sequential injection, that is, it supplies two unique samples to two systems independently. In contrast to integrated IC systems, modular ion chromatographs are more expensive but offer application flexibility by reconfiguring them for future applications requiring different separation or detection techniques. Modular ion chromatographs are field upgradable from a single-channel to a dual-channel instrument. They are the instrument of choice for method development work, though the ROI is more difficult to calculate.

What advice would you give to a young chromatographer considering specialization in IC, and how does the field stay vibrant and innovative going forward?
I would highly recommend specializing in IC to a young chromatographer, as there are not that many experts left in this field of science today.

The scientists in industry and academia who were driving the development of IC during the past decades are about to retire or have already done so. To avoid a knowledge drain, it is necessary to pass on the accumulated experience to the younger generation of chromatographers. For a young chromatographer considering a specialization in ion chromatography, it is important to develop a solid understanding not only of the fundamental separation and detection principles, but also of the latest advances in hardware and software. It is also crucial to gain practical experience to be able to understand the wide applicability of IC.

This year, we are celebrating the golden jubilee of ion chromatography, as this analytical method has matured over the past 50 years. Although “revolutionary” developments may not be occurring within the foreseeable future, the field will stay vibrant as stationary phase materials, detection methods, and automation techniques are continuously improved. Moreover, the demand for precise analyses in almost all IC application areas is steadily increasing. If a young chromatographer is willing to invest in self-education and is open to new technologies, this field can be further advanced and innovative solutions can be created.

References
1. EFSA Journal. Peer Review of the Pesticide Risk Assessment of the Active Substance Glyphosate. EFSA J. 2023, 21 (3), 8164. https://doi.org/10.2903/j.efsa.2023.8164.

2. U.S. EPA. The Determination of Inorganic Anions in Water by Ion Chromatography, Methods 300.0 and 300.1; U.S. Environmental Protection Agency: Cincinnati, OH, USA, 1993.

3. DIN EN ISO 10304-3. Water Quality — Determination of Dissolved Anions by Liquid Chromatography of Ions — Part 3: Determination of Chromate, Iodide, Sulfite, Thiocyanate and Thiosulfate; 1997.

4. Japanese Industrial Standards (JIS). JIS K0101:1998 Testing Method for Industrial Water; JIS K0102:2022 Testing Methods for Industrial Water and Industrial Wastewater – Part 2: Inorganic Anions, Ammonium Ion, Organic Nitrogen, Total Nitrogen and Total Phosphorus; JIS M0202:1999 Analysis of Inorganic Anions in Mine Water and Wastewater.

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 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.