Handbook of Ion Chromatography

By Joachim Weiss and Oleg Shpigun

This three-volume handbook is the standard reference in the field, unparalleled in its comprehensiveness. It covers every conceivable topic related to the expanding and increasingly important field of ion chromatography. The fourth edition is completely updated and revised to include the latest developments in the instrumentation, now stretching to three volumes to reflect the current state of applications.

Ion chromatography is one of the most widely used separation techniques of analytical chemistry with applications in fields such as medicinal chemistry, water chemistry and materials science. Consequently, the number of users of this method is continuously growing, underlining the need for an up-to-date reference.

A true pioneer of this method, Joachim Weiss studied chemistry at the Technical University of Berlin (Germany), where he also received his PhD degree in Analytical Chemistry. In 2002, he did his habilitation in Analytical Chemistry at the Leopold-Franzens University in Innsbruck (Austria), where he is also teaching liquid chromatography. Since 1982, Dr. Weiss has worked at Dionex (now being part of Thermo Fisher Scientific), where he currently holds the position of Technical Director for Dionex Products within the Chromatography and Mass Spectrometry Division (CMD) of Thermo Fisher Scientific, located in Dreieich (Germany).

• Language: English
• Publisher: Wiley
• Release date: Jun 21, 2016
• ISBN: 9783527651634

Book preview

Foreword

Since the introduction of ion chromatography in 1975, this method has not only developed into the most powerful tool for the determination of inorganic and organic ions of low and high molecular weight but also found its application for the analysis of a wide variety of ionizable organic compounds. In the past decade, numerous new technologies introduced in the field of ion chromatography were focused on increasing the speed of analysis, sensitivity, and resolution of separations. For everybody using this method nowadays for either scientific research or routine analysis, it is very important to have a comprehensive source of information covering all recent developments in this field together with some theoretical background and practical applications. The fourth edition of the Handbook of Ion Chromatography by Dr. Joachim Weiss completely fulfills this demand.

The present edition is considerably updated and expanded, covering all aspects of ion chromatography and all developments in this field that have been introduced over the past 10 years. This includes the application of hydrophilic interaction and mixed-mode liquid chromatography for the determination of ionic and ionizable compounds, the introduction of novel detection methods and hyphenated techniques, the design of new separation media for capillary and monolithic columns, columns packed with particulate ion exchangers having smaller particle diameters, and mixed-mode stationary phases.

A significant part of this new edition is devoted to various applications of ion chromatography for the determination of ions in a wide range of simple and complex matrices and to practical problems that an analytical chemist might face. It makes this book an indispensable tool for everybody using ion chromatography for everyday routine analysis or doing research work in this field. Since a lot of attention is paid to the determination of biologically relevant organic compounds such as amino acids, carbohydrates, proteins, and nucleic acids, this book will also be of great interest and importance for scientists and practitioners working in biochemistry and biopharmaceutical industries.

There is no doubt that the fourth edition of Handbook of Ion Chromatography will gain great popularity and wide recognition as an important contribution to analytical science and industry, and I wish the author and the publishing house a lot of success with this outstanding work.

Moscow, October 2015Prof. Dr. Oleg Shpigun

Chemistry Department

Lomonosov Moscow State University

Preface to the Fourth Edition

With the publication of Hamish Small's legendary paper in Analytical Chemistry in 1975, 2015 marks the 40th anniversary of the introduction of ion chromatography (IC). Over these four decades, ion chromatography not only became the most dominant method in ion analysis but also developed into a significant chromatographic technique within the field of separation science. While in its earliest embodiments, IC was focused primarily on the analysis of inorganic anions, today IC plays an important role in the analysis of organic and inorganic anions and cations. Although separations of ions by ion-exchange chromatography prevail, other liquid chromatographic techniques such as ion-exclusion chromatography, reversed-phase liquid chromatography in the ion-suppression mode, and even hydrophilic interaction and mixed-mode liquid chromatography are also used today. Thus, the definition of the term ion chromatography became much broader over the years to be an umbrella term today for all liquid chromatographic techniques that are suitable for separating and detecting ionic and ionizable species.

More than 10 years have passed since the publication of the third edition of this book in 2004. Although the method of ion chromatography was already well matured and widely accepted at that time, the past decade has seen a number of exciting developments in ion chromatography that are described in this new edition and that further established this analytical technique. Of particular importance are the new hyperbranched condensation polymers developed for anion-exchange chromatography with improved selectivities and chromatographic efficiencies. With the introduction of 4 µm ion-exchange packing materials, the pathway of method speedup in ion chromatography follows the one used in conventional HPLC by applying UHPLC techniques. In ion chromatography, however, the pathway of using smaller particle sizes and smaller column formats can be followed only to a certain extent due to the limited back pressure tolerance of metal-free components in the fluidic system of IC instruments. Progress in the design of cation exchangers has been made toward new stationary phases for improved separations of amines. A new section has been devoted to monolithic separation materials in anion-exchange chromatography. A considerable effort has been focused on their development, because monolithic media offer the potential benefit of faster analysis while maintaining chromatographic resolution, thus following the trend toward shorter analysis times in an alternative way. In the past, polymer monoliths were used only for the separation of biomolecules such as peptides, proteins, and nucleic acids. Only very recently has progress been made in developing polymer monoliths for the separation of small-molecular weight ions, first in the form of aggregated particle monoliths to avoid PEEK column wall adhesion and finally in the form of nanobead-agglomerated monoliths covalently bonded to the inner column wall.

Over the past decade, electrolytic eluent generation (RFIC™) has been well established as an alternative to manually prepared eluents. RFIC not only facilitates the use of gradient elution techniques in ion chromatography but also provides the user with more consistent data. Electrolytic eluent generation was also a prerequisite for the development of capillary ion chromatography. As an analytical tool, capillary IC offers several important advantages, including higher mass sensitivity, compatibility with smaller sample volumes, and significant reduction of eluent consumption and associated waste disposal. In addition, the very small flow rates used in capillary IC enable the permanent operation of the system over a long period of time, thus eliminating time-consuming and error-prone steps such as system startup and equilibration as well as manual eluent preparation. The enhanced stability of capillary IC systems increases laboratory productivity, as fewer calibration sequences are required and the system can quickly be verified for system performance by just running check standards, which is of utmost importance for the pharmaceutical industry (see Section 10.7). Along with the RFIC technology, hydroxide eluents, which are particularly suitable for concentration gradients in anion-exchange chromatography, are increasingly replacing classical carbonate/bicarbonate buffers predominantly used so far. In contrast to carbonate/bicarbonate buffers, which will still be used for relatively simple applications that only require isocratic elution, higher sensitivities are achieved with hydroxide eluents. This trend is supported by an exciting development of hydroxide-selective stationary phases for anion-exchange chromatography.

A growing number of applications are based on hyphenation, thus coupling ion chromatography with ICP–OES, ICP–MS, and ESI–MS. The advantage of coupling various application forms of ICP with ion chromatography includes the ability to separate and detect metals with different oxidation states. The analytical interest in chemical speciation is based on the fact that the oxidation state of an element determines toxicity, environmental behavior, and biological effects. Hyphenation with ESI–MS provides the analyst with mass-selective information. Challenging applications such as the determination of haloacetic acids in water at trace levels by IC–ESI-MS/MS or the identification and the quantification of metabolites by coupling capillary IC to an Orbitrap MS clearly demonstrate the need for MS hyphenation to achieve the required sensitivity and specificity. The updated and expanded section on hyphenated techniques underlines this importance. Because amino acids, carbohydrates, proteins, and oligonucleotides are also analyzed by ion-exchange chromatography, the respective sections have been updated and expanded in this new edition. In combination with integrated pulsed amperometry as a direct detection method for amino acids and carbohydrates, anion-exchange chromatography revolutionized these two application areas. Thus, ion chromatography has become almost indispensable for the analysis of low- and high-molecular weight inorganic and organic anions and cations.

Since the publication of the third edition, all these developments have made it necessary to rewrite major parts, so this fourth edition can be confidently regarded as a new text. With the exception of Chapters 2 and 9, every chapter has been renewed or significantly revised. Due to the unmanageable number of existing and newly developed stationary phases for anion- and cation-exchange chromatography, the respective Sections 3.4 and 4.1 on stationary phases have been completely reorganized for better clarity. Chapter 7 on hydrophilic interaction liquid chromatography (HILIC) has been added for the first time, because nowadays this technique is also used for separating ionic and ionizable compounds. The general structure of this book proved to be of value, and has thus remained unchanged. Sections 3.10.5, 3.12, and 3.13 in Chapter 3 and Section 10.9 in Chapter 10 were originally written by an expert, Dr. Dietrich Hauffe, to whom I expressed my sincere gratitude at that time. However, due to important developments over the past 10 years, these sections have been updated and expanded by myself. In addition, the chapters on detection and applications have been considerably expanded with new material and with numerous practical examples in the form of chromatograms. The ever-growing number of applications in the petrochemical industry, including biofuels, was the reason to devote a separate section to this subject.

The objective of this fourth edition is the same as that of the previous three editions: the author addresses analytical chemists doing research work in this field, who wish to familiarize themselves with this method, as well as practitioners, who employ these techniques for everyday routine analysis and are looking for a reference book that can help facilitate method development and provide an overview on the existing applications.

At this point, I would like to express my sincere gratitude to many of my colleagues in all parts of the world, who contributed their experience and knowledge to the preparation of this edition. I am particularly grateful to Dr. Kelly Flook (Sunnyvale, USA) for her willingness to review the section on monolithic separation media and for her valuable suggestions, and to Emma Ramirez and Jennifer Ambra (Sunnyvale, USA) for their incredible patience to provide me with electronic versions of the huge number of chromatograms needed for this edition. I am also very grateful to Dr. Andrea Wille (Metrohm, Herisau, Switzerland), Karl-Heinz Jansen (Sykam, Fürstenfeldbruck, Germany), Dr. Maria Ofitserova (Pickering Laboratories, Mountain View, USA), and Volker Nödinger (Tosoh Bioscience GmbH, Stuttgart, Germany) for their unrestricted cooperation in providing me with IC methodologies and corresponding chromatograms. Last but not least, I would be grateful for any criticisms or suggestions that could serve to improve future editions of this book.

Finally, many thanks to my wife and children for all their support and amazing amount of understanding and tolerance during the period when I had to spend most of my available spare time at the computer for preparing this new edition.

NiedernhausenJoachim Weiss

February 2016

1

Introduction

1.1 Historical Perspective

Chromatography is the general term for a variety of physicochemical separation techniques, all of which have in common the distribution of a component between a mobile phase and a stationary phase. The various chromatographic techniques are subdivided according to the physical state of these two phases.

The discovery of chromatography is attributed to Tswett [1,2], who in 1903 was the first to separate leaf pigments on a polar solid phase and to interpret this process. In the following years, chromatographic applications were limited to the distribution between a solid stationary and a liquid mobile phase (liquid solid chromatography, LSC). In 1938, Izmailov and Schraiber [3] laid the foundation for thin-layer chromatography (TLC). Stahl [4,5] refined this method in 1958 and developed it into the technique known today. In their noteworthy paper of 1941, Martin and Synge [6] proposed the concept of theoretical plates, which was adapted from the theory of distillation processes, as a formal measurement of the efficiency of the chromatographic process. This approach not only revolutionized the understanding of liquid chromatography but also set the stage for the development of both gas chromatography (GC) and paper chromatography.

In 1952, James and Martin [7] published their first paper on gas chromatography, initiating the rapid development of this analytical technique.

High-performance liquid chromatography (HPLC) was derived from the classical column chromatography and, besides gas chromatography, is one of the most important tools of analytical chemistry today. The technique of HPLC flourished after it became possible to produce columns with packing materials made of very small beads (≈10 µm) and to operate them under high pressure. The development of HPLC and the theoretical understanding of the separation processes rest on the basic works of Horvath et al. [8], Knox [9], Scott [10], Snyder [11], Guiochon [12], Möckel [13], and others.

Ion chromatography (IC) was introduced in 1975 by Small et al. [14] as a new analytical method. Within a short period of time, ion chromatography evolved from a new detection scheme for a few selected inorganic anions and cations to a versatile analytical technique for ionic species in general. For a sensitive detection of ions via their electrical conductance, the separator column effluent was passed through a suppressor column. This suppressor column chemically reduces the eluent background conductance, while at the same time increasing the electrical conductance of the analyte ions.

In 1979, Fritz et al. [15] described an alternative separation and detection scheme for inorganic anions, in which the separator column is directly coupled to the conductivity cell. As a prerequisite for this chromatographic setup, low-capacity ion-exchange resins must be employed so that low-ionic strength eluents can be used. In addition, the eluent ions should exhibit low equivalent conductances, thus enabling detection of the sample components with reasonable sensitivity.

At the end of the 1970s, ion chromatographic techniques began to be used to analyze organic ions. The requirement for a quantitative analysis of organic acids brought about an ion chromatographic method based on the ion-exclusion process that was first described by Wheaton and Bauman [16] in 1953.

The 1980s witnessed the development of high-efficiency separator columns with particle diameters between 5 and 8 µm, which resulted in a significant reduction of analysis time. In addition, separation methods based on the ion-pair process were introduced as an alternative to ion-exchange chromatography because they allow the separation and determination of surface-active anions and cations.

Since the beginning of the 1990s, column development has aimed to provide stationary phases with special selectivities. In inorganic anion analysis, stationary phases were developed that allow the separation of fluoride from the system void and the analysis of the most important mineral acids as well as oxyhalides such as chlorite, chlorate, and bromate in the same chromatographic run [17]. Moreover, high-capacity anion exchangers have been developed that enable the analysis of, for example, trace anionic impurities in concentrated acids and salinary samples. Problem solutions of this kind are especially important for the semiconductor industry, seawater analysis, and clinical chemistry. In inorganic cation analysis, simultaneous analysis of alkali and alkaline-earth metals is of vital importance, and can be realized only within an acceptable time frame of less than 15 min by using weak acid cation exchangers [18]. Of increasing importance is the analysis of aliphatic amines, which can be carried out on modern cation exchangers without adding organic solvents to the acid eluent.

Since the publication of the third edition in 2004, considerable effort has been focused on the development of monolithic separation materials for use in ion chromatography. Monolithic media offer the potential benefit of faster analysis or improved resolution with comparable analysis speed, thus following the trend toward shorter analysis times observed in conventional liquid chromatography. While method speedup in conventional liquid chromatography (UHPLC) is achieved by utilizing smaller particle sizes and smaller column formats, this pathway can be followed only to a certain extent in ion chromatography due to the limited back pressure tolerance of metal-free components in the fluidic system of IC instruments. Most research in the area of monolithic sparation media has been devoted to silica-based materials [19], which are not very suitable for ion chromatography, especially for anion separations due to pH limitations. Polymer monoliths, on the other hand, were so far used only for the separation of biomolecules such as peptides, proteins, and nucleotides [20]. Only very recently has progress been made in developing polymer monoliths for the separation of small-molecular weight ions, first in the form of aggregated particle monoliths to avoid PEEK column wall adhesion [21] and finally in the form of nanobead-agglomerated monoliths covalently bonded to the inner column wall [22].

The scope of ion chromatography was considerably enlarged by newly designed electrochemical and spectrophotometric detectors. A milestone of this development was the introduction of a pulsed amperometric detector in 1983, allowing a very sensitive detection of carbohydrates, amino acids, and divalent sulfur compounds [23,24]. A recent development in the field of electrochemical detection is 3D amperometry. The relationship of 3D amperometry to conventional amperometry is in some ways similar to the relationship of diode array detection to single wavelength UV absorbance detection. Three-dimensional amperometry enables the continuous acquisition of current throughout the entire waveform period rather than only during a predefined period within the waveform when current is integrated. The complete data set enables, among other things, postchromatographic current integration. Because different chemical compounds oxidize differently at a given applied oxidation potential, subtle differences in the amount of current generated through a waveform can provide additional information about the identity and purity of the substances being analyzed.

Applications utilizing postcolumn derivatization in combination with photometric detection opened the field of polyphosphate, polyphosphonate, and transition metal analysis for ion chromatography, thus providing a powerful extension to conventional titrimetric and spectrometric methods.

A growing number of applications are based on hyphenation, thus coupling ion-exchange chromatography with ICP–OES, ICP–MS, or ESI–MS. The advantage of coupling the various application forms of ICP with ion chromatography includes the ability to separate and detect metals with different oxidation states. The analytical interest in chemical speciation is based on the fact that the oxidation state of an element determines toxicity, environmental behavior, and biological effects. Hyphenation with ESI–MS provides the analyst with mass-selective information. Depending on the type of MS (single quadrupoles, triple quadrupoles, ion traps, etc.) coupled to IC, molecular weight and/or structural information can be obtained. The recently published EPA Method 557 [25] for determining haloacetic acids in water at trace levels by IC–ESI-MS/MS, for instance, clearly demonstrates the need for MS hyphenation to achieve the required sensitivity and specificity for challenging applications.

These developments made ion chromatography an integral part of both modern inorganic and organic analyses.

Even though ion chromatography is the dominating analytical method for inorganic and organic ions, ion analyses are also carried out with capillary electrophoresis (CE) [26], which offers certain advantages when analyzing samples with extremely complex matrices. In terms of detection, spectrometric methods such as UV/Vis and fluorescence detection as well as contactless conductivity detection [27] are commercially available today. Because inorganic anions and cations as well as aliphatic carboxylic acids cannot be detected very sensitively, applications of CE for small ion analysis are rather limited compared to IC, with its universal suppressed conductivity detection being employed in most cases.

Dasgupta and Bao [28] and Avdalovic et al. [29] independently succeeded to miniaturize a conductivity cell and a suppressor device down to the scale required for CE. Since the sensitivity of conductivity detection does not suffer from miniaturization, detection limits achieved for totally dissociated anions and low-molecular weight organics competed well with those of ion chromatography techniques. Even though the works of Dasgupta and Bao, and Avdalovic et al. have never been commercialized, capillary electrophoresis with nonsuppressed conductivity detection can be regarded as a complementary technique for analyzing small ions in simple and complex matrices.

1.2 Types of Ion Chromatography

This book only discusses separation methods that can be summarized under the general term ion chromatography. Modern ion chromatography as an element of liquid chromatography is based on three different separation mechanisms, which also provide the basis for the nomenclature in use.

Ion-Exchange Chromatography (HPIC)

This separation method is based on ion-exchange processes occurring between the mobile phase and the ion-exchange groups bonded to the support material. In highly polarizable ions, additional nonionic adsorption processes contribute to the separation mechanism. The stationary phase typically consists of polymeric resins based on styrene, ethylvinylbenzene, methacrylates, or polyvinyl al-cohols modified with ion-exchange groups. With the exception of polyvinyl alcohols, the resins are usually copolymerized with divinylbenzene for high mechanical and chemical stability. Ion-exchange chromatography is used for the separation of both inorganic and organic anions and cations. Separation of anions is accomplished with quaternary ammonium groups attached to the polymer, whereas sulfonate, carboxylate, phosphonate, or mixtures of these groups are used as ion-exchange sites for the separation of cations. Chapters 3 and 4 deal with this type of separation method in greater detail.

Ion-Exclusion Chromatography (HPICE)

The separation mechanism in ion-exclusion chromatography is governed by Donnan exclusion, steric exclusion, sorption processes and, depending on the type of separator column, by hydrogen bonding. A high-capacity, totally sulfonated cation-exchange material based on poly(styrene-co-divinylbenzene) is typically employed as the stationary phase. In case hydrogen bonding should determine selectivity, significant amounts of methacrylate are added to the styrene polymer. Ion-exclusion chromatography is particularly useful for the separation of weak inorganic and organic acids from completely dissociated acids that elute as one peak within the exclusion volume of the column. In combination with suitable detection systems (postcolumn chemistry, RI, ELSD, and Corona CAD), this separation method is also useful for determining amino acids, aldehydes, and alcohols. A detailed description of this separation method is given in Chapter 5.

Ion-Pair Chromatography (MPIC)

The dominating separation mechanism in ion-pair chromatography is adsorption. The stationary phase consists of a neutral porous divinylbenzene resin of low polarity and high specific surface area. Alternatively, chemically bonded octadecyl silica phases with even lower polarity can be used. The selectivity of the separator column is determined by the mobile phase. Besides an organic modifier, an ion-pair reagent is added to the eluent (water, aqueous buffer solution, etc.) depending on the chemical nature of the analytes. Ion-pair chromatography is particularly suited for the separation of surface-active anions and cations, sulfur compounds, and transition metal complexes. A detailed description of this separation method is given in Chapter 6.

Alternative Methods

In addition to the three classical separation methods mentioned above, reversed-phase liquid chromatography (RPLC) can also be used for the separation of highly polar and ionic species. Long-chain fatty acids, for example, are separated on a chemically bonded octadecyl phase after protonation in the mobile phase with a suitable aqueous buffer solution. This separation mode is known as ion suppression [30].

Chemically bonded aminopropyl phases have also been successfully employed for the separation of inorganic ions. Leuenberger et al. [31] described the separation of nitrate and bromide in foods on such a phase using a phosphate buffer solution as the eluent. Separations of this kind are limited in terms of their applicability, because they can be applied only to UV-absorbing species.

Moreover, applications of multidimensional ion chromatography utilizing mixed-mode phases are very interesting. In those separations, ion-exchange and reversed- phase interactions equally contribute to the retention mechanism of ionic and polar species [32,33]. These alternative techniques are also described in Chapter 6.

1.3 The Ion Chromatographic System

The basic components of an ion chromatograph are shown schematically in Figure 1.1. It resembles the setup of conventional HPLC systems.

Figure depicting the basic components of an ion chromatograph that include eluent, pump, sample injection valve, separator (ion-exchange reaction), detector (suppressor reaction and conductivity cell), and output (integrator/data system).

Figure 1.1 Basic components of an ion chromatograph.

A pump delivers the mobile phase through the chromatographic system. In general, dual-piston pumps are employed. A pulse-free flow of the eluent is necessary for employing sensitive conductivity, UV/Vis, and amperometric detectors. Therefore, a sophisticated electronic circuitry (sometimes in combination with pulse dampeners) is used to reduce residual pulsation as much as possible.

The sample is injected into the system via a valve injector, as schematically shown in Figure 1.2. A three-way valve is required, with two ports being connected to the sample loop. Sample loading is carried out at atmospheric pressure. After switching the injection valve, the sample is transported to the separator column by the mobile phase. Typical injection volumes are between 5 and 100 µL, but smaller and larger injection volumes are used for capillary-scale ion chromatography and large-volume direct injections, respectively.

A schematic diagram representing a loop injector for injecting sample into the system. A three-way valve is required, with two ports being connected to the sample loop.

Figure 1.2 Schematic representation of a loop injector.

The most important part of the chromatographic system is the separator column. The choice of a suitable stationary phase (see Section 1.5) and the chromatographic conditions determine the quality of the analysis. The column tubes are manufactured from inert materials such as PEEK (polyether ether ketone). In general, separation is achieved at room temperature. Only in very few cases – for example, for the analysis of long-chain fatty acids – an elevated temperature is required to improve analyte solubility. An elevated column temperature is also recommended for the analysis of inorganic and organic cations on weak acid cation exchangers for selectivity reasons. Very rarely column temperatures below ambient are used to avoid analyte degradation.

The analytes are detected and quantified by a detection system. The performance of any detector is evaluated according to the following criteria:

Sensitivity

Linearity

Resolution (detector cell volume)

Noise (detection limit)

The most commonly employed detector in ion chromatography is the conductivity detector, which is used with or without a suppressor system. The main function of the suppressor system as part of the detection unit is to chemically reduce the high background conductivity of the electrolytes in the eluent and to convert the sample ions into a more conductive form. In addition to conductivity detectors, UV/Vis, amperometric, charge, fluorescence, and MS detectors are used, all of which are described in detail in Chapter 8.

Chromatographic signals are displayed in the form of a chromatogram. Quantitative results are obtained by evaluating peak areas or peak heights, both of which are proportional to the analyte concentration over a wide range. In the past, this was performed using digital integrators that were connected directly to the analog signal output of the detector. Due to the lack of GLP/GLAP conformity, digital integrators are hardly used anymore. Modern detectors feature USB ports that enable the connection to a personal computer or a host computer with a suitable chromatography software. Computers also take over control functions, thus allowing fully automated operation of the chromatographic system.

Because corrosive eluents such as diluted acids and bases are often used in ion chromatography, all parts of the chromatographic system being exposed to these liquids should be made of inert, metal-free materials. Conventional HPLC systems with tubings and pump heads made of stainless steel are only partially suited for ion chromatography, because even stainless steel is eventually corroded by aggressive eluents. Considerable contamination problems would result, because metal ions exhibit a high affinity toward the stationary phase of ion exchangers, leading to a significant loss of separation efficiency. Moreover, metal parts in the chromatographic fluid path would make the analysis of analytes such as orthophosphate, complexing agents, and transition metals more difficult.

1.4 Advantages of Ion Chromatography

The determination of ionic species in solution is a classical analytical problem with a variety of solutions. Whereas in the field of cation analysis both fast and sensitive analytical methods (AAS, ICP, polarography, and others) have been available for a long time, there was a lack of corresponding, highly sensitive methods for anion analysis before ion chromatography was introduced in the mid-1970s. Conventional wet-chemical methods such as titration, photometry, gravimetry, turbidimetry, and colorimetry are all labor-intensive, time-consuming, and occasionally troublesome. In contrast, ion chromatography offers the following advantages:

Speed

Sensitivity

Selectivity

Simultaneous detection

Stability of the separator columns

Speed

The time necessary to perform an analysis becomes an increasingly important aspect, because enhanced manufacturing costs for high-quality products and additional environmental efforts have led to a significant increase in the number of samples to be analyzed.

With the introduction of high-efficiency separator columns for ion-exchange, ion-exclusion, and ion-pair chromatography in recent years, the average analysis time could be reduced to about 10 min. Today, a baseline-resolved separation of the seven most important inorganic anions [34] requires less than 5 min. Quantitative results are obtained in a fraction of the time previously required for traditional wet-chemical methods, thus increasing sample throughput.

Sensitivity

The introduction of microprocessor technology, in combination with modern high-efficiency stationary phases, makes it a routine task to detect ions in the lowest microgram/Liter concentration range without preconcentration. The detection limit for simple inorganic anions and cations is about 5 µg/L based on an injection volume of 25 µL. The total amount of injected sample lies in the low nanogram range. Even ultrapure water, required for the operation of power plants or for the production of semiconductors, may be analyzed for its anion and cation content after preconcentration with respective concentrator columns. With these preconcentration techniques, the detection limit could be lowered to the lowest nanogram/Liter range. However, it should be emphasized that the instrumentation for measuring such incredibly low amounts is more sophisticated than that required for milligram/Liter concentrations. In addition, high demands have to be met in the creation of suitable environmental conditions. The limiting factor for further lowering the detection limits is the contamination by ubiquitous chloride and sodium ions.

High sensitivities down to the femtomole range are also achieved in carbohydrate and amino acid analysis by using integrated pulsed amperometric detection.

Selectivity

The selectivity of ion chromatographic methods for analyzing inorganic and organic anions and cations is ensured by the selection of suitable separation and detection systems. Regarding conductivity detection, the suppression technique is of vital importance, because the counterions of the analyte ions as a potential source of interference are exchanged against hydronium and hydroxide ions. A high degree of selectivity is achieved by using solute-specific detectors such as a UV/Vis detector to analyze nitrite in the presence of high amounts of chloride. New developments in the field of postcolumn derivatization show that specific compound classes such as transition metals, alkaline-earth metals, polyvalent anions, orthosilicate, and so on can be detected with high selectivity. Such examples explain why sample preparation for ion chromatographic analyses usually involves only a simple dilution and filtration of the sample. This high degree of selectivity facilitates the identification of unknown sample components.

Simultaneous Detection

A major advantage of ion chromatography – especially in contrast to other instrumental techniques such as photometry and AAS – is its ability to simultaneously detect multiple sample components. Anion and cation profiles may be obtained within a short time; such profiles provide information about the sample composition and help to avoid time-consuming tests. However, the ability of ion chromatographic techniques for simultaneous quantitation is limited by extreme concentration differences between various sample components. For example, the major and minor components in a wastewater matrix may be detected simultaneously only if the concentration ratio is <1000 : 1. Otherwise, the sample must be diluted and analyzed in a separate chromatographic run. Modern chromatography data systems recognize samples with component concentrations outside the calibrated range, which are then automatically reinjected either after an appropriate dilution or through a second injection loop of a smaller volume.

Stability of the Separator Columns

The stability of separator columns very much depends on the type of packing material being used. In contrast to silica-based separator columns commonly used in conventional HPLC, resin materials such as styrene-based polymers prevail as support material in ion chromatography. The high pH stability of these resins allows the use of strong acids and bases as eluents, which is a prerequisite for the widespread applicability of this method. Strong acids and bases, on the other hand, can also be used for rinsing procedures. Meanwhile, most organic polymers are compatible with typical HPLC solvents such as methanol and acetonitrile, which can be used for the removal of organic contaminants (see also Chapter 10). Hence, polymer-based stationary phases exhibit a low sensitivity toward complex matrices, such as wastewater, foods, or body fluids, so a simple dilution of the sample with deionized water prior to filtration is sometimes the only sample preparation procedure.

1.5 Selection of Separation and Detection Systems

As previously mentioned, a wealth of different separation techniques is summarized under the term ion chromatography. Therefore, what follows is a survey of criteria for selecting stationary phases and detection modes suitable for solving specific separation problems.

The analyst usually has some information regarding the nature of the ion to be analyzed (inorganic or organic), its surface activity, its valency, and its acidity or basicity. With this information and on the basis of the selection criteria outlined schematically in Table 1.1, it should not be difficult for the analytical chemist to select a suitable stationary phase and detection mode. In many cases, several procedures are feasible for solving a specific separation problem. In these cases, the choice of the analytical procedure is determined by the type of matrix, by the simplicity of the procedure, and, increasingly, by financial aspects. Two examples illustrate this.

Table 1.1 Schematic representation of selection criteria for separation and detection modes.

Various sulfur-containing species in the scrubber solution of a flue-gas desulfurization plant (see also Section 10.2.4) are to be analyzed. According to Table 1.1, nonpolarizable ions such as sulfite, sulfate, and amidosulfonic acid with pK values below 7 are separated isocratically by HPIC using a conventional anion exchanger and are detected via electrical conductivity. A suppressor system should be used to increase the sensitivity and specificity of the procedure. Often, scrubber solutions also contain thiocyanate and thiosulfate in small concentrations. However, due to their polarizability, these anions exhibit a high affinity toward the stationary phase of conventional anion exchangers. Three different approaches are feasible for the analysis of such anions. A conventional anion exchanger may be used with a high-ionic strength mobile phase. Depending on the analyte concentration, difficulties with the sensitivity of the subsequent conductivity detection may arise. Alternatively, a special methacrylate-based anion exchanger with hydrophilic functional groups may be employed. Polarizable anions are not adsorbed as strongly on this kind of stationary phase and, therefore, elute together with nonpolarizable anions. Taking into account that other sulfur-containing species such as dithionate may also have to be analyzed, a gradient elution technique with a hydroxide eluent has to be employed, which allows all compounds mentioned above to be separated in a single run utilizing a high-efficiency separator column and conductivity detection. However, the required concentration gradient makes the use of a suppressor system inevitable. Concentration gradients on anion exchangers reach their limit when extremely polarizable anions such as nitrilotrisulfonic acid have to be analyzed. In this case, ion-pair chromatography (MPIC) is the better separation mode, because organic solvents added to the mobile phase determine analyte retention.

The second example is the determination of organic acids in soluble coffee. According to Table 1.1, aliphatic carboxylic acids are separated by HPICE on a totally sulfonated cation-exchange resin with subsequent conductivity detection. While this procedure is characterized by a high selectivity for aliphatic monocarboxylic acids with a small number of carbon atoms, sufficient separation cannot be obtained for the aliphatic open-chain and cyclic hydroxy acids that are also present in coffee. Only after introducing a new stationary phase with specific selectivity for hydroxycarboxylic acids did it become possible to separate the most important representatives of this class of compounds in such a matrix. Ion-exclusion chromatography is not suited for the separation of aromatic carboxylic acids, which are present in coffee in large numbers. Examples are ferulic acid, caffeic acid, and the class of chlorogenic acids. Due to π–π interactions with the aromatic rings of the organic polymers used as support material for the stationary phase, aromatic acids are strongly retained and thus cannot be analyzed by HPICE. A good separation is achieved by reversed-phase chromatography using chemically bonded octadecyl phases with high chromatographic efficiencies. These compounds are then detected by measuring their light absorption at 254 nm.

Further details on the selection of separation and detection modes are given in Chapters 3–7.

References

1. Tswett, M. (1903) Trav. Soc. Nat. Var., 14, 1903.

2. Tswett, M. (1906) Ber. Deut. Botan. Ges., 24, 385.

3. Izmailov, N.A. and Schraiber, M.S. (1938) Farmatsiya, 3:1, 2.

4. Stahl, E. (1956) Pharmazie, 11, 633.

5. Stahl, E. (1958) Chemiker Ztg., 82, 323.

6. Martin, A.J.P. and Synge, R.L. (1941) Biochem. J., 35, 1358.

7. James, A.T. and Martin, A.J.P. (1952) Analyst, 77, 915.

8. Horvath, C., Melander, W., and Molnar, I. (1976) J. Chromatogr., 125, 129.

9. Knox, J.H. (1976) Theory of HPLC, Part II: solute interactions with the mobile phase and stationary phases in liquid chromatography, in Practical High Performance Liquid Chromatography (ed. C.F. Simpson), Heyden & Son, Chichester.

10. Scott, R.P.W. (1976) Theory of HPLC, Part II: solute interactions with the mobile phase and stationary phases in liquid chromatography, in Practical High Performance Liquid Chromatography (ed. C.F. Simpson), Heyden & Son, Chichester.

11. Snyder, L.R. (1965) Chromatogr. Rev., 7, 1.

12. Guiochon, G. (1980) Optimization in liquid chromatography, in High Performance Liquid Chromatography, vol. 2 (ed. C. Horvath), Academic Press, New York.

13. Möckel, H.J. Lecture: Instrumentelle Analytik I. Technical University Berlin, 1974–1984.

14. Small, H., Stevens, T.S., and Bauman, W.C. (1975) Anal. Chem., 47, 1801.

15. Gjerde, D.T., Fritz, J.S., and Schmuckler, G. (1979) J. Chromatogr., 186, 509.

16. Wheaton, R.M. and Bauman, W.C. (1953) Ind. Eng. Chem., 45, 228.

17. Weiss, J., Reinhard, S., Pohl, C.A., Saini, C., and Narayaran, L. (1995) J. Chromatogr., 706, 81.

18. Jensen, D., Weiss, J., Rey, M.A., and Pohl, C.A. (1993) J. Chromatogr., 640, 65.

19.Unger, K., Tanaka, N., and Machtejevas, E. (eds) (2011) Monolithic Silicas in Separation Science, Wiley-VCH Verlag GmbH, Weinheim.

20.Svec, F., Tennikova, T.B., and Deyl, Z. (eds) (2003) Monolithic Materials: Preparation, Properties, and Applications, Elsevier, Amsterdam.

21. Kuban, P., Dasgupta, P.K., and Pohl, C.A. (2007) Anal. Chem., 79, 5462.

22. Pohl, C.A., Saini, C., and Agroskin, Y. (2010) A new monolithic anion-exchange column for fast separation of inorganic and organic anions in a variety of sample matrixes. Presentation at the Pittcon 2010, Orlando, FL, USA.

23. Rocklin, R.D. and Pohl, C.A. (1983) J. Liq. Chromatogr., 6 (9), 1577.

24. Martens, D.A. and Frankenberger, W.T. (1992) J. Liq. Chromatogr., 15, 423.

25.US EPA (2009) Method 557: Determination of Haloacetic Acids, Bromate, and Dalapon in Water by Ion Chromatography Electrospray Ionization Tandem Mass Spectrometry. Technical Support Center, Office of Ground Water and Drinking Water, US EPA, Cincinatti, OH, USA.

26. Jandik, P. and Bonn, G. (1993) Capillary Electrophoresis of Small Molecules and Ions, VCH Publishers, Inc., New York.

27. Zemann, A.J., Schnell, E., Volgger, D., and Bonn, G.K. (1998) Anal. Chem., 70, 563.

28. Dasgupta, P.K. and Bao, L. (1993) Anal. Chem., 65, 1003.

29. Avdalovic, N., Pohl, C.A., Rocklin, R.D., and Stillian, J.R. (1993) Anal. Chem., 65, 1470.

30. Johnson, E.L. and Stevens., B. (1978) Basic Liquid Chromatography, Varian Associates Inc., Palo Alto, CA, USA, p. 92.

31. Leuenberger, U., Gauch, R., Rieder, K., and Baumgartner, E. (1980) J. Chromatogr., 202, 461.

32. Stillian, J.R. and Pohl, C.A. (1990) J. Chromatogr., 499, 249.

33. Zhang, K., Dai, L., and Chetwyn, N.P. (2010) J. Chromatogr. A, 1217, 5776.

34.DIN 38 405 Part 19 (1988) Die Bestimmung der Anionen Fluorid, Chlorid, Nitrit, Bromid, Nitrat, Orthophosphat und Sulfat in wenig belasteten Wässern mit der Ionenchromatographie.

2

Theory of Chromatography

2.1 Chromatographic Terms

Chromatographic signals are usually registered in the form of a chromatogram. A typical chromatogram is schematically depicted in Figure 2.1.

Figure illustrating a general chromatogram where on the left-hand side is the starting point, and on moving rightward, following a time interval of tm is a small minute peak. From here at a time interval of ts is a large bell-shaped curve. Distance between start and the peak of bell-shaped curve denotes tms (tms = tm + ts).

Figure 2.1 General illustration of a chromatogram.

Two different components are separated in a chromatographic column only if they spend different amounts of time in or at the stationary phase. The time in which the components do not travel along the column is called the solute retention time, ts. The column dead time, tm, is defined as the time necessary for a nonretained component to pass through the column. The gross retention time, tms, is calculated from the solute retention time and the column dead time, as shown in Eq. (2.1):

(2.1) equation

The chromatographic terms used in the characterization of a separator column can be seen in Figure 2.1.

In a first approximation, the shape of a chromatographic peak is described by a Gaussian curve (Figure 2.2).

Figure depicting the Gaussian curve represented by a bell-shaped curve where s is the standard deviation. The y-axis represents the peak height at maximum, while the x-axis represents the peak width at the baseline.

Figure 2.2 The Gaussian curve.

The peak height at any given position x can be derived from Eq. (2.2):

(2.2) equation

2.1.1 Asymmetry Factor As

The signals (called the peaks) due to elution of a species from a chromatographic column are rarely perfectly Gaussian. Normally, the peaks are asymmetrical to some extent; this is expressed by Eq. (2.3) (see also Figure 2.3):

(2.3) equation

Figure depicting a graph defining the asymmetry factor where the peak shape is characterized by a rapid increase of the chromatographic signal followed by a comparatively slow decrease. A dashed vertical line from the center of the peak divides it into two unequal parts such that the left portion is smaller than the right portion. h denotes the height of the peak.

Figure 2.3 Definition of the asymmetry factor.

At As values higher than 1, this asymmetry is called tailing. The peak shape is characterized by a rapid increase of the chromatographic signal followed by a comparatively slow decrease. Adsorption processes are mainly responsible for such tailing effects. At As values lower than 1, the asymmetry is called leading or fronting. This effect is characterized by a slow increase of the signal followed by a fast decrease. The leading effect occurs if the stationary phase does not have a sufficient number of suitable adsorption sites and hence, some of the sample molecules (or sample ions) pass the peak center. For practical applications, separator columns are considered to be good when the asymmetry factor is between 0.9 and 1.2.

2.2 Parameters for Assessing the Quality of a Separation

2.2.1 Resolution

The objective of a chromatographic analysis is to separate the components of a mixture into separate bands. The resolution R of two neighboring peaks is defined as the quotient of the difference of the two peak maxima (expressed as the difference between the gross retention times) and the arithmetic mean of their respective peak widths, w, at the peak base.

(2.4) equation

As shown in Figure 2.4, these parameters can be obtained directly from the chromatogram. If the peaks exhibit a Gaussian peak shape, a resolution of R = 2.0 (corresponding to an 8σ separation) is sufficient for quantitative analysis. Thus, the two peaks are completely resolved to baseline because peak width at the base is given by Eq. (2.5):

(2.5) equation

Higher values of R would result in excessively prolonged analysis times. At a resolution of R = 0.5, two sample components can still be recognized as separate peaks.

Figure 2.4 Parameter for assessing resolution and selectivity.

2.2.2 Selectivity

The decisive parameter for the separation of two components is their relative retention, which is called selectivity α. The selectivity is defined as the ratio of the solute retention times of two different signals, as shown in Eq. (2.6):

(2.6) equation

According to Figure 2.4, these parameters may also be obtained from the chromatogram. The selectivity is determined by the properties of the stationary phase. In HPLC, the selectivity is also affected by the mobile phase composition. If α = 1, there are no thermodynamic differences between the two sample components under the given chromatographic conditions; therefore, no separation is possible. At equilibrium, which is established in reasonably close approximation in chromatography, the selectivity α is a thermodynamic quantity. At a constant temperature, selectivity α depends on the specific properties of the sample components to be separated and on the properties of the mobile and stationary phases being used.

2.2.3 Capacity Factor

The capacity factor, k, is the product of the phase ratio Φ between stationary and mobile phases in the separator column and the Nernst distribution coefficient, K, as shown in Eq. (2.7):

(2.7) equation

The capacity factor is independent of the equipment being used, and is a measure of the column's ability to retain a sample component. Small values of k imply that the respective component elutes near the void volume; thus, the separation will be poor. High values of k, on the other hand, are tantamount to longer analysis times, peak broadening, and a decrease in sensitivity.

2.3 Column Efficiency

A fundamental disadvantage of chromatography is the broadening of the sample component zone during its passage through the separation system. Peak broadening is caused by diffusion processes and flow processes. Peak broadening can be measured by the plate number, N, or the plate height, H.

The height of a theoretical plate, in which the distribution equilibrium of sample molecules between stationary and mobile phases is established, is related to the plate number via the length of the separator column, as shown in Eq. (2.8):

(2.8) equation

Based on chromatographic data, the theoretical plate height, H, which is defined as the ratio of the peak variance and the column length L, can be calculated via Eq. (2.9):

(2.9) equation

The term 8 ln 2 arises from the approximation of a peak as a Gaussian curve.

Using Eq. (2.8), the number of theoretical plates is shown in Eq. (2.10):

(2.10) equation

Two sample components may be separated from each other only if their k values are different. The effective plate number, Neff, or the effective plate height, Heff, is used to describe the separation efficiency of a column, as shown in (Eqs. 2.11) and (2.12).

(2.11) equation

(2.12) equation

The resolution of a column can be coupled to multiple parameters, including (i) efficiency of the separator column, (ii) selectivity, and (iii) capacity factor in a single equation. These parameters are defined by

(2.13) equation

(Equation 2.13) may be reordered, as shown in Eq. (2.14), so that the plate number required to afford the desired resolution may be calculated for any given values of k and α:

(2.14) equation

2.4 The Concept of Theoretical Plates (van Deemter Theory)

Based on the work of Martin and Synge [1], van Deemter et al. [2] introduced the concept of the theoretical plate height, H, as a measure for relative peak broadening in correlation with the terminology used in distillation technology. In general form, the plate height, H, is given by Eq. (2.15):

(2.15) equation

(van Deemter equation)

The individual terms of the sum vary depending on the mobile phase velocity, v.

Accordingly, term A is independent of the flow velocity and characterizes the peak dispersion caused by the Eddy diffusion. This effect considers the different pathways for solute molecules in the column packing. The longitudinal diffusion is described by the term B/v. The term C·v comprises the lateral diffusion and the resistance to mass transfer between mobile and stationary phase. These effects depend linearly on the flow velocity.

With the peak width expressed in terms of length unit σl, it follows by taking Eq. (2.8):

(2.16) equation

or

(2.17) equation

Various portions σi contribute to the broadening of a peak. The sum of their variances gives the total band spreading:

(2.18) equation

The portion of the peak broadening that arises due to the irregularity of the column packing is called Eddy diffusion. It is approximated by Eq. (2.19):

(2.19) equation

All molecules present in the mobile phase at time tm may diffuse in and against the flow direction. The contribution of the longitudinal diffusion in the mobile phase is described by Eq. (2.20):

(2.20) equation

Lateral diffusion and resistance to mass transfer are the predominating effects for the total peak broadening and thus mainly determine the efficiency of the separator column. Any one sample molecule that interacts with the column packing diffuses back and forth between the stationary and the mobile phase. It is retained at the stationary phase and, therefore, trails the center of the peak, which passes through the separator column. This effect is illustrated in Figure 2.5 [3]. In the mobile phase, on the other hand, the sample molecule travels with the eluent. The mass-transfer effect causes a peak broadening, because sample molecules pass through the column ahead of, as well as behind, the peak center. Due to the eluent flow through the column, the equilibrium between the solute concentration in the mobile phase and in the adjacent stationary phase is not attained. Both phases contribute to the resistance to mass transfer. Therefore, two terms must be considered for the peak broadening. The peak broadening by the stationary phase is given by the following equation:

(2.21) equation

Figure illustrating the mass-transfer effect where the upper and lower sides of the horizontal axis (interface) denote mobile and stationary phases, respectively. In the stationary phase an inverted bell-shaped curve is present and corresponding to it in the mobile phase two bell-shaped overlapping curves are present. The dashed curve denotes equilibrium concentration and the solid line curve denotes actual concentration. A rightward arrow denotes flow direction and a bidirectional arrow is present on the horizontal axis.

Figure 2.5 Illustration of the mass-transfer effect.

Instead of the capacity factor, k, the capacity ratio, ke, which is calculated by Eq. (2.22), is used in the equation above:

(2.22) equation

The surface-functionalized column packing materials used in ion chromatography minimize the contribution to the total peak broadening caused by the stationary phase, because solute molecules are not able to penetrate into the packing material.

The dependence of the peak broadening on the mobile phase is given by Eq. (2.23):

(2.23) equation

The column coefficient, ω, is a measure for the regularity of the packing. The contribution to the total peak broadening is significant, but may be substantially influenced by decreasing the particle diameter, dp. The use of eluents with low viscosity also leads to a reduction in the peak broadening, , because the diffusion coefficient of the solutes in the mobile phase increases accordingly.

The total plate height, H, may be expressed by combining Eqs. (2.19)–(2.23):

(2.24)

equation

The first experimental results published by Keulemans and Kwantes [4] confirmed the applicability of the equation to gas chromatography, but it soon became apparent that the equation introduced by van Deemter et al. is of only limited validity for liquid chromatography.

In 1961, Giddings [5] proposed an HETP equation, which may be considered as a special case of the van Deemter equation:

(2.25) equation

Giddings' main criticism of the van Deemter equation was that a finite contribution to the peak broadening by the Eddy diffusion term is predicted even for zero flow velocity. However, at the flow velocities encountered in practical applications, the equation proposed by Giddings reduces to the van Deemter equation, because all other terms remain the same.

The equation introduced in 1967 by Huber and Hulsman accounts for this phenomenon [6].

(2.26) equation

They introduced a coupling term that causes the Eddy diffusion term to vanish if the flow velocity approaches zero. In contrast to van Deemter and Giddings, the resistance to mass transfer in the mobile phase is described by an additional term D · v¹/². However, this factor resembles the coupling term proposed by Giddings both in its physical interpretation and in its dependence on the flow velocity.

In the early 1970s, Knox et al. [7,8] suggested another HETP equation based on their extensive data. Their equation differed significantly from the equation discussed thus far. This equation was derived by curve fitting on the authors' extensive data:

(2.27) equation

Finally, Horvath and Lin [9,10] developed an equation very similar to the one introduced by Huber and Hulsman:

(2.28) equation

The only difference between the two equations is the description of the resistance-to-mass-transfer effect, which Horvath and Lin interpret to depend on the square of the cubic root of the flow velocity instead of a quadratic root dependence.

Although the various HETP equations differ significantly from each other, Scott et al. [11] showed that, on the basis of their extensive experimental data, the dependence of the theoretical plate height H on the linear flow velocity may be satisfactorily described by the van Deemter equation in the range between 0.02 and 1 cm/s. In Scott's tests, porous silica materials with four different particle sizes were employed as stationary phases, on which nine solute compounds were separated using six different eluent mixtures.

Although the experimental data for H and v may be depicted by any hyperbolic function, not all of them provide a meaningful physical insight into the dispersion process. According to Scott et al. [11], the van Deemter equation in the form given as follows is applicable to liquid chromatography under normal operating conditions:

(2.29)

equation

The coefficients λ and γ are numbers that describe the quality of the packing; in case of well-packed columns, they are between 0.5 and 0.8. The coefficients a, b, and c were calculated by the authors to be 0.37, 4.69, and 4.04, respectively.

The coupling term introduced by Giddings seems to be particularly significant only for surface-functionalized packing materials. This is due to the low porosity of these materials, which reduces the volume of mobile phase being retained in the packing. Hence, the term describing the resistance to mass transfer is smaller, so that the mass-transfer effect between the particles gains significance.

Instead of the total plate height H and the linear flow velocity v, often the reduced plate height, h,

(2.30) equation

and the reduced flow velocity, u, are used

(2.31) equation

The graphical representation of ln h as a function of ln u (Figure 2.6) is known in the literature as the Knox plot. The dependence of the curve's position on the retention of the compound is disadvantageous. Minima in this kind of illustration are only obtained for compounds having no retention (k = 0).

Figure illustrating Knox plot where a graph is plotted between ln h on the y-axis and ln u on the x-axis. Three curves are present and a vertical line passing through all the three curves denotes ln uopt.

Figure 2.6 General illustration of a Knox plot.

2.5 van Deemter Curves in Ion Chromatography

The terms for the various contributions to the peak broadening combined in Eq. (2.24) give the impression that they are independent of each other. In practice, however, interdependence exists between these terms. This leads to a much smaller decrease in separation efficiency than predicted by simplifying the van Deemter theory.

By plotting the theoretical plate height calculated via (Eqs. 2.8) and (2.10) as a function of the flow rate, u, the dependence obtained for an anion-exchange column (IonPac AS4) is shown in Figure 2.7.

A graph is plotted between HETP on the y-axis and linear velocity u on the x-axis for an IonPac AS4 anion separator using tms(SO42-). The data plots are plotted and a slightly curved line joins few points in a line. It is indicated from the graph that the plate height is almost invariant at higher flow rates.

Figure 2.7 HETP versus linear velocity u for an IonPac AS4 anion separator using tms .

It is clear from Figure 2.7 that the plate height is almost invariant at higher flow rates. Similar dependencies have also been observed by Majors [12] for silica-based HPLC columns with smaller particle size. Such dependencies show that higher flow rates may lead to drastically reduced analysis times without any significant loss in chromatographic efficiency.

Cation separator columns exhibit a more pronounced dependence of the plate height on the flow rate (Figure 2.8).

A graph is plotted between HETP on the y-axis and linear velocity u on the x-axis for an IonPacCS1 cation separator using tms(K+). The data plots are plotted and all points are joined by a line. It is indicated from the graph that the cation separator columns exhibit a more pronounced dependence of the plate height on the flow rate.

Figure 2.8 HETP versus linear velocity u for an IonPac CS1 cation separator using tms (K+).

In this case, a compromise between separation efficiency and required analysis time has to be made. Flow rates between 2.0 mL/min and 2.3 mL/min have proved to be most suitable for practical applications.

References

1. Martin, A.J.P. and Synge, R.L.M. (1941) Biochem. J., 35, 1358.

2. van Deemter, J.J., Zuiderweg, F.J., and Klinkenberg, A. (1956) Chem. Eng. Sci., 5, 271.

3. Johnson, E.L. and Stevenson, B. (1978) Basic Liquid Chromatography, Varian Associates Inc., Palo Alto, CA, USA.

4. Keulemans, A.I.M. and Kwantes, A. (1956) Vapor Phase Chromatography (eds D.K. Desty and C.L.A. Harbourn), Butterworths, London, p. A10.

5. Giddings, J.C. (1961) J. Chromatogr., 5, 46.

6. Huber, J.F.K. and Hulsman, J.A.R.J. (1967) Anal. Chim. Acta, 38, 305.

7. Kennedy, G.J. and Knox, J.H. (1972) J. Chromatogr. Sci., 10, 549.

8. Done, J.N. and Knox, J.H. (1972) J. Chromatogr. Sci., 10, 606.

9. Horvath, C.S. and Lin, H.-J. (1976) J. Chromatogr., 126, 401.

10. Horvath, C.S. and Lin, H.-J. (1978) J. Chromatogr., 149, 43.

11. Katz, E., Ogan, K.L., and Scott, R.P.W. (1983) J. Chromatogr., 270, 51.

12. Majors, R.E. (1972) Anal. Chem., 44, 1722.

3

Anion-Exchange Chromatography (HPIC)

3.1 General Remarks

Ion exchange is one of the oldest separation processes described in the literature [1,2]. The classical column chromatography on macroporous ion-exchange resins was a precursor of modern ion-exchange chromatography. The major differences between both processes are the method of sample introduction and the type of separation and detection systems being used. In classical ion-exchange chromatography, a column (typically 10–50 cm long) was filled with an anion- or cation-exchange resin having a particle size between 60 and 200 mesh (0.075–0.25 mm). After the sample was applied to the top of the column, it migrated down the column driven by gravitational force, and became more or less separated. Individual fractions of the eluent were collected using a fraction collector, and subsequently analyzed in a separate work step. Due to the high ion-exchange capacity of the columns, high electrolyte concentrations were necessary to ensure the elution of the sample ions from the column. In many cases, several liters of eluent had to be worked up.

The enormous improvement in the performance of modern ion-exchange chromatography is attributed to the pioneering work of Small et al. [3]. Their major achievement was the development of low-capacity ion-exchange resins of high chromatographic efficiencies that could be prepared reproducibly. The required injection volume was reduced to 10–100 µL, which resulted in an enhanced resolution with very narrow peaks. Another important improvement was that of automated detection, which allowed continuous monitoring of the signal. The introduction of conductivity detection for ionic species added a new dimension to ion-exchange chromatography.

3.2 The Ion-Exchange Process

The resins employed in ion-exchange chromatography carry functional groups with a fixed charge. The respective counterions are located in the vicinity of these functional groups, thus rendering the whole entity electrically neutral. In anion-exchange chromatography, quaternary ammonium bases are generally used as ion-exchange groups; sulfonate groups are used in strong acid cation-exchange chromatography. Weak acid cation exchangers are usually functionalized with carboxylate or phosphonate groups, or with a mixture of both.

When the counterion of the ion-exchange site is replaced by a solute ion, the latter is temporarily retained by the fixed charge. The various sample ions remain in the column for different periods of time due to their different affinities toward the stationary phase, and thus, separation is brought about.

For example, if a solution containing bicarbonate anions is passed through an anion-exchange column, the quaternary ammonium groups attached to the resin are exclusively in their bicarbonate form. If a sample with anions A− and B− is injected onto the column, these anions are exchanged for bicarbonate ions according to the reversible equilibrium process given by Eqs. (3.1) and (3.2):

(3.1)

equation

(3.2)

equation

The separation of anions