Chelation and its Role in Contemporary Liquid Chromatography

1.1 Basic Chromatographic Principles

The performance of any separation system depends on two main factors, namely, separation efficiency and separation selectivity. As a rule, high separation efficiency can be achieved through the correct optimisation of the physical and chemical properties of the separation media (in the case of column liquid chromatography – column length and diameter, particle size and porous structure of the stationary phase), and through design and control of the separation conditions (column temperature, pressure, viscosity of the mobile phase etc). Obtaining greater or alternative separation selectivity is a significantly more difficult challenge, as it is predominantly associated with the precise adsorption mechanism, which is dependent upon the chemistry of either the adsorbate or adsorbent, and in most practical circumstances, both of them.

Contemporary high-performance liquid chromatography (HPLC) includes all possible combinations of non-specific and specific interactions between separated analytes (molecules or ions) and different adsorbents or stationary phases to achieve maximum separation selectivity. Chromatographic methods based on non-specific, usually weak interactions (van der Waals forces, induction and dispersion), have been reported for many years as reversed-phase (RP-HPLC) and classical normal-phase (NP-HPLC) modes of HPLC (Table 1.1). However, more specific and higher energy interactions (hydrogen bonding, π–π interactions, coordinate bonding and others) can provide a significantly higher degree of separation selectivity, the ultimate examples of which could be a β-cyclodextrin bonded phase for the use in inclusion chromatography or a highly specific bioaffinity phase. Consequently, the long-established trend in chromatographic research and development is the search for new and highly selective stationary phase materials, and their subsequent exploitation in innovative and emerging modes of HPLC, such as chiral phase chromatography, zwitterionic chromatography and others.

Complexation and chelation represent another category of specific interactions, here between metal ions and ligands, which have long been used within the mobile phase in liquid chromatography, through the addition of reagents for greater control and optimisation of separation selectivity. However, achieving desired separations via complexation at the surface of the adsorbent, with such complexation being the sole or dominant separation mechanism, has not been demonstrated too frequently in liquid chromatography. Historically, this has been due to practical and synthetic difficulties in the preparation of high efficiency chelating substrates, and additional problems resulting from the slow kinetics associated with the reversible bonding of ions within these chelating stationary phases. However, such difficulties are now well understood and documented, with considerable improvements having been demonstrated in this area, which collectively have resulted in the emergence of new high-performance modes of liquid chromatography based upon pure stationary phase chelation (or chelating ion exchange) interactions. This current monograph details these developments, in particular detailing the various methods of stationary phase preparation, exhibited and exploited stationary phase properties and selectivity, discussion of the theoretical and experimentally determined retention mechanisms, and finally the various applications of such chelating stationary phases for the separation and determination of metal ions.

1.2 Chelation as a Mechanism for Obtaining Separation Selectivity

As alluded to previously, separation selectivity in a chromatographic system can be increased, or modified, through the exploitation of multi-point or multi-bond interactions between the adsorbate molecules and the adsorbent, in combination with molecule specific effects, such as molecular size/weight etc. Chelation is an example of such an interaction, here being defined as the formation of two or more simultaneous and spatially separate covalent binding events between a single polydentate ligand and a central metal ion. The corresponding thermodynamically based 'chelation effect' results in a dramatic increase in the observed affinity of any such polydentate ligand towards specific metal cations. Thus stationary phase chelation has been utilised to provide the all important mechanism for achieving enhanced separation selectivity in many different modes of liquid chromatography, such as for example, ligand-exchange chromatography, or so-called immobilised metal ion affinity chromatography (IMAC).

For the chromatographic separation of metal ions, methods showing chelation taking place simultaneously in both the mobile and stationary phases have been developed. However, the chromatographic system exploiting chelation in only the mobile phase is much simpler, both theoretically and experimentally. The addition of various chelating reagents to the mobile phase has become a common way to regulate the separation selectivity of metal ions in ion exchange chromatography, and also within RP-HPLC, where stable metal–ligand complexes have been separated based upon differences in overall complex charge or hydrophobicity.

Ion exchange chromatography (IEC) is based on electrostatic interactions between ion exchangers and solvated ions. Such electrostatic interactions comprise ion–ion, ion–dipole and dipole–dipole interactions, with interaction energies of 100–350 kJ mol-1, 50–200 kJ mol-1 and 5–25 kJ mol-1, respectively (Table 1.1). In some cases, cation–p interactions with energies of 5–80 kJ mol-1 can also take place in certain instances/examples of IEC. However, when considering the chelating effect, the formation energy for chelates on the surface of a chelating stationary phase, exhibiting coordinate bonding or dative covalent bonding, should be considerably higher under optimum conditions. Additionally, as chelating stationary phases may be neutral (e.g. β-diketone functionalised phase), positively charged (e.g. 8-hydroxyquinolinol bonded phases) or negatively charged (e.g. iminodiacetate resins), it is often probable, and in fact inevitable, that such phases exhibit some degree of mixed mode retention, including both chelation and ion exchange interactions occurring simultaneously, but of different relative strengths, dependent upon the nature of the solvated metal ion. Where both interactions are known to occur simultaneously without inhibition of the other, the term 'chelating ion exchange' may be a more fitting description of the retention mechanism than simply chelation. In most modes of liquid chromatography it is well known that multiple retention mechanisms interacting simultaneously are unlikely to result in achieving high chromatographic efficiency. Therefore, to obtain efficient chromatographic separations using a chelating stationary phase, it is important to have chelation as the dominant retention mechanism. In chelation chromatography this can be achieved by suppression of any electrostatic interactions occurring between the solvated metal ions and the charged functional groups of the stationary phase. The simplest way to do this is through increasing the ionic strength of the mobile phase, typically using an alkali metal salt.

1.3 Terminology and Definitions

Historically, most modes of liquid chromatography are named systematically according to the dominant nature of the adsorbate–adsorbent interaction. Exceptions to this rule have occurred where names have been adopted according to the stationary phase used, such as chiral phase chromatography (CPC) and immobilised artificial membrane chromatography (IAMC), or according to the kind of separated analytes, as in standard ion chromatography (IC), which actually can also be found described under the traditional rules as high-performance ion exchange chromatography (HPIEC). Where chelation in the stationary phase is the dominant mechanism providing for the retention and separation of ions, clearly the technique cannot be described using the term 'ion exchange', as this clearly contradicts the recommendations of IUPAC, who define ion exchange as the

equivalent exchange of ions between two and more ionised species located in different phases, at least one of which is an ion exchanger, without the formation of new types of chemical bonds.

For similar reasons, the use of the term 'adsorption-complexing chromatography' is also not well suited to describe the chelation based separation mechanism under consideration. One of the earliest papers, published in 1979 by Jezorek and Freiser, utilising a chelating stationary phase (silica immobilised 8-hydroxyquinoline) for the chromatographic separation of metal ions, described the new approach as metal-ion chelation chromatography (MICC). However, this term does not properly distinguish the technique from low efficiency classical chromatographic methods used for metal ion extraction using chelating resins and so was not adopted universally. Thus, with the lack of suitably accurate accepted terminology available, a new name for chromatographic methods utilising stationary phase chelation as the dominant retention mechanism for the efficient (high-performance) chromatography of metal ions was presented, based upon a combination of the above nomenclaturial traditions. The name high-performance chelation ion chromatography (HPCIC) places a correct emphasis on the exact adsorbate–adsorbent interaction and the nature of the adsorbate itself. The name also correctly describes the technique as specifically referring to high-performance applications of chelating phases and as a sub-discipline of the more widely applicable term ion chromatography, which now is accepted as a term describing any modern high-performance liquid chromatographic method for the separation of ions.

A similar term, complexation ion chromatography, was suggested by Time-rbaev and Bonn. However, according to them, this definition should include

all ion chromatographic modes in which complexation is exploited for the separation and detection of metals in different ways.

Obviously, in this interpretation complexation ion chromatography would cover too broad an area, including many with many different approaches in various fields of liquid chromatography, so it would be difficult to consider it as a unique separation technique.

A possibly confusing factor connected with the terminology prescribed above may have arisen from the emergence in the early 1990s of the so-called chelation ion chromatography (CIC) system from the Dionex Corporation and the associated trade name. The automated technique developed utilised switching valves within a complex ion chromatographic system, incorporating short chelating and ion exchange columns for on-line metal ion extraction and preconcentration prior to their separation on coupled ion exchange columns. Obviously, the correct description/terminology for this method should be simply 'ion chromatography of metal ions with on-line preconcentration of trace metals', as no actual separation of metal ions is achieved on the short chelating column. This example does however further justify the inclusion of the 'high-performance' preface to the name for the work described herein, to distinguish it from all other methods using chelating resins for simple preconcentration or extraction of metal ions (of which there is a considerable amount of literature references available, describing low-pressure columns packed with large particle low efficiency chelating resins, typically of 0.2–0.3 mm diameter).

1.4 Historical Developments

The basic principles of coordination chemistry were developed more than 120 years ago by the Swiss chemist Alfred Werner who was awarded the first Nobel Prize in chemistry. The term chelate was introduced in 1920 by Gilbert Morgan and Harry Drew who gave a very clear description for the specific type of complexes formed by polydentate ligands:

The adjective chelate, derived from the great claw or chela of the lobster or other crustaceans, is suggested for the caliper like groups, which function as two associating units and fasten to the central atom so as produced heterocyclic rings.

This description was strongly supported by later discoveries, such as the important 'rule of rings' described by Lev Chugaev, who found that chelates containing five- to six-membered rings are usually the most stable. These very early breakthroughs paved the way for the extensive exploitation of the extraordinary selectivity of the chelate effect in many varied applications of analytical chemistry, including chromatography.

One element of chelation based chromatographic methods which has historically received considerable attention is the nature of the chelating phase itself, and in particular the approach to the immobilisation of what are often relative complex structures (as compared to simple ion exchange functional groups). One of the first chromatographic techniques reported which utilised chelation as an element of the separation mechanism was given the name, precipitation chromatography, as described by Erlenmeyer and Dahn in 1939. In this very early work a micro-crystalline powder of a pure chelating ligand was used as the column packing, thus completely eliminating the need for any immobilisation strategies. The authors explored the differences in solubility of metal chelates formed at the surface of 8-hydroxyquinoline crystals as the basis for their separation. There is a well-defined correlation between solubility and stability constants of certain metal chelates, so this work could be considered as the first example of a form of chelation ion chromatography. The principles and further development of precipitation chromatography have been documented in an early monograph and a more recent review. However, clearly, the use of chromatographic columns packed with pure organic reagents as demonstrated by Erlenmeyer and Dahn was not the most cost effective or practical approach, so the use of various support materials, having developed chelating surface chemistries, including alumina, titania, silica, calcium carbonate, and various ion exchangers, became more popular phases in the technique, which continued into the late 1980s to be reported under the name 'precipitation chromatography'. The use of paper impregnated with organic reagents such as thiocyanates, 8-hydroxyquinolinol, dimethylglyoxime and others, quickly gained popularity in precipitation chromatography because of simplicity and improved sensitivity, resulting from distinctive colourful chromatographic bands (Figure 1.1).

Most precipitates produced during the separation of metals in precipitation chromatography are formed with complexing ligands which can themselves be retained to some extent on the support. With this in mind, a different separation mechanism known as adsorption–complexing chromatography was suggested in 1954 by Gurvich and Gapon. In adsorption–complexing chromatography, the adsorbent retains both complexing reagent and its associated metal complexes, such that the difference in stability of these metal complexes was used as the basis for their separation, and hence the separation of the metals themselves. Technically the proposed method is very reminiscent of precipitation chromatography, but here no new solid phase (precipitate) is formed. However, it should be noted that the complexing stationary phase was often prepared by adsorption or immobilisation of complexing reagents on the surface of the support material prior to separation. This extremely useful approach is still widely used today for the separation or isolation of specific metals, particularly if the selective reagent itself cannot be easily covalently attached to the surface of the support.

The slow (or in some cases rapid) bleeding of the chelating ligand from the column is a significant drawback of adsorption–complexing chromatography, such that the idea of using partition chromatography with two immiscible liquid phases was developed as an alternative approach. Extraction chromatography, based on this principle, received quick recognition following its first description by Siekierski and Fidelis, detailing the separation of lanthanides (Figure 1.2) in 1960. (Also see Dietz et al.) In extraction chromatography, a hydrophobic reagent, often a bulky organic chelating ligand, is strongly retained in the immobilised liquid stationary phase, providing the desired separation selectivity. The technique is still regularly reported for application in the isolation and separation of radionuclides and heavy metals. A similar mechanism exists in countercurrent chromatography (CCC) when applied to metal ion separations. As a rule, both of these separation techniques explore chelation in the liquid stationary phase, in a similar fashion to multi-step liquid–liquid extraction, to achieve selective separations from complex mixtures of metal ions. Of course, the large volume of experimental data obtained on extraction of metal ions with various organic reagents provided a good basis for the fast development and application of both extraction chromatography and CCC to the separation of metal ions.