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...