Paul R. Haddad
Australian Centre for Research on Separation Science, School of Chemistry, University
of Tasmania, GPO Box 252-75, Hobart 7001 (Australia)
Research conducted at the University of Tasmania involves the general field of separation science applied to the separation and quantification of ionic species. Studies have been undertaken using high performance liquid chromatography (HPLC), ion chromatography (IC), capillary electrophoresis (CE) and capillary electrochromatography (CEC). With each technique, the general aim of the research has been to study separation mechanisms and methods of detection, with a view to improving fundamental understanding of these aspects and to apply this understanding to the development of new chromatographic and electrophoretic methods of analysis. For the study of separation mechanisms, the approach has been to make detailed measurements of the retention or migration of analytes in the desired system, to derive mathematical models which describe these observations, and to use these models to devise strategies for the computer-assisted optimization of separations for particular applications. A recurring theme has been the investigation and manipulation of separation selectivity. In the study of detection methods, emphasis has been placed on potentiometric detection using reactive metallic electrodes and the theory and application of indirect methods of spectrophotometric detection. The separation of complexed metal ions and sample handling methods have comprised further major research themes.
The research programs can be divided broadly into chromatographic methods and electroseparation methods. These are discussed separately below and some illustrative examples of recent projects in each area are given.
The separation of inorganic anions by IC has been studied extensively. The first aspect of this
work has involved the investigation of fundamental separation mechanisms in ion-exchange, ion-exclusion and ion-pair systems, with a view to designing software for computer-assisted selection of optimal separation conditions. Mathematical retention models capable of accurate prediction of retention behaviour in eluents containing two competing ions have been formulated  and alternative modelling approaches using expert systems and artificial neural networks have been investigated. These studies have led to the development of a comprehensive computer program for simulation and optimization of IC separations . This program, called "Virtual Column 2" allows the selection of the best columns and eluents for the separation of a desired group of analytes. In addition, many parameters can be varied (such as the eluent composition, the relative concentrations of analytes in the sample, the degree of tailing exhibited by each peak, etc) and the resultant chromatogram can then be simulated. Figure 1 shows the optimized separation of 12 analytes on a Dionex AS4A column using a carbonate-bicarbonate eluent. The top box in the figure is a contour map showing the quality of all possible separations, with the darkest areas representing the best separations. The chromatogram in the bottom box is the optimized separation. A version of this software suitable for teaching the principles of IC is available free of charge at www.virtualcolumn.com.
Fig. 1. Output from Virtual Column 2 software showing contour map of the quality of all
possible chromatograms (top) and the optimized separation of 12 analytes (bottom).
Other studies in IC are concerned with new detection modes, especially potentiometric detection using solid metallic electrodes (particularly copper) and indirect spectrophotometric detection. In the former approach, metallic copper is permitted to establish a diffusion layer of copper ions (both copper(I) and copper(II)) at the electrode surface and therefore a Nernstian potential. Solutes exerting any change to the concentration of copper ions in this layer will produce a change in potential, which can then be used as the basis for detection . The latter approach allows the use of conventional HPLC instrumentation for IC separations.
Studies on methods for the separation of metal complexes have comprised a continuing theme of research which has involved both fundamental and applied studies. Emphasis has been on the separation of dithiocarbamates and 8-hydroxyquinolinates, metallo-cyanides, Th(IV) and U(VI), Nb(V) and Ta(V), and the use of chelation IC. A strong focus has been applied to the separation of metallo-cyanide complexes because of the importance of these species in the extraction of gold from its ores. Methods for monitoring of metallo-cyanides, free cyanide and cyanide degradation products (cyanate and thiocyanate) have been developed and have been used on operating gold mines . The methodology allows the complete speciation of cyanide and its degradation products in both process liquors and tailings dams, which permits cyanide consumption and release to be minimised. Combined with an automated on-line preconcentration method, the technology can be used for speciation of cyanide at ultra-trace levels in environmental samples so that the forms in which cyanide is distributed in an environmental sample can be specified. Figure 2 shows the output from the HPLC. The chromatogram on the left is from a UV detector and shows UV-absorbing species (metallo-cyanides and thiocyanate) in the sample. The middle chromatogram is an enlarged view of the gold peak, whilst the chromatogram on the right is measured in the visible region after a post-column reaction to visualise further analytes, especially free cyanide.
Fig. 2 Separation of metallo-cyanides, free cyanide and thiocyanate .
Electroseparation methods (capillary electrophoresis [CE] and capillary electro-chromatography [CEC]) have been examined as separation tools for the same group of analytes studied in IC. Again, particular attention has been paid to modelling the separation processes and the development of optimization software to assist the design of separation methods. Another strong theme to this work has been the study of separation selectivity (i.e., the order in which analytes appear at the detector). In CE, parameters which have been examined have included the composition and pH of the carrier electrolyte, the use of surfactant mixtures, added organic solvents and the manner in which reagents are prepared. These studies provide a sound basis for design of a separation having a desired selectivity.
Research undertaken in IC and CE has laid the foundation for an intensive new research program in CEC using ion-exchange stationary phases. The objective has been to develop CEC separation systems for ionic analytes which show separation selectivity that can be manipulated systematically between the extremes of ion-exchange and electrophoresis. To this end, an ion-exchange stationary phase (or pseudo-stationary phase) has been introduced into a CE separation as packed particles , a suspension of particles added to the electrolyte , an adsorbed layer of nanometre-sized particles on the capillary wall , or a soluble ion-exchange polymer added to the electrolyte . These approaches have been developed theoretically and migration behaviour has been modelled mathematically and optimized. All of the above approaches have proven successful, but the most promising results have been obtained using wall-coated systems or with the use of soluble polymers. These studies show that viable separation systems can be developed in which peaks for desired analytes can be moved throughout the chromatogram in order to eliminate interferences, to speed up the separation, or to improve resolution. The methodology developed in this work represents a simple means for achievement of a desired separation selectivity.
A further advantage of the wall-coated approach is the ability to perform an on-line enrichment of a sample during the CEC analysis. The basis of this method is to form a preconcentration zone in the capillary (approx 10 cm in length) comprising of a layer of 75 nm anion-exchange functionalised latex stationary phase particles adsorbed electrostatically to the capillary wall. The separation zone comprises the remainder of the capillary, which is not functionalised. Pressure-assisted injection of several capillary volumes of the sample results in the adsorption of anionic analytes onto the ion-exchange stationary phase and preconcentration of the sample (Fig. 3(a)). The capillary is then filled from the outlet end with a electrolyte have weak ion-exchange properties (Fig. 3(b)). In the next step, the electrolyte vials at each end of the capillary are filled with an electrolyte that contains a strong ion-exchange competing ion. On applying the separation voltage this electrolyte moves into the capillary and elutes the analytes from the preconcentration zone (Fig. 3(c)). These analytes are then separated by CE in the remainder of the capillary (Fig. 3(d)). Preconcentration factors of several thousand have been achieved using this method and the technique has been applied to the determination of anions in Antarctic ice cores, for which a sample volume of only 50 ?L is required for a duplicate analysis.
Fig. 3. Steps involved in on-line preconcentration in CEC
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8. M.C. Breadmore, P.R. Haddad and J.S. Fritz. Electrophoresis, 21 (2000) 3181-3190.
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