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The need to develop and retain practitioners skilled in the use of "classical" 20th century analytical tools was a common theme highlighted by speakers at the symposium, as was the broad applicability of analytical skills that might be derived from mastering such classical techniques. Further benefits of many classical techniques included the flexibility to deal with "atypical" samples and the ability to complement the information derived from mainstream "modern" analytical techniques.
Don't rush to crushMr GARY NICHOLS (Pfizer Central Research, Sandwich) reflected that analytical chemists had a tendency to crush or dissolve their samples. But examining the sample first before taking such actions had many potential benefits for pharmaceutical research and development. Mr Nichols went on to give examples of microscopy that illustrated how the "humble" light microscope was a powerful tool when in the hands of a skilled practitioner.
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New Technologies Forum: challenging the status quo
The New Technologies Forum recently established by the Royal Pharmaceutical Society played a key role in accelerating mutual understanding of new technologies between the pharmaceutical industry and regulatory bodies, the symposium heard.
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The strengths and weaknesses of classical "wet" chemistry for pharmaceutical analysis were characterised by Mr RON ROONEY (Rooney Labs). He presented examples to highlight that, in some circumstances, the precision of results obtained from volumetric and gravimetric techniques could be better than those from instrumental techniques such as atomic absorption spectroscopy. However, pharmacopoeial methods often lacked the flexibility that would allow classical approaches to be used as alternatives to instrumental methods. Typically, sample sizes were prescriptively defined without consideration for the implications of having inappropriately small reaction volumes when titrating manually.
Mr Rooney also stated that one of the most valuable aspects of classical methods was that they taught real-world chemistry. In his experience, analysts who had been trained in good volumetric and gravimetric techniques were able to turn their hand to virtually any method with good results, whereas the reverse was not necessarily true.
A further advantage of the classical approach was the reduced reliance upon certified reference materials.
The disadvantages of classical methods were generally better understood than the advantages. Common limitations were that they lacked specificity, were not stability indicating and required lengthy, skilful sample preparation. Hence it was considered that, in the modern analytical laboratory, classical analytical techniques could provide one cornerstone in the strategy to be able to respond to the challenges of non-routine analyses.
Thin layer electrochromatography (TLE) was presented as an analytical tool for the future, based on the intrinsic advantages of the established technique of thin layer chromatography (TLC), Dr ALAN HOWARD (University of Southampton) told the meeting. Typically, TLC had the capability to retain and reveal all components, it was relatively cheap and fast to perform, and allowed the simultaneous analysis of up to 10 samples. These strengths could be enhanced by adding the influence of both electrophoretic and electro-osmotic forces.
A major advantage of TLE was the speed with which an analysis could be performed. Electro-osmotic induced solvent flow resulted in separation characteristics that were similar to those obtained by TLC, but five to 10 times faster. For example, while conventional TLC of compounds related to pirimicarb took about 18 minutes to complete, the same elution sequence was obtained by TLE in 90 seconds.
Planar electrophoresis was widely used by the biochemistry disciplines, but not in the analytical chemistry laboratory. This might be attributed, in part, to the frequent need for non-aqueous solvents to affect dissolution. It was possible, however, to achieve acceptable ionisation of solutes in non-aqueous media such as ethanol and acetonitrile. Several examples were presented to highlight the use of electrophoretic solute migration to influence the selectivity and elution order of chromatographic separations.
Dr Howard noted that it was possible to achieve robust TLC separations in both horizontal and vertical modes; the significant evaporation and drainage effects observed in TLE required that all separations be performed using horizontal plates. Commenting on future possibilities for TLE, he predicted the use of two-dimensional separations combining TLC with TLE.
Opportunities for applying chiroptical analysis within the pharmaceutical industry were described by Dr ALEX DRAKE (King's College, London), who discussed the technique's applications in chirality, in biomacromolecular conformation and interactions and in pharmaceutics.
Dr Drake said that optical activity was the result of the differential interaction of left and right circularly polarised light with a chiral molecule. This resulted in differential absorption (circular dichroism) and differential speed (optical rotation).
Focusing on circular dichroism (CD), Dr Drake commented that, while it was relatively easy to collect CD spectra (eg, on-line as a high performance liquid chromatography detector), the interpretation of the data generated required a high level of expertise. Chemical groups around the chromophore significantly influenced the CD spectra generated. From theoretical considerations and experimental observations it had been possible to classify chiral drugs into four groups so far as optical activity was concerned. Understanding the relationship between spectroscopic moments and the molecular environment could thus aid the elucidation of absolute stereochemistry and R,S nomenclature.
In biotechnology, CD spectroscopy was an essential tool to monitor biopolymer conformation (secondary structure). Often, CD was the only spectroscopic (and physical) technique to distinguish between active and non-active proteins. Dr Drake used ricin as an example to show that subtle changes to disulphide components of the molecule caused a loss in biological activity, but that these changes did not alter the secondary structure. Chiroptical spectroscopy was able to detect such changes, particularly in aqueous solution, which emphasised the potential value of this technique to the rapidly expanding biotechnology sector of pharmaceutical analysis.
As an example of the role of CD spectroscopy in pharmaceutics, Dr Drake highlighted how the problem of gel formation of calcitonin hormone formulations had been tracked using this technique. Similarly, given the sensitivity to conformation and the molecule's environment, circular dichroism was a key tool for understanding factors such as the ability of a protein or an excipient to bind drugs.
The current technology and future challenges for capillary electrochromatography were described by Dr PAUL FERGUSON (formerly at Imperial College, London, and now at Pfizer Central Research, Sandwich), who said that the emerging value of this technique was based on the bringing together of key attributes associated with both liquid chromatography and capillary electrophoresis.
The ability to separate neutral compounds via partitioning between the mobile and stationary phases was complemented by electrophoretic flow to separate charged species. Moreover, the use of electro-osmotic rather than hydraulic flow of the mobile phase promoted much improved plate counts, and thus sharper peak shapes.
In addition to outlining how various parameters could be employed to optimise a CEC method, Dr Ferguson commented on a "focusing effect" that had been achieved with some basic compounds. Incredible peak efficiencies of between six and eight million plates per metre had been observed when analytes were apparently "focused" by the combination of forces employed. To date, detailed investigations had been unable to generate a clear understanding of how such an effect was generated, and why the effect was neither rugged nor readily reproduced.
Presenting future directions for the development of CEC, Dr Ferguson said that macroporous particles and polymer monoliths had recently been used to expand the types of stationary phases used for achiral molecules. In addition, chiral compounds had been successfully resolved by highly selective chiral phases, such as those created from molecular imprinted polymers. These examples highlighted an area of ongoing investigation.
It was also postulated that column frits were a contributory factor to the poor peak shape observed for some analytes. Hence fritless columns were currently being evaluated.
Finally, looking ahead to the final topic of the meeting, Dr Ferguson described the possibility of performing CEC on a polystyrene derivatised chip and presented experimental data.
The final emerging technology for the 21st century discussed at the meeting was that of miniaturised chemical analysis, the so called "laboratory-on-a-chip". Dr ANDREW DE MELLO (Imperial College, London) presented the development towards miniaturised total analysis systems as a marriage between the fields of molecular chemistry and microelectronics.
The advantages of miniaturisation were summarised as a combination of superior analytical performance, enhanced integration/automation with microelectronics, and higher throughput. Moreover, as noted with the established technique of microscopy, small scale systems only required small samples. Portability was also as an attribute likely to grow in importance, enabling the laboratory to go to the sample.
Superior analytical performance was possible through a combination of fast separations (due to the physical dimensions) and use of high voltages for electrophoretic separations (due to excellent heat dissipation within microchips). Dr de Mello presented examples of a two-component separation achieved within 700 microseconds.
To complete the work towards miniaturised total analysis systems it was necessary to combine separation modules with those able to perform fast chemical reactions. Dr de Mello thus highlighted some of the current challenges being addressed to undertake classical chemistry reactions on chips. One example was a lack of turbulent flow, and there was a need to design reactors based on diffusion within laminar flow to mix reagents. - Contributed.