10
November/December 2009
Enabling facile, rapid and successful chromatographic Flash purification
Stéphane Dubant and Ben Mathews* Chemical Research & Development, Pfizer Global Research & Development, Sandwich, Kent, CT13 9NJ, UK. *Corresponding Author
Flash liquid chromatography (Flash-LC) is an established technique that can have a very positive impact on productivity in the pharmaceutical research and development chemistry laboratory - provided it is used efficiently. Finding the optimal conditions for a normal phase or reverse phase Flash-LC separation can be a laborious process. The aim of this work was to develop a screening system to enable automated, rapid and reliable method development for preparative Flash chromatography. Implementation of these method screening systems into the chemists workflow has had beneficial productivity and quality impacts at different stages of drug development, and on scales ranging from laboratory to pilot plant.
Key words: Flash chromatography, automated method development, normal phase, reverse phase.
Introduction Since first being introduced in 1978 by Still et al.1
, Flash chromatography, a medium
performance liquid chromatographic (MPLC) purification technique, has evolved considerably, and has become a common tool for synthetic chemists in their everyday work. This is particularly true in modern industrial environments such as the pharmaceutical industry where tight timelines demand high productivity and quality. Flash- LC is a purification method of choice for large scale preparative separations and is used routinely on a laboratory scale (g-kg) and pilot plant scale (10’s of kg). Advances made by instrument manufacturers have given access to reliable automated Flash-LC purification hardware for the laboratory and dependable pre-packed columns. Whilst most chemists are familiar with this technique, it is appropriate method development that remains the main hurdle in making the best use of it. Developing fit for purpose methods for routine laboratory work can be time consuming. Developing an optimised method to efficiently execute the more demanding applications of Flash-LC within the pharmaceutical industry, such as purification of materials to be used in non- clinical or clinical trials to tight purity requirements, can be particularly testing for those not skilled in the art.
Flash-LC employs particles with dimensions on order of 50 µm which give relatively low column efficiencies (N) compared with other LC techniques such as the ubiquitous high performance liquid chromatography (HPLC).
Column efficiency cannot be relied upon in order to achieve a meaningful preparative Flash-LC separation. Instead a high selectivity (alpha) is required to achieve the resolution required to translate a Flash chromatographic method into pure product. The optimisation of selectivity is a key factor for successful method development for preparative applications. One very practical and effective means to affect selectivity for normal phase (NP) Flash-LC is by careful solvent choice - selecting solvent systems composed from a combination of solvents from the eight NP solvent selectivity families (based on Snyder’s et al.2
and Glajch et al.3
solvent selectivity descriptors). For reversed phase (RP) Flash-LC the selectivity may be most affected by organic modifier choice or pH. Conventionally the method development process involves screening solvent combinations using TLC to determine the best solvent(s) and relative proportions in order to achieve the required separation. However, in order to achieve the required selectivity it is often necessary to use binary, ternary, or sometimes even quaternary solvent systems composed of a combination of solvents from the eight NP solvent selectivity families. Hence, even a routine method can involve a vast array of solvent combinations and proportions. Once the selectivity effects are known, other factors such as the chromatographic band shape, solubility, stability, solvent cost and environmental issues can be considered. The method development process can be arduous and the consistency and quality of
the optimised method is dependant on the skill set of the individual.
The key to enabling facile, rapid and successful Flash-LC purifications is to be able to quickly identify the best conditions to maximise selectivity for a given separation problem using minimal effort and achieving consistent results independent of a chemist’s experience with the technique. The aim was to deliver method development systems to rapidly and easily develop a method for normal or reverse phase Flash chromatography. The method screening systems would be implemented into intuitive and integrated purification workflows in order to encourage and enable the use of robust and successful purifications.
Experimental
All solvents used in this work are HPLC grade from Sigma-Aldrich (Gillingham, UK). The screening instruments were Agilent (Stockport, UK) 1100 HPLC systems (quaternary pumps) equipped with a solvent selection valve to enable screening of more solvents on the normal phase system. Detection was performed with a diode array UV/Vis detector. The evaporative light scattering detector used with the normal phase screening system was an ELS 1000 from PolymerLabs (Church Stretton, UK). The instruments were controlled using Chemstation Rev. B.03.01 and Easy-Access Rev. A.05.01 software. All Flash chromatography experiments were performed on a Biotage (Uppsala, Sweden) Isolera One equipped with a variable wavelength UV detector and two collections beds.
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