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10 August / September 2016


Peak Distortions in Preparative Supercritical Fluid Chromatography – a More Complete Overview


Torgny Fornstedt


Department of Engineering and Chemical Sciences, Karlstad University, SE-651 88 Karlstad, Sweden Torgny.Fornstedt@kau.se + 46 73 271 28 90 (www.separationscience.se)


Supercritical Fluid Chromatography (SFC) has re-emerged as a first-rate technique for purification of candidate drugs in the pharmaceutical industry; fuelled by new and improved instrumentation and a rapidly growing interest in the scientific community. SFC is considered much more complex and difficult than HPLC by new users but that is largely compensated for by strong advantages such as lower environmental impact and the much shorter separation times and thus larger production rates. However, there are remaining challenges and difficulties with packed column SFC, the most studied are those resulting from the compressibility of the mobile phase, leading to the concept that SFC is a ‘rubber variant’ of HPLC, where everything considered constant in HPLC, is not in SFC. In this article we will discuss and review, with new and review materials another challenge not often addressed in SFC - the fact that sample components cannot be dissolved in the mobile phase, but have to be dissolved in a solvent, and what type of peak distortions that may generate.


Introduction


Supercritical Fluid Chromatography (SFC) in the Preparative mode (Prep-SFC) is a most environmentally friendly purification method [1]. Instead of using harmful solvents as in Preparative Liquid Chromatography (Prep- LC), environmentally friendly supercritical fluids, such as carbon dioxide which already exist in the bio-cycle, and is used in SFC. However, in SFC we have not reached the level of expertise and knowledge achieved in the area of nonlinear separation theory in LC. There are many issues encountered with preparative packed column SFC we still do not fully understand, or that we simply apply Prep-LC knowledge to.


The main reason why Prep-SFC is more complex than Prep-LC is due to the increased compressibility of the mobile phase. It can be said that SFC is a ‘rubber variant’ of LC where everything considered constant in LC is not constant in SFC [1-5]. This ultimately results in radial and axial density and temperature gradients in the column that affect the thermodynamics of adsorption and cause a volumetric flow rate gradient through the column [1-5]. Therefore, the ‘set’ operational conditions do not necessary reflect the correct ‘real’ conditions experienced in practice. The differences of certain parameters such as external operational and column conditions such as column temperature, pressure, flow, density, eluent composition external to the column are irrelevant while other conditions related to the internal column conditions are


of utmost importance [2,3]. Fundamental studies therefore require the use of external sensors for pressure, temperature and mass flow, especially if the studies are aimed at reliable method transfer and scale up from analytical to preparative scale.


A recent study on the dependences of the adsorption on the mobile phase composition were investigated using a chemometric Design of Experiments (DoE) approach [3]. Using this approach, we were able to study the combined effect of temperature, pressure and co-solvent fraction in analytical and preparative SFC. More specifically, by using DoE, careful measurements of the experimental conditions and properly selected racemic model compounds, we could investigate how productivity, selectivity and retention in chiral SFC depended on pressure, temperature and co-solvent fraction [3]. Among others we found that the productivity for preparative SFC was most influenced by the co-solvent fraction and the column temperature, where high co-solvent fraction and temperature gave maximum productivity in the studied design space [3]. For reliable computer-assisted optimisation of a preparative SFC units’ production, a reliable method for the determination of adsorption isotherms in SFC are required along with proper mathematical models for the SFC experiments. Some recent work applying well-known LC adsorption isotherm determination methods to SFC found that


the methods that worked best for SFC were often the worst ones for LC [4,5].


A recent investigation on performing reliable method transfer from analytical pilot scale to large scale SFC purification was also performed [6] where we came across the fact that most analytical instruments are volume-controlled while most preparative scale units are mass-controlled. This problem was solved by measuring the mass flow, the pressure and the temperature on the analytical unit using external sensors. The analytical scale SFC experiments were performed in our research laboratory and then we used these experiments to calculate the correct operational conditions for scale- up. Our calculations were verified using a large scale process unit at AstraZeneca R&D Mölndal, Sweden and the results from the large scale unit agreed well with those from our analytical unit [6].


It was mentioned above that the compressibility of the mobile phase is a main reason for SFC being more complex than LC but there is another important problem, resulting in complex behaviour, in SFC due to the fact that we cannot easily dissolve the solute component in a carbon dioxide (CO2


) based mobile phase, only in the co- solvent. This has the consequence that the sample solvent introduced at the top of the column will have a composition that deviates strongly from that of the mobile phase. The fact that the sample cannot be readily dissolved in the mobile phase in SFC and


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