Addressing the Need for Faster Screening and Fraction Collection for Chiral and Achiral SFC

Introduction

Supercritical Fluid Chromatography (SFC) has become a well established technique in modern laboratories and is the preferred technique [1, 2] for both analytical and preparative enantiomeric separations for many reasons, including its speed and resolution advantages over normal phase HPLC. In addition to chiral applications, there has been steady growth over the past decade in the use of SFC for achiral analysis in numerous industries [2]. The continued expansion of applications employing SFC has led to expansive achiral column development and the optimisation of hardware platforms to enable more rigorous screening of  solvent and column combinations for analytical SFC.
In reversed-phase HPLC the C18 column is so widely used it is often identified as the universal column [3]. In SFC there is a lack of universal or ‘go to’ column and at least 70 columns to choose [4]. Therefore, chiral and achiral SFC analysis requires column screening to narrow down the many available columns to determine the best column to achieve the desired resolution of the peaks of interest. The growing number of possible solvent [2]:column [4] combinations available today, combined with the increase in the number of samples requiring analysis, means higher throughput is required to keep pace with the demand. When column screening is performed to determine the selection of the most efficienct chromatographic condition to scale up, time is of the essence and the answer is desired as rapidly as possible.
The rapid analytical SFC screening provides the solvent and column combination for direct scale up to purification of the target analytes. Preparative SFC enables rapid purifications, using minimal solvents, along with easy fraction recovery due to the physical properties of the CO₂ enabling that portion of the mobile phase to convert to the gaseous state upon exiting the column and encountering pressure less than its critical pressure. In enantiomeric purifications, only two fractions are needed to collect each of the enantiomers. The use of SFC for achiral purification requires an increase in the number of fraction vessels available for use which can be accomplished through utilisation of an open-bed fraction collector. The use of such an open-bed fraction collector allows for greater capacity which ensures selection of fractions to satisfy the user’s desired purity or recover criteria.

Method and Materials

Flavanone, ketoprofen, sulphamethazine, methanol, ethanol, acetonitrile and tetrahydrofuran were purchased from Sigma-Aldrich (St. Louis, MO, USA). The test compounds flavanone, ketoprofen and sulphamethazine were identified by a pharmaceutical customer as the ones they use for internal chiral and achiral SFC system testing. The CO₂ cylinder was purchased from Airgas (PA, USA). Columns were acquired from multiple suppliers: Princeton Chromatography (Princeton, NJ, USA), Phenomenex (Torrance, CA, USA) and Chiral Technologies (PA, USA). The Jasco Analytical SFC (CO₂ pump, co-solvent pump, automsampler, column oven, PDA detector and back pressure regulator) and Parallel SFC system (CO2 pump, co-solvent pump, solvent selector valve, autosampler, column oven, column selection valves, 4 UV detectors and back pressure regulator) were used for the analytical screening and development. The parallel SFC injects the sample and splits the sample and flow to 4 columns simultaneously as shown in Figure 1 with the screening capacity of 20 columns and 10 solvents. The Jasco Open-Bed Preparative SFC (CO2 pump, co-solvent pump, automsampler, column oven, UV detector, back pressure regulator and open-bed fraction collector) was used for the preparative purifications.

Conclusions

Parallel screening of 4 columns offers a 4-fold reduction in screening time in SFC compared to traditional single column screening. This time savings can provide faster turn-around time to move to scale up on preparative or allows more columns and solvents to be screened when the initial do not provide the resolution. With more achiral samples being analysed on SFC, increased fraction capacity of an open-bed fraction collector provides collection of the principle compounds as well as potential impurities. The ability to automate the purification of multiple chiral and achiral samples with immediate sample dry down is critical to increasing sample throughput. As the number of potential drug compounds each year increases, faster sample processing is required to keep up with the modern pharmaceutical SFC lab.