Supercritical fluid chromatography

Abstract

In the last decade, supercritical fluid chromatography (SFC) has emerged as a complementary technique to high performance liquid chromatography (HPLC) and gas chromatography (GC). Initially reported as high-pressure GC in the 1960s, it hibernated for many years due to technical challenges. Finally, with the latest generation of instrumentation, robust conditions have been achieved that deliver reliable solutions in analytical chemistry. In SFC, supercritical fluids are used as the mobile phase, with almost exclusive use of carbon dioxide under supercritical conditions (scCO₂). Like HPLC, modern SFC-based methods complement the scCO₂ mobile phase with organic co-solvents as modifiers to adapt the elution strength. Initially considered to be similar to normal-phase HPLC, the use of modifiers has drastically broadened the applicability of the technique.

Due to the lesser viscosity and greater diffusivity of supercritical fluids compared with liquid solvents, fast and efficient separations can be achieved even for challenging target analytical separations such as enantiomer analysis, but is applicable also beyond chiral separations.

The Green Chemistry Group has organised scientific conferences on SFC now for several years to provide a platform for networking between scientists and to promote fruitful exchange between groups working in this field. Additionally, a strong focus is given to young scientists interested in separation science, with preconference workshops on SFC fundamentals.

In 2023, diverse topics from pharmaceutical and bioanalytical fields have been covered at this conference, and the evaluation of the applicability of SFC played a significant role, especially in comparison with HPLC-based methods.

This special issue covers manuscripts out of both fields. Schmidt et al. report the development of an SFC-UV-based method for purity assessment of active pharmaceutical ingredients using quality-by-design principles is exemplified by the active pharmaceutical ingredient carbamazepine.¹ Method development was discussed in the light of regulatory compliance, which is of paramount importance in pharmaceutical analysis. Especially with the recently published guidelines of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) and the general chapter of the US Pharmacopeia (USP) on life cycle management, Analytical Quality-by-Design (AQbD) considerations are increasingly considered in method development. In addition to achieve robust and reliable analytical methods, AQbD helps to increase method understanding significantly. Sources of variability have been identified and are classified as critical quality attributes, which need strict control within the identified limits. Additionally, continuous method improvement (if necessary) is facilitated under the umbrella of AQbD, including the integration of potentially new target analytes. A comparison with the currently monographed purity method for carbamazepine in the European Pharmacopoeia (Ph. Eur.) found a drastic reduction of the turn-around times for sample analysis with a reduction in time factor >20.

The second application we cite in this issue is a method from Jambo and co-workers for impurity profiling of vitamin D3 in drug products.² In this case, mass spectrometric detection was used in combination with SFC, specifically a single quadrupole mass spectrometer. A design-of-experiments (DOE) approach was chosen for method development. In addition to the optimisation of chromatographic separation, DOE was also performed for the MS conditions.

In most of the recently used designs in SFC-MS, hyphenation offers the possibility of separate optimisation of chromatographic conditions and analyte ionisation, the latter aided using an additional make-up flow solvent. This use of hyphenation is also applied in the bioanalytical methods reported by Hansen et al. and by Bredendiek and Parr in this special issue.^{4, 3} Two manuscripts focus on the analysis of isomeric drugs or drug metabolites, respectively. Even if combined with MS/MS detection, the target analytes must be chromatographically separated to allow their separate determination, as ion transitions do not allow for their discrimination. SFC-based analysis enabled the required selectivity for both analytical tasks. In both manuscripts, chiral stationary phases are used to achieve separation of all target analytes. The applicability of SFC-MS/MS for pharmacokinetic investigations is thereby proven.

Finally, Gavrilovic et al. discuss the routine applicability of SFC-MS/MS was investigated for multiple analyte methods used for wide screening for prohibited substances intended for use in doping control laboratories.⁵ Up to ~200 target analytes were amenable in a single analytical run with turn-around times of less than 20 min. Robust determination of analytes of a broad range of polarities was possible using a mobile phase gradient from minimal to >50% of modifier added to the scCO₂. Covering large modifier amounts certainly results in nonsupercritical state of the total mobile phase in the pure physicochemical sense but nicely shows the flexibility of modern SFC systems.

Overall, SFC is reported as a sustainable, efficient and versatile alternative to traditional chromatographic methods. Its orthogonality in separation may help to overcome limitations observed by other chromatographic methods. More laboratories are advised to evaluate this innovative approach for advanced pharmaceutical and biological research and development.