(W1130-02-12) Sustainable Separation: Supercritical Fluid Chromatography as a Modern Replacement for Normal Phase Chromatography

Purpose: Despite significant advancements in chromatographic techniques, normal phase chromatography (NPC), the progenitor of modern chromatography, persists in current pharmacopeial and standardized analytical methodologies. This continued reliance on NPC necessitates the use of volatile organic compounds (VOCs) such as hexane and ethyl acetate. These solvents present considerable occupational health and safety hazards, contribute to environmental pollution, and incur substantial financial burdens associated with procurement and regulated disposal. Using Cholecalciferol as a representative molecule, this study investigates the superior efficacy and environmental benefits of using Supercritical Fluid Chromatography (SFC) as a contemporary, greener alternative to conventional NPC.

Methods: The current USP monograph for Cholecalciferol employs traditional normal phase conditions, such as 0.3% amyl alcohol in hexane as the mobile phase. An experimental SFC method was developed to transition toward more environmentally friendly solvents. Given that supercritical CO₂ (ScCO₂) has a polarity similar to hexane, it was selected as a greener alternative, used in combination with methanol as the modifier to separate Cholecalciferol and its derivatives under isocratic conditions. The sample analyzed was the USP Vitamin D Assay System Suitability Reference Standard, which contains Cholecalciferol, pre-Cholecalciferol, and trans-Cholecalciferol dissolved in peanut oil. This standard was prepared at a concentration of 25 mg/mL in a 1:1 mixture of toluene and hexane. The analytical SFC method was designed to run isocratically with 1% methanol for 4 minutes to achieve separation of the Cholecalciferol components, followed by a ramp to 50% methanol at a flow rate of 1.2 mL/min for 4 minutes to flush the peanut oil diluent from the stationary phase. These conditions were successfully applied across different SFC instruments from various vendors, with only minor modifications—such as reducing the methanol concentration to 0.5% and the injection volume from 2μL to 1μL—to maintain a comparable separation profile (Table 1).

Results: SFC separation of Cholecalciferol and its derivatives yielded highly efficient and reproducible results. Under SFC conditions, clear resolution was achieved between pre-Cholecalciferol, trans-Cholecalciferol, and Cholecalciferol, with significantly reduced retention times compared to traditional normal phase chromatography. In the normal phase method using 0.3% amyl alcohol in hexane, the total run time was approximately 60 minutes, with the main Cholecalciferol peak eluting at 28 minutes (Figure 1). In contrast, the SFC method reduced the total run time to just 12 minutes, an 80% decrease, while achieving comparable or superior separation. The Cholecalciferol peak eluted at 2.26 minutes and 3.35 minutes under SFC conditions (Figure 2A), demonstrating a substantial improvement in throughput. Moreover, the method proved robust across different SFC platforms from various vendors. Minor adjustments, such as reducing the methanol modifier concentration from 1% to 0.5% and decreasing the injection volume from 2 μL to 1 μL, were sufficient to maintain consistent separation profiles and retention times (Figure 2B). This cross-platform consistency underscores SFC instrumentations adaptability and potential for broader implementation.

Conclusion: This study definitively showcases the substantial benefits of Supercritical Fluid Chromatography (SFC) over traditional normal phase chromatography (NPC), exemplified by the effective separation of Cholecalciferol and its derivatives. By substituting hazardous solvents like hexane with a sustainable supercritical CO₂/methanol mobile phase, SFC offers an environmentally friendly and highly efficient approach, reducing run times by 80% without compromising analytical performance. The method's robust reproducibility across various vendor platforms further highlights its adaptability. Beyond these advantages, SFC enables cost-effective solvent management and easy integration with universal detectors for non-UV active compounds, and for experienced users, training is straightforward. These findings offer compelling evidence for the widespread adoption of Supercritical Fluid Chromatography (SFC) as a modern, environmentally conscious alternative in pharmaceutical and analytical laboratories. While opportunities exist to enhance SFC-UV sensitivity for trace impurities and streamline interlaboratory transfers, SFC's overarching sustainability, efficiency, and robustness underscore its transformative potential. Addressing integration into Quality Control (QC) laboratories and fostering sponsored method development are crucial next steps to fully realize SFC's capabilities and establish it as the gold standard for future chromatography.

References: 1. United States Pharmacopeia and National Formulary (USP-NF). Cholecalciferol. In: USP-NF Online. Rockville, MD: United States Pharmacopeial Convention. Updated September 20, 2024. Accessed September 20, 2024. https://doi.usp.org/USPNF/USPNF_M17120_03_01

Acknowledgements: Disclaimer: “Certain commercial equipment, instruments or materials may be identified in this abstract/poster to specify adequately the experimental procedure. Such identification does not imply approval, endorsement, or certification by USP of a particular brand or product, nor does it imply that the equipment, instrument, or material is necessarily the best available for the purpose or that any other brand or product was judged to be unsatisfactory or inadequate."