Supercritical Fluid Chromatography: Advances, Applications, and Sustainability

Three leading experts in supercritical fluid chromatography (SFC)—John Reilly (Novartis, Basel, Switzerland), Maria Kristina Parr (Freie Universität Berlin, Germany), and Larry Miller (Novartis, Cambridge, Massachusetts, USA)—discuss the evolution of the technique and where it delivers the most value today. Modern SFC instrumentation has resolved the robustness and sensitivity issues that once confined it to niche use, and the technique now plays an established role in chiral separations, biopharmaceutical workflows, and environmental and anti-doping analysis. According to the panelists, SFC's advantage lies less in a single broad "green" claim and more in specific, demonstrable gains—in solvent reduction, cycle time, and separation power—that continue to expand its reach beyond traditional applications.

How does modern SFC differ from traditional SFC when instruments were launched? Have issues associated with robustness been resolved? Have there been any other significant advances worth mentioning? Have there been any advances in the understanding of the fundamentals of SFC, such as retention, selectivity, and brand broadening in SFC conditions?

John Reilly: I remember when, in the mid-1990s, Eli Lilly's Separations group, led by Joe Kennedy, evaluated SFC and found it lacking in robustness for routine analytical use. However, by 2003, with the availability of Berger analytical and prep instruments, Craig White's UK team at Eli Lilly took the risk and invested heavily in SFC instrumentation and CO₂delivery infrastructure for early-phase drug discovery. This led to SFC becoming the routine process for all chiral purifications at the UK site, eventually supporting large-scale chiral purifications for global teams. This could only have been delivered by management taking the risk in investment for bulk tank delivery, where the CO₂ is compressed to liquid and delivered to the laboratory by means of a booster pump. We must thank these pioneers of the early 21st century, as SFC has now been widely implemented in Big Pharma drug discovery for not only chiral but also achiral purifications. This is due not only to the robustness of the instrumentation but also to the robustness of the delivery of CO₂ to the laboratory.

Maria Kristen Parr: In terms of modern analytical SFC instrumentation, the robustness of separations is drastically improved. With the current generation of technical realization, it may be considered as robust as other chromatographic separations. This seems to be mainly related to the innovations in backpressure regulation within tight limits.

Larry Miller: When you compare modern SFC instruments to those from the early 2000s, the main breakthrough was reduced detector noise and increased sensitivity. This advance now allows SFC to be used in regulated environments in pharmaceuticals and other industries. Another advance over the last 20 years is the expansion of SFC beyond low- or medium-polarity compounds and beyond research laboratories in the pharmaceutical industry.

JR: I think there have been many innovations in column technology for SFC to cover the expanding range of polarities of small molecules, particularly for achiral applications. However, I think there is still more scope to deliver more “bespoke” columns for larger molecules such as degraders, peptides, and radioligand therapies (RLTs) to cover more complex separation challenges.

MKP: Another important change in modern SFC understanding refers to its use with modifier amounts up to 60% or even more. This makes the technique more versatile and no longer considered a separate technique clearly different from high performance liquid chromatography (HPLC). Unfortunately, there is still a lack of extended pressure ranges in commercial SFC instrumentation. Pressure ranges up to 1200 bars are used in in-house-built solutions, for example, at Boehringer, but the scientific community has been hoping for a new generation of instruments for quite some time.

The conditions now employed in SFC are broader than the “supercritical fluid domain” would allow. How does that expand the application domain? How do you see SFC evolving in the future? Are there any technological developments that would make the technique more widely available?

JR: Operating instruments below the critical point introduces new modifiers for studying complex mixtures. Recent improvements include adding small amounts of water to enhance sample solubility and mobile phase polarity. Peptide, oligonucleotide, and nucleoside/nucleotide separations are being validated for both analytical and preparative use. Balancing the time spent on SFC method development within industrial laboratories may be better served by seeking further academic collaborations in this field. Although challenging, it is an opportunity to utilize SFC further for more complex new modality separations.

MKP: In that regard, SFC for chiral separation of synthetic oligos seems to be an obvious field to explore further. This field seems to combine the advances seen in SFC for chiral separation with the newly extended range of analytes. Furthermore, SFC was possible even for the analysis of highly polar analytes, ions, and even inorganic salts in recent years. With the extended range of recent SFC methods, a broad range of analyte polarities can be covered in a single analytical run. This may also help to overcome limitations of HPLC-based methods for metabolomics or biological applications.

LM: The vast majority of the SFC work performed operates outside of supercritical conditions, employing high modifier percentages (up to 80%), which allows compounds of higher polarity, such as those mentioned by John, to be analyzed or purified using SFC. While SFC uses less organic solvent and generates reduced waste compared to HPLC using the same columns, one major limitation of current state-of-the-art SFC equipment is the larger extracolumn volumes compared to ultrahigh-pressure liquid chromatography (UHPLC), which limits SFC to 3.0-mm-internal diameter (i.d.) columns or larger. With UHPLC routinely using 2.0-mm-i.d. columns, this limitation greatly eliminates the reduced solvent advantage of SFC. The last major advance in SFC equipment was almost 10 years ago with the introduction of high-performance SFC equipment. The development of analytical SFC equipment that can routinely use 2.0-mm-i.d. columns would expand the use of SFC.

What specific advantages does modern SFC offer over traditional HPLC when analyzing chiral compounds in pharmaceutical development?

JR: The integration of SFC for chiral separation has allowed for productivity increases, decreases in toxic/hazardous solvent consumption, and a greener chromatographic approach. Ultimately, this leads to a decrease in time for the “drug-design-test” cycle for drug discovery projects.

MKP: Particularly for preparative applications, SFC methods allow for easy product recovery. The analytical-scale method development helps to later upscale methods without a huge amount of effort. In addition, in the analytical scale, the decrease in solvent use and waste generation is considered beneficial. It has also been demonstrated that SFC may resolve chiral drugs and chiral impurities or metabolites in one single run, where HPLC may sometimes struggle.

How is SFC currently being applied in biopharmaceutical workflows, such as the analysis of peptides, oligonucleotides, or biologics?

JR: At Novartis, we are currently investigating where SFC “orthogonality” can provide benefits for more complex modalities. We have demonstrated that for some cyclic peptides, SFC is beneficial, although we need to invest more time in this space to have more definition on the prediction of when SFC may be of more value here than traditional reversed-phase (RP) HPLC approaches.

MKP: Our data on the separation of antibiotic peptides clearly demonstrate that SFC is a useful tool in peptide mixture separation. Also, in the case of insulins, we have demonstrated that SFC is a very useful technique. Extending the approach to larger peptides and proteins is worth exploring, particularly when using high modifier levels that enable gradients spanning both classical SFC and HPLC conditions within a single run—covering amino acids, small and large peptides, and even intact proteins, for improved impurity profiling. As mentioned above, I foresee SFC as also being very useful for chiral separations in oligo analysis.

LM: SFC is being explored for more molecules beyond small molecules, including peptides and oligonucleotides. While some interesting work has been published on SFC for peptides and oligonucleotides, because of the high polarity of some of these molecules, it is far from a universal technique for the analysis of these types of molecules. LC is still the go-to technique for the analysis and purification of most biotherapeutic molecules.

In drug discovery pipelines, how does SFC contribute to high-throughput purification and screening of lead compounds?

JR: It has been fundamentally demonstrated throughout the industry that within drug discovery, SFC speeds up the purification step, which ultimately will lead to more efficient screening of lead compounds.

LM: Most purification laboratories in pharmaceutical research are using SFC to increase productivity and reduce cycle time for chiral and achiral high-throughput workflows. The use of SFC reduces purification times, solvent usage, and waste generation, and results in lower purification costs.

Can SFC be effectively coupled with mass spectrometry (SFC–MS) for metabolomics or lipidomics applications in biomedical research?

JR: There have been a range of publications to demonstrate SFC–MS applicability for bioanalysis applications within drug discovery; however, I still believe that this opportunity has not been taken up as much because RP LC–MS has such a broad applicability here.

MKP: The high chromatographic separation capability of SFC may ease proper compound identification, especially in cases where large numbers of potential isomers are expected. In that sense, lipidomics, lipid profiling, and characterization of nanoparticle composition may be fields of interest in the future.

LM: Coupling MS to SFC is very easy and has been used for almost 30 years. Significant research has been published over the past 15 years on the use of SFC–MS in the lipidomics and metabolomic fields.

How is SFC being adopted in environmental or food analysis, particularly in screening for trace-level contaminants or complex natural products?

JR: I haven’t been close enough to these SFC applications in environmental or food analysis to comment in detail, although we recently hosted an African scholar, Susana Dufie-Boatey, here at Novartis, where we demonstrated the applicability of SFC for purification of crude extracts of Ghanian natural products. We hope to be able to publish our findings in the future, as SFC was a very efficient way to separate these polar products.

MKP: SFC has been investigated in the field of anti-doping analysis, where ultra-trace levels of high numbers of suspect analytes must be covered, allowing for ultra-trace level identification. Several multiple analyte methods have been published and evaluated by different groups.

LM: There have been several presentations and publications of SFC in environmental and food analysis. The coupling of supercritical fluid extraction (SFE) to SFC can assist with trace-level analysis of complex mixtures.

Are there emerging industrial or academic research areas where SFC is gaining traction due to sustainability benefits, such as reduced solvent use or lower carbon footprint?

JR: The sustainability “Green Chemistry” approach for SFC should gain more traction, although within academic settings, I believe the more compelling argument is to be able to demonstrate more “new modality” pharmaceutical complex separations on SFC, whether this is by modification of stationary phase or by using more mixed modifiers.

MKP: In my view, the carbon footprint of analytical methods is still not the focus of industrial research due to its relatively low amount compared to preparative applications. The latter is certainly the focus in the industrial area. In academia, I see more of the general applicability of SFC in the focus of researchers, even if the sustainability aspect of SFC separations is very often mentioned in the introduction of scientific publications dealing with SFC separations. With green analytical chemistry becoming more of a focus of researchers as well, this aspect is also gaining more attention.

LM: Within the pharmaceutical industry, there is a push to make our analytical footprint more sustainable. Most of the waste generated during analysis is from the development and manufacturing groups. As SFC is used infrequently in these areas, most of the research on sustainable analytical methods is related to high performance liquid chromatography (HPLC) or gas chromatography (GC).