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Food analysis, column selection, HILIC-SFC, and more
The 6th International Symposium on Packed Column SFC (SFC 2012) this past October included plenary lectures, scientific talks, poster sessions, and award presentations. Key areas addressed include supercritical fluid chromatography (SFC) for food analysis, medicinal chemistry purifications, hydrophilic interaction liquid chromatography SFC, SFC column selection, green SFC, and analytical and preparative SFC.
The 6th International Symposium on Packed Column SFC (SFC 2012) was dedicated to bringing scientists together to discuss the latest advances in supercritical fluid chromatography (SFC). The symposium is organized by the Green Chemistry Group and alternates between the United States and Europe. The 2012 conference was held in Brussels, Belgium, from October 3 to 5. SFC 2012 attracted more than 150 scientists from 18 countries. In addition, eight exhibitors displayed equipment and stationary phases for SFC use. The three-day conference included 19 oral presentations, 41 poster presentations, and two vendor workshops. A short course dedicated to SFC theory and achiral, chiral, and preparative SFC was held before the conference. The program and many of the oral and poster presentations from SFC 2012 and previous SFC conferences can be found at www.greenchemistrygroup.org/index.html.
On Thursday and Friday mornings, the conference started with a plenary lecture from a recognized expert in SFC. Here is a brief description of those two lectures.
SFC for Food Analysis
Terry Berger (Aurora SFC) led off the first day of the conference with a discussion of separation problems that are not currently addressed by SFC. Over the past 20 years SFC has become a technique dominated by pharmaceutical applications. In the late 1980s, SFC had a much wider base of applications including polymers, fuels, food, and environmental contaminants, as well as pharmaceutical applications. Berger's talk focused on the potential of SFC for food and nutritional analysis. SFC separations have been developed for a number of food-related analyses including sulfa residues in milk, preservatives, natural food colorings, antioxidants, and vitamins. In those examples, SFC afforded shorter analysis times and used less solvent. Berger also reported on the first baseline separation of eight sulfonamides by SFC. The SFC method was four times faster than the reported high performance liquid chromatography (HPLC) separation. Recent advances in analytical SFC equipment and sensitivity have allowed sulfamethazine to be quantitated to 9 ng/mL (below the legal limit).
SFC in Medicinal Chemistry Purifications
The second plenary lecture of the conference was given by Eric Francotte (Novartis Institutes for Biomedical Research), who reported on the expanding role SFC is playing in medicinal chemistry purifications. Novartis has implemented a worldwide initiative to promote the use of SFC purifications to increase medicinal chemistry productivity. The company is promoting the use of SFC over reversed-phase HPLC whenever possible. This approach shortens the time necessary to purify final compounds and frees up medicinal chemists' time by routing the time-intensive purification process to a specialty group. There is currently no generic achiral SFC stationary phase, thus an efficient screening process must be developed to minimize analysis time while maximizing purification success. Francotte reported on the use of eight column chemistries and a parallel SFC–mass spectrometry (MS) system to efficiently develop analytical SFC methods suitable for purification. In the two years since this initiative began, the purification approach has moved from 90% reversed-phase HPLC in 2010 to 80% SFC in 2012.
Although purification is still the main application for SFC, the use of analytical SFC has been increasing. This was evident by the higher number of talks discussing analytical SFC compared to past conferences. Didier Thiébaut (ESPCI Paris Tech) presented his recent work on exploring two-dimensional SFC to separate complex mixtures. He presented work concerning the impact of various instrument parameters on the number of peaks observed and reported on the 2D SFC separation of a vacuum distillate from coal tar, showing comparable results to 2D gas chromatography (GC). Two-dimensional SFC allows for the use of long columns and can be used for heavier (higher molecular weight and more polar) samples; it is not restricted to oil samples.
Claudio Brunelli presented work from Pfizer's Sandwich, UK, facility where they are working to move SFC from a generic approach to a critical tool for analytical method development. He reported on the use of SFC–time-of-flight (TOF) MS for the identification of minor enantiomers in chiral SFC methods without requiring enantiomer standards. The use of SFC for analytical method development for Dacomitib (a development candidate for non-small cell lung cancer) was also presented. Dacamotib and its intermediates presented numerous analytical challenges, including low solubility and stability in aqueous and acidic mobile phases. SFC methods were quickly developed for step 1 and 2 products that eliminated the stability issues observed with reversed-phase HPLC. The largest separation problem for this project was the separation and quantitation of up to 47 genotoxic impurities that required limits of quantitation <1 ppm. SFC was explored for this separation and has shown impressive initial results.
Sub-2-μm particles are routinely used in HPLC but, because of equipment design, they have not been routinely used in SFC. The latest analytical SFC designs have reduced extracolumn and dwell volumes such that the advantages of sub-2-μm particles can be realized with SFC. Alexandre Grand-Guillaurne-Perrenoud of the University of Geneva presented a critical analysis of sub-2-μm columns for SFC and their application for the analysis of pharmaceutical compounds. His study showed the extracolumn volume for the new generation of SFC equipment (60 μL) is higher than that for ultrahigh-pressure liquid chromatography (UHPLC) (13 μL), and the smallest column internal diameter that should be used on the new generation of SFC equipment is 3 mm. A higher value for height equivalent to the theoretical plate (HETP) was seen for SFC compared to HPLC (2.8 vs. 2.2), possibly because of packing issues or decompression cooling. In SFC, as pressure increases, mobile-phase density changes and temperature gradients are generated that can affect selectivity, making system pressure drop a critical parameter for method transfer. Grand-Guillaurne-Perrenoud also reported on SFC analysis of basic pharmaceutical compounds. Using 2-ethylpyridine columns, poor peak shape was observed, especially for compounds with pKa > 8. The use of a hybrid silica column with a volatile additive (ammonium hydroxide) gave good peak shapes for all compounds.
The main use of SFC for the past 15 years has been the preparative resolution of enantiomers. Although SFC is expanding into other areas, the preparative arena is expected to remain prominent because of the advantages of SFC over HPLC such as speed, lower costs, and reduced solvent usage. Arvind Rajendran of Nanyang Technological University presented work on the two injection approaches in preparative SFC (mixed stream and modifier stream injection). Mixed-stream injection introduces the sample in the carbon dioxide or modifier stream, whereas modifier stream injection introduces the sample in the modifier stream before mixing with carbon dioxide. It has been shown that large-volume injections in the mixed-stream injection mode can result in peak distortion. Further experiments were performed in Rajendran's laboratory to explore this effect. Large-volume injection studies under mixed-stream injection and a low modifier percentage yielded an increase in retention with increased injection volume. He proposed that this impact was caused by the large viscosity difference between the injection plug and the SFC mobile phase, which resulted in increased pressure and reduced linear velocities. Further experiments are planned to confirm this theory.
Alexander Bozic reported on the role of SFC for pharmaceutical purification at Sanofi-Aventis. He discussed limitations of the commercially available equipment in their laboratories that ultimately resulted in the design and construction of an in-house preparative SFC system. The main advantage of this system was a gas–liquid separator that allowed open-bed fraction collection at higher flow rates (up to 350 mL/min) without the need for a make-up pump, while affording higher product recoveries than currently available gas–liquid separators. Manny Ventura of Amgen presented an investigation on the coupling of achiral and chiral columns during SFC method development. Often, samples submitted for chiral separation have achiral impurities. If those impurities are coeluted with either enantiomer, an achiral separation must be done before or after the chiral purification. Ventura evaluated the coupling of achiral and chiral columns to resolve all sample components in one step, thus increasing the process efficiency.
Larry Taylor of Virginia Tech reported on the analysis of hydrophilic analytes using water-rich modifiers. He showed that adding 5–10% more water to the modifier allows the elution of polar molecules, even those with high water solubility. For all this work he used bare silica stationary phases. Taylor calls this approach hydrophilic interaction liquid chromatography (HILIC) SFC. This approach was demonstrated through multiple examples, including polar pharmaceutical compounds and unprotected dodecaptide isomers.
SFC Column Selection
Pat Sandra of the Research Institute for Chromatography presented a review of column selection for SFC. In his opinion, silica is the first choice for achiral SFC method development. Sandra also presented work on using small internal diameter columns in SFC. He showed that 2-mm i.d. columns could not achieve the same efficiency under SFC operation that is seen under HPLC conditions.
Caroline West of the Universite d'Orleans gave a thought-provoking talk on the "greenness" of SFC. SFC is referred to as a green technology; West reduced the "12 principles of green chemistry" to six principles of green chromatography and evaluated SFC for each of these principles. Her conclusion was that although SFC is green, it could be made even greener. The main areas to consider are the amount of organic modifier required for separation, which results in increased waste, and the energy required for the production and recycling of carbon dioxide. She suggested the use of appropriate stationary phases that would allow elution at lower modifier percentages. One example of a seven-component mixture showed that solvent usage could be reduced from 10.5 mL to 0.5 mL per sample with only a stationary phase change. Currently, carbon dioxide recycling is used only for high-flow-rate preparative SFC systems. To increase energy efficiency for carbon dioxide production, she recommended its use for smaller SFC systems.
Poster Sessions and Best Poster Awards
As with previous SFC meetings, many attendees chose to present their work in poster format. Topics covered all areas related to SFC, including analytical and preparative SFC and stationary phase development and evaluation. Best poster awards consisting of cash prizes were given to the top two posters as judged by the scientific committee.
This year's winning poster was "Effects of Modifier Nature and Percentage on Retention and Selectivity in SFC" by Caroline West and coauthors from the Universite d'Orleans. This work investigated the changes in retention and selectivity that occur when the modifier (methanol, ethanol, isopropanol, or acetonitrile) is varied. Seven stationary phases with different column chemistries were studied using chemometric methods. This study showed that selectivity was only marginally affected by the modifier, with acetonitrile showing the most significant differences. In addition, these results support the theory of a "HILIC-like" retention mechanism in SFC in which the adsorbed mobile-phase molecules have an active role in the retention process as they interact with the analytes.
The second place winner was "Development of a High-Throughput Lipid Profiling Method by Using a Quadrupole Orbitrap Mass Spectrometer and an Automated Lipid Identification Software" by Takeshi Bambi and coauthors from Osaka University. This poster reported on the use of SFC–MS for high-throughput lipid analysis and the use of lipid search software for identification of the separated lipids. With this approach, more than 200 lipid molecular species were detected and automatically identified from mouse plasma.
The 7th annual Symposium on Packed Column SFC (SFC 2013) will be held in Boston, Massachusetts, from July 10 to 12, 2013. Location and timing were chosen to precede Prep 2013, allowing scientists to attend both meetings. Additional information on SFC 2013 can be found on the Green Chemistry Group web site: www.greenchemistrygroup.org.
Larry Miller is with Amgen in Cambridge, Massachusetts.
Larry Taylor is with the Department of Chemistry at Virginia Tech in Blacksburg, Virginia.
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