Highlights of SFC

Highlights from the oral and poster programs of the 9th International Symposium on Packed Column SFC (SFC 2015) are reviewed in this synopsis.

The 9th International Symposium on Packed Column Supercritical Fluid Chromatography (SFC 2015) was held in Philadelphia, Pennsylvania, on July 22–24, 2015. Selected highlights of both the oral and poster SFC 2015 programs are reviewed in this synopsis.

Scale-Up

SFC has long been used for chiral analysis in support of pharmaceutical development, but implementation of the technology in a regulated good manufacturing practice (GMP) production has its challenges. Daniel Markowitz from Johnson Matthey Pharmaceutical Material and Services presented benefits of this green alternative to conventional solvent-based crystallizations and low-pressure chromatography. The effects of injection loops, stacked injections, UV scaling, resin selection as well as solvent and CO₂ recycle at the plant scale were discussed. A new system was described that contained two 20-cm columns in parallel.

Jeffrey Kiplinger from Averica Discovery Services reminded the audience that the only important criteria for adoption of a technology are economic. In this regard, batch-to-batch cross contamination and system clean-out protocols and alternative fractionation or collection design were considered. Direct measurement of economic impact can result in better instrument design and better planning for new technology implementation.

Theory

Interest concerning various phenomena taking place in a column used for packed-column SFC separations continues to be relatively high. Don Poe of the University of Minnesota discussed the Joule–Thompson coefficient as a criterion for efficient operating conditions in SFC using porous and superficially porous packings in a convective air environment. The efficiency for elution of n-alkylbenzenes on 250 mm x 4.6 mm columns packed with 5-μm fully porous and superficially porous particles at optimum flow rates in a convective oven at 20 °C to 60 °C to 80 °C and pressures from 90 bar to 250 bar, with CO₂ mobile phase containing 5%, 10%, and 20% methanol (v/v) was measured.

In a separate study, Ruben De Pauw from the Vrije Universiteit Brussel identified and quantified the different contributions to extracolumn band broadening in packed-column SFC, such as the influence of sample solvent, injection volume, extracolumn volumes, and detector cell volume or design. Abhijit Tarafder from Waters Inc., investigated how density gradient along a packed SFC column affects the overload band profiles of preparative SFC. A computer program that simulates changes in overloaded peak shapes as a function of density gradient at a given operating condition was used. Tarafder considered if having a steep density gradient in preparative SFC is always deleterious as in analytical situations, or if there could be some advantage in having density gradients in prep-SFC.

Both Poe and Tarafder preceded their technological presentations with a personal tribute to Professor Georges Guiochon, formerly of the University of Tennessee, with whom many manuscripts concerning packed-column SFC had been coauthored within the past five years.

Hyphenated Methods

The increasing demand for shortening development time lines in the pharmaceutical industry has made throughput analysis techniques very popular. Mohammad Al-Sayah from Genentech Inc., described the development of an on-line two-dimensional (2D) chromatographic system utilizing reversed-phase liquid chromatography (LC) in the first dimension and SFC in the second dimension. The 2D LC–SFC system could achieve simultaneous achiral and chiral analysis of pharmaceutical compounds. The peaks of interest from the first reversed-phase LC dimension column were effectively focused as sharp concentration pulses on a small-volume C18 trapping column and then injected onto the second-dimension chiral packed-column SFC column. Full automation of the system was achieved.

Christine Aurigemma from Pfizer Inc., discussed early efforts to implement open-access analytical packed-column SFC–LC in the medicinal chemistry laboratory that would allow chemists to work more efficiently. Adapting the technology to align with the work habits of chemists will be a key factor in facilitating the adoption of SFC to boost chemist productivity and efficiency.

Stationary Phases

Daniel Armstrong of the University of Texas at Arlington provided a lucid discussion of recent directions in packed-column SFC concerning chiral separations including new chiral selectors and small-particle-diameter column packings that will have a significant impact on the field. Another presentation by Vincent Desfontaine of the University of Geneva focused on the evaluation of new stationary-phase chemistries in sub-2-µm and core–shell material for the analysis of basic compounds in SFC. Various representative sets of basic drugs were injected such as mixes of active pharmaceutical ingredients with their respective pharmacopeia impurities. Different analytical conditions were also compared such as absence of additive, additive in the mobile phase, and additive in the injection solvent.

A lecture by Oleg Pokrovsky from the Kurnakov Institute of General and Inorganic Chemistry concentrated on stationary phases where the separation of closely related compounds was not based on hydrogen bonding. In these instances, the separation required involvement of other types of interaction such as electrostatic, dipole-induced dipole, or dispersion. A survey of several cases of non-hydrogen-bonding-driven separations of closely related compounds was considered such as xylenes, dichloroanilines, and methoxy-derivatives of psoralen. All ortho-substituted compounds were eluted earlier than “ortho-free” isomers. A distinctly stronger retention of 3,4-dichloroaniline compared with 3,5- and other isomers confirmed the significance of dipole–dipole and other electrostatic intermolecular interactions in the separation of this model compound. Standard column screening revealed that no achiral column was able to separate meta- and para-xylene in packed-column SFC except porous graphitic carbon (PGC), which suggested that PGC differs substantially from both hydrogen bonding and non-hydrogen bonding silica-based phases in the elution order of dichloroanilines.

Polysaccharide-based chiral stationary phases (CSPs) are well recognized as a powerful tool in chiral separation. To clarify the potential and selectivity feature of these phases in achiral separations, Tohru Shibata and associates from Daicel Corp., have systematically studied achiral isomer separations with polysaccharide-based phases under both high performance liquid chromatography (HPLC) and SFC conditions. The retention under both conditions roughly correlated, but inversion of elution order between isomers was sometimes observed. Nevertheless, these CSPs exhibited excellent potential for achiral separations and analysis in packed-column SFC as well as HPLC.

Applications: Cannabinoids

The cannabis industry is one of the fastest growing industries in the United States. Christopher Hudalla from ProVerde Laboratories Inc., presented the development of a workflow based on supercritical fluid technologies for the analysis, extraction, and purification of cannabinoids for the preparation of cannabis-based therapeutics. Complementing this presentation was the lecture of Ira Lurie from George Washington University on ultrahigh performance SFC for the analysis of synthetic cannabinoids. Presently, there are more than 20 synthetic cannabinoids under permanent or temporary federal control in the U.S. The effects of cosolvents, additives, pressure, temperature, and gradient slope on optimizing the separation were presented.

Applications: Medicinal

Productivity of modern medicinal chemistry requires instrumentation for automated synthesis and high throughput purification that can process a large number of samples within a meaningful timeframe. Gerard Rosse from Dart Neuroscience LLC discussed the decision-making process for selecting packed-column SFC coupled to mass spectrometry (MS) as the prevailing method for compound purification. Instrumentation for analytical and preparative SFC–MS, infrastructure, logistics, workflows, and robotics to support the purification of >10,000 compounds each month was presented.

The application of SFC for chiral metabolite separations in a DMPK environment was discussed by Hermes Licea Perez from GlaxoSmithKline. A complex mixture of 14 stereoisomeric metabolites provided important data on which species circulate in the human body. Packed-column SFC in combination with chemical derivatization was proven superior for the separation of four diastereoisomeric species of another drug development compound. The method was fully validated and applied to evaluate potential in vivo chiral conversion in pooled clinical and preclinical samples.

Implementation of SFC in regulated GMP laboratories has been somewhat slow, owing to limitations in instrument sensitivity, reproducibility, accuracy, and robustness. Michael Hicks from Merck Inc., reported on an investigation into the use of modern packed-column SFC for enantiopurity analysis of several pharmaceutical intermediates and compared the results with conventional HPLC approaches historically used for analysis in a GMP setting. The findings clearly illustrated that modern packed-column SFC now exhibits a degree of precision, reproducibility, accuracy, and robustness comparable to that of HPLC.

Alexandre Grand-Guillaume Perenoud of the University of Geneva focused on the evaluation of a modern packed-column SFC–HRMS (quadrupole time-of-flight [QTOF]) platform as a potential key analytical tool to support bioactive identification. The preliminary screening step involved 15 different state-of-the-art stationary phases and over 100 natural compound standards. The author highlighted the applicability of SFC to the natural compound space, which included highly polar and very apolar molecules. In parallel, selected column chemistries have been identified as particularly well suited for the analysis of specific compound subclasses. Finally, optimized analytical conditions were applied for the full characterization of several plant extracts.

Applications: General

Edgar Naegele from Agilent Technologies lectured on the quantitative determination of multipesticide residues in vegetables by packed-column SFC coupled to triple-quadrupole MS. The final optimized method was performed on an amino column at a flow rate of 3 mL/min in a gradient with methanol as the organic modifier. The multipesticide sample comprised 17 pesticide compounds. For all compounds, the achieved linearity was better than 0.999, limits of quantification (LOQs) were typically below 2.9 ppm, retention time relative standard deviations (RSDs) were below 0.4%, and area RSDs were below 4%. Matrix effects were found in the recovery range of 70–120% and LOQs were at 10 ppb, which met typical requirements for quantitative determination of pesticides in a food matrix.

To minimize internal corrosion of carbon steel, chemical corrosion inhibitors are most frequently used. These materials are amine-based and as such exhibit high toxicity to aquatic organisms, hence stricter regulations have been imposed regarding the use and subsequent discharge of such chemicals in the environment through produced water. John Langley of the University of Southampton reported on the preparation of a model corrosion inhibitor comprising quaternary amines, imidazolines, and imazolines for qualitative analysis using HPLC–MS and ultrahigh-performance SFC–MS (UHPSFC–MS). The use of modern packed-column SFC as the chromatographic separation decreased analysis times by eliminating the sample preparation step before analysis, especially in the case of crude oil, and reduced elution times by a factor of four when compared to HPLC.

Robert Campbell from Dow Chemical Company noted further applications in a study of the composition of co-polymeric surfactant materials via packed-column SFC–MS with electrospray ionization. Frequently, the spectra and chromatograms are too complex for interpretation with acceptable degrees of confidence. Software-assisted characterization has been used for deconvolution of the complex data sets. Factors such as adduct formation, multiple charging, and the degree of ionization were shown to complicate spectral interpretation. The resulting data analysis made the elucidation of detailed structural information possible with a high degree of confidence.

Supercritical Fluid Extraction

The U.S. Food and Drug Administration (FDA) requires that at least 97% of caffeine be removed in order to call the product decaffeinated coffee. Supercritical carbon dioxide is an ideal fluid to apply to this process. Extraction is always performed on the green beans. Dried green beans are quite hard and dense so a wetting process, causing some swelling of the beans, is necessary before supercritical fluid extraction (SFE) can be performed. John Langley from Waters Corp., demonstrated various extraction techniques as well as a method for the rapid and convenient determination of residual caffeine levels using SFC.

Best Posters

The subject of this year’s winning poster dealt with SFE. Jacquelyn Runco from Waters Inc., demonstrated an SFE–SFC workflow to isolate target flavor compounds from vanilla beans and ground cinnamon. The use of automated processes such as solvent selection, mobile-phase composition, and vessel switching allowed for quick extraction method screening. For both samples, multiple extraction parameters were evaluated to determine optimal yield and extract complexity.

The award for second place best poster presentation went to Takato Uchikata from Shimadzu Corp., for extraction and analysis using on-line SFC–SFE–MS. The hyphenated system was capable of simultaneous multicomponent analysis with on-line automation of everything from sample pretreatment to separation and analysis. For example, in the analysis of pesticides in food products, the system took only 5 min for a complete analysis involving sample pretreatment when compared to at least 35 min for conventional systems. Additional applications included the analysis of biomarkers from dried blood spots and extraction of trace additives in polymers.

SFC 2016

A one-day SFC conference will be held in San Diego, California on May 23, 2016. SFC 2016 will be held in Vienna, Austria, on October 5–7, 2016. Oral and poster presentations will be solicited in the areas of SFC and SFE in the spring of this year. Information on both conferences can be found at 
www.greenchemistrygroup.org.

Larry Taylor is an emeritus professor of chemistry at Virginia Tech, in Blacksburg, Virginia, program co-chair for SFC 2015–2016, and a member of the Green Chemistry Group.