In 1964, University of Utah chemistry professor J. Calvin Giddings enunciated a theoretical platform, “unified separation science,” that could confer the resolving power of GC to LC. Giddings’ model combined the higher mobile phase diffusion and efficiency of GC with LC’s higher selectivity via orthogonal separation modes. His vision has been made a reality through supercritical fluid chromatography (SFC), which uses supercritical or subcritical carbon dioxide as the mobile phase.
Professor Larry Taylor at Virginia Tech has described the continuum between, or “unification” of, GC and open-column LC as follows:
- High-pressure GC
- Solvating gas chromatography
- Supercritical fluid chromatography
- Subcritical fluid chromatography
- Enhanced fluid chromatography (high pressure)
- Liquid chromatography
Supercritical CO2 is an inexpensive, low-viscosity, highly compressible, green solvent that improves chromatographic efficiency for a given stationary phase particle size and linear velocity
SFC is greener than any form of HPLC, even low-volume UHPLC. Before it is cooled and squeezed into its supercritical or subcritical physical state, the carbon dioxide mobile phase is extracted from the atmosphere, to which it is subsequently vented. Thus, despite it being a “greenhouse” gas, the extraction and evaporation of carbon dioxide contributes zero net greenhouse gases.
By contrast, conventional HPLC solvents are toxic and expensive in both their acquisition and disposal. Acetonitrile, the most-used organic solvent in HPLC, has become prohibitively expensive in recent years. Users pay “both ways” for the luxury of using it: more than $100 per liter for HPLC-grade material, and approximately the same amount for disposing of the diluted eluent.
Waters has recently adapted SFC to its ACQUITY® instrument platform in a product branded UltraPerformance Convergence Chromatography (UPC2®).
Chiral, normal-phase-like separations
Waters’ original target market was chiral analysis, where SFC already enjoyed a solid reputation. Waters has demonstrated chiral SFC separations that require one-thirtieth the time and consume one-eightieth the solvent compared with standard normal phase HPLC. “When you consider this savings on the scale of hundreds to thousands of injections, the financial impact to an organization can be quite exceptional,” says Waters’ John van Antwerp.
Yet SFC may not remain king of the hill for chiral separations. Christopher Welch, PhD, who manages Merck and Co.’s (Rahway, NJ) New Technologies Review and Licensing Committee, has commented that reverse-phase chiral LC is improving. “It is not bad—it’s something to watch,” he advised Lab Manager. Chiral Technologies, Sigma, Phenomenex, Regis Technologies, and other vendors supply reverse-phase chiral columns.
Since its selectivity overlaps significantly with normalphase chromatography, SFC is orthogonal to reversephase LC. The technique is applicable to a diverse range of compounds, including most organic-soluble compounds, most salts of organic acids and bases, strong organic acids and bases, small lipophilic peptides, and nonpolar solutes (e.g., waxes and oils). In addition to being the go-to method for chiral separations, SFC also separates positional isomers and diastereomers and is compatible with most popular detection modes.
“UPC2 is the combination, or convergence, of GC and LC,” says Ken Fountain, director of Chemistry Applied Technology and Global UPC2 Applications. It exploits the advantages of carbon dioxide-based mobile phases in either supercritical or subcritical mode, while retaining the ability to run gradients with common organic solvents such as methanol or acetonitrile.
The most formidable hurdle to “instrumenting” SFC in the form of UPC2 was to make the new platform as robust and reliable as traditional analytical chromatography. “Laboratory scientists know deep down that GC and LC are robust. They trust those technologies. We wanted to bring that same level of trust to SFC,” Fountain says.
Martin Vollmer, SFC product manager at Agilent Technologies, describes SFC as “a sleeping beauty” that has been grossly underutilized considering its analytical capabilities. Reasons for this were lack of sensitivity, low instrument robustness, and absence of appropriate knowledge and infrastructure for supercritical fluids.
Analytical instruments require a standard CO2 flask, which can be installed in any lab. “This is a door opener for a much wider application space beyond chiral analysis, where SFC is known to provide superior selectivity,” Vollmer adds. SFC has become an “intriguing alternative” for replacing lengthy and toxic normal phase separations, and the most dependably orthogonal method to reverse phase analysis. Fat-soluble vitamins, sterols, food additives, organic light-emitting diodes, pyrethroid insecticides, petrochemicals, and vegetable oils are some analytical targets for which SFC provides robust analysis.
Robustness, ease of use, and performance are key elements of an instrument to be accepted as a mainstream analytical tool. The Agilent and Waters SFC systems are based on proven LC platforms, with the added twist of employing CO2 as an additional solvent. Not much extra knowledge is required, and safety issues in analytical mode are minimal.
“SFC is no longer considered an academic-only instrument,” says Martin Vollmer. “Many pharmaceutical, food, and chemical companies are investing in SFC technology and anticipated growth rates are higher than for other analytical instruments.”
Lab managers are beginning to view SFC as an alternative to normal-phase HPLC. Some growing areas for SFC include fuel analysis, including traditional petroleum products and biofuels. Pharmaceutical companies are beginning to notice that SFC works not just for chiral separations, but for non-chiral normal-phase separations. “CO2 behaves similarly to hexane or heptane; they use the same columns and modifiers,” notes D.J. Tognarelli, chromatography product specialist at JASCO (Easton, MD). “People are going out of their way to exploit the method’s speed and environmental benefits. The ability to switch from chiral columns to non-chiral normalphase columns, on one instrument, is a big plus.”
Vendors still have work to do to educate potential buyers about the advantages of SFC for many applications. “Many lab workers know the term supercritical fluid chromatography, but to them the instruments are like black boxes. They have no idea how they work,” says Tognarelli.
JASCO convinces customers through dialogue, training, and education. “SFC sounds complex, but when you explain that the only significant difference is replacing hexane with carbon dioxide, and that you still retain the ability to use polar modifiers, they begin to understand,” Tognarelli says. “Bridging the gaps in knowledge and understanding is our main focus.”
Like the other major SFC system vendors, JASCO relies heavily on its already successful HPLC platforms to sell its SFC systems. The column, oven, autosamplers, co-solvent administration, and modifier pump are identical, and the detector is “virtually the same,” according to Tognarelli. The main operational differences are that SFC systems are cooled and the system maintains pressure throughout
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