Thirty years ago, supercritical fluid chromatography (SFC) was described optimistically as the mode of the future, unifying and replacing GC and HPLC for many applications. Although this has not happened yet, Waters (www.waters.com) clearly has the vision now. Waters has enjoyed 20/20 vision in the past, so let’s check back in 2020.
Waters added a Pittcon Editors’ Gold Award to its collection of plaques from prior years. The new ACQUITY UPC2™ (UltraPerformance Convergence Chromatography™) is showing greatly improved and truly useful detection sensitivity using SFC. Last year, I reported on six new SFCs on the floor at Pittcon®, including the ACQUITY® SFC from Waters. These clearly demonstrated that improved technology was reigniting market interest.
Despite introducing a new analytical SFC chromatograph in 2011, chromatographers at Waters saw that ACQUITY SFC was only the first step in a long staircase. They decided to take the express elevator to the top. UPC2 combines SFC with Waters UPLC® technology and expertise in fluidics, kinetics, and thermodynamics to deliver fast, reproducible separations. For example, supercritical CO2 has very low viscosity, which improves mass transfer kinetics, and hence column efficiency, even compared to hexane or water. The SFC region offers another degree of freedom to the operating space. Thus, one can vary pressure and temperature independently to optimize a separation. With CO2, pressure can change solubility of analytes significantly. Plus, CO2 is more compatible with MS than most any other mobile phase, and is much less expensive.
During the Q&A following the formal introduction, Art Caputo, President of Waters, predicted that UPC2 will grow quickly and relegate HPLC and GC to niche techniques in eight years. He pointed out that ACQUITY technology was introduced eight years ago, and today the LC business is dominated by ultrahigh-performance liquid chromatography (UHPLC). Point made!
Let’s look at the advantages first: Waters believes that UPC2 is ideal for tough-to-analyze compounds, including hydrophobics and chirals, lipids, fuels, natural products, surfactants, and thermally labile analytes. As an example, Waters showed an 18-peak chromatogram of mixed analytes complete in less than 8 min. The peaks are very narrow. A side-by-side comparison of an impurity profile with reversed-phase liquid chromatography (RPLC) confirms that SFC can be much more efficient, and also has orthogonal selectivity.
Now for the limitations: SFC of polar molecules needs to be worked out. SFC still works best for chloroform-soluble analytes. Perhaps someone will develop SFC-HILIC (hydrophilic interaction chromatography) technology for polars. Also, SFC has not shown utility for analytes larger than about 10,000 Da. Will it work for membrane proteins? This is a difficult application segment for HPLC and UHPLC. Then there is the cost of instruments—SFC is a bit more expensive than UHPLC. This difference could be made up by improved productivity and lower mobile phase cost. But an SFC costs four times as much as a comparable GC. The cost of mobile phase in GC is low, especially if one uses hydrogen generators, so the economics are not favorable. Historically, the rate of conversion to a superior technology (e.g., capillary columns replacing packed columns) in GC is only about 3% per year.
New preparative SFC
Chiral separations are the most often-cited advantage of SFC, both on the analytical and preparative scale. JASCO (www.jascoinc.com) introduced the Prep-2088 preparative SFC, which features a circular dichroic (CD) detector, optimized mobile phase pumps, and backpressure regulator. The CO2 pump has a cooled pump head. Maximum flow is 120 mL/min with a Pmax of 5000 psi. The modifier pump has a maximum flow of 80 mL/min. The new backpressure regulator keeps the system stable, irrespective of the flow. Constant pressure is essential to control solubility.
One example in the press conference that introduced the Prep-2088 showed a chiral separation of two enantiomers that provided resolution of about 0.7, which means that the valley between the two-peak maxima is a mixture. Using the CD signal as a guide, the operator collected the leading edge of the first peak and the trailing edge of the last peak. The middle valley, which is a mixed fraction, was collected for reinjection. This is not a new concept in prep, but the convenience and performance of the 2088 appears to qualify as a major advance.
In the past, chromatographers chose columns for SFC from the long list of HPLC columns. This worked, but it is now apparent that SFC can be improved with columns that have been designed, packed, and tested for SFC.
BEH 2-EP, CSH Fluoro-Phenyl, and HSS C18 SB
In support of the UPC2, Waters introduced three columns and promised one more. Each is specifically optimized for SFC. 1) The BEH 2-EP (ethylpyridine) is bonded to a 1.7-µm ethylene-bridged hybrid particle. This is a general-purpose phase that provides good retention, peak shape, and selectivity. 2) The BEH phase provides stronger interactions with polar groups, such as phospholipids, than the 2-EP phase. 3) The CSH Fluoro-Phenyl column uses Waters charged surface hybrid technology to bind fluorophenyl ligand. This phase is recommended for weak acids, bases, and neutrals. Columns range in size from 2.1 × 50 to 150 mm. Particle size is 1.7 µm. Midyear, Waters plans to introduce the HSS C18 SB surface chemistry on 1.8-µm high-strength silica. All four phases will also be available in 3.5-µm diameter.
GreenSep™ Basic SFC
Last year, ES Industries (www.esind.com) also introduced columns optimized for SFC, which offer superior selectivity, peak shape, and durability. The most recent introduction, GreenSep™ Basic, binds imidazole to 3- or 5-µm silica spheres. Pore diameter is 120 Å. Compared to GreenSep Ethyl Pyridine, the imidazole phase provides much stronger retention and improved selectivity for separation of amine-containing analytes.
Silica is a commonly used stationary phase for SFC. All too often, the separations show tailing or broad peaks. This may be due to trace metal impurities in the silica. ES Industries selected high-purity silica for the GreenSep Silica columns. Packed columns are available with nominal particle size as small as 1.8 µm and as large as 20 µm.
Nacali USA, Inc. (www.nacalaiusa.com) introduced the COSMOSIL 3-hydroxyphenyl column, specifically optimized for SFC of beta blockers. Compared to the 2-ethylpyridine stationary phase, the elution orders are reversed for pindolol and atenolol with the same SFC mobile phase.
SFC column classification
It appears that the ideal stationary phases for SFC may be different than HPLC. The problem is how to scout and classify them. To help in this curation, David Kohler and Matt Przybyciel of ES Industries developed a range of test probes that help classify and select columns with maximum diversity in selectivity. If SFC is in your future, you should consider this as part of column scouting for method development.
Click here to read Dr. Stevenson's reviews on advances in liquid phase separtions from Pittcon 2012 and gas chromatography instruments and columns.
Robert L. Stevenson, Ph.D., is a Consultant and Editor of Separation Science for American Laboratory/Labcompare; e-mail: firstname.lastname@example.org.