product review
The SFC Comeback Pharmaceuticals give supercritical fluid chromatography a fighting chance. Cheryl M. Harris
S
even years ago, Karen Phinney attended her first conference as a freshly hired chemist at the National Institute of Standards and Technology (NIST). She recalls speaking excitedly with a fellow scientist about the latest supercritical fluid chromatography (SFC) system that NIST, headquartered in Gaithersburg, Md., had just purchased. “I’m doing SFC,” Phinney told him. “Oh, science fiction chromatography,” he replied. Phinney knew the comment represented what some thought of SFC, a separations technique that was considered revolutionary when first introduced but whose reputation had slowly ebbed over the years. But she kept her cool. “I was new in my career, so I wasn’t going to have real snide remarks, and I still had some doubts about it at that time, too.” Rather, she politely explained to her colleague that although some people thought of SFC as more of a scientific frivolity than a novelty, she nonetheless had obtained good results with it. And that was the truth. Today, Phinney, an admitted former SFC skeptic who now defends the method, uses SFC to study chiral separations with a focus on pharmaceutical compounds. “I really didn’t have very much knowledge of SFC capabilities,” recalls Phinney. “The only things that I did know came from graduate school, and it was a very superficial view of SFC.” Although this separations technique lives under the shadow of HPLC, the potential uses of SFC are indeed worth looking into, say researchers and company representatives. Not only is SFC
more versatile than HPLC, but it is also more cost-efficient in the long run, they claim. And SFC’s speedy separations are quite an attractive asset. Phinney is among a growing group of analytical chemists who believe the technique will move forward—maybe not by “leaps or bounds,” she says—but enough for it to be a stable analytical method, especially for the pharmaceutical industry. Tom Chester, a research
fellow at Procter & Gamble, headquartered in Cincinnati, Ohio, agrees. He has seen fairly significant changes in attitude about the technique among his industrial peers. “There’s hope, and there’s real growth occurring,” he says. Experts say that in the beginning, there were great expectations for SFC. Now, decades after its invention, SFC has again attracted serious attention and has been given a second chance. In this
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Table 1. Selected SFC systems. Product
BergerSFC
Series SF3 System
Jasco 1500 Series SFC/SFE System
SuperSep
Sem i-Prep SFC 50 and Prep SFC 200
Com pany
Berger Instruments, Inc. 130 Executive Dr. Newark, DE 19702 302-266-8201
Gilson 3000 W. Beltline Hwy. P.O. Box 620027 Middleton, WI 53562 608-836-1551
Jasco Inc. 8649 Commerce Dr. Easton, MD 21601 410-822-1220
NovaSep, Inc. 23 Creek Circle Boothwyn, PA 19061 610-494-0447
Thar Technologies 100 Beta Dr. Pittsburgh, PA 15238 412-967-5665
URL
www.bergersfc.com
www.gilson.com
www.jascoinc.com
www.novasep.com
www.thartech.com
Approxim ate price (USD)
$60,000+
$55,00–80,000
$60,000–90,000 depending on detector(s)
$60,000 (base price)
$95,000 and $195,000
Type
Analytical
Analytical and preparative
Analytical and semipreparative
Analytical and preparative
Semipreparative and preparative
Colum n type (m m )
Packed (1–10)
Packed (1–20)
Packed (2–10)
Prepacked (up to 30 i.d.), Packed (customer provides Prochrom dynamic axial column) compression columns from 50 to 450 i.d.
Fluid delivery
Single-pump module for GC detection only; dual reciprocating pump module to add modifier for UV detection
Pump A module for CO2; Peltier-cooled CO2 pump Diaphragm pump for CO2 CO2 up to 50 g/min; cosolpump B module for organic (PU-1580-CO2) and modifier with feedback control, pis- vent to 50 mL/min modifier (0–100% in CO2) pump (PU-1580) ton, or diaphragm pumps for feed and cosolvent (depends on size)
Pressure control
Electronic back-pressure regulator
Electronic back-pressure regulator; programmable
Automated back-pressure regulator
Com patible detectors
UV–vis (DAD, VWD), FID, MS, and analog input
UV–vis (DAD, VWD, scan), ELSD, and FID
UV–vis (DAD, VWD), circu- UV standard; option of any UV–vis, circular dichroismlar dichroism-UV chiral additional detector availUV chiral detector, ELSD, detector able for analytical SFC or FID full flow
Num berof detectors
Space for two GC detecMultiple detectors can be tors inside oven; more than configured one LC detector can be attached externally
Pressure range Analytical: up to 400 atoutlet(bar) preparative: 100–200
90–400
Analog control valve
PC-controlled pressure regulator
Multiple detectors can be configured
1 (standard)
One UV included, chiral detector optional
Up to 350
250 max (standard)
Up to 600
0–60 (standard)
Up to 150
Tem perature range (°C)
Ambient + 50 (when used with cryogenic cooling, ambient – 50)
Ambient + 3 to 200 (option- Ambient – 15 to 80 al subambient conditions) ambient + 10 to 80
Totalliquid flow rate
10–50 mL/min
0.2–25 mL/min
PU-1580-CO2: 0.1–10 mL/min SuperSep 10: 45 g/min; Su- 1–50 mL/min PU-1586 with cooling jack- perSep 20/30: 125 g/min; et: 0.1–20 mL/min SuperSep 50: 500 g/min; SuperSep 100: 2 kg/min; SuperSep 150: 5 kg/min
Special features
Autosampler with solidstate heating and cooling; optional column and solvent switching valves; two preparative systems: Prep SFC and MultiGram (microtiter plate in test tube or liter bottle); bundled analytical system for chiral methods development and analytical SFC/MS; company manufactures its own SFC columns
Independent programming of mobile-phase pressure, composition and flow rate; components are compatible with HPLC
Patented back-pressure regulator allows wide range of fluid flow (up to 50 mL/min) and modifier composition (0–100%)
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“Smart” automated fraction collection, automated clean-in-process; cosolvent gradient, CO2 recycling, full qualification
Current good manufacturing practices (cGMP) validation available; can be scaled to move up to the company’s 1 kg/min cGMP
product review
issue, Analytical Chemistry looks at the method and its comeback into the analytical arena. Table 1 lists some examples of instruments that are available to prospective buyers. If there ever was a “survivor” story, it’s been that of SFC; and experts say the technique’s endurance lies in its ability to get the job done.
SFC survival instinct SFC was first developed in the 1960s, and expectations were high when commercially available instruments were finally put on the market in the early 1980s. Unlike GC and LC, researchers were able to vary three parameters— composition, temperature, and pressure—with the mobile phase. But scientists expected grandiose results, and the hoopla over SFC turned out to be its Achilles heel. “There was a lot of big enthusiasm, initially thinking that SFC was going to replace LC, and obviously that didn’t happen,” says Phinney. Muneo Saito, a director at Jasco, Inc., says that his company had more modest expectations and thought that packedcolumn SFC (pSFC) would replace just normal-phase HPLC. “It took a long time before this happened,” he says. In the 1990s, many SFC users were disappointed by open-tubular (or capillary) SFC (cSFC) because of its poor reproducibility and limited application range, says Saito. This bad impression lingered and discouraged some from trying out SFC until the last quarter of the 1990s, as researchers began seeking higher analytical throughput and lower solvent consumption, he adds. Now, pSFC is replacing normal-phase HPLC, says Saito. Misconceptions about SFC’s abilities have dimmed its reputation over the years, explains Phinney. Even today, experts say it’s hard to convince analysts that the technique is “user-friendly”. “I think people feel that SFC is a bit more complex than LC as far as routine operations; and therefore, they’re less willing to incorporate it into their laboratories,” she says. But such fears over SFC are unfounded, say experts. “I think the gap of complexity is decreasing,” says Jean Blehaut, chief executive officer at NovaSep Inc., USA, in Boothwyn, Penn., which acquired SFC supplier Prochrom in
1999. In time, SFC will be almost as quick as HPLC to set up, he says. The price of SFC systems has also discouraged potential buyers, but they have to think of the long-term savings, say experts. SFC systems are more expensive than comparable HPLC units. Phinney, for example, recently priced an SFC system with all the necessary detectors at almost $80,000, which is about double the cost of an HPLC system. But the instrument does pay for itself, primarily because of higher throughput and speed, says Chester. “We calculate we can save 70% in operating costs compared to HPLC.” A changing market also hindered acceptance of the separations method. Phinney says she still has trouble knowing who’s in the business and who’s not. Just when she was getting comfortable with her Hewlett-Packard (HP) SFC system, she was told the company was abandoning the product. HP sold the rights to its SFC instrument in 1995 to Terry Berger, a former HP employee who started Berger Instruments, which was then bought in 2000 by MettlerToledo, based in Greifensee, Switzerland. Other companies that tried and abandoned the SFC market include Isco, Inc., and ABB Process Analytics. Today’s surviving SFC suppliers are small firms up against far larger HPLC companies, which have the edge in marketing and sales staff, explains Chester. “With an SFC representative, you might not even be able to see this person live in your own location because there are so few of them and they have such big areas to cover,” he says. However, SFC companies say things are changing for the better. Berger officials say that SFC now accounts for less than 5% of the HPLC market, but they estimate the method has the potential to go as high as 25%. “So, there’s plenty of room to grow,” says Debra Harrsh, director of marketing. Because the SFC market share is growing, says Harrsh, Berger is building a bigger business space and increasing staff. “We grew twice as fast as people thought we would,” she says. Todd Palcic, manager of sales and marketing for Thar Technologies in Pittsburgh, Penn., has seen growth in the SFC
market as well. In 1999, Palcic thought preparative SFC had reached its “nadir”. “I wouldn’t have given this [technique] the time of day two years ago,” says Palcic. That’s no longer the case. The company received about 100% more inquires in 2001 about its SFC system from prospective clients than in 2000, he says. “Today, we are witnessing a resurrection.” The pharmaceutical industry brought about this resurrection. “The drug area represents new applications for us,” confirms Chester, adding that he knows of one pharmaceutical company with about 20 SFC instruments. Once, SFC was traditionally used for low-molecular-weight polymeric materials and nonionic surfactants, say Chester and David Pinkston, a principal scientist who also works at Procter & Gamble. “Now things have evolved to the point where we spend about half of our time in our lab with pharmaceuticals and the other half with small polymers and surfactants,” says Pinkston. While the pharmaceutical industry has given SFC a boost, the method appears to be overlooked in academia, according to experts. The academic community tends to “grab onto” the latest technique, says Phinney. “So, for them,” she says, “SFC is kind of an old story.” Chester is worried that the problem may even be broader. “There’s just not that many professors with chromatography programs compared to the many thousands of people in industry [who] are doing chromatography,” he explains, adding that the phenomenon seems to be specific to the United States.
Sizing up SFC Even those who stand by SFC will say that for many things, HPLC can be the best choice. Most of the time, when working with compounds that are only water-soluble, reversed-phase HPLC is the first choice, say Chester and Pinkston. HPLC is still the choice for reversed-phase separations with aqueous organic mobile phases, says Phinney. But for certain applications, such as chiral separations and high-throughput screening or analysis, SFC has some advantages, she says. Because the composition, temperature, and pressure can be varied, she continues, SFC gives the re-
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product review
searcher more options to alter the solute retention time and selectivity. SFC also produces better resolution of complex mixtures and faster analysis times than general LC methods, says Phinney. Chester says that SFC is typically 3–5 times faster than HPLC. “If you have hundreds, or thousands, or tens of
that are not quite resolved well enough, then instead of trying to find a new column or new mobile phase, change the temperature by 5 or 10 °, see if they move relative to each other, and frequently they do, if they’re dissimilar.” Next, SFC instrumentation is versatile. Many analysts mistakenly see SFC as a
“When I took the same column and used it with SFC, I could be ready to start injecting samples within 30 min.” —Phinney thousands of [samples] to get through, we’re talking about real money,” he adds. Phinney also finds that she saves time waiting for a column to equilibrate. With HPLC, she remembers having to wait up to 3 h to get a nice baseline before injecting any samples. “When I took the same column and used it with SFC, I could be ready to start injecting samples within 30 min,” she says. In addition, longer columns can be used in SFC than with HPLC, says Chester. “We’ve used long columns to separate very complex polymer mixtures,” says Pinkston. For example, they’ve used meter-long packed columns to separate complex polymers in ways that can’t be done with HPLC, he says. In HPLC, column length is typically 25 cm, but Pinkston routinely puts four columns together for SFC. This makes it easier to generate 100,000 theoretical plates or more; and with the speed advantage, the longer column separation run doesn’t require a lot of additional analysis time, adds Chester. Also, the selectivity in SFC often matches that found with reversed-phase HPLC, but SFC is much more easily adjustable, says Chester. The temperature effect in SFC is also much larger than that of HPLC, which means researchers can use temperature to finetune the selectivity. “That is really cool,” he says, “because if you get a technique that’s working really well, except you [have] just a pair of peaks
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separate technique, says Chester. In fact, HPLC applications can be run on an SFC instrument, but not the other way around without modifications. The difference, says Chester, comes down to controlling the pressure of the column outlet. In HPLC, for example, the default is 1 atm; but in SFC, the exit pressure is controlled. “We routinely do ordinary HPLC and high-temperature and ultrahightemperature HPLC [experiments] on our SFC [instrument],” says Chester. SFC also offers a much wider selection among mobile phases, explains Chester, because the technique is not required to stick only to liquids that are well behaved at ambient conditions. Although CO2 is commonly used as the SFC mobile phase, Chester says, in his laboratory, liquid and supercritical CO2 are used interchangeably, and often the researchers don’t know when they’ve changed between the two. “Our approach here is completely different,” adds Chester. “We regard SFC and HPLC as just being two cases in a larger chromatography that we call ‘unified chromatography’.” He argues that there’s nothing particularly special about supercritical liquids anymore. “What is special is that we can pick mobile phases that don’t work under HPLC conditions, and we can find the conditions that yield real optimal separations. . . . It just doesn’t matter whether the mobile phase is supercritical or subcritical or near-critical or anything else, as long as we’ve
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achieved the optimal separations.”
Choosing the right SFC system SFC systems are divided into two categories: analytical SFC for quantitative analysis and preparative SFC for qualitative and purification work. Most SFC work at Procter & Gamble is analytical, says Pinkston, although some preparative SFC is being considered. Phinney and Blehaut believe vendors are moving more toward preparativescale applications. “I think there is really a much larger demand for prep SFC in, for example, medicinal chemistry departments, where they need 100 mg to a gram of sample,” says Blehaut. Industry researchers can now do large-scale, preparative separations using HPLC. “The problem with doing that is you generate lots and lots and lots and lots of solvent waste,” says Phinney, “and you also have to isolate whatever material you want to collect from that volume of solvent. . . . That obviously is time consuming and not very environmentally friendly.” Companies are trying to develop preparative SFC systems that reduce solvent waste and make it easier to isolate the compound, says Phinney. Laboratories are also cutting down on organic solvents because of high disposal costs, she adds. George Bednar, senior vice president of Gilson in Middleton, Wis., agrees, saying that SFC will continue to grow in the fast, preparative, purification market. SFC systems can use packed columns, open-tubular (or capillary) columns, or both. In cSFC, pressure is controlled at the column inlet using a pump, and automated programming of the mobilephase composition isn’t available. On the other hand, the composition of the mobile phase can be programmed in pSFC. In its early stages, pSFC suffered from pressure changes that researchers had to manually adjust with a pressure relief valve. The result was irreproducible pressure gradients. In 1992, automated downstream pressure control improved pSFC. This made the generation of pressure gradients independent from the mobile-phase flow rate and composition. Some companies have SFC systems that also electronically cool the pump for
product review
CO2. Phinney prefers the electronic control because she feels it’s a bit simpler. Although most labs prefer pSFC, there are some applications where cSFC makes sense, experts say. Phinney still sees a place for cSFC in foods and nutrition research for analyzing fatty acids or compounds that are not volatile enough or too thermally unstable for GC. cSFC is more easily interfaced to a flame ionization detector (FID) than pSFC, explains Pinkston. “You can analyze more analytes using cSFC with pure CO2 coupled to an FID without having to modify those analytes, because the surface area of the open-tubular column is so much lower,” he says. Moreover, with cSFC, analytes have less opportunity to interact irreversibly with the stationary phase, Pinkston says. Having some flexibility in SFC detection is important, says Phinney. Highpressure cells are required for SFC; so one can’t use a standard HPLC detector to do SFC, but an SFC detector can be used for HPLC, Chester says. Phinney typically uses UV–vis detectors, such as diode array detectors (DADs). More researchers are now interfacing their SFC instruments to a mass spectrometer, says Pinkston, adding that he’s been doing SFC with MS for 16 years. Other detectors used with SFC include variable wavelength detectors (VWDs), evaporative light scattering detectors (ELSDs), as well as electron capture, flame photometric, fluorescence, chemiluminescence, and FT-IR detectors.
Other factors to consider Often, columns that are really designed for HPLC are used for SFC, says Pinkston. “You need to make sure those components, such as tubing, columns, and injectors, are used within their pressure and temperature limits, he says. For example, says Pinkston, an SFC column can be heated to up to 250 ºC, but if that column is only designed to go up to 60 ºC, “you’re going to have a mess on your hands.” One can make some mistakes choosing mobile phases, says Phinney. She tends to use CO2 in conjunction with an organic modifier, which changes the polarity of the mobile phase. Methanol
as a modifier strengthens the CO2 and raises the polarity of the mixture, making it perform better for more polar solutes. It’s possible to choose conditions where the CO2 and methanol are not a single fluid and actually separate into a gas and liquid, says Phinney. One can also choose parameters incorrectly and possibly cause leaks, she says, which can be more dramatic in SFC than in HPLC because higher pressures are involved. In addition to CO2, fluorocarbons and even purified hot water have been used as
columns they’re using for SFC in the laboratory are HPLC columns. “We’re kind of stuck using HPLC columns,” says Chester. “There really hasn’t been much work to optimize stationary phases for SFC.” He and Pinkston say they’d like to see more work to improve the bonding chemistry and surface preparation of SFC columns.
The future of SFC Last August, about 200 researchers convened for the 10th International Sym-
“You can analyze more analytes using cSFC with pure CO2 coupled to an FID without having to modify those analytes. . . .” —Pinkston mobile phases, say experts. For Phinney, a pressure of ~300 atm is enough because she’s using modifiers. With pure CO2, varying the pressure can vary the solvating power of the mobile phase. But Phinney warns that choosing a bad grade of CO2 can defeat the purpose of using the technique. “I’ve even heard of people taking dry ice and using that for SFC, with less than successful results,” she says. The outcome could be a lot of noise in the baseline. Most companies that sell gases for chromatography or other scientific applications also sell quality CO2. Phinney’s advice: Save yourself some headache and buy it from them. Another thing to watch out for is proper temperature control, adds Pinkston. If it varies a few degrees, the separation can vary, he says. Pumps for SFC are as reliable as HPLC pumps, adds Phinney, and the same types of failures in the pump seals or check valves tend to occur. Occasionally, the back-pressure regulator of the SFC system will leak, and it will not reach the desired pressure, she explains. The various seals and check valves also have finite lifetimes in both SFC and HPLC systems, says Phinney. They haven’t had any serious problems with the commercial columns, say Chester and Pinkston. In fact, the packed
posium and Exhibit on Supercritical Fluid Chromatography, Extraction, and Processing. While some may say that an attendance of 200 is small, Chester, Pinkston, and Phinney, are quick to call it a success. The conference attracted new faces and growing interest, says Chester, who helped coordinate the event. “I certainly came away from that meeting having a very positive view of the future of SFC,” says Phinney. Industry is pushing SFC to the forefront, say experts. Pinkston says he knows of a number of industrial groups who work with SFC that aren’t encouraged to publish for competitive reasons. Despite the tough obstacles SFC has faced in the analytical market, it’s gotten through them nonetheless. Phinney’s initial doubts about SFC seven years ago have definitely been replaced with excitement for the future. “I think it appears that the vendors are making investments in instrumentation, supporting the instrumentation, and making improvements in response to their customer needs,” she says. “It’s interesting to hear that now things are kind of changing around again.” Cheryl M. Harris is an assistant editor with Analytical Chemistry.
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