Two leaders in separation science are developing technologies that will save big pharma time and money. The environment should benefit, too.

Large-volume chromatography is poised for change. Prevailing methods are causing unwanted side effects and forcing both small contract laboratories and large pharmaceutical companies to look at new ways to run separation routines safely and efficiently.

Gas and liquid chromatography (GC and LC) rely on the use of organic solvents to isolate analytes for study. These solvents are toxic to both the environment and users, however, and the growth of chromatography has exposed these disadvantages to the general public. Governments are continuing to push for environmentally friendly disposal methods and recycling measures that will continue to increase the expense of using these solvents. Already, LC users have been compelled to seek less toxic and more expensive alternatives to materials such as hexane.

Thar Instruments’ SFC-MS Prep 30 mass-directed system was launched this year at Pittcon. Image: Thar Instruments Inc.

To avoid these costs, some laboratories are adopting conservation practices by reducing the size of their analysis systems or establishing recycling procedures. Others are circumventing organic solvents through the use of supercritical fluid chromatography (SFC), which has gained a wide acceptance in the pharmaceutical industry. Superior resolution, faster separation, and overall higher throughput than high-performance liquid chromatography (HPLC) have already convinced some companies to make the switch. A growing reputation as a “green” technology is icing on the cake. SFC equipment and supply manufacturer Thar Instruments Inc., Pittsburgh, Pa., has attracted the attention of global LC and mass spectrometry (MS) leader Waters Corp., Milford, Mass., and the two have embarked on a mission geared to popularize SFC technologies in the drug discovery realm. In 2005, the companies established a partnership to market and sell new SFC systems that incorporate Waters’ expertise in MS and software.

Recently, that OEM partnership expanded to give Thar access to Waters’ demonstration facilities and corporate R&D resources. The result of this work has been integrated SFC-MS systems announced at Pittcon 2007. Two new systems are scheduled for launch in early 2008.

“The purpose of the agreement is to give our customers access to top of the line software and top of the line equipment,” says Todd Palcic, vice president of Thar Instruments. “SFC is useful not just in chiral applications. More and more, companies are looking for fast analysis techniques,” and SFC promises to address this demand.

The criticality of supercritical

Advantages in resolution, separation speed, and higher overall throughput than many other chromatography methods, including HPLC, are possible because supercritical fluids behave in a particular way. Induced by high pressure and usually high temperature, supercritical compounds have the ability to diffuse through solids like a gas and dissolve materials like a liquid. They are also tunable and can dramatically change density with minor changes in temperature or pressure.

Thar Instruments is leveraging Waters’ expertise in mass spectrometry in developing complete SFC/MS systems. Waters’ 3100 MS, pictured here, is a good match for Thar’s SFC equipment. Image: Thar Instruments Inc.

These properties all have ramifications in analyte extraction. The low viscosity and high diffusivity of carbon dioxide speed up flow rates and reduce back pressures. This creates a faster mobile phase. LC systems use very high temperatures to detect the analyte. Carbon dioxide, however, naturally nebulizes at ambient temperature and high pressure. The sample is more easily analyzed by the detector without the need for extreme temperatures. This is a major reason why SFC is used in decaffeination procedures—it preserves the flavor of the coffee.

Water is a common supercritical fluid, but commercial SFC systems typically use carbon dioxide. Non-volatile, this gas requires comparatively little energy to reach subcritical or supercritical fluidity. That level for CO2 is 31.1°C and 7.2 MPa.

Not every SFC routine requires supercriticality—many of Thar’s systems rely on subcritical flows, according to Palcic—but what’s notable here is the temperature requirement for CO2, which is far lower than with other materials.

Strengthening the case for SFC/MS

Thar Instruments has been developing SFC systems since 1990 and remains the only company in the world designing, fabricating, and selling analytical, preparative, and process scale SFC systems. Why hasn’t the method attracted more development interest?

One of the problems, according to both Thar and Waters, is that researchers have previously focused running SFC as a GC by using capillary columns. As a result, the belief has arisen that SFC only works well on certain applications such as chiral separation. That is not the case as proven by the successful economic scale-up of an SFC purification of fish oil in Europe and palm oil in Malaysia, says Palcic.

Another reason for the misconception has been the higher capital cost of an SFC system and a general lack of robust equipment. Demand for higher throughput has existed since the 1990s, when the pharmaceutical boom began. The focus for development of the largest segment of chromatography, LC, was centered on automation and software rather than fundamental changes in process technology.

Thar was instead hoping to address major bottlenecks in separation—both preparative and analytical—by pursuing SFC. Chiral separation processes would often take months to complete, but SFC equipment cut such procedures down to a week. Companies began to realize that with SFC-MS systems they could move from 1.5 mL/min to 4 to 5 mL/min, a throughput jump of three times. In new mass-directed configurations, which Thar has already launched and sold to customers in the form of the SFC-MS Prep 30, overall throughput increases further.

Currently, Palcic says, the strongest motivation for SFC adoption is cost. The use of SFC for analytical separations and analysis alone could save about $25,000 per instrument per year. In addition, preparatory separation routines are growing rapidly at a much higher rate than analytical processes. Because SFC is suitable for both purposes, Thar is estimating savings of up to $200,000 per year per instrument based on preparative scale separations. The initial premium for SFC-MS is 25% higher than other systems, but these costs could drop as SFC adoption rates rise.

Much of these savings stem from the use of CO2 in purification because SFC methods typically require about 80% CO2 and only 20% organic solvent. Thus, SFC drastically reduces the need for organic solvents. It also eliminates aqueous waste, which is difficult to dispose of and will likely become more problematic as government restrictions increase in the future. The purchase cost of solvents has also increased as the cost of oil and ethanol increases.

Because SFC is closely related to LC, the instrumentation and methods will be familiar to researchers. Thar’s SFC-MS were developed to work seamlessly with Waters’ MS systems, such as the 3100 MS and the 2998 Photodiode Array, and with Waters’ software, MassLynx.

In addition, CO2 is non-flammable and cheap, and reusing this greenhouse gas helps establish a positive “green” image for the technique.

“It’s green for the body, too,” says Palcic. “There are toxicity concerns about leaks with preparative HPLC systems,” and complications regarding storage and handling of volatile organic compounds are growing. Fire marshals in cities such as Cambridge, Mass., San Francisco, Calif., and even Shanghai, China, have taken a close and critical look at what is being used in chemistry laboratories.

Insurance firms have also taken notice, and Palcic says the fire hazard involved with solvents has changed the attitudes of many pharmaceutical companies. In some cases, particularly smaller contract companies, laboratories simply have no available room for more systems and are looking to replace existing machines with higher throughput levels.

Then there is the human factor.

“Companies understandably don’t want to pay chemists to do mundane work like drying down solvent,” says Palcic. “They want them to devote more time to drug discovery.”

Finally, energy is an important driver. Drying down solvent or aqueous waste often requires a rotary evaporator, which consumes a lot of electricity and often needs special power-supply equipment. Using n-heptane in lieu of hexane reduces volatility concerns but requires more energy to dry from the material of interest because it has the boiling point of water. Personnel costs for monitoring this equipment must also be factored in.

“The first and most important aspect (of SFC growth) is the economic driver. Pharmaceutical companies decided in this decade they needed to start saving money. They buy SFC to save money. That’s clear,” says Palcic, who points to Regis Technologies Inc., Morton Grove, Ill., a maker of custom synthetic compounds for clients. They are an example, he says, of a company that has switched entirely from LC to SFC for contract purification services.

In the coming years, Thar anticipates more wholesale shifts in chromatography practice, especially in drug development. In 2008, Thar will launch a new mass-directed system, the SFC-MS Prep 100, which will use Waters’ FractionLynx software.

“The real significance of the agreement will come in the next few months as more companies move into mass preparative SFC. We’ve already shipped out a couple of these systems. This is certainly the most exciting time in our company’s history,” says Palcic.

—Paul Livingstone

Resources
• Thar Instruments Inc.,
Pittsburgh, Pa., 412-967-5665, www.thartech.com

• Waters Corp.,
Milford, Mass., 508-478-2000, www.waters.com