product review
Prep LC systems for chemical separations Chromatography is not just for analysis. Steve Miller
O
riginally introduced in the 1980s, preparative HPLC (prep LC)— along with its bigger sibling, process HPLC—has become a major tool for the production of specialty chemicals. In the past 10 years, prep LC has evolved from a fairly expensive curiosity to a routine chemical isolation technique in many labs because of its speed and efficiency. It is not surprising that many of these labs are in the pharmaceutical industry, where fast turnaround and rapid development correspond to profits. “Prep LC is an expensive method,” says Georges Guiochon of the University of Tennessee and the Oak Ridge National Laboratory. “You can get excellent results if you are willing to pay for [them]. That is why so many of the applications are in the pharmaceutical industry. The FDA [U.S. Food and Drug Administration] demands purity, and the additional cost is justified in order to obtain it.” The killer application for pharmaceutical processing has been the separation of chiral compounds, in which one enantiomer has desirable pharmacological activity while its partner can be useless or even harmful, and chiral stationary phases provide rapid separations of the two. The most basic definition of prep LC comes from Cecilia Mazza of Waters Corp. “Depending on the application, prep LC involves sample recoveries from micrograms for analytical standards to kilograms for clinical evaluation or production,” she says. “Actually, prep LC can be done on any scale as long as you © 2003 AMERICAN CHEMICAL SOCIETY
are collecting fractions. Some labs are going to smaller-scale prep to evaluate new products.” On the pilot and full production scales, process LC systems isolate compounds at rates of thousands, or even millions, of kilograms per year in columns as large as 2 m in diameter. One industry insider estimated that the current annual market for prep LC instruments is in the $100–200 million range, and the annual prep LC column consumption tops $300 million. Annual growth is 5–15%, according to company representatives. Table 1 lists selected manufacturers of prep LC instruments. Some experts believe that prep LC can be applied to a much wider range of chemical syntheses. “The economies of scale for chromatography are enormous compared to other purification methods, but [prep LC] is often overlooked because it requires a large capital investment and because of a lack of familiarity with the technique,” says Anita Katti at Kennesaw State University. “The biggest problem that I see for prep LC is the need
to educate synthetic chemists and chemical engineers [about] its capabilities.”
Not your analytical chemist’s HPLC The basic principles of prep LC are the same as those of the instrument that inhabits most modern analytical chemistry labs. A liquid eluant is pumped through a column containing a stationary solidsorbent phase. The chemistry of each component of an injected mixture determines the ratio of the time a molecule spends adsorbed on the stationary phase to the time it spends dissolved in the moving liquid phase. The retention differences cause the individual com-
N O V E M B E R 1 , 2 0 0 3 / A N A LY T I C A L C H E M I S T R Y
477 A
product review
Table 1. Manufacturers of selected prep LC systems. Company
Flow rates (mL /min)
Detectors
Target markets
Agilent Technologies www.chem.agilent.com
10 –100
UV, MS
Pharmaceuticals and proteomics
Amersham Biosciences Corp. www.amershambiosciences.com
10 –100
UV, conductivity
Research instruments for pharmaceuticals and therapeutics
Biotage, Inc. www.biotage.com
25 mL/min to 17 L/min
UV, pH, conductivity, RI
Pharmaceuticals, peptides, and fine chemical process chromatography
Gilson, Inc. www.gilson.com
1–200
UV–vis, MS
Analytical to prep-scale chromatography
IRIS Technologies www.iristechnologies.net
30 mL/min to 1000 L/min
UV, pH, conductivity, RI
Process-scale pharmaceuticals, biotech, and fine chemicals
Knauer GmbH www.knauer.net
100 –1000
UV, IR, fluorescence, ELSD, conductivity, amperometric
Universal HPLC applications
NovaSep www.novasep.com
250 mL/min to 250 L/min
UV
Process LC systems at all scales
ELSD: evaporative light-scattering detector
RI: refractive index
pounds to separate into bands as they move through the column. In practice, analytical and prep LC techniques are quite different. “In scaling up from analytical to prep LC, you have to forget everything that you have learned,” says Guiochon. “At very high concentrations, you obtain broad bands instead of sharp Gaussian peaks, and the high concentration of one component can affect the retention of other compounds.” There can also be problems at the higher concentration if you exceed the solubility of the compound in the eluting solvent, he adds. David Keller of Sonntek agrees. “Analytical versus prep is night and day—you can’t just scale up from an analytical method,” he says. “Prep requires a mindset change. You saturate the column with a big slug of material and keep trimming off the edges to get as much material through as possible and still get the purity.” And unlike the trial-and-error process common in analytical method development, people design prep LC separations by thermodynamic calculations before starting, he notes.
Basic instruments An across-the-board comparison of the prices and functions of prep LC instruments is not possible because most man478 A
SFC: supercritical fluid chromatography
ufacturers offer a wide range of pump, detector, and software combinations that vary significantly in cost. In general, a small system designed to isolate gram quantities of product using a UV or refractive index detector will fall somewhere in the price range of $35,000– 75,000. Fractionation is triggered as the sample passes through these nondestructive detectors, and the collected fractions are analyzed to determine the purity of the product. Although some basic instruments are available off the shelf, most manufacturers prefer to work with the customer, designing an instrument to the specifications of the proposed application. Systems designed to isolate milligram-to-gram quantities of material—often designated as semiprep chromatography—operate at flow rates of up to ~100 mL/min. Despite the size of these columns—up to 1 in. diam—the operating conditions, flow rates, and chromatographic conditions will seem familiar to the analytical chromatographer. Larger benchtop units, designed to deliver compounds in gramto-kilogram quantities, operate at solvent flow rates up to ~1000 mL/min. These systems incorporate columns that are several inches in diameter and packed with larger particles than those used for conventional analytical methods. LC systems designed for kilogram or larger
A N A LY T I C A L C H E M I S T R Y / N O V E M B E R 1 , 2 0 0 3
preparation scales, with pumps delivering more than a liter per minute of solvent, are generally too large for benchtop use and are delivered as freestanding skid systems.
Stepping it up a notch The earliest prep LC systems were considered difficult and touchy because they needed intensive hands-on monitoring—sometimes even manual operation of the fractionating valves. But prep LC today is becoming a black box instrument. “We see the key to successful applications as designing a system that is easy to use,” says Christian Grotenfels at Agilent Technologies. “The synthetic chemist does not need to also be an instrument expert. Ideally, you just need to type in the sample name and click a button.” The entire process may be a bit more complex than that, but not much. Numerous instrument manufacturers, including Gilson, Agilent, Waters, and Varian, have adapted extremely selective detectors, such as MS, and extremely sensitive universal detectors, such as evaporative light-scattering detectors, to prep LC applications. Because these detection systems are destructive, a small stream must be diverted from the eluant flow to the detector. According to Mazza, an automated, high-
product review
Table 1. Manufacturers of selected prep LC systems (continued). Company
Flow rates (mL/min)
Detectors
Target markets
Sonntek www.sonntek.com
100 –1000
UV, RI, fluorescence, chiral, amperometric
Pharmaceuticals, biotech, and protein analyses
SSI/Lab Alliance www.laballiance.com
12–250
UV, RI, fluorescence, chiral, amperometric
Life sciences markets
TechniKrom www.technikrom.com
20 mL/min to 200 L/min
UV
Pharmaceutical and nutraceutical prep and process LC
Thar Technologies www.thardesigns.com
50 –350
UV, chiral
SFC systems
Varian, Inc. www.varianinc.com
50 –3200
UV, RI, pH, conductivity
Pharmaceutical prep and process LC to multi-kilogram scale
Waters www.waters.com
10 –300
UV, MS, ELSD, conductivity, fluorescence
Pharmaceutical and peptide production from microgram to multigram scale; MS-controlled fractionation; total solution, including user training
ELSD: evaporative light-scattering detector
RI: refractive index
throughput system with MS control of the fractionation can process 10,000 samples per month. This ability does not come cheap, however. High-end benchtop prep LC systems can cost as much as half a million dollars.
Specialized techniques Several manufacturers offer instruments that incorporate techniques beyond normal or reversed-phase chromatography. Thar Technologies, specializing in supercritical fluid chromatography, offers a prep-scale supercritical fluid chromatography system that costs $100,000– 250,000. According to the company’s Todd Palcic, supercritical carbon dioxide offers substantial savings in the mobile phase, compared to typical solvents such as methanol. Not only is the solvent less expensive, it avoids the laborintensive stripping of solvent from the product. But, Guiochon notes, “Technically, HPLC with a good solvent offers a better separation, and it is less expensive. On the other hand, when you perform the separation in carbon dioxide and water, and maybe a small amount of ethanol, you avoid any possibility of toxic solvent residues in your product.” Simulated moving bed (SMB) systems perform a continuous separation, as opposed to the batch separation of most chromatographic processes. In a
SFC: supercritical fluid chromatography
true moving bed system, the sorbent phase travels in a direction opposite the flow of the solvent. Prep LC systems incorporating SMB simulate this process by switching the flows in a bank of half a dozen or more prep columns. In practice, SMB is applicable only to binary separations, so it is particularly useful for separating enantiomers with no other impurities, according to Guiochon. One component is more retained in the stationary phase and the other is more soluble in the mobile phase, so as the flows are switched within the series, one goes with the liquid and the other stays with the column. “A simulated moving bed continuously purifies the moving stream,” says Keller. “It is exceptionally efficient because it uses the whole column, all the time.” Several fullscale SMB production methods have received FDA approval. And for a really big separation . . . process-scale LC moves beyond the lab and into a manufacturing environment, using flow rates as high as hundreds of liters per minute. Two companies that specialize in process equipment, Biotage and NovaSep, also manufacture prep LC systems. Because of the scalability of LC separations, these instruments are particularly useful for lab separations or pilot operations for manufacturing processes.
Coming up Most manufacturers have cited recent interest in prep LC for the production of nutraceuticals, pharmacologically active natural products. Applications range from small-scale prep LC for isolation and testing of active compounds to full-scale production facilities to purify kilograms or tons of material. A second growth field for prep LC is large biological molecules, such as proteins and oligonucleotides. Because these compounds are found in extremely complex mixtures and are generally heatsensitive, LC may be a particularly useful technique for isolating and identifying them. “The future potential for prep LC is very big, but it has not arrived yet,” says Keller. “The biggest problem is getting past the large initial capital investment to take advantage of the savings and benefits of liquid chromatography.” Steve Miller is a freelance science writer based in State College, Pa.
N O V E M B E R 1 , 2 0 0 3 / A N A LY T I C A L C H E M I S T R Y
479 A
