Shrinking the
LC Landscape
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More than a decade ago, chip-based technology made its debut. Now, researchers are waiting to see its impact on the LC market.
O
ANTHONY FERNANDEZ
n a June afternoon inside the Palais des Congrés in Montreal, Canada, Karen Hahnenberger of Eksigent Technologies, based in Livermore, Calif., greeted onlookers with a smile. She caught their attention with her poster presentation at the HPLC 2002 conference and explained the technology of nanobore separation columns with 75- to 150-µm i.d. and electrokinetic flow controllers that provide 50- to 500-nL/min flow rates. At the end of the day, she had about 40 business cards, which pleased her. “I think [the concept of electrokinetic flow control] was new to quite a few people,” recalls Hahnenberger. “I did spend quite a bit of time on the technology, the capability of miniaturizing and multiplexing [HPLC].” When miniaturization of total analysis systems (µTAS)—a.k.a. “lab-on-a-chip” research—made its debut more than a
Cheryl M. Harris
F E B R U A R Y 1 , 2 0 0 3 / A N A LY T I C A L C H E M I S T R Y
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COURTESY FRED REGNIER
One of the early chip-based HPLC systems was presented by Fred Regnier in 1998. It was an ~8-cm quartz wafer separated by 1.5 10-µm rectangular channels. Monolithic support structures acted as particles packed in a column.
decade ago, analytical chemists quickly began applying the technology to CE, capillary eletrochromatography (CEC), and GC. Although miniaturizing those techniques quickly proved fruitful, doing the same to LC was a different story. The difficulties of microscaling pressure-based LC systems, of fabricating efficient separations devices on which nanoliter volumes flow in an area the size of one’s hand, became far more challenging to solve. Nevertheless, researchers pressed on, especially in industry. “The HPLC market is huge, and if one can capture that [through chip-based HPLC], there’s a big win associated with it,” says David Rakestraw of Eksigent. But unless companies can reach or exceed the performance of conventional HPLC, there’s limited value in it, he adds. Companies must come up with a “missionary sell” for the pharmaceutical and proteomic laboratories demanding faster systems with higher throughput, adds Fred Regnier of Purdue University. “The people that made the first instrument are like the missionaries . . . [who] will win a bunch of people over. The question is: How many will they win over?”
Opening the floodgates When Andreas Manz, D. Jed Harrison, and the late H. Michael Widmer published their groundbreaking work on µTAS in 1992 (1), Harrison realized their experiments with electrophoresis on chips were “the start of a new industry” (2). 66 A
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Soon after, J. Michael Ramsey’s group at Oak Ridge National Laboratory published its fundamental papers on µTAS (3, 4), and the floodgates opened. What followed were reams of research papers on chip-based separations from laboratories around the world. The earliest work on µTAS involved CE and CEC, because it’s easier to apply a high voltage to a chip than pressure, say experts. “The main concern with pressure-driven separations is how to make the pressure connection,” says Ramsey. CEC also turned out to be more forgiving with poorly packed columns, add researchers. But with a CE system, researchers aren’t free to choose the buffers they want, says Manz, who is now at Imperial College (England). “And the beauty of [pressure-driven systems] is you can choose the solvents or the buffers you like.” Injection and detection problems, along with nonuniform heating across the columns, also diminished some of the excitement for CE, says William LaCourse of the University of Maryland–Baltimore. “CE was supposed to kill all of LC and put it out of business,” says LaCourse. “It never did.” Despite its drawbacks, electrochromatography continues to be explored on chips, which puts its research ahead of pressuredriven, chip-based systems. Experts say relatively little is currently published about chip-based LC, and the research being done is mostly kept secret within company walls. “People are definitely cautious with what they disclose to the public market,” says Chris Phillips of Nanostream, Inc., in Pasadena, Calif. The limited number of academic papers could be attributed to the overall arduous task of miniaturizing the liquid world onto a chip, say researchers. “The GC guys are just able to boost the temperature up with a little heater you put on the chip. Well, it’s hard to make the LC gradient,” says Regnier. “And it turns out that mixing two liquids on a microscale is not a trivial process, believe it or not.” Evaporation when handling samples is also a big problem with miniaturizing LC, says LaCourse. In addition, he adds, at such small sizes, detection becomes a problem, and LC doesn’t have the diversity of detectors that GC has. Despite the obstacles facing them, some researchers are forging ahead to downscale LC, especially for the pharmaceutical and proteomic industries where the HPLC market continues to be strong and profitable. While GC is applicable for gas-phase and other small molecules found in chemical agents—making it ideal for environmental analysis—LC is the best approach for analyzing biological toxins and larger molecules, especially proteins, explains Rakestraw. Manz says he’s currently experimenting with a chip-based LC device that has three or four columns attached end-to-end in a loop, which increases the peak capacity. The molecules would be pumped around the loop, but at different speeds, depending on their chromatographic properties, and a detector would see the same component multiple times, he says. “I think that’s
F E B R U A R Y 1 , 2 0 0 3 / A N A LY T I C A L C H E M I S T R Y
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COURTESY OF NANOSTREAM
one of the beauties that chip technology could bring into the chip, researchers in government laboratories also toiled away. picture . . . that you could do multidimensional separations During the late 1990s, scientists from Sandia National Laboraeventually, or that you could feed back a sample in a clever way tories experimented with miniature-scale detection systems for chemical and biological warfare agents in their MicroChemLab to get these cyclic separations.” One of the early systems to attract attention was presented project. Currently, the research involves chip-based electrophoreby Regnier at the 1998 Micro Total Analysis Systems confer- sis, but scientists are in the throes of exploring chip-based LC, ence in Banff, Canada. The device had an elecroosmotic pump- says Duane Lindner of Sandia. “The ultimate objective is to ing system, a 100-pL mixer, and monolithic columns on a ~8-cm quartz wafer separated by 1.5 10-µm rectangular channels (5). The combined cross-sectional area of channels across the width of a column was ~500 µm2. The overall system functioned as a chromatography column, says Regnier. The cross-sectional area served as the column, and the monolithic support structures acted as particles packed in a column. One could fabricate 10 million structures on a single wafer simultaneously, he adds. Regnier’s work impressed many researchers and company officials. Applied Biosystems later licensed the technology from Purdue University. “Basically, [Regnier] made . . . many ‘islands’ on a chip. By this, he simulated this particular packing exactly,” says Jean-Pierre Chervet, founder of LC Packings, Inc., headquartered in The Netherlands. “This is a very nice approach Nanostream’s 24-plex LC chip uses on-chip splitters to multiplex solvent flow down channels. Each because it’s fully controlled. It’s channel has an injector port (left) allowing ~400-nL samples to be introduced at ~10 µL/min/column. very homogenous and has definite advantages.” But Regnier and other researchers know this system isn’t marketable. The deep-reactive make a device that incorporates both gas and liquid separaion etching on quartz chips made the technology too labor-in- tions.” Researchers are developing low-volume injectors, he tensive and expensive. The chip-based systems being produced says, and they’re looking at how the geometry of a chip-based now are made of less-expensive polymeric materials, such as LC system affects the separations process. For example, says polydimethyl-siloxane, says Regnier. Also, the fabricated Lindner, a “serpentine” column is more effective than a straight monolithic support structures that act as packed particles in his one, but if the curves in a coiled column aren’t designed propdevice are nonporous, so the system doesn’t have a lot of sur- erly, dispersion can occur. Rakestraw, who had been working at the national laboratoface area, he says. Then there is the problem of the system being too small to couple with detection systems. “When it’s ry then but left when he and a group of researchers formed Ekthat small, it may be a little bit harder to couple it to electro- sigent in 2000, recalls Sandia spending about $25 million to spray and things like that,” says Regnier. Finally, the 10-µm develop microchannel-based separation techniques that had channel depth poses problems for UV detection, “but that’s multiple channels running in parallel. Now working privately, he and his co-workers are finding out just how competitive the true of all chip-based systems,” he adds. While scientists in academia experimented with LC on a race to get the right chip-based LC system to market can be.
COURTESY OF EKSIGENT TECHNOLOGIES
Agilent offers capillary-column LC systems with 75- to 100-µm i.d. that are coupled to an ion trap MS. About four years ago, Agilent and Caliper Technologies Corp. joined together to market a microfluidic device for bioanalysis that’s similar to CE-on-a-chip, says Curt Novak of Agilent. But Novak says company officials won’t disclose whether or not they are working specifically on chip-based LC technology. However, he adds, “we do want to follow it closely and participate in the trend in the marketplace.” Brian Murphy of Waters says the company is interested in downscaling LC further but realizes that even miniaturizing the capillary LC column is fairly new. Things take One of Eksigent Technologies’ HPLC chips (above) contains an integrated sample injector and a time, he adds. “Currently, the iner15-cm separation column. A version of the company’s nanoscale HPLC system (not shown) uses tia in this market is huge, and peoelectrokinetic flow controllers to generate flow rates of 50–500 nL/min. ple take baby steps,” says Murphy. “People are not ready to drop what they’re doing and transfer over to “The companies who have put effort into [chip-based LC] chip-based LC. . . . It’s a leap—it’s a big leap from where we aren’t really that interested in sharing with the world what the are today.” secrets are.”
Pumps: Working under pressure Building the perfect product To face the challenge of developing the right chip-based system, microfluidic companies monitor the changing LC landscape and listen carefully to potential buyers already heavily invested in LC products. The government, for example, spends a few hundred million dollars in research, whereas the giant pharmaceutical companies will spend billions, says Rakestraw. “We don’t want to say to pharmaceutical companies, ‘Hey, change your technology to fit what we do,’” says Surekha Vajjhala of Nanostream. “We’re saying we are going to make sure that you can do things the way you’ve always done them, just faster.” Ultimately, one wants to have multiple separations running in parallel and have that in a compact format, adds Rakestraw. Start-up companies, such as Eksigent and Nanostream, are experimenting in planar nanoscale LC systems for the market, and older companies like LC Packings, Agilent Technologies, Inc., and Waters Corp. have already come out with commercial systems in which only the LC capillary column has been miniaturized. Chervet of LC Packings says today’s miniaturized columns are mainly used in proteomics and offer high sensitivity. “In other words, instead of using 5 mL of blood from a patient, we can do it with a droplet and get the same information,” he says. 68 A
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The most interesting part of chip-based LC is the interface between the chip and the outside world, particularly the pump, or whatever generates the pressure, says Manz. The analytical chemist won’t care how the pressure is generated on an LC system as long as the separation is not affected by it, he adds. One of Eksigent’s pumping approaches is to use electrokinetic pumps, or flow controllers, that are ~3–4 cm in length. “Having a pump that allows you to accurately control flow rates at these levels is a key component,” says Rakestraw. The electrokinetic flow controllers are small capillary tubes with porous material in them, and a voltage is applied across them. The company’s chips are about a few centimeters in length and ~5 mm wide. Electrokinetic pumps can theoretically generate pressures in the range of 100,000 psi, whereas traditional HPLC systems, with column lengths generally of 10–25 cm, would typically provide 5000 psi, says Rakestraw. Electrokinetic pumps are known to generate very high pressures, which allows researchers to use smaller particle sizes and longer columns for better separations, he says. But high pressures aren’t necessarily needed for chip-based LC systems, adds Rakestraw. Nanostream researchers have developed parallel LC-on-achip that allows multiple chromatograms to be performed simultaneously. One critical element is the splitter, say Phillips
and Vajjhala. By using several splitters to separate the solvent into precise volumes, a single pumping source can be used, they say. Researchers can operate at pressures
