Nearly a decade after the debut of high-pressure, sub-two-micron- based liquid chromatography, the trend toward UHPLC (ultra-high-performance liquid chromatography) persists. “This is amply demonstrated by our sales numbers, as well as by independent market studies and customer surveys,” says Michael Frank, senior director, LC Global Marketing at Agilent Technologies (Santa Clara, CA). The principal advantages of UHPLC are faster separations and higher resolution. However, many UHPLC customers still use conventional column diameters such as 3.0mm to 4.6mm ID for their sub-2μm particle columns. “Narrower column IDs are primarily seen in LC-MS setups,” Frank explains. Only a few UV-LC labs have moved on to narrow-bore columns. “The reason is probably that the larger ID columns are still more forgiving in point of sample preparation (i.e., less susceptible to clogging).”
On the system side, Frank sees a “higher loading” compared with the past—more users per system, more samples, and longer system operation times— to the point of overnight and weekend operation. “We know several big customers who continuously examine the loading of their analytical systems with the goal of improving them—an exercise that is purely cost-driven.” This leads to higher demands on instrument uptimes; for example, in total solvent volumes pumped through before exchanging out a seal or in a higher number of valve switches before initiating maintenance.
Since Waters’ (Milford, MA) debut of its branded UPLC®, the trend toward columns employing ever-smaller particle sizes has continued. “Users were looking for speed and efficiency, with resolution equal to or better than older methods,” says Michael McGinley, bioseparations product manager at Phenomenex (Torrance, CA).
According to McGinley, the more recent trend is toward “geometrically designed” stationary phases. Available from many vendors, these are known as superficially porous, porous shell, or fused core, or as Phenomenex’s own variety, Core-Shell.
The general construction of these columns is similar: a homogeneous porous shell is grown around a solid silica particle. Materials diffuse only into the shell, versus into the core of the particle as in traditional HPLC. The result is more rapid, efficient mass transfer into and out of the particle, thus faster separations. Proponents claim that superficially porous columns provide many of the advantages of UHPLC without the extremely high backpressures.
An interesting aspect of porous shell technology is that columns are available for all pressure regimens, from older HPLC models up to the most modern UHPLC systems. Phenomenex, for example, produces Core-Shell columns with particles as small as 1.3 microns. “We’ve gone past the issue of higher pressure,” McGinley explains. “Users can pick a column that’s appropriate for the pressure their system can handle, even down to 200-bar.”
Users are still restricted by the laws of physics, however: smaller-particle porous shell columns work only on high-pressure systems, while largeparticle columns work on any system.
According to Simon Robinson, HPLC product manager at Shimadzu (Columbia, MD), HPLC is experiencing something of a lull in terms of radically new technology. While fused core columns are emerging with novel bonded phases, Robinson says this platform is underutilized. “It could one day live up to its promise, but the customer base has not embraced it as enthusiastically as it could have. It looked like it was really going to take off two years ago, but now it appears to be holding steady.” Robinson says that most users he encounters employ either legacy methods at five microns or sub-twomicron but have not had the opportunity to explore superficially porous technology.
HPLC purchase decisions
HPLC purchase decisions depend strongly on the application. However, since chromatographic equipment is typically a sizable investment, users expect a long service life. Practically speaking, users should consider the ease with which they upgrade systems as newer technologies are commercialized. “Performance is typically a key decision point, but here it is critical to look at the big picture; for example, 1200 vs. 1300 bar pressure capability translates to approximately 2 percent higher chromatographic efficiency,” says Agilent’s Michael Frank. “Whereas more sensitivity or linear range on the detector might make a difference in terms of hours of analysis time spent per samples, since additional runs or other detectors might be involved.”
System flexibility is another factor, as many labs demand rapid changeover between different chromatographic conditions, to the point of unattended changes.
Especially for regulated labs, the capability to run legacy methods and obtain exactly the same results as with conventional HPLC is critical. This means new methods, utilizing the increased power of new systems, that run on the same system as legacy methods, without the need for expensive method changes and revalidations.
Robinson believes that potential buyers should consider pressure flexibility as a significant feature when performing due diligence on new HPLC instrumentation. Systems capable of both high- and low-pressure operation can save direct costs and minimize the use of precious laboratory space. “You’re looking at one set of parts, one software platform, and a single set of service and technical knowledge.” In modern labs, particularly highthroughput establishments, the issue is not necessarily instrument productivity but getting more from limited benchtop space.
“An LC-MS system dedicated to high-pressure operation is a very expensive investment,” Robinson observes. “The time and cost of method revalidation is another concern for many companies, which is why many HPLC methods are specified for standard pressures below 5000 PSI.”
Lest one assume that the trend toward faster, higher-pressure, more expensive instruments has left labs of modest means in the dust, Eric Anderson, general manager at Buck Scientific (East Norwalk, CT) assures us that the market for entry-level LC instruments is alive and well. “Our niche market is for the little guy who doesn’t need everything,” Anderson says. “People who buy from us are more concerned about their budget than anything else.”
Customers include smaller schools and laboratories, some of which require three or four instruments but lack the resources for more than one or two. High-end users, for example, pharmaceutical and biotech companies, are not typical purchasers of Buck’s HPLCs.
Buck sources components like pumps and detectors from the 15 to 20 OEMS who specialize in these parts. It then assembles HPLC systems into systems run and controlled by Buck’s proprietary software. “The software interface is everything,” Anderson says. “Anyone can put a system together. Making all the pieces work well together is much more difficult.”
Buck can customize in terms of detectors and pumps, but it does not get involved with very highpressure systems or with mass detectors. Over the years the company has fashioned rebuttals to most arguments for sourcing only top-of-the-line LC systems. “It should come down to whether our specifications accommodate your application,” Anderson advises. “It’s pretty rare when they don’t.”