Although less than a decade old, UHPLC—ultra high performance liquid chromatography—has become the de facto performance standard despite lagging far behind conventional HPLC in total systems sold. UHPLC refers to about a dozen rapid, very high-pressure (above 5,000 psi) LC systems employing stationary phase particle sizes of around two micrometers or smaller. Hence the equivalence of “sub-2-micron” and UHPLC.
The terms UHPLC and UPLC™ are often confused. UPLC, which stands for “ultra performance liquid chromatography,” is a trademark of Waters Corporation (Milford, MA), but the technology is generically known as UHPLC. So while every UPLC is UHPLC, the reverse is not true.
Continuous improvement
UHPLC debuted in 2003 with Agilent’s 1200 series, which used 1.8-micron particle “rapid resolution high throughput” (RRHT) columns. The next year Waters introduced its Acquity UPLC system, which employed 1.7-micron bridged ethane-silicon hybrid particles. Today every major HPLC manufacturer manufactures a highpressure system: Thermo Fisher (Accela), Jasco (Xtreme- LC), Shimadzu (UFLC-XR), Hitachi (LaChrom Ultra), Scientific Systems (Ultra HP), Dionex (RSLC), Knauer (Platin Blue), and PerkinElmer (Flexar). Each product introduction builds on the strengths of previous systems.
According to Bill Foley, director of LC Product Marketing at Waters, UHPLC has entered the mainstream. “UHPLC is pretty much the standard or benchmark today, particularly in quality control and for repetitive HPLC workflows.”
Customer input has led to numerous upgrades in detectors, autosamplers, column chemistry, and methods. Most mass spectrometers that Waters sells, for example, are sold with UPLC systems. Other detector modes today include optical detectors, photodiode array detectors (PDAs) operating at extended wavelengths, and enhanced fluorescence detectors. These innovations result from the need for speed, throughput, and sensitivity required by evermore- complex samples and low-abundance analytes.
Surface chemistries, which give columns their selectivity, are somewhat more limited for UHPLC compared with HPLC; but UHPLC is catching up, with numerous vendors offering a full range of column chemistries. Waters has recently introduced charged surface hybrid (CSH) chemistry, which applies a uniform positive or negative surface charge (in acidic and basic mobile phases, respectively,) in addition to a surface chemistry. The company also recently added size exclusion/gel permeation in sub- 2-micron format, raising its UHPLC column chemistry offerings to eleven.
HPLC physics, particularly with respect to how particle size and flow affect resolution, apply to UHPLC, and predict that UHPLC will provide superior resolution, sensitivity, and speed. But obtaining these benefits requires taking a holistic approach to LC system design, which must be matched to the chemistry, bandspread, and dispersion. For example, the size of flow cells and the length and internal diameter of the tubing is reduced, data acquisition rates for the detectors must be higher, and filter time constants must be optimized.
Cost versus performance
Capital outlays for UHPLC—today, between 20 percent and 40 percent higher than for HPLC—remains a sticking point for potential adopters. Yet one can make a reasonable case that sub-2-micron HPLC can save many labs money in the long run.
The most obvious way is through its higher throughput and productivity. “For some labs you can replace two, even three HPLCs with one UHPLC,” Foley says. Slower flow rates translate to significantly lower solvent consumption as well. LC-grade solvents are notoriously expensive, but labs pay twice for them: on acquisition and through waste generation. Disposal costs for some solvents are almost as high as the purchase price.
The flip side of replacing several instruments with one higher-throughput system is the “all your eggs in one basket” argument: Downtime and repairs can be disastrous when the only HPLC available stops working.
“Some customers see a dramatic payback,” notes Foley. Considering the productivity gains, savings in solvent and disposal, reduction in bench space and controlled environments, and ongoing operating costs, UPLC is priced “pretty reasonably.”
The payback argument becomes even more compelling when the value of high-quality data is factored in. Foley provides several examples where the difference between good data and bad data can cost millions of dollars: quality control for pharmaceutical batches, pesticide levels in foods, quantities of incriminating evidence at crime scenes. “Everyone in the analytical chemistry business is looking for an edge, an advantage,” Foley tells Lab Manager Magazine. “Clearly, UPLC allows them to see things in their samples they could not otherwise detect.”
The naysayers
UHPLC’s introduction caused something of a schism—that persists (although not as strongly) to this day—among HPLC experts.
UHPLC promised much faster throughput and the potential for higher resolution. But detractors noted that speed and resolution were often at odds in very highpressure systems. The other major knock against UHPLC was that methods were not easily transferred from 3- or 5-micron particle systems to the newer sub-2-micron formats. Methods remain a sticking point for UHPLC, particularly in environmental, safety, pharmaceutical, and forensics industries where data integrity is paramount.
There were many other bones of contention as well, including cost, whether UHPLC was overkill given the capabilities of current 3-micron particle columns, and operational issues.
Foley admits that UHPLC may not be for everybody, as conventional HPLC systems still strongly dominate LC sales. “It might not make sense to revalidate or reconfigure legacy methods,” he says, particularly in highly regulated industries such as pharmaceuticals/biotech, or where data is routinely presented in a court of law.
Nevertheless, developing UHPLC methods or porting over HPLC methods to UHPLC is easier than ever. Most vendors now offer kits, consisting of a sub-2-micron and conventional column version of the same chemistry, to facilitate method transfer.
Methods development groups, which make dozens or hundreds of injections for one project, love UHPLC because the shorter run times, coupled with automation, allow them to conduct experiments that are more thorough in less time than with conventional HPLC.
Where research labs are inclined to adopt UHPLC, standard analytical labs have been reluctant to jump on the sub-2-micron bandwagon in large numbers. UHPLC is found in only about 10 percent of QA labs, according to Wilhad Reuter of PerkinElmer. “UHPLC demands more from the operator—more vigilance and savvy. It’s very effective, but there’s a learning curve.”
UHPLC solvents must be carefully filtered using a 0.2-micron membrane, and those who take shortcuts with their solvents sooner or later pay for it, Reuter says. Samples are filtered as well. Users should avoid opening purge valves and system leaks that at UHPLC pressures can cause serious damage to the instrument.
While no one argues that UHPLC runs are much shorter, some are dissatisfied with equilibration time for column chemistries and pressures, particularly when using gradients. To reduce waiting time, pumps are being designed without pulse dampers or with lower-volume dampers. Pulse dampers are components that smooth out pump pulses to create a uniform flow.
Against the backdrop of UHPLC’s alleged shortcomings and notable achievements, one should remember that traditional column packings, particularly 5- and 3-micron particles, still dominate the HPLC market, with a share of at least 80 percent. Even 10-micron particle columns are still serving legacy methods.
Tom Jupille, an HPLC trainer and consultant with LC Resources (Walnut Creek, CA), likens 5-micron particles to a Toyota Camry, and sub-2-micron particles to a Ferrari Testarossa. “The Ferrari is faster and sexier, but it’s also phenomenally expensive, requires a much more skilled driver, and spends more time in the shop.”
He argues that most users would do better to optimize their standard-particle HPLC systems than to invest in UHPLC. The reasons he gives are a higher standard of operator training, cost, and the established base of equipment and methods.
Jupille admits the technological brilliance of UHPLC and that some users will definitely benefit. Yet he believes that sub-2-micron is little more than another stop in the continuing evolution of HPLC. Jupille also has nothing but praise for the leaders in UHPLC systems, calling Waters’ UPLC “brilliant… their original Acquity system blindsided the competition.” But with the introduction of the Agilent 1290, he says, that company finally managed to surpass Waters’ specs on pressure and flow. “Not enough to make a Waters shop switch, but it should prevent Agilent shops from defecting.”
Agilent’s 1290 Infinity was the first LC system, according to the company, capable of running standard HPLC as well as UHPLC columns. Agilent’s advertising campaign for the 1290 recognizes the lingering controversy surrounding very high-pressure LC: The 1290, according to the company, “ends the debate.”
Early on one could argue that UHPLC was overpriced, says Helmut Schulenberg-Schell, Ph.D., Agilent’s worldwide LC marketing manager, based on cost and a narrow application range. “But with the introduction of the 1290, there is no longer a reason not to try UHPLC since the investment hurdle is eliminated.”
More recently, in June, 2010, Dionex (Sunnyvale, CA) announced that henceforth all its LC systems, including its entry-level units, will be capable of both HPLC and UHPLC. All that is required to switch between modes is changing the column swap. “Users can run legacy five-micron particle methods just as easily as sub-2-micron methods,” explains Phil DeLand, market development manager for pharmaceuticals.
Dionex is retaining its original line and model designations for the new “UHPLC+” instruments, as well as physical presentation and – most importantly – the original pricing. This essential eliminates “capital costs” as a barrier to UHPLC acquisition.
Dionex has also upgraded oven temperatures, to 110º C, to reduce mobile phase viscosity and improve flow rates. And, through its acquisition of ESA Biosciences last year, it can provide UHPLC-capable charged aerosol and electrochemical detectors in addition to conventional detection modes.
Early UHPLC spent a lot of effort promoting the technique, but chromatographers perceived the UHPLC- HPLC choice as an either-or decision, says Phil DeLand of Dionex. “But there’s no fundamental reason it has to be that way. Nothing prevents making every module UHPLC- and HPLC-capable.”
Shimadzu (Columbia, MD) was one of the last major manufacturers to introduce a UHPLC instrument. For years the company’s position seemed to be that a lot of life remained in supra-2-micron systems.
For example, by employing columns with 2.2-micron particle sizes operating at 10,000 psi, Shimadzu’s XR systems provide the performance of sub-2-micron UHPLC without the pressure-related drawbacks, says Simon Robinson, HPLC product manager at Shimadzu.
Robinson recently clarified his company’s positions, past and present. The HPLC instrument business can only go as far as current column technology will take it, he explained. Shimadzu was not initially impressed with the available choices of sub-2-micron particle columns. “But that has changed. Today the market is much more customer-driven,” and it’s clear that at least a subgroup of users prefer the higher-pressure systems. “The [column] situation is still not ideal, but it has opened up quite a bit.”
Although it is not universally adopted and its drawbacks are often exaggerated, UHPLC is recognized by all as chromatography’s crowning achievement. Is a UHPLC system in the stars for your next LC purchase? That depends on your lab’s workflow and the relative value you place on performance and cost or familiarity.
“You’ve got to enter this market cautiously,” reminds Robinson.
Fast alternatives at modest pressures
Several column technologies promise something approximating sub-2-micron performance but without the extreme pressures or need to purchase new systems. One such technique is core shell technology from Phenomenex (Torrance, CA). Agilent (“superficially porous”) and Supelco (Fused Core™) have similar products.
Core shell technology uses silica particles consisting of a solid inner core, with chemistries bonded atop a porous outer shell. Standard silica particles are porous throughout, in a configuration that David Schock, marketing manager at Phenomenex, calls “a spherical sponge.” With conventional media, the mobile phase and solutes diffuse throughout the entire particle. In core shell, solutes remain mostly on the particle surface, experiencing anywhere from 20 percent to 50 percent less stationary phase during their travels down the column and, therefore, faster elution.
According to vendors, core shell extends the capabilities of conventional HPLC equipment by providing separations as good as those achieved on sub-2-micron columns, but without the high backpressures and need to purchase new equipment. Core shell’s only drawback is relatively low capacity, meaning it is not suitable for preparative LC.
Another “fast” method employs monolithic columns, which are essentially a single rod of porous silicon or polymer encased in a metal or plastic jacket. Monoliths are intriguing for the numerous selectivity possibilities and rapid elution. Numerous vendors now supply them, including Phenomenex (Onyx), Bia Separations (CIM Disk, CIM Tube), Agilent (Bio-Monolith), Merck Chemicals (Chromolith), and Dionex (ProSwift).
For a complete UHPLC resource, visit our UHPLC page on Lab Manager.
