It’s been known for decades that using smaller particles and higher pressure increases the capabilities of liquid chromatography (LC). It has only been in about the past five years, though, that advances in technology made it easier and more economical for researchers to move from high-performance liquid chromatography (HPLC) to ultrahigh-performance liquid chromatography (UHPLC). Some platforms actually perform both technologies. To make this transition more economical, researchers can even turn to columns that generate UHPLC-like performance from an HPLC platform.

“Liquid chromatography is one of the biggest tools used in drug discovery,” says Joe DiCeasare, PhD, fellow scientist at PerkinElmer, which is headquartered in Waltham, Mass. “Depending on the size of the company, it could have dozens to hundreds of liquid chromatographs in R&D through quality assurance.”

In moving from HPLC to UHPLC, says DiCeasare, “a big opportunity is the improvement in productivity and throughput, plus a reduction in solvent consumption.” Overall, UHPLC uses shorter and smaller inside-diameter columns, as well as smaller particles. Typically, HPLC uses particles that are about 5 ?m in diameter, and UHPLC uses smaller particles, sometimes below 2 ?m in diameter. In general, all of the components—column size, solvent, and so on—are reduced in moving from HPLC to UHPLC.

click to enlarge Some instruments, such as the Thermo Scientific Accela shown here, can run HPLC or UHPLC protocols. (Image: Thermo Fisher Scientific)

Although UHPLC is not really new to the market, Michael Frank, PhD, product manager for HPLC systems and solutions at Agilent Technologies, in Santa Clara, Calif., says that UHPLC is just starting to gain real penetration in drug discovery and development. “With UHPLC, you can get a separation in a minute not hours,” Frank says.

As one might expect, all of the benefits of UHPLC also create a few obstacles. “UHPLC’s biggest challenge is that it generates much higher back pressure on your system, and that requires more-sophisticated instrumentation, which typically comes with a higher price,” Frank says.

In addition, HPLC retains some strong points. “One is that it’s been available much longer, so providers offer a broad choice of columns,” Frank says. “The number of columns with different selectivities or stationary phases for different types of separations is not as big for UHPLC, yet.”

Getting more with less
In today’s LC environment, companies give customers a range of options, often distinguished by their maximum performance pressure.

For example, PerkinElmer offers its Flexar FX-10 and FX-15 UHPLC systems, which work at pressures up to 10,000 and 18,000 pounds per square inch (psi), respectively. (Originally, the FX-15 only went up to 15,000 psi.)

When moving applications to higher-pressure LC, some researchers expect increased complexity. But DiCeasare says, “It’s easy. All companies have a method-transfer application, and it lets you take parameters from HPLC and convert that to new parameters to use with UHPLC.”

click to enlarge To ramp up the pressure in liquid chromatography, researchers can turn to tools like the Flexar FX-15, which works at pressures as high as 18,000 pounds per square inch. (Image: PerkinElmer)

The efficiency of LC or the ability to distinguish narrow peaks is typically defined as plates/meter. With a UHPLC system relying on sub–2 ?m particles, says Michael McGinley, bioseparation product manager at Phenomenex (Torrance, Calif.), the efficiency is 200,000 to 250,000 plates/meter. By using a conventional HPLC system, though, and Phenomenex’s Kinetex Core-Shell LC columns with 2.6-?m particles, McGinley says, that it’s possible to get more than 275,000 plates/meter. He adds, “You can’t walk up to a stock HPLC and get 275,000 plates/meter with these columns. It takes some minor modifications, including going to tubing with a smaller inside diameter and replacing the flow cell.” Still he adds that, with these changes, “We get performance on par with or better than sub–2 ?m fully porous media.” Besides the physical changes to the instrument, says McGinley, “the system will be pretty much the same, and you shouldn’t need to change protocols.”
For customers already using UHPLC, Phenomenex offers Kinetex Core-Shell columns with 1.7-micron particles. “UHPLC users can get well over 300,000 plates/meter,” says McGinley.

Other companies aim to increase efficiency by focusing on flow. Reducing the flow in LC offers a range of potential benefits, including higher throughput and reduced sample volume requirements. For instance, Don Arnold, PhD, founder and general manager at Eksigent, which has its headquarters in Dublin, Calif. and is now a division of AB SCIEX, says, “We’ve carved out our area of expertise in the micro- and nano-flow areas.” Nonetheless, Arnold points out that though microflow HPLC offers some nice advantages, there “has been a reluctance to operate in the micro-flow regime, because earlier generations of the technology were less robust.” Because of that, Eksigent started off by taking on the most difficult challenge first, which is nano-flow. “That’s where the most challenging problems lie,” Arnold explains. “With a very small diameter column, you need to be more sensitive to extra-column variance,” such as inconsistencies introduced by variations in tubing, fittings, and so on.

The reduced column diameter also works best with high-tech pumping. “We have a unique pumping approach,” Arnold says. “At these low flow rates, delivering accurate and repeatable flow is not trivial.” To solve that issue, Eksigent uses a pneumatic amplification pumping system with flow meters on every pump that measure the flow rate and provide adjusting feedback tens of times per second.

Keeping results accurate, however, also depends on having an accurate flow cell. When this cell only contains enough volume for a hundred nanoliters or so, small geometric variations make significant differences in the volume. To control the accuracy of the flow cell, Eksigent turned to computer chip-like microfabrication allowing excellent dimensional precision.
Accuracy is key when LC gets small in size and high in pressure. For example, Eksigent’s ExpressLC-Ultra uses columns with a 0.5-mm internal diameter and can handle pressures up to 10,000 psi to deliver the advantages of UHPLC and microflow HPLC in a single system.

Help from hybrids

click to enlarge Some of today’s instruments for liquid chromatography also help researchers automate processes. As shown here, for example, a scientist is loading the autosampler in Agilent’s 1290. (Image: Agilent Technologies)

Like cars running on petroleum or electricity, some LC systems—including Agilent’s 1200 Infinity Series and the Thermo Scientific Accela—can take on HPLC or UHPLC columns. Agilent’s 1220 and 1260 Infinity LCs, for instance, can work with pressures up to 600 bar (about 8,700 psi), and the 1290 Infinity LC can go as high as 1,200 bar (over 17,000 psi). The latter, says Frank, “is required for narrow-bore UHPLC columns.”

In making these hybrid LC systems, the engineers at Agilent changed only the parts that needed to be changed. “The others have been kept constant,” says Frank, and “this makes the system able to be 100% compatible with conventional HPLC methods, but also being able to run UHPLC methods.”

Despite the added capabilities of Agilent’s 1200 Infinity Series, it remains economical. As Frank says, “We can offer the UHPLC performance, in the 600 bar systems, in the same price range as our previous HPLC systems. 1,200 bar systems are still at a higher price point.”

The Thermo Scientific Accela systems also bring researchers the hybrid option. “With such a flexible instrument, you can keep the current methodology you are using in HPLC and—little by little—transfer the methods to UHPLC,” says Sergio Guazzotti, PhD, global product marketing manager, liquid chromatography at Thermo Fisher Scientific, headquartered in Waltham, Mass. He adds, “This is particularly important in pharma, where methods are validated.”

In the past, explains Guazzotti, no one had the technology to put HPLC and UHPLC in one instrument without sacrificing performance. To maintain performance in hybrid instruments, Thermo Fisher Scientific combined a pumping technique with force feedback control. “That allows the measurement—in real time—of the compressibility of the solvent with every piston stroke in the pump,” Guazzotti explains. Moreover, this leads to reduced pulsation without the need for pulse dampeners, which Guazzotti says, “gives a better discrimination of peaks from baseline, thereby improving the limits of detection.”

In drug discovery and development, researchers will continue to move to UHPLC, if for no other reason than speed. “With sub–2 ?m particles,” says Guazzotti, “we can perform a run in one minute.” That speed really adds up by the end of the clinical trial phase, which Guazzotti says can take about 90,000 LC runs for just one compound.

Parts beyond pressure
Although increasing pressure pushes LC ahead, other factors come into play, as well. To keep LC systems cranking up the sensitivity, the systems need to detect smaller concentrations of compounds. Accordingly, Agilent introduced it 1260 Infinity Diode-Array Detector, which, according to the company, offers up to a 10-fold higher sensitivity at the same price as its predecessor. Such an increase in sensitivity enhances the limit of detection or lets a user reduce the sample volume.
As pressure increases and components improve, even more advanced forms of LC will make their way into drug discovery and development.

About the Author
Mike May is a publishing consultant for science and technology based in Houston, Texas.