Product Focus: UHPLC

Improving Throughput at Both Ends

All major vendors of ultra high-performance liquid chromatography systems (UHPLC) have overcome significant problems related to detection, speed, column life (although users still grumble), usability, interface, and the special circumstances of working at very high pressures. The next frontier in rapid chromatography is one every analytical scientist will recognize.

“We’re now capable of performing super-short UHPLC runs, but sample prep has failed to keep up. It has become a bottleneck,” says April DeAtley, product planning manager for LC at PerkinElmer (Shelton, CT).

The solution, which top instrument firms are working on, is integrating UHPLC systems with online or inline sample preparation.

There are many ways to streamline sample prep, limited only by available robotics, automated liquid handlers, and affinity resins. The only hard-and-fast requirement is that it should involve as little input from users as possible and not interfere with the chromatography.

Realizing the goal of automated chromatography sample prep will require that instrument manufacturers work closely with companies that they are not used to dealing with. DeAtley stresses that solving “the customer’s puzzle” will involve much more than good chromatography systems or outstanding automation. “This requires us to think more laterally than we normally do as LC product managers.” The same could be said for designers of automation or sample prep disposables.

DeAtley believes the other extreme of the sample life cycle—detection— could use improvement as well. She notes that outside of pure pharmaceutical R&D, samples and matrices tend to be complex. Analytes exist in low concentrations, and their concentrations (or even their presence) are not always predictable.

The good news is that UHPLC is more versatile and modular compared with other analysis methods. Modern systems utilize multiple detectors where appropriate, and systems already accommodate a good deal of automation.

DeAtley is confident that by stepping outside their comfort zones UHPLC system manufacturers will eventually “develop the answers that customers really need.”

Reconsidering conventional HPLC

Unlike other UHPLC vendors, Jasco Analytical Instruments (Easton, MD) does not do most of its business with pharmaceutical and biotech companies. The reason, says chromatography product specialist D.J. Tognarelli, is software compatibility. Large-volume customers employ data systems that sometimes control and run a dozen or more UHPLC systems. “When you control 100 instruments with one software package, acquiring a stray instrument, with its own learning curve for instrument and software, doesn’t make much sense,” Tognarelli says.

Two related factors affecting purchase decisions are experience and compatibility, whereby customers become “XYZ-vendor shops.”

Jasco prefers to vie for customers of one-off systems within academia or smaller biotech companies. Here, the company can compete in terms of cost and features, particularly in offering instruments along the continuum of HPLC and UHPLC.

What makes this possible is the emergence of porous shell (also known as superficially porous and trademarked Core-Shell and Fused-Core) column technology. “With these columns, users can obtain performance similar to that of UHPLC but on more standard HPLC systems,” Tognarelli explains. “Purchasers can save some money on the initial system purchase and not have to deal with the high pressures of UHPLC.” Aside from cost, the principal pressure-related advantage of porous shell technology is that columns last longer.

According to Tognarelli, porous shell columns have resparked interest in his company’s core HPLC/UHPLC customers. “Users are beginning to reconsider the older HPLC platform.”

Yet the trend toward higher-pressure systems employing sub-two-micron particle size columns is in no danger of subsiding, at least for pharmaceutical and biotech customers. “It’s hard not to be impressed with the performance of UHPLC systems,” Tognarelli admits. “Cutting analysis times by 90 percent is significant. Unless they’re absolutely strapped by budget constraints, labs that have a UHPLC from a major vendor will probably stick with that product line.”

One area in which Jasco is doing quite well is supercritical fluid chromatography. The company offers units for routine analytical applications (with a specialty in chiral separations), semi-preparative, prep, supercritical fluid extraction, and a system dedicated to fuels analysis.

Taming pressure pulses

Aside from expensive solvents, analytical columns are the single largest ongoing expense in UHPLC. This issue has a long history.

As column chemistries other than C18 came online, users began to observe poor column life. The problem is particularly acute in the case of 2D HPLC and its variants. “Since the frits are really small on these columns, and the columns themselves are expensive, users employ track columns followed by a back flush step,” notes Simon Robinson, HPLC product manager at Shimadzu Scientific Instruments (Columbia, MD). “This cuts down on sample preparation that no one enjoys and reduces consumables while generating data that is more reproducible.”

Column life has, to some degree, held back 2D LC applications. When switching columns in a high-pressure system, the dead volume increases and, as the valve switches, the system experiences a pressure pulse. “Each time you do that the column takes a very hard hit,” Robinson says. Plate counts and separation ability degrade fairly quickly as the solid phase rapidly compresses and expands, smashing into the column frits and breaking apart.

Degradation caused by pressure spikes in part explains why a column would last 500 injections on one system but only 80 injections on another, or similar discrepancies between similar columns from different vendors installed on the same system.

According to Robinson, the problem is not pressure per se, but pressure fluctuations. “We discovered five years ago that if you leave a column in place at fifteen to twenty thousand psi for a week, it’s fine. If you cycle the same column at much lower pressure through 100 injections, you see an immediate degradation of performance.”

System designers have solved many of these problems through reduction of dead volumes and use of makebefore- break valves when applicable. With these valves, flow is not interrupted between load and inject operations. Column construction has also improved. Through a combination of these improvements, chromatographers can now perform multidimensional separations without having to worry about valves and columns. This applies as well for method scouting systems that might hold five or six columns.