Product Focus: HPLC Systems

Employment Trends, Modularity, UHPLC, and New Life for Liquid CO₂

Many labs today face downsizing, a trend that began before the economic downturn of 2008. “Employers are more likely to retain analytical scientists who can not only press buttons and achieve the appropriate results, but who can also quickly perform unaided troubleshooting, develop methods, and generate consistently reliable data,” says Ade Kujore, marketing specialist at Cecil Instruments (Cambridge, UK). HPLC systems offering ease of control, diagnostics, repair, maintenance, and system testing help scientists meet these goals.

Modular HPLC systems provide versatility in terms of troubleshooting and repair as well as choice of mobile phase (through the ability to swap out columns rapidly). Consequently, some experts predict a decline in the all-in-one instruments that have dominated HPLC for the last two decades. The downside for vendors, says Ms. Kujore, will be higher expectations for customer support.

She suggests that before purchasing a new system, users consider:

• Service response time and average downtime
• Detector specifications, such as low drift, noise, and stray light
• Pump specifications, such as low pump pulsation and speedy and accurate gradient mixing
• Autosampler specifications, carryover, injection precision, and availability of accessories
• Column heater/chiller specifications; for example, compartment size, temperature ranges, speed of temperature changes, and the accuracy and stability of required temperatures
• Modularity, including for thirdparty autosamplers and liquid handling systems
• Frequency of routine maintenance, time involved in mobile phase or column changes, and cost of consumables

The eternal question

The dilemma over HPLC versus UHPLC has not disappeared, despite some vendors declaring the matter settled and a few stragglers insisting that anything that occurs on UHPLC is achievable at five microns or higher.

While column particle diameters of three to five microns still dominate, the lures of high sensitivity, speed, and low sample/injection volumes tug at every chromatographer’s heart. UHPLC involves more complex sample preparation, more frequent maintenance, and a greater attention to detail than conventional LC. Methods do not often transfer faithfully between the platforms. Laboratories— particularly those with legacy HPLC systems and methods— should ask, frankly, whether they have the time and the will to switch over and revalidate all their critical methods.

The introduction of superficially porous (“core shell”) particle columns promising UHPLC performance on an HPLC-class instrument has further complicated purchase decisions. Columns based on superficially porous materials may not be available in all separation formats, however, and they do require tweaking the instrument for optimal performance.

Finally, some experts, who will remain unnamed due to their dwindling numbers and influence, still believe that anything that occurs on a UHPLC is achievable on an ordinary HPLC.

Bottom line: conventional supra- 2-micron HPLC and UHPLC are both here to stay and are still vying to find their optimal niches.

Gaining momentum

According to Simon Robinson, HPLC product manager at Shimadzu (Columbia, MD), the switch to UHPLC is gaining momentum. “People are putting in the time and doing the transition,” he says. HPLC Systems But Robinson warns that method transfer remains an issue that will not go away “with a couple of mouse clicks.” The issue is not so much difficulty as it is the time investment, which Robinson describes as a “hardship” whose rewards are speed and throughput. Users can get lucky and scale directly to fast LC, but most of the time this will involve “some degree of method development.”

Several systems today provide software and other tools that ease method transfer. At Pittcon last year, Agilent introduced ISET (Intelligent System Emulation Transfer), which promises seamless transfer of methods between LC and UHPLC systems. At the time, Christian Gotenfels, Agilent’s 1290 Infinity LC product manager, predicted that ISET would “revolutionize LC method transfer” by minimizing cost and effort involved in development, validation, and execution of UHPLC/HPLC methods.

Many labs in regulated industries still employ old columns and systems that occasionally become scarce, even in the used equipment marketplace. The large, concentrated injections made on older LC systems can hide a multitude of sample preparation and matrix effects, while exceeding a certain volume or concentration on UHPLC results in “a lot of tailing and some ugly-looking chromatography,” Robinson says. Achieving the superior results promised by UHPLC “requires a change in mentality. Analysts need to be more precise and careful with sample prep.”

Another go at CO₂ mobile phases

Waters (Milford, MA) first commercialized sub-2-micron particle HPLC in 2004 and remains a leader in both UPLC® (its trademark) and larger particle technologies. Senior product manager David De- Pasquale tells Lab Manager Magazine that adoption of UPLC was slow at first but is picking up. “Only a few vendors took the holistic approach to sub-2-micron, where modules were specially built to handle the high back pressures.” Today, with nearly every manufacturer offering UHPLC (the generic term for sub- 2-micron), DePasquale believes that instruments claiming pressures significantly higher than 15kpsi to 18kpsi will face diminishing returns for the back pressures involved.

One factor fostering adoption of small particle LC is the appearance of verifiable methods in the U.S. Pharmacopeia, as well as among food and environmental methods. Before these methods arose, users had to choose between older technology and the burden of validating methods.

At Pittcon 2012 Waters introduced a new twist on UHPLC that uses supercritical CO₂ as the primary mobile phase. Ultraperformance Convergence Chromatography™(UPC²™) separates nonpolar molecules that would normally be candidates for normal phase. Many companies are reluctant to run these, however, due to the flammability and purchase/disposal costs of hydrocarbon solvents

Waters prefers not to use the term “supercritical” when discussing the system because of supercritical CO₂’s reputation of being difficult to work with. But DePasquale offers assurance that “we’ve solved all the issues with liquid CO₂.” Waters hopes that the new format will achieve greater acceptance than supercritical LC because it tackles both chiral chromatography and more conventional normal phase separations.

For additional resources on hplc systems, including useful articles and a list of manufacturers, visit www.labmanager.com/hplc-systems. For more information on UHPLC, go to www.labmanager.com/UHPLC