“Instrumentation flexibility, automation, and ease of use to improve the overall efficiency in processing samples is as strong today as it has ever been,” says Ed Long, strategic marketing manager at Thermo Fisher Scientific (www.thermofisher.com). To Long, the most significant HPLC trend of late has been the use of triple quadrupole mass spectrometry, a technique he describes as once specialized and sophisticated but now common.
Traditionally, technologies are adopted by research labs first, or by smaller companies, where they undergo trial by fire before larger firms or manufacturers accept them as standard. Long notes that manufacturing-worthy instrumentation has requirements that go beyond accuracy, reliability, and operational robustness.
“In manufacturing situations, customer needs evolve beyond simple instrument performance and capabilities.,” adds long. “Significant issues include data-handling software for turnkey reporting, ease of use, improved automation, and greater connectivity to other software applications such as LIMS, batch and lot records management, and instrument maintenance and validation.”
Thermo Fisher Scientific’s layered applications such as LCQUAN software provide a secure data acquisition and computational package for regulated (GLP/GMP) companies. LCQUAN 2.5, the latest version of the product, is a 21 CFR Part 11-compliant data acquisition and archiving package that operates with the company’s LC/MS products.
A good deal of pioneering work on HPLC continues in academic and government research labs. Frantisek Svec, Ph.D., lead scientist at Lawrence Berkeley National Laboratory, has been at the forefront of HPLC work since the early 1990s. Dr. Svec’s research interests include novel stationary HPLC phases, including monolithic/capillary phases for both HPLC and capillary electrochromatography. Another research focus, with potentially tremendous but probably distant interest for biomanufacturers, are enzyme-immobilized stationary phases that will digest proteins within the column before separating peptides and amino acides for proteomics studies.
Dr. Svec sees greater use of high temperature and pressure, originating from the adoption of ultrasmall (less than 2-micron) particle sizes in stationary phases. The smaller the particle, the faster and more efficient the separation, which raises an interesting physical/mechanical issue for chromatographers.
In HPLC, the diffusion of analyte molecules from the mobile phase onto the solid phase, where they interact with the surface chemistry, and back again into the solvent is critical. The smaller the particles, the shorter the diffusion path, and the faster the separation. “However, we have reached the point where the interparticle voids are also smaller, about 20% of the particle size,” says Dr. Svec. Pushing mobile-phase flow through these voids requires higher pressure to achieve a desirable flow or column velocity. Another approach is to increase the column temperature, which causes mobile-phase viscosity to fall while speeding up molecular diffusion.
Monolithic columns, another specialty of Dr. Svec, can overcome high pressure requirements. Monoliths are formed not by packing particles into preformed columns but in situ from precursor materials generated within the column. Monoliths are attractive for small-bore columns as well as microfluidic HPLC devices.
Dr. Svec collaborates with several chromatography vendor companies on HPLC, including Dionex (www.dionex.com), which holds an exclusive license from Cornell University for polymeric monolithic columns (Dr. Svec worked at Cornell from 1992 to 1997). Dionex manufactures columns as well as instruments and systems.
At “Pittcon 2007” the company introduced numerous columns and products for sample preparation, auto-sampling, and eluent regeneration. The company’s monolithic columns include reverse-phase and ion-exchange formats. Interestingly, Dionex is pushing HPLC as a medium for process analytics.
Another player in monolithic columns is Merck (www.merck.de), which has licensed technology from Japan’s Kyoto University for manufacturing silica monolithic columns. These are created by oxidizing silanes in situ in the presence of a porogen (pore-forming material) such as polyethylene glycol. A 2002 U.S. patent describes the manufacturing process, which encases the monolith in a stainless steel or fiber-reinforced polymer casing.
The only downside of monolithic columns is their relative newness. A report from Bristol-Myers Squibb (www.bms.com) from 2003 suggested that monolithic columns work as well as traditional particle-packed columns for pharmaceutical analysis. But, as Dr. Svec points out, packed-column HPLC has been around for more than 30 years. “The industry is conservative. If something works people are reluctant to change it. Plus, many analytical methods are on file with regulators, which can also slow down adoption of new technologies.”