Like many other analysis platforms, HPLC was once considered a sophisticated method dominated by expert analysts or, at the very least, scientists willing to delve into an instrument’s innards. Today, HPLC’s commodity status is generally viewed as a welcome development.
HPLC trends track closely with trends in life science instrumentation: Instruments are becoming more compact and feature-laden as prices stay steady; customers demand higher resolution, sensitivity, throughput, and reliability. Speed is of the essence, particularly for pharmaceutical and biotechnology laboratories and manufacturing environments. Instruments easily mastered by non-instrument specialists or junior-level technicians are highly desirable. For busy life science labs, the goal is to get the job done and concentrate on the science, not the analysis—“shortening the development lifecycle” if you will.
“This is what we hear from customers all the time,” notes Michael Kraft, worldwide product marketing manager for new business opportunities at Agilent (www.agilent.com), “and this is what drives our instrument development.”
Emerging preferences for smaller-footprint analysis paths represent compactness, economy, higher sensitivity, and a “greening” of laboratory work through lower solvent and (sometimes) energy consumption.
Agilent’s HPLC-Chip system, a lab-on-a-chip implementation of liquid chromatography with a mass spectrometer (MS) detector, is an example. The microfluidic analysis chip in the 1200 Series HPLC-Chip/MS integrates columns, connection capillaries, and nanospray emitter directly on a reusable polymer substrate.
According to Kraft, the only limitations of chip-sized analyzers is their marriage to mass detectors (not really a bad thing) and the perception that they are still “only” research-grade instruments. “People aren’t rushing to install them into quality control labs.” The same could be said for HPLC, generally, with respect to process analytic technology (PAT) deployments.
The quest for a generic HPLC detector continues. Today, companies employ specific detectors (UV-VIS, fluorescence, light scattering, mass) based on the analyte of interest and/or the experiment. Even within one detector type, mass, there exists a panoply of designs and ion sources. “In an ideal scenario, HPLC users would prefer one detector for quantifying known and unknown compounds over a wide dynamic range,” says Kraft.
Although HPLC use is widespread in biomanufacturing for purity analysis, verification of release specifications, and stability studies, HPLC applications for real-time analysis has not caught on as rapidly as it might have. HPLC analysis more often than not involves manual sampling and is performed off-line.
Agilent recently teamed up with Groton Biosystems (www.grotonbiosystems.com) on PAT-worthy instrumentation employing Agilent’s 1200 Series LC system and Groton’s Automated Reactor Sampling technology. Together, the products will automate sampling and analysis of amino acids and proteins and provide a closed-loop system for monitoring nutrients in bioreactors. The idea is to tie a continuous, real-time, or near real-time, analyzer to a bioprocess while avoiding manual sampling, off-location analysis, and human intervention wherever possible.
“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.”
Micro to Nano Flow
Eksigent (www.eksigent.com) provides HPLC systems and fluid delivery mechanisms that enable rapid, super-accurate HPLC at micro- or nano-flow scale. The flow controller for the company’s NanoLC product line, for example, precisely controls solvent gradients at flows as low as 20 nL/min; the NanoLC2D does the same for 2-D chromatography. For methods development, the company offers the ExpressLC800 system, with eight HPLC columns operating in parallel.
According to Mark Atlas, marketing director, the eight-column instrument is ideal for users who need to devise a new separation method from fermentation samples. Users can test an analytical run on eight different columns or up to eight unique combinations of column and elution conditions, for example, temperature, solvent, gradient.
The LC800 uses a common autosampler but can direct samples to any of the columns, each of which has its own pump. Aided by a robust data-management platform, operation is a one-person job.
One LC800 instrument currently resides in a bioproduction facility but not strictly within GMP manufacturing. Atlas says this customer uses the instrument for “final product evaluation.” He believes adoption of HPLC for PAT is “just a matter of time,” but that that time, alas, is not today.
“PAT is evolving,” Atlas adds. Users are still evaluating the best options available and not committing to a single technique like HPLC.” HPLC analytics are evolving, too. “People are reluctant to adopt anything too rapidly, even if it appears to be the best solution.”
Companies are interested in nanoscale HPLC for QC and during process development, he says. “Because of the low volumes, nano-LC can pick out very low levels of contaminants from a fermentation, even when one component is present at a high level.”
The development of true nano-level fluidics has been the key to nano-LC’s success. Solvent stream splitting simply will not do for reproducible, reliable protein analysis using nanospray MS. “People used to think they could just use a splitter. That’s not true. When doing nanospray MS, you need a true nanoflow system.”
For example, Eksigent’s ExpressRT-100 HPLC instrument performs rapid, integrated sampling and diluting, with direct injection into a microflow HPLC. The ExpressRT is used in process optimization and monitoring, but Eksigent markets the device for synthetic organic reactions.