Unlike many other mature analytical technologies, HPLC seems to have entered a period of intense innovation and competition. During the last decade we have witnessed the debut and evolution of ultra high-performance LC (UHPLC), widespread adoption of mass detectors, and greater appreciation for rapid methods based on novel or revived column technologies.
Dr. Stefan Schuette, Sr. Marketing Director for Liquid Phase Separations at Agilent Technologies (Waldbronn, Germany), identifies the major trends in HPLC as:
- UHPLC via both totally porous sub-2µ particle columns and superficially porous columns
- Multi-method/walk-up or “open access” systems that allow method-switching
- Bio-inert, metal-free UHPLC systems for sensitive biopharmaceuticals
- 2D LC
- Automated, online sample and standards preparation
- The revival of supercritical fluid chromatography (SFC) for both chiral and achiral applications
- “Green” LC achieved through solvent-sparing small-diameter columns’ supercritical mobile phases
- Mobile HPLC by which instruments are brought to the sample
Yet the features most users look for in HPLC have not substantially changed, according to Schuette. “Users continue to seek performance, productivity in terms of speed and cost per analysis, data quality, and backward/forward compatibility. In other words, faster, cheaper, better.”
Analytical labs were at one time interested primarily in reducing the cost per sample. No longer, says Simon Robinson, HPLC Product Manager at Shimadzu (Columbia, MD), who sums up the overriding trend in HPLC instrumentation as: “Speed, speed, speed.” Companies are most concerned, he says, with getting through large numbers of samples, generating data quickly, and effectively managing time and human and physical resources.
For years the major technological trends in HPLC were instrument-related, says April DeAtley, Product Planning Manager for LC at PerkinElmer (Waltham, MA) and to a significant degree they still are. “Everyone was concerned with who had the highest pressure systems, or the latest bells and whistles.” Today, at least from PerkinElmer’s perspective, usability has moved to the top, or close to the top, of manufacturers’ priorities.
“Current users are less experienced in chromatography, but more comfortable around ‘technology,’ than were previous generations,” Ms. DeAtley says. “That is why HPLC, and analytical chemistry itself, are trending toward touch technology where users interact less with the instrument itself and more with the computer.” She predicts that in the future methods will be “dialed up” rather than developed and tweaked by the user, similar to the way users operate consumer electronics. We have not quite reached the point of iPodlike control, “but within a few years we will definitely see instruments that are ‘applicated,’ where users select a method and go.”
Frank Steiner, Ph.D., Manager for Small Molecule Solutions at Thermo Fisher Scientific (Munich, Germany), concurs that system developers need to design UHPLC for accessibility and ease of use. “Customers don’t want to have to undergo a lot of training to exploit these instruments fully,” he says.
One could ask if the apparent decline in analytical skill may be in part caused by the growing reliance on advanced interfaces, or is it the other way around—users simply don’t need to know as much about the inner workings of their instruments?
“It’s probably a bit of both,” Ms. DeAtley says. “In school we learn manual calculations in class and never carry them out again. To some extent the experience factor has declined because users just don’t need to know or do some of these things anymore.”
Compared with HPLC, UHPLC provides improved resolution, sensitivity, and throughput through the use of sub-2µ particles, typically packed in 2.1mm or 1.0mm ID (internal diameter) columns. UHPLC is characterized by very high back-pressures resulting from the mobile phase passing through ultra-small particle beds packed tightly in long, thin columns. These factors result in a reduction in resolved peak elution volume—provided the instrument is optimized to reduce unnecessary volumes along the sample’s flow path.
UHPLC speeds separations, which generates more data per unit time than conventional HPLC. Acquiring, managing, and reporting that data demands a faster data acquisition rate and chromatography data systems with scalable capabilities.
The success of sub-2µ UHPLC has been a vindication for Waters’ (Milford, MA) strategy to introduce fast, low volume, very high pressure LC. Waters shipped the first such instrument, trademarked UPLC© (Ultra Performance Liquid Chromatography), in 2004, and all major vendors have followed suit. Generic sub-2µ particle LC is referred to as UHPLC or u-HPLC.
UHPLC shows the highest uptake in QC labs, where the majority of installed LCs are located. Contributing to this ongoing momentum will be changes to USP Chromatography and equivalent chapters in other pharmacopoeias, which allow greater flexibility for changing column dimensions and/or particle size.
Method development becomes less time-consuming with the improved workflows that faster LC provides, and this has led to enhanced software for rapid method screening, statistical analysis, and the rapid, iterative generation of robust methods. “This process was not feasible before UHPLC,” observes Elizabeth Hodgdon, Senior Product Manager at Waters (Milford, MA).
Stefan Schuette defines UHPLC in terms of column technology rather than system, pressure, or detector speed. “We refer to all types of LC employing stationary phase particle sizes of less than 2µ as UHPLC, for example columns packed with 1.7µ particles, with a 3mm or 4mm internal diameter and a length of 15mm, which can achieve very rapid runs at high resolution,” but at pressures normally associated with conventional HPLC.
Next to the ability to withstand very high pressures, the single most critical design feature for UHPLC systems, according to experts, is minimizing dispersion or band spreading. Dispersion arises from volumetric factors within the instrument that cause peaks to elute in larger volumes, thus eroding the high resolving power of small-particle columns. For this reason, vendors trim extraneous volumes when designing instruments.
Users need to consider and control factors affecting band spreading as well. Dispersion is tolerable in relatively large-volume HPLC systems, but in UHPLC, tubing IDs and lengths should be minimized, and care should be taken in making connections. For example, 5µ particle columns have void volumes of about 3mL, which provides acceptable resolution for HPLC. But void volumes for most sub-2µ columns are just 10 percent as large. “Failure to optimize the system to the requirements of the new column will result in no gain, or even worse performance than with conventional HPLC columns,” notes Bill Letter, Sr. Scientist and Consultant at Chiralizer Services (Newtown, PA).
The need for speed, as exemplified by UHPLC systems, brings other benefits that are now taken for granted, such as reduced usage of mobile phase and smaller sample injections. But these present their own challenges, for example the precision of injection volumes, carryover, system maintenance, column selection, and temperature stability. These issues caused a significant backlash against UHPLC during the mid-2000s and persist to this day.