Enhancing Speed and Sensitivity
A new version of HPLC, ultra-high-pressure liquid chromatography or U-HPLC, is sparking explosive interest in the scientific community, especially those in drug discovery, according to Ed Long, LC and LC/MS marketing manager, Thermo Fisher Scientific (www.thermo.com). His colleagues will put up a poster on the novel technique at Pittcon.
“Many scientists are finding compelling benefits to this technology, almost as if it’s a magic elixir, because it can be not only faster, but better. Conceptually, both HPLC and U-HPLC are based on similar principles of instrumentation and chromatographic separation science,” explains Long. What has changed is the use of small-particle columns (silica-based packing with particle dimensions of less than two microns in diameter).
“These require higher-pressure systems to suitably handle the higher backpressures. With Fast LC or U-HPLC, however, chromatography separations can now be reduced to run times in seconds instead of minutes or hours. This dramatically increases the number of samples processed per unit time, increasing chromatographic throughput.”
Long notes that the use of smaller particles and/or shorter columns can lead to increased sensitivity and better resolution of components. “The smaller particles provide increased theoretical plate generation, which results in higher efficiencies over a broader range of mobile phase linear velocities. The high efficiency results in more peak capacity, or peaks per unit time, with sharper peaks that typically generate higher signal-to-noise values. Many pharmaceutical companies have been the early adopters of the U-HPLC or Fast LC technology. By exploiting improved sensitivity and resolution, these systems have been applied to difficult separations and small molecule analysis, including impurity profiling, metabolite screening and identification, and pharmaceutical development.”
When laboratories transfer their methods to these newer and faster systems, Long suggests that care must be taken to ensure that operating flow rates, gradient profiles, and injection volumes are appropriately scaled to obtain an equivalent or superior separation. “Almost everyone will have to make some changes, but this impact depends on where you are in the process. For some, this can be direct and simple, but others, such as in QA/QC, will need to prepare thorough documentation. However, at the end of the day, the benefits for doing this far outweigh the time spent performing method transfer.”
In addition to being a complete supplier of these technologies, Thermo Fisher now offers Turboflow™ technology. Rohan Thakur, Ph.D., strategic marketing manager, says, “Turboflow is a technology that provides a significant benefit for atmospheric-pressure-ionization-based bioanalysis or LC-MS/MS. TurboFlow columns thoroughly clean up protein debris and other macromolecules responsible for causing ion suppression from biological samples. So, it’s a valuable and synergistic technology to minimize sample preparation, enhance sensitivity, and reduce matrix effects.”
For scientists preferring to work within the HPLC environment, upgrades and enhancements are available. Curtis R. Campbell, Ph.D., product manager for Shimadzu Scientific Instruments (www.shimadzu.com), says, “Scientists can benefit from new and improved HPLC systems and specialty software programs. These programs are wrappers for traditional chromatography data systems and allow high-quality data to be collected by less technically savvy users.
“Shimadzu offers one example in Prominence MD, a method-development program. This system lends itself to drug discovery because it allows the user to submit many screening runs with just a few mouse clicks—easily selecting from up to ten different column and mobile-phase combinations for these runs. The system automatically switches between columns, mobile phases, etc. and monitors the system until such time that it is re-equilibrated before moving to the next run. Once the run is complete, it is automatically emailed to the user for evaluation.”
Dr. Campbell notes that scientists can also improve separation and analysis through the choice of columns. “For example, HPLC performance can be pushed even further using the Shimadzu XR 2.2-µm particle-size columns.
“The choice of this particle size, along with stringent QC on the silica support for optimal packing efficiency, gives performance equal to sub-2-µm particle columns at system pressures of conventional HPLC equipment, expanding the capabilities of existing lab equipment. The Prominence Series HPLC is ideal for this kind of application.”