Featured Article
Wednesday December 14, 2005
Working with biomolecules—particularly proteins—is a tricky process, as sample sizes are often small or very dilute. So scientists who want to prepare their proteins for mass spectrometric analysis frequently use capillary or nanoscale liquid chromatography (nano-LC), which both have the high sensitivity and low flow rate capacity required for such applications.
Although these systems generally work well, they do have some drawbacks—most notably, the complexity of the instruments themselves. In conventional nanoscale HPLC systems, a pump delivers fluids at nanoflow rates through an autosampler connected to a small (100 micron internal diameter (ID)) enrichment column. This column is then connected to a 50-150 mm, 75 micron ID capillary analytical column that’s interfaced to a nanospray MS source. “The problem with that approach is basically all of the small connections and tubings and fittings that you have to put together … the problems with leaking, troubleshooting, band broadening caused by dead volumes in the connections—all of those are issues that both compromise the reliability of that setup and the robustness, as well as the chromatographic performance of the column,” says Agilent product manager Georges Gauthier.
Given the recent boom in proteomics, it’s little wonder that a number of companies have either improved existing—or developed entirely new—nanoscale chromatography systems. Eksigent Technologies, for instance, developed the NanoLC 1D and 2D HPLC systems, which use microfluidics technology to control flow, eliminating the need for splitters and associated plumbing. And all system components—including the pump, injection valve, and columns—are completely integrated, reducing chromatographic band broadening and decreasing run time, according to the company.
Agilent has also developed a microfluidic system called the HPLC-Chip that alleviates many of the problems associated with conventional HPLC, including dead volumes and multiple connections, by integrating the enrichment and separation columns, hydraulic connections, and electrospray MS interface in a single device. According to the company, the HPLC-Chip reduces the number of connections and fittings required for the typical nano-LC setup by 50 percent. Says Gauthier: “Everything is put together now with seamless connections, there’s no connective tubing, there’s no capillaries, there’s no fittings, there’s no dead volume,” he explains. The system’s first release is designed to work with Agilent’s ULTRA Ion Trap mass spectrometer.
Users looking for a high-throughput microfluidic LC system may want to consider the Veloce system from Nanostream, which employs ready-made, 24-column Brio cartridges that contain all the necessary plumbing. These cartridges, in conjunction with the Veloce system’s eight-needle autosampler for 384-well plates, allow users to run multiple samples in parallel very quickly, and are ideal for drug discovery applications, according to company literature.
Another alternative, Waters Corporation’s nanoACQUITY UPLC (ultra-performance liquid chromatography) system, is designed to be used with column packing particles that, at 1.7 microns, are significantly smaller than the standard 5 micron beads. Because the smaller particle size means the columns work best when run at higher flow rates and higher pressure, Waters developed a completely redesigned system that includes an ultra high pressure (15,000 psi) pump and tubings and fittings that can handle volumes under 200 µl. “You can’t just crank down on the fittings and put on new tubings; it’s designed from the ground up,” says product manager Patricia Young of Waters. “[The UPLC system] is engineered for maximum peak capacity, high efficiency, and high resolution separation.” The company estimates that the UPLC technology increases throughput 10-fold over conventional HPLC.
Although the systems mentioned above improve upon the existing technology in several ways, conventional nanoLC systems will likely remain on the market for some time. Some, in fact, are sold by the very companies that are working to improve the technology; Agilent, for example, still sells the 1100 Series Nanoflow system. Product managers in the field say that such instruments will continue to have a following. “There’s people who will probably want to do [HPLC] the conventional way, just because it would offer them more flexibility in designing different valve approaches or column approaches … I don’t think that it will ultimately disappear,” says Agilent’s Gauthier.
Even if your particular application doesn’t require a nanoscale LC system, there are plenty of HPLC instruments on the market to meet a variety of needs. Take a look below for a few examples.