HPLC and UPLC
“HPLC has been around for a long time, but aside from minor refinements, there weren’t many advances in the field until UPLC® came along,” alleges John Gebler, Ph.D., director of biopharmaceutical sciences at Waters (www.waters.com). “We saw improvements in column design and improved lab software over the years, but these were incremental in nature and not groundbreakers.”
In conventional HPLC, samples are forced through the column under pressure, which decreases their diffusion, allowing sharper resolution of peaks as they elute from the column.
In UPLC, much smaller particles, less than 2.0 mm, are used to pack the column and a greater pressure is applied, resulting in improvements in separation profiles, according to Dr. Gebler. “The 1.7 micron particles are composed of silica in the form of a bridged ethyl hybrid, an appropriately durable material for the separation procedures. Our UPLC systems are designed to work under pressures of up to 15,000 psi, so this increased pressure provides greater speed, resolution, and sensitivity.”
Waters offers two options, the Acquity UPLC System and the nanoAcquity UPLC System. Both can be coupled to a mass spectrometer to give the investigator increased peak concentrations in less time. “With UPLC you increase sensitivity and you maximize the signal-to-noise ratios of your mass spec instrument.”
The Waters system can be employed in research protocols for antibody characterization in which translational modifications are of special interest. According to Dingyi Wen, Ph.D., principle scientist at Biogen Idec (www.biogenidec.com), “We use the Waters Acquity system for determining the primary sequences of antibodies as well as glycosylation and other secondary modifications. The UPLC separation provides sharp peaks, even when the quantities available are small.”
Dr. Wen investigates antibody candidates by digesting them with endoproteases, followed by separation, and finally identification of peptides in the mass spectrometer. HPLC does not provide the resolution or sensitivity needed for the research protocols, and its use of splitting can result in loss of material, she explains.
Dr. Wen has two Waters UPLC systems in her laboratory and plans to add a third. She finds the Acquity UPLC HSS T3 columns for retaining and separating polar compounds via reversed-phase HPLC and UPLC useful.