Technology Heats Up
In May, Waters announced a partnership with NIBRT focused on creating a database for glycan analysis based on the company’s UltraPerformance Liquid Chromatography® (UPLC®) platform. NIBRT will develop, maintain, and license the database, which should launch in 2011 and will be co-marketed by NIBRT and Waters.
The database will contain chromatographic retention times for sets of glycan structures associated with biotherapeutic compounds. It is intended for use by biopharmaceutical manufacturers as a tool for evaluating the structure of glycosylated proteins and as a means of monitoring product integrity and bioprocess control. Researchers will use the Acquity UPLC system with an ethylene bridged hybrid glycan separation column and fluorescence detection to separate the glycans released from glycoproteins in the form of 2-aminobenzamide derivatives.
Peter Carr, Ph.D., professor in the department of chemistry at the University of Minnesota, Minneapolis, describes three main technological advances in HPLC “that have come together to push it forward”: the instrumentation that enabled ultrahigh pressure LC (UHPLC), which was based on the work of Jim Jorgenson, professor in the department of chemistry at the University of North Carolina, Chapel Hill; the development of micropellicular particles, composed of a solid inner core that is chromatographically inactive and impermeable, surrounded by a thin crust of porous material that is chromatographically active; and the use of high temperatures, which has been the focus of Dr. Carr’s research.
Dr. Carr describes temperature as “the third dimension in HPLC.” He is using higher temperatures to decrease fluid velocity, which yields an increase in the flow rate without the need for higher pressures and raises the diffusion coefficients to enhance mass transfer and limit peak broadening.
To achieve the benefits of increased temperature without compromising the quality of the chromatographic separation, Dr. Carr’s group has developed thermally stable stationary phases—zirconia-based and hyper-crosslinked silica-based phases. They are using these media to perform ultrafast high-temperature LC (UFHTLC) as the second dimension in comprehensive 2-D LC/LC for analytical applications, in which all of the material that exits the first column goes into the second column.
The increased speed enabled by the high temperatures is not only necessary to handle the large number of samples produced by the first-dimension LC separation, but it also provides another, unexpected benefit. “As you do the second dimension faster, your overall resolving power improves,” says Dr. Carr. If the speed of the second dimension is less than optimal and, consequently, “you don’t take enough fractions out of the first dimension,” the fractions will be too big, remixing can occur, and “you lose information that is already there.”
By running the second dimension at optimal speed, which Dr. Carr estimates from experimental data as a separation every 15–20 seconds, it is possible to achieve maximal peak capacity.
In 2-D LC/LC, the cycle time of the second dimension has a greater impact on the overall 2-D peak capacity than does the first dimension, explains Dr. Carr, and he recommends the use of strategies such as higher temperatures to optimize the speed of the second dimension rather than options such as microparticle-based media combined with high pressures, or long monolithic columns, to accelerate the first-dimension separation.