Authored by Kim Baughman, Ph.D., Director of Development, Microbac Laboratories, Inc.

While high-performance liquid chromatography (HPLC) is a mature, widely-used analytical technique, the advent of ultra-high pressure liquid chromatography (UHPLC) has energized disciplines that use the technique extensively.  In UHPLC, columns packed with sub-2 µm particles are used, and when combined with the elevated operating pressures, can result in a significant reduction in retention times.  With current UHPLC systems, analysis times can be decreased by a factor of 9 when compared to HPLC analysis.  Thus, a 30-minute cycle time is reduced to less than four minutes, while maintaining equivalent efficiency and resolution.   This has proven useful for improving throughput for high-volume analyses and for improving method development time cycles, where it is advantageous to experiment with various method conditions.  Other advantages of UHPLC include greater sensitivity because of the sharper peak profile and reduced solvent consumption due to the shorter cycle times.  This latter advantage has increased in significance as global prices for acetonitrile, one of most common mobile phase components, have skyrocketed.  Solvent consumption reductions of greater than 80% are frequently achieved with UHPLC.

Several modifications of the instrumentation have been required to support the use of UHPLC.  The most obvious is the construction of systems capable of handling the increased pressure. The new UHPLCs are capable of operating at pressures up to 15,000 PSI, compared to about 2,000 PSI for traditional HPLC.   It is also important to reduce the tubing volume and other dead-volume sites in the system, including detector cell volume, to limit peak broadening.  Electronics have also been upgraded to accommodate faster data collection as the peak widths can be as much as 10 times smaller than that seen with traditional HPLC systems, thus requiring a much more rapid data collection interface.

As new analytical methods are developed, particularly those expected to be high-throughput, UHPLC is becoming more and more popular.  There is also the issue of whether to convert current HPLC methods to UHPLC methods.  Laboratories are often hesitant to change methods for current products and applications, particularly in highly regulated environments, but migration to UHPLC may be an option.  In addition, methods developed using UHPLC may be back-transferred to conventional lower pressure HPLCs. For example, UHPLC may be used to rapidly scout a variety of columns and conditions to achieve a method development goal.  Then, after appropriate parameter adjustments, conditions for a conventional HPLC method are developed for use in QC laboratories that may not have the capability of UHPLC.

Systematic approaches are also becoming better understood for reliable and rapid transfer of conventional HPLC methods to UHPLC methods.  Several parameters must be adjusted to accommodate the transfer.  The injection volume is scaled down to avoid overloading the column and the mobile phase flow rate may be adjusted to maintain the resolution of lower-pressure methods under UHPLC conditions, while still achieving shorter run times.  In the case of gradient profiles, the gradient may be programmed at a much faster rate, but the improved efficiency is also dependent on the reduced dead volume in the system and on the mixing chamber, so the final gradient program is dependent on the instrument design factors.

Versatility is another advantage of the newer models of UHPLC instrumentation.  In addition to operation in the UHPLC mode, these instruments can be used in HPLC mode for methods already in house that have not been upgraded.  Thus, one instrument serves dual purposes.

The Southern Testing and Research Division of Microbac Laboratories, Inc. is currently using UHPLC for a number of LC/MS/MS and UHPLC/UV and PDA applications.  Please contact us at microbac_info@microbac.com regarding your UHPLC and method development needs.

For more information, please contact: microbac_info@microbac.com.