Running Isocratic HPLC Methods on a UPLC instrument
Recently, we investigated the transfer of a size exclusion chromatography (SEC) method from an HPLC instrument to a Waters Acquity UPLC® H-Class Bio. In an SEC separation, the main driving force of separation is size, or more specifically the molecule’s hydrodynamic radius. In other words, no other secondary interactions occur or should occur, and the order in which molecules elute is from high molecular weight to low molecular weight. For SEC assays, the mobile phase is often a biological buffer with a predetermined pH (typically around 7), and the temperature of separation is often ambient. Hence, the remaining variables that can affect separation are flow rate and column length, both of which are directly related to chromatographic resolution.
If the same SEC column is used on both HPLC and UPLC instruments, any differences observed between the HPLC-acquired chromatogram and the UPLC-acquired chromatogram are likely related to two possible differences, one being flow rate, and the other being post-injector volume. Differences can occur in the flow rate of each instrument, despite identical flow rates itemized in each method; however, this is unlikely. Differences in the volume of the chromatographic system from the injector to the detector can also exist. If this volume is dramatically different, changes in analyte retention time will be observed.
To evaluate the factors that influence an isocratic SEC method transfer, we first used a mixture of four protein standards of varying molecular weight from approximately 14 kDa to 700 kDa on a Waters Biosuite SEC column (10 µm, 250 Å, 7.5 mm × 300 mm) using an HPLC and the Acquity UPLC H-Class Bio and discovered a slight shift in retention times between the HPLC instrument and the UPLC instrument (Figure 1).
The UPLC instrument demonstrated consistently earlier retention times for each of the identified peaks, suggesting that the programmed flow rates differed or there was a volume difference between the sample manager and the detector. We found that the flow rates for the instruments were identical. Based on this data, the difference in peak retention times were attributed to differences in system volume.
In support of this conclusion, a noticeable improvement in resolution was observed between critical peak pairs, indicating less post column dispersion resulting from lower post column volume. In this case, no change to the existing HPLC SEC method was required in order to achieve near identical chromatography on the UPLC instrument.
Running Gradient HPLC Methods on a UPLC Instrument
The practice of running gradient HPLC methods on UPLC instruments requires evaluation of additional parameters not typically considered with isocratic methods. This is particularly important when separating a large number of components in a complex mixture, such as performing a peptide map of a therapeutic protein.