November 1, 2013 (Vol. 33, No. 19)

Details on How Two Methods Were Transferred with Minimal Adjustment

As analytical technology continues to advance, both regulatory agencies and pharmaceutical companies are keen to avail themselves of these enhancements. In the case of chromatography, evolution from HPLC toward UPLC provides numerous benefits. These include cost savings, higher throughput, and improved resolution, to name a few.

The path from HPLC to UPLC is not entirely without difficulty as many companies have HPLC-based methods filed with regulatory agencies, particularly those used in quality control departments related to product release.

A mechanism for overcoming the technology lag between labs is to implement instrumentation in the QC laboratory with the capability of running both HPLC and UPLC methods on the same instrument. In this scenario, the new instrumentation will have the ability to perform existing legacy HPLC methods while providing the capacity to perform newly developed UPLC-based assays arriving from development labs. This adoption strategy can help to provide familiarity with UPLC instrumentation ahead of UPLC method deployment.

In this article, we will discuss data that demonstrates the ability of UPLC to run legacy HPLC methods.

While this concept is straightforward, how would such a scenario actually be implemented? After all, HPLC instruments and UPLC instruments are significantly different. The ideal situation would be one where the HPLC method can be run directly on the UPLC instrument. There are several key properties that differentiate HPLC from UPLC, and knowledge of these differences greatly helps with the process of transferring HPLC methods to UPLC.

Despite all the innovations in liquid chromatographic technology, the principles of chromatography remain unchanged. The factors that affect selectivity and retentivity in HPLC are the same in UPLC.

Case Studies

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.

Figure 1. SEC chromatography run on UPLC is highly similar to that run on HPLC. To evaluate the transferability of an SEC method from HPLC to UPLC, a protein standard containing four proteins was separated using a Waters Biosuite SEC column. (A) Resulting chromatogram from HPLC SEC performed on an Agilent 1200. (B) Resulting chromatogram from SEC performed on a Waters Acquity UPLC H-Class Bio.

To evaluate the factors that potentially influence a complex gradient separation, we established an HPLC peptide-mapping method and performed the chromatographic analysis using either a legacy HPLC instrument or the Acquity UPLC H-Class Bio.

We first evaluated the separation of a sample of trypsinized ribonuclease B using a Waters XBridge C18 column (3.5 µm, 4.6 mm × 100 mm) on each instrument where an obvious, but consistent, retention time shift was observed in each chromatographic peak (Figure 2A). The UPLC instrument generated chromatographic peaks at earlier retention times compared to the HPLC, which was not entirely surprising considering the differences in dwell volumes between the instruments. To quantify the dwell volume differences, each system’s precise dwell volume was determined, and the volume difference between the two instruments was found to be 360 µL.

As this difference must be accounted for, one option was to adjust the isocratic hold step and delay delivery of the gradient by the calculated difference in instrument dwell volumes. This requires a change to the time component in each line of a gradient table and in some cases could be considered a change in the chromatographic method. An alternative to this approach is to program a gradient start offset into the instrument method while leaving the gradient table unchanged.

For our study, we chose to program a gradient start offset to avoid making changes to the gradient table. To test this adjustment, we separated the same ribonuclease B peptide mix on each instrument, but included the gradient start offset in the HPLC method conducted on the UPLC. Comparison of the results showed nearly identical chromatography, indicating that this approach is a good candidate for performing large molecule gradient method transfers (Figure 2B).

Figure 2. Programming a gradient start offset aligns peptide-mapping chromatograms on systems with different dwell volumes. To evaluate the transferability of a peptide mapping method, trypsinized ribonuclease B was separated using a Waters XBridge C18 column coupled to either a Waters Acquity UPLC H-Class Bio (top chromatograms) or an Agilent 1200 (bottom chromatograms). (A) The legacy HPLC peptide-mapping method was used without any modification on each instrument. (B) The HPLC method was adjusted with a gradient start offset equivalent to the difference in dwell volumes between the Agilent 1200 and Waters Acquity UPLC H-Class Bio.

Admittedly, a simple peptide mixture such as the ribonuclease B digest does not accurately represent a complex peptide map. To more rigorously test our strategy, we trypsinized infliximab and separated the mixture using the approach described above. Our initial inspection of the separations indicated nearly identical chromatography. To quantify the similarity in the separations, we integrated each of the chromatograms and identified 65 peaks to monitor comparability across each instrument. The consistency between the two instruments was very good, with only minor differences observed in retention times (Figure 3).

In this particular example, issues relating to column temperature, flow rate, and solvent delivery configuration were evaluated and determined to have insignificant influence on this peptide mapping separation. This should not suggest that these same parameters would not affect method transfer for other samples. As with the earlier recommendation, each method transfer should be taken on a case-by-case basis, and the analyst should be prepared to consider several separation parameters to achieve equivalent chromatography.

In this example, we were able to transfer a complex peptide mapping method from an HPLC instrument and achieve nearly identical chromatography on a UPLC instrument by making one simple modification to the instrument method, namely adding a gradient start offset. This approach left the gradient table intact as well as all other aspects of the instrument method.

Figure 3. A gradient start offset generates equivalent chromatography with complex peptide map samples. To determine the ability of a gradient start offset on more complex peptide samples, infliximab was trypsinized and separated using a Waters XBridge C18 column coupled to either (A) an Agilent 1200 or (B) a Waters Acquity UPLC H-Class Bio. (C) Relative retention times from each peptide map were plotted for each identified peak.


Adoption of new analytical instrumentation across an organization can be a concern for those worried about the direct and indirect costs associated with migration to new technology. Using a step-wise approach where new UPLC-based instrumentation is acquired and used for running legacy HPLC methods is one strategy where companies can separate the cost of new instruments from the future cost of method refiling following the update of legacy HPLC methods.

Eoin F. J. Cosgrave ([email protected]) is senior scientist and Sean M. McCarthy is senior manager, scientific operations at Waters.

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