HPLC method transfer
Method transfer is adapting a method of analysis to a different HPLC system. The more similar the two instruments are, the more straightforward the process.
Common types of method transfers include converting HPLC methods to UHPLC methods to optimize speed and throughput. Other situations involve transferring a validated method from one lab to another, like research and development to operations or a clinical laboratory.
Regardless of the system similarity, instrument-to-instrument transfers often cause variability due to differences in the hardware and software technologies, particularly for systems from different manufacturers.
Hardware parameters affecting method transfer
Parameters like the gradient delay volume, pump types, column heating modes, detector flow cell volume, detector settings, and extra column volumes impact the reproducibility of a transferred method. The best way to overcome challenges during method transfer and achieve equivalent results with both instruments is to consider the complete parameters of a system during transfer.
Gradient delay volume (GDV)
The gradient delay volume is the volume from the mixing point of the eluents to the column head.
- The instrument GDV plays a central role in a successful HPLC or UHPLC method transfer. Learn more ›
- Modifications in the GDV between instruments can alter resolution and retention time, so changing the GDV is a standard solution in method transfer. Learn more ›
Since the GDV is variable for all instruments, matching the difference in volume is not always an easy task.
Factors affecting the gradient delay volume
The GDV is affected by all volumes between the point of mobile phase mixing and column head, but the pump usually contributes the most. Besides mixer volumes, the pump type is vital since low-pressure mixing pumps exhibit significantly higher GDVs than high-pressure mixing pumps.
Mixing effects of sample volume and surrounding mobile phase are also relevant for peak shape reconstruction at the new system. Small tubing dimensions can help reduce peak dispersion in front of and behind the column. Larger volumes improve pre-column mixing of the sample plug, positively affecting especially early eluting peaks if sample solvent is of high elution strength.
Detector flow cell volumes are noteworthy when transferring a method because all volumes influence peak dispersion before detection. Flow cell volume should be small compared to the peak volume, and detector settings must be consistent and capable of projecting the proper shape.
Effect of heating modes
The role of thermostatting or heating the column and mobile phase is often under-looked during method transfer.
Different column heating modes like still air and forced air, along with pre-column heating, diversely affect separation selectivity due to radial or axial temperature gradients. This effect is especially true for separations at pressures above 400 bar where frictional heating of the column material occurs.
Extra-column volume effects
Ensuring the extra-column volume (ECV) of the new instrument matches the original system will enable you to transfer methods successfully without re-validation.
Pre-column volume broadens the sample plug and smooths the gradient. Post-column volume only impacts analyte band broadening. Both pre- and post-column volumes affect analyte retention times.
Notably, data variability is more pronounced in UHPLC versus HPLC methods. Be sure to account for the variation in ECV during method transfer, especially when transferring an established method between different system models and manufacturers.
One should not overlook the complexity and likelihood of encountering retention time reproducibility and other issues in method transfer. To avoid setbacks in the process, you should carefully analyze method parameters, system features, and compatibility. Below are some expert resources to help you prepare for method transfer and troubleshoot any issues.
Method transfer is adapting a method of analysis to a different LC system. Common situations include converting HPLC methods to UHPLC methods to optimize speed and throughput. Other scenarios involve transferring a validated method from R&D to operations or a clinical laboratory.
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