Transfer of established reversed-phase methods across HPLC, UHPLC and UPLC chromatographic instrumentation requires careful consideration of the operating parameters and design of each instrument. For example, gradient formation can be influenced by binary or quaternary mixing, dwell volume, viscosity changes in the mobile phase, the gradient shape, residual volumes, and many other factors that can vary across different pumps. The mechanism for sample injection, as well as the detector, can influence linearity and quantitation. Extra column dispersion can impact the resolution and efficiency of a separation. To understand the effect these factors may have on methods transfer, both instrument characteristics and specific method conditions must be factored and evaluated when transferring HPLC and UHPLC methods.
In this presentation, studies will evaluate methods transfer, for a variety of samples and methods, including USP assays. The first set of studies will evaluate the same column and method across multiple instruments from different manufacturers. The impact of instrument attributes- including solvent delivery and temperature control - on the separation fidelity will be evaluated. The system dwell volume and extra column dispersion of each system will be characterized and its impact assessed on the separations, specifically on the relative retention and resolution of the analytes. The transferability of the method will be assessed through system suitability criteria (relative retention, % area, etc.).
An additional set of experiments will evaluate the impact of system attributes on method scaling, in which the L/dp ratio remains constant, and the stationary phase ranges from 5 μm to –sub-2-µm particles. The flow rate and gradient steps for these separations will be scaled accordingly. In both sets of examples, consideration will be made to conduct method transfer in accordance with regulatory guidelines for allowable adjustments to compendial methods.
Transfer of methods across different systems illustrates the impact characteristics such as dwell volume or gradient formation can have on a chromatographic separation. For isocratic separations with low k' - where extra column effects are a major contributor to band broadening, higher values can significantly impact resolution, sensitivity and efficiency (Figure 1). In this case, keeping the l/dp constant and matching a column's dimensions with the systems dispersion enable the efficiency of the column to be achieved. At the other extreme, when using larger ID columns and larger injections, specifically those samples in organic diluents, strong solvent effects can be observed resulting in poor peak shape for those early eluting peaks.
The studies will culminate in guidelines for successful methods transfer of HPLC methods as well as considerations for scaling or of methods from 5 to sub-2um columns. The impact of the instrument characteristics on a separation will be described and illustrate how an understanding of the LC system aids in methods transfer and method migration or translation.