What are the allowed adjustments in USP General Chapter 621 for HPLC Column Parameters and Flow rates in Isocratic Elution? (During Method Development)

 

Stationary phase: No change of the identity of the substituent (e.g., no replacement of C18 by C8); the other physicochemical characteristics of the stationary phase, i.e., chromatographic support, surface modification and extent of chemical modification must be similar; a change from totally porous particle (TPP) columns to superficially porous particle (SPP) columns is allowed provided the above-mentioned requirements are met.

Column dimensions (particle size, length): The particle size and/or length of the column may be modified, provided that the ratio of the column length (L) to the particle size (dp) remains constant or in the range between −25% to +50% of the prescribed L/dp ratio.

Adjustments from totally porous to superficially porous particles: For the application of particle-size adjustment from totally porous to superficially porous particles, other combinations of L and dp can be used, provided that the plate number (N) is within −25% to +50%, relative to the prescribed column. These changes are acceptable, provided that system suitability criteria are fulfilled, and selectivity and elution order of the specified impurities to be controlled are demonstrated to be equivalent.

Internal diameter: In absence of a change in particle size and/or length, the internal diameter of the column may be adjusted.

Caution is necessary when the adjustment results in smaller peak volumes due to a smaller particle size or smaller internal column diameter, a situation that may require adjustments to minimize extra-column band broadening by factors such as instrument connections, detector cell volume and sampling rate, and injection volume.

When the particle size is changed, the flow rate requires adjustment, because smaller-particle columns will require higher linear velocities for the same performance (as measured by reduced plate height). The flow rate is adjusted for both the change in column diameter and particle size using the following equation:

F = flow rate indicated in the monograph (mL/min)

F = adjusted flow rate (mL/min)

dc = internal diameter of the column indicated in the monograph

dc = internal diameter of the column used (mm)

dp = particle size indicated in the monograph (µm)

dp = particle size of the column used (µm)

When a change is made from ≥3-µm to <3-µm particles in isocratic separations, an additional increase in linear velocity (by adjusting the flow rate) may be justified, provided that the column performance does not drop by more than 20%. Similarly, when a change is made from <3-µm to ≥3- µm particles,an additional reduction of linear velocity (flow rate) may be justified to avoid reduction in column performance by more than 20%. After an adjustment due to a change in column dimensions, an additional change in flow rate of ±50% is permitted.

Column temperature: ± 10°C, where the operating temperature is specified, unless otherwise prescribed