In prior instalments of this series on method adjustment, we’ve looked at the guidelines from the European Pharmacopoeia (EP) and United States Pharmacopoeia (USP) for allowable adjustments in pH (Part 1), mobile phase (Part 2), and column dimensions (Part 3). In this article we’ll look at flow-rate and particle-size adjustments.
Both the EP and USP allow a change in the flow-rate of up to ±50% from the normal method conditions. A footnote in the USP states that this is in addition to the adjustments discussed last week, which were made to correct the linear velocity when the column diameter was changed. There’s nothing complicated about this guideline. A method that normally runs at 1 mL/min can be adjusted in the range of 0.5-1.5 mL/min. My guess is that these guidelines were instituted primarily for methods in which a specific retention time is given as part of the system suitability test. There is a note in the EP that indicates that a flow-rate reduction is not allowed if the plate number is part of the system suitability test. Although with the 10 µm particle size columns popular in the past, a flow-rate change often would affect the separation because of a change in the plate number, a factor of two in flow rate is unlikely to change the separation in real applications of today’s columns of ≤5 µm particles, so I expect that this restriction in adjustments will change in some future EP revision.
As just noted, a change in flow-rate with today’s columns is unlikely to affect the separation, but it will affect other aspects of the method. The pressure will change directly with the flow-rate, whereas the retention time will change with the inverse of the flow rate. And these changes only apply to isocratic separations – changes to flow rate in gradients can give unexpected changes in peak spacing.
Both the EP and USP agree in the allowable reduction in column packing particle size of 50%. This would allow a change from a 5 µm particle to a 3.0 or 3.5 µm one or from 3 µm to 1.7-2 µm. However, it does not allow for a change from 5 µm to 1.7-2 µm. In my opinion, this is overly restrictive. It may have made sense in the days when the low-purity, type-A silica columns were used widely, and column chemistry changes were common when the particle size was changed. But with today’s high-purity, type-B silica columns and the improved manufacturing controls that are in place, the chemistry should not change with particle size as long as the same phase (e.g., C18) and same product line (e.g., Ascentis) are used. As a result, I expect that the EP and USP will eventually allow the use of any particle size, so long as the selectivity does not change.
This blog article series is produced in collaboration with John Dolan, best known as one of the world’s foremost HPLC troubleshooting authorities. He is also known for his research with Lloyd Snyder, which resulted in more than 100 technical publications and three books. If you have any questions about this article send them to TechTips@sepscience.com