I’m using gradient elution on an LC-MS (Waters 2695) for PPCP separation and analysis. Currently, with my column volume and flow rate, I allow 12-13 minutes for re-equilibration between injections, extending the overall method time to about 36 minutes. Increasing the flow rate slightly to 0.4 or 0.5 mL/min could potentially shorten re-equilibration time and I can adjust that time in between somewhere to allow more effective separation during the gradient steps, or simply decrease the overall method duration. However, I’m considering whether this adjustment could impact column or system performance, such as creating back pressure issues.
My current gradient is set at 20% methanol from 0 to 2 minutes, then linearly increases to 80% at 8 minutes, 95% at 9 minutes (held isocratic until 20 minutes), followed by a return to 20% methanol at 23 minutes, with a 13-minute re-equilibration period.
Increasing flow rate during re-equilibration in your LC-MS gradient method can shorten method time. Raising it to 0.4–0.5 mL/min could reduce re-equilibration without significantly affecting separation. However, caution is essential, especially if altering flow during the gradient phase. Higher flow rates may lead to back pressure issues, which could affect both system components and column life. Ensure that the increase stays within your column’s recommended range to prevent impact on separation efficiency and peak shape. Testing minor adjustments will help confirm that separation quality and system stability remain intact before fully implementing any flow rate changes.
While you can change the flow rate during the analysis (or equilibration) phase of the run, it is generally recommended to use the optimized flow rate for the method, then adjust the remaining method parameters (e.g. composition, time, temperature etc) to find the best method parameters. Changes to the flow rate directly change the system back-pressure on the system (often, not a concern) and analysis efficiency. If changed during the method, may change the detector signal and result in 'drifting' which requires a great deal of time to stabilize (depends on type of detector, column dimensions, change in flow rate and sensitivity). Instead, optimize the analysis method for better throughput. This includes optimizing the flow rate for the analysis method (use the correct linear rate) as well as the mobile phase composition, tubing, flow path, detector cell etc. Equilibration time will 'take as long as it takes'. If your equilibration time is too short, then late eluters, col-elution and/or invalid data may be collected which should be avoided.
Note: It sounds like you are new to chromatography so you may not know which parameters are adjusted, and in which order, to develop a method. When asking for help with HPLC questions, please note that we need some basic information about the proposed method to provide constructive advice. You did not include the basic information in your post (which tells us you are new, and do not yet know what these parameters are and how they impact the method). The most important item missing from your question is that of the COLUMN dimension (L x ID) and the particle size used. The column info AND flow rate are two of the most important parameters to know, BEFORE discussing any HPLC question. Those two parameters allow us to determine the linear flow rate, estimated column void volume (and Tzero) plus, with sample Retention times, Kprime values.
- Badges
- Science topic
- Similar topics
- Linguistics
- Composition Studies
- Publication