Developing HPLC Methods for Speed and Longevity
Based on customer calls for support on method development and method transfers, here are our top recommendations for developing a swift, trouble free method.
- If you can use isocratic rather than gradient elution, do so. It avoids column re-equilibration time and dwell or delay time issues which will inevitably happen when the method is moved to a different instrument or lab. Be clear about how you make the mobile phase, not just its nominal composition. A 50/50 solution of methanol and water might be produced by mixing 500mL of methanol and 500mL of water, taking 500mL of water and filling to 1L with methanol, or taking 500mL of methanol and filling to 1L with water. All three will produce different actual ratios of methanol to water.
- If you need a gradient to ensure good separation of your analytes, measure and record the gradient delay time or volume in the instrument(s) used to develop and validate the method. The delay time / volume is an inherent isocratic hold at the beginning of the method, and duplicating that hold is critical for method transfer of fast gradient methods, or gradient methods with early eluting compounds.
- Take positive control of pH and temperature for your methods. Use buffers rather than straight acid / base addition to modify pH, and run your column at a specific temperature using your column oven. Zirchrom’s Buffer Wizard™ is a great tool for picking buffer systems to match your pH, ionic strength and buffering capacity requirements. Rather than starting method development at “room temperature” pick 25°C or 30°C for a standard starting point. If your method is transferred to a lab that is warmer or colder than your own, they’ll still be able to get the same results.
- Make your method backwards compatible, but don’t go overboard. Pick the smallest column that will still be compatible with all the instruments you expect the method to be run on. Today a 3µm fully porous particle packing or 2.7µm core-shell column packed in a 3mm ID column is reasonably safe for transfer to HPLCs less than 20 years old. These columns will run methods 40% faster and consume 70% less solvent per run than a 5µm packing in a 4.6mm ID column (stationary phase staying the same). Few labs are limited to instruments that need to run at over 0.5mL/min, so there’s little benefit and much cost to sticking to 5µm columns.
- When deciding how fast your method needs to be, always look at the choke points in the overall process. If HPLC analysis is your rate limiting step for that product, develop a method that takes advantage of the fastest instrument(s) you’ll have access to. If the HPLC analysis is not the limiting step, then running on a slightly slower but less expensive / more common / easier to maintain instrument has long term pay offs.
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