Attention Method Development Scientists! Learn about novel tools for replicating your existing methods
Webinar Highlights | Simplifying Methods Transfer: Novel Tools for Replicating Your Established Methods on an ACQUITY Arc System
I’m Dr. Paula Hong, Principal Scientist at Waters Corporation. I recently conducted a webinar that talked about the instrument characteristics that can impact transfer of established reversed-phase methods across both HPLC and UHPLC systems. These factors include dwell volume, extra-column dispersion and mobile phase pre-heating or temperature control. We also discussed ways to evaluate your instrument and some novel tools to assist in transferring established HPLC methods from previous generations of LC equipment to modern UHPLC systems.
If you missed it, watch the webinar on demand.
We had many great questions. Here are some of the highlights from our Q&A:
- Any suggestions when using HPLC columns (300 x 7.8 mm) on a low dispersion system like the UPLC H-Class system?
For low dispersion systems in general, HPLC columns may require larger bore tubing from the injector to the column and from the column to the detector to reduce backpressure and alleviate any strong solvent effects. Specifically, the ACQUITY UPLC H-Class system can accommodate larger columns with a 30 cm column compartment. This column compartment comes in various configurations, with either active or passive preheating. For HPLC columns and higher flow rates, the configuration with passive preheating may be more appropriate as it has larger id tubing. This would help reduce strong solvent effects and lower backpressure.
- Do you recommend solvent exchange when you are in a strong solvent?
Yes, – as discussed previously, when strong solvent effects are observed, one solution is to match the sample diluent to the initial mobile phase conditions. However, you can also add volume between the injector and the column to improve mixing and reduce strong solvent effects. This can include adding a piece of tubing or an column filter. In addition, scaling your method, and with it the injection volume, to a smaller column configuration also reduces strong solvent effects.
- Could you please comment on the effect of pre-column heating on the resolution and efficiency of these different column/system variants?
Solvent pre-heating is important to ensure adequate temperature control. Poor temperature control can result in thermal mismatch effects or loss of efficiency of a separation. However, preheating may be affected by flow rate due to residence time in the pre-heater. Many instruments have active and/or passive preheating. As long as the mobile phase temperature control is adequate, both can provide similar chromatographic performance.
- You mentioned reducing flow cell volume. Does that lower signal intensity?
No, not in itself. Actually if the flow cell volume were reduced, and everything else kept constant, extra-column dispersion would be lower. This would result in a smaller peak width and greater peak height to increase sensitivity. However, there are other factors. We know that UV absorbance is based on Beer’s law, which is derived from the molar extinction coefficient, path length and concentration. A lower volume flow cell may have also have a different path length. Both will contribute to the absorbance. For example, we have found changing from an analytical flow cell with a 10mm path to a microbore flow cell with an 8 mm path did not change sensitivity. While the lower dispersion flow cell produced sharper peaks and greater resolution, the shorter path length negated any increase in sensitivity.
- Should I put an isocratic hold in my method?
Since we never know if we are transferring to a system with larger or smaller dwell volume, it makes sense to add an isocratic hold in our method. When we do that we can simply change the duration of that hold to adjust for dwell volume differences across systems.
- Do I need to measure my system’s dwell volume?
Well that depends. Most manufacturers list dwell volume values in the instrument specifications. This gives you a good starting point. Even if you don’t have a general value, you can test your method and then make minor (or major) adjustments to the method based on the retention times. If you are still struggling, then measuring dwell volume may help you understand how your system is performing.