HPLC Method Transfer Failures? 5 Ways to Prevent Them

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HPLC Method Transfer Failures? 5 Ways to Prevent Them

Team TFS
Team TFS

“I love using outdated and unreliable instruments,” said no scientist ever.

There is nothing more frustrating than spending hours prepping for your experiment only to be shut down because the instrument broke, again. Ugh.



If you’ve ever worked with old and flaky things, you can relate to my frustration. I recall countless times in graduate school when I was unable to move forward with the next step of my ten-step synthesis because the geriatric NMR and HPLC instruments were down. Hours and sometimes even days of research progress was lost during those downtimes.

About three years into my studies, our department received a grant and upgraded our instruments with the funds. Talk about the best gift ever. What chemist does not like using dependable, quick, and robust tools? The only real sidestep was time spent learning the new software and adjusting my method to the new HPLC instrument. Once I got everything worked out, the quality of my separations and purity of my target compounds improved.

Transfer your methods seamlessly with the built-in technology of the Thermo Scientific ™ Vanquish™ Core HPLC system

If you’ve never transferred an HPLC method before, you might not be aware of the potential roadblocks. Method transfer at times is challenging because changes in the temperature, extra column volume, and detector settings can alter the resolution, peak separation, and retention times. Here are five parameters to consider when transferring a validated method from your old HPLC to your new Vanquish Core HPLC system to ensure the transfer is fast, smooth, and painless.

1. Adjust for differences in the gradient delay volume to maintain retention times

Not all HPLC runs require a gradient for separation. But if your analysis requires the detection of compounds in a complex mixture with a broad range of polarity then your method is likely to involve a gradient, and you need to account for the gradient delay volume when switching to a new instrument. A change in this volume affects retention time and selectivity of early eluting compounds run on a gradient method. To mitigate this issue, the Vanquish Core HPLC system is equipped with a tunable gradient delay to ensure a headache-free method transfer.

2. Retain resolution by matching temperature settings

The use of eluent preheaters and column compartment thermostatting has the largest impact on the temperature inside the column. A thermal mismatch between the incoming solvent and column may cause retention times to shift, compounds to co-elute, or an inversion in selectivity (i.e., peaks 4 and 5 switches). You can gain further control of your separation temperature with the Vanquish column compartment’s still-air and forced-air modes to improve efficiency or reduce frictional heat, respectively, to mimic the thermal settings of different vendor’s instruments.

3. Align the flow cell volume to conserve method sensitivity

An increase in the ratio of the flow cell volume to the peak volume impacts the efficiency of separation by causing peaks to spread or overlap, reducing the method sensitivity and signal-to-noise ratio. Standard practice is to keep the flow cell volume within ten percent of the peak volume of the smallest peak to prevent the mixing of peaks due to an excess flow cell volume.
Save time by taking note of the flow cell volume in your old HPLC and match these to your new one to preserve the quality of separation.

4. Maintain spectral intensity by adjusting the detector settings

Beer Lambert’s law states absorbance is directly proportional to the concentration of an analyte in solution times the path length of the cell and molar absorption. Based-on this definition both the path length of the flow cell and detection wavelength settings will influence sensitivity. A decrease in the path length or shifting of detection wavelengths to a range of lower molar absorptivity reduces the amount of light seen by the detector, lowering sensitivity. Retain maximum absorbance and confirm the detector settings of your new HPLC parallel to the old instrument.

5. Prepare eluent mixtures fresh to preserve separation and retention times

The last thing you want to do is use old eluents when running an experiment on new instruments. Over time, buffer solutions dissolve oxygen and carbon dioxide, and aromatic solvents evaporate, giving rise to a change in the pH of your eluent buffers and reducing the strength of organic-mixed eluent solutions. These chemical modifications will throw off the separation quality and reproducibility of retention times. At the end of the day, good lab practice saves time, money, and most importantly your sanity.

Advance Your Science with the Vanquish Core HPLC system

Quality matters in science. Working with outdated and unreliable equipment limits your ability to make discoveries, progress research, grow your business—along with the lost time and money spent on repairs. Don’t let the fear of change of having to use a new instrument, learn new software, or the cost of upgrading impede your business’s future.

We have an endless supply of literature resources and technical support to set your method transfer up for success when you decide to upgrade to our Vanquish Core HPLC. Now that you have the tools to ensure successful method transfer, are you ready to make the change?

For more tips, watch this on-demand webinar on approaches to method transfer, optimization and speedup. You can check out this case study from Patheon and blog on LabCompare to learn how Vanquish LC systems empower success in pharmaceutical manufacturing.

P.S. Don’t Forget to Vote in the Select Scientist Choice Awards by February 8th, 2021!

We are honored to announce the nomination of our Vanquish Core HPLC as the Best New Separation Product of 2020 in the Scientists‘ Choice Awards (happy dance). Will you vote for us to show your appreciation for innovation in chromatography? Hurry, the last day to cast your vote is February 8th, 2021!