liquid-chromatographyThe one parameter in analytical liquid chromatography that has been consistently increasing has been the pump pressure of HPLC (high performance liquid chromatography) and UHPLC (ultra-high performance liquid chromatography) instruments. Switching from HPLC to UHPLC brings obvious benefits in throughput, reduced solvent usage and increased resolution, to name a few. The Thermo Scientific™ Vanquish™ Horizon UHPLC system now offers 1500 bar of pump pressure, but what does 1500 bar offer you and is it required?

My personal opinion is, yes, 1500 bar is required or at least having the option to use all that pump pressure is always beneficial. It’s a bit like home air conditioning in the United Kingdom where I reside; it would be only sparingly used, but it would be good to have it available for those few days a year when it’s required. What 1500 bar of pump pressure offers you is the ability in certain applications to push the boundaries of what is currently possible and either perform faster separations and increase throughput and productivity, increase resolution or peak capacity or a combination of both. It also future-proofs your separations for those new applications and advances in column technology. You might not use 1500 bar every day, but it is always good to have it in reserve for when you do.

Faster Separations

I’m sure everyone would like faster separations, as long as there is no compromise on other parameters such as resolution or peak shape. Column technology has been evolving to be able to perform faster separations by offering smaller particles and reduced column diameters. With increased flow rates these do offer faster separations, but the trade-off is higher backpressures and, ultimately, it is the backpressure that prevents the use of smaller particles and diameters. What 1500 bar offers is the ability to push the limits of column technology further to achieve faster separations. In this example, Ibuprofen impurity analysis time was reduced from 2.5 to 1.5 minutes, without loss of resolution or peak shape by using a 1.5 µm Thermo Scientific™ Accucore™ Vanquish™ column compared to a 4 µm Thermo Scientific™ Accucore™ XL column. Another example is from peptide mapping where the flow rate on the Vanquish Horizon is increased that allowed for the separation to be reduced from thirty to five minutes without any loss of sequence coverage as measured by mass spectrometry. The increased flow rate used to perform the five minutes separation led to a system pressure of over 1300 bar – well within the 1500 bar capabilities of the Vanquish Horizon.

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Greater Resolution and Peak Capacity

The alternative to faster separations is to use the higher pressure capabilities to improve resolution, peak capacity and sensitivity / dynamic range. The obvious way to do this is to use longer columns or to couple columns together in series, but this again increases the backpressure. This poster note shows how coupling three 250 mm columns together to essentially deliver a column of 750 mm allowed the high resolution separation and detection of peptides across a broad dynamic. By connecting three 250 mm columns together, more than 60 µg of digested mAb could be injected per run and increased the number of identified UPS proteins spiked in the digested NIST mAb from 9 to 23. The consequence of using of an effective column length of 750 mm was backpressure over 1300 bar, within the capabilities of the Vanquish Horizon.

Do you need 1500 bar?

If you are looking for faster separations or improved resolution and peak capacity of any combination in-between, then the option to be able to run at pressures approaching 1500 bar is required and can only be offered by the Vanquish Horizon. 1500 bar also prepares you for the next advances in column technologies.

Additional Resources

  • Take a closer look at the Vanquish Horizon and the complete Vanquish UHPLC family on the Vanquish hypersite
  • Interested in switching from HPLC to UHPLC? Watch these on-demand webinars that review the factors you need to consider. These webinars are available in both English and German