shutterstock_276016517This year I have attended a number of conferences across a variety and routine and applied markets and whilst viewing the many posters at these events, something struck me; nearly all of them we focus very much around the mass spectrometer, whilst the liquid chromatography (LC), or more specifically HPLC / UHPLC (High Performance Liquid Chromatography / Ultra High Performance Liquid Chromatography) hardly got any mention or attention. That’s why I am writing this article, to defend UHPLC and make sure it’s duly appreciated in the future.

It seems that I am not alone in my thinking as this recent article by Shishkova et al, entitled ‘Now, More Than Ever, Proteomics Needs Better Chromatography’ illustrates that while a lot of the focus in proteomics has been on mass spectrometry and the advances in instrumentation, little attention has been paid to chromatography and it’s now chromatography that is key to drive proteomics and the rapid and deep analysis of mammalian proteomes. Whilst this article is focused on nano-flow LC, the same applies to traditional HPLC and UHPLC.

How Does Liquid Chromatography Aid Mass Spectrometry?

There are a number of ways in which liquid chromatography supports mass spectrometry and ensures that the mass spectrometer is working to its full potential and delivering the data you require; in my view the following three are the most important.

A mass spectrometer analyses spectra sequentially. Thus it relies on liquid chromatography to deliver the analytes for analysis in a timely and workable fashion. Over the past decade the acquisition speed of mass spectrometers has increased over twentyfold using instruments such as Orbitrap-based mass analysers. Such speed is required when dealing with complex samples such as proteomes where thousands of peptides need identifying, and in routine analysis where time-to-result is critical such as multiple pesticide residue identification. Such acquisition speeds thus require a liquid chromatography system capable of delivering the analytes to the mass spectrometer at the required rate to ensure the mass spectrometer is working to its full potential. If the UHPLC system is not delivering them at a fast enough rate then your expensive mass spectrometer is not being fully utilised resulting in longer analysis times.

The peak capacity and resolution of your liquid chromatography also affects the mass spectrometry. With highly complex samples, you want to reduce the number of co-eluting analytes introduced into your mass spectrometer as this generally causes ion suppression during ionization. Avoiding co-elution will thus increase sensitivity since analytes of difference abundance are well separated. There are a number of ways in which liquid chromatography can be optimised to increase resolution, however the most common method is to increase the column length and reduce particle size, although this generally leads to higher back-pressures.

The final way that liquid chromatography enables your mass spectrometer to deliver optimal results is by performing accurate, precise and reproducible separations. This is especially important for quantitation where the accurate retention time of the peaks is required and when comparing across a number of samples you need the confidence that the exact peak is going to elute off the column at exactly the same time each time you perform the separation.

Liquid Chromatography Considerations

Before beginning your LC-MS experiments you need to consider not only the mass spectrometer, but also your HPLC / UHPLC system because your whole workflow will suffer if this is not appropriate.

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Modern mass spectrometers can analyse many spectra per second and so you need a UHPLC system with sufficient power and resolution to be able to deliver the resolved analytes for the mass spectrometer to run at full capacity and shorten analysis times. Two examples are illustrated here, the first looking at peptide mapping and the second at pesticide analysis. In both cases they use the power of the Thermo Scientific™ Vanquish™ UHPLC system to dramatically reduce separation time and fully utilise the acquisition capabilities of the mass spectrometer.

To increase resolution and peak capacity often requires longer columns and smaller particle columns which both contribute to higher back-pressures, thus your UHPLC system will be required to deal with such pressures. Resolution can also be increased by other means such as adjusting the column temperature and optimising eluent conditions, again these need to be factored in to the UHPLC system choice.

Finally, whether connected to a mass spectrometer or not, a UHPLC system should always offer reproducible separations with very low standard deviations in retention times, preferably below 0.1%, as illustrated in this technical note.

On the subject of which UHPLC gives the optimal performance in combination with mass spectrometry under different conditions and applications, this poster was presented at the recent International  Society of Chromatography conference in Cork which highlights the UHPLC considerations depending on your application and specific requirements around throughput, resolution and robustness.

Summary

The next time you consider performing LC-MS, please consider both parts of the equation and not focus all your attention on the mass spectrometer. Because no matter how sophisticated your mass spectrometer is, if your HPLC / UHPLC is not sufficient then you will not be using your mass spectrometer to its full potential, resulting in slower and poorer results. Your choice should be guided by the intended application and scientific question, but remember to focus on the complete LC-MS system.

One final intriguing thought is if you are analysing known compounds or analytes and your HPLC / UHPLC system is giving high resolution and highly reproducible and precise separations, then potentially there is no need to use a mass spectrometer, but an alternative lower cost detector option such as UV or Charged Aerosol Detection might suffice.

Resources

  • See how the Thermo Scientific Vanquish UHPLC platform can enable you to get the most out of your mass spectrometer by visiting our Vanquish site.
  • For nano-flow applications, such as proteomics, information on the EASY nLC™ 1200 with columns up to 75 cm for ultra high resolution separations and the Ultimate™ 3000 RSLCnano can be found via the links.
  • Visit our mass spectrometry product page for more information on our range of mass spectrometry solution.