Columns Matter, Too: Choosing the Best Stationary Phase for NanoFlow and Microflow LC-MS

Markus Wanninger

Reading Time: 5 minutes

Part 2: Considering stationary phases for trap-and-elute configurations

In part 1 of this series, I brushed over some basic mechanisms, advantages, and considerations when implementing trapping in a proteomic workflow. The volumetric challenge of nano- or microflow LC-MS and its trap-elute configuration play a vital role when setting up the proteomics separation.

Both volume and elution direction can cause band broadening, which can deteriorate the chromatography. This is why I want to discuss here the impact of the retentivity and characteristics of the stationary phases for reversed-phase proteomic separations and how you can use them to your advantage.

The advantages of focusing a sample

Understanding the retentivity of your stationary phases used in a trap-and-elute setup for peptide separations will give you a significant advantage when optimizing your method. The difference in retentivity is the basis for call chromatographic separations.

Peptide retentivity is influenced by many factors:

  • Mobile phase
  • Every aspect of the stationary phase (base particle properties, ligand, etc.)
  • Characteristics of the analyte and how it interacts with the stationary phase

In reversed-phase chromatography, hydrophilic peptides will not retain very well on any C18 stationary phase, whereas hydrophobic peptides will be strongly retained and elute later in the separation. The hydrophobic retentivity is thus the deciding factor to separate different peptides.

To further optimize the separation and ensure the best peak capacity of the nanoflow LC separation delivered to the MS, the peak dispersion should be minimized. This adverse effect of peak broadening can be mitigated by a “refocusing effect,” where the injected analytes are strongly retained at the inlet of the column and form a narrow band.

In order to focus an injected sample volume, the stationary phase needs to be able to retain the analytes of interest at the start of the gradient. When doing a trap-and-elute separation, the stationary phase of the analytical column needs to be more retentive than the stationary phase of the trap column in order to refocus. For background knowledge on the theory involved, I recommend reading Waters’ HPLC primer.

The contenders

Before jumping head-first into the detail of peptide separations and retentivity, I want to briefly introduce the stationary phases we generally use for peptide separations in peptide bioanalysis and in peptide and protein quantitation in general.

Bridged Ethylene Hybrid (BEH) and Charged Surface Hybrid (CSH) are stationary phases based on the same base particle. The CSH particle has a positive surface charge, which gives it different properties for peptide separations compared to the BEH particle. High Strength Silica (HSS) offers the retentivity and selectivity of a silica-based particle for use at higher pressures where traditional silica particles would have mechanically failed. Lastly, there is a silica-based Symmetry trap columns, which are also silica based.

Stationary phases used for peptide separations

How is all this relevant for your peptide separation? Let’s keep going.

Peptide retentivity

We looked at the peptide retentivity of common stationary phases used in nanoEase M/Z columns and iKeys. We did this by injecting a peptide mixture under gradient conditions and monitoring the chromatography of 12 individual peptides. The percent of acetonitrile required to elute each of the 12 peptides was compared, and showed us the differences in retentivity for each of the phases.

Peptide retentivity of common stationary phases
Peptide retentivity of common stationary phases.

When looking at the individual stationary phases, we can discern the hydrophobicity of the individual peptides. At a first glance, you can also differentiate the retentivity of the individual stationary phases. That’s a great set of data to have already.

When we further simplify this graph and average the percent organic (acetonitrile, ACN) required to elute the 12 peptides, we are able to paint a very simple yet understandable picture.

Percent organic required to elute 12 peptides
Percent organic required to elute 12 peptides.

This graph shows us that Symmetry C18, the material used in trap columns shows – on average – the lowest peptide retentivity of all phases. BEH C18 and HSS T3 require about 5% or so more acetonitrile than Symmetry to elute peptides. That’s great news.

Suppose we elute an analyte from a Symmetry C18 trap at 15% acetonitrile. The same analyte in the same mobile phase (15% acetonitrile) will still be retained on a downstream BEH C18 or HSS T3 analytical nanoLC column to benefit from the refocusing effect. And that will make for some great chromatography and well-separated peaks – which will lead to more protein and peptide identifications.

But, looking closely, this also suggests that CSH, a stationary phase with excellent loadability and peak shape for peptides may not be the best candidate for trap-and-elute proteomics. That is why I generally recommend using CSH for direct-inject peptide separations.

Of course, none of this is absolutely true for every compound of your sample, but it is a fantastic starting point when choosing the right stationary phase for a proteomics project.

In more detail

In essence we found that the ability to pair different stationary phases to best suit the needs of your separation is a significant advantage and can have a very positive impact on your results.

There is a lot more to say about the details behind this and what it means for your proteomics nanoflow LC-MS analysis. My colleague Moon Chul Jung summarized all his findings and comparisons in an excellent white paper about choosing the right stationary phase for nano- and microflow LC-MS for proteomics.

Also, in case you missed it, I wrote a short piece about general trapping mechanisms such as forward and reverse trap elute.

 

Additional resources