Columns Matter, Too: Choosing Optimal Conditions for Trap-and-Elute Nanoflow and Microflow LC-MS

Markus Wanninger

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Part 3: Optimizing Trapping Conditions

The previous installment of this series discussed the details, effects, and advantages of stationary phases for proteomic peptide analysis and how the retentivity of the trap and analytical column can improve your peptide separation. Here, I wanted to briefly touch on trapping conditions such as flow rate and trapping volume since, in my experience, it is an often neglected parameter.

The very basics of trapping

Figure 1.

Trap columns tend to have a larger inner diameter and are packed with larger particles than their analytical counterpart. This allows for loading a larger volume of sample at higher flow rates, as discussed in an earlier post. Effective trapping captures all analytes of interest in a larger sample while discarding interfering compounds. This requires the most hydrophilic compounds to still be retained during the trapping step, and the ability to elute all analytes in small volumes to successfully re-focus the bands on the analytical column.

Generally speaking, using trapping may increase the dynamic range of your analyses by allowing to inject more sample and reducing the negative effects of the sample matrix.

Considerations when optimizing trapping conditions

Peptide loss of hydrophilic peptides during trapping step is not uncommon, as peptide recovery is largely dependent is dependent on the retention. Peptide recovery can be optimized by adjusting the trapping conditions accordingly.

As the retention of the trap column is ideally lower the retention of the analytical column and is therefore fixed, one of the parameters that should be adjusted is the solvent strength during the trapping process. Lower strength solvents will help to capture less retentive species better on the trap column.

Also, the loading volume needs to be large enough to transport the sample from the injection loop onto the trap and to wash off unwanted compounds, such as salts and other ionic interferences. But the trapping volume needs to be small enough to avoid washing off those weakly retained peptides. This also requires considering the solvent composition of the injected sample and how it may affect the final solvent composition during the loading process. Additionally the flow rate during the trapping process needs to be optimized.

Figure 2 shows how the flow rate and trapping duration and thus the resulting overall trapping volume affects peptide recovery when using one solvent concentration. When the sample is being trapped with too much volume, chances of the loss of hydrophilic peptides drastically increases.

Figure 2.

Changing the solvent composition can also have an impact on peptide recovery, with stronger solvents being more prone to washing of weakly retained peptides.

Figure 3.

Optimizing the trapping methodology should not be a neglected step when developing a trap-and-elute method for nano- or microflow LC-MS analyses. It is beneficial that you understand the overall retentivity and selectivity of the stationary phases of the trap column as well as the analytical column. A well-adjusted trap-and-elute method will increase the dynamic range of your method since it allows you to inject significantly more sample and it will also increase the overall sensitivity of the analysis due to decreased matrix effects.

 

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