Discovering Analyte Breakdown On-Column

I completed my winter internship at Restek and my main task was to expand the Pro-EZGC libraries.
So I created a library for Nitrosamines on the Rxi-1301Sil MS and while acquiring data we noticed a foot on a couple of my peaks.

Figure 1: On-column breakdown can be observed for the late eluting nitrosamine compounds as indicated by the foot at the leading edge of the peak.

I attempted to translate the simulation to a column with the same phase, but with a thicker film. Since the Rxi-624Sil MS and Rxi-1301Sil MS are the same stationary phase, I modeled a run with the Rxi-1301Sil MS Library using the column dimensions of an Rxi-624 Sil MS. As I ran the conditions provided by EZGC we noticed a strange rise of the baseline.

Figure 2: Rise in the baseline is observed which is most likely caused by analyte breakdown on-column. Breakdown components remain in the stationary phase and are observed as carryover.

One of my colleagues immediately suggested that the nitrosamines could break down as a function of elution temperature. In order to test this theory, I prepared three different run conditions with different elution temperatures for the late eluting nitrosamines on the Rxi-624 Sil MS:

Figure 4: Adjusting the final hold changes the elution temperature of the last three compounds. If the compound response drops as elution temperature increases it will support our theory of on-column breakdown.

These are the compounds I used (sorted by elution order):

Figure 5: List of nitrosamines found in the chromatogram where the last three compounds are labeled as 1, 2 and 3.

Figure 5: Three chromatograms illustrating the three different programs with increasing elution temperature of the last three nitrosamines. Notice the significant increase in breakdown.

I labeled the last three peaks; the ones that break down appear to have the most activity on-column. Thanks to the MS I was still able to determine the retention times and it became evident that the breakdown increases with higher elution temperature. Was this a general problem with these compounds on this phase, or was this specific to the Restek column?


Figure 6: Chromatograms with three different oven conditions. The last three nitrosamines have increasing elution temperatures from top to bottom. These chromatograms were performed on a competitor column to verify the on-column breakdown is most likely related to the cyano stationary phase and not specific to a manufacturer.

We only had a 60m competitor column available, so it’s not a direct comparison, however, we see similar breakdown. We can tell that the nitrosamines break down on-column on this phase in general. But if you use a film that is thin enough and run the compounds with a low elution temperature it will minimize on-column breakdown. Using an Rxi-1301Sil MS is still faster than using a “5-type” phase, but you will have to enter your nitrosamines into ProEZGC and find out for yourself.

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