Our Technical Service group periodically fields questions about 4-nitrophenol disappearing during semivolatiles analysis while other nitrophenols, such as 2,4-dinitrophenol and 4,6-dinitro-2-methylphenol, still have strong responses. I think the topic is interesting enough to warrant a standalone article.
Figure 1 - Classic exaggeration used by Jaap de Zeeuw to teach basic chromatographic principles
Figure 1 is a slide on column activity that I have shamelessly stolen from one of Jaap’s webinars. It looks like a gross exaggeration meant to hammer the point home. That was my thought, at least, until I looked at the series of extracted ion chromatograms for 4-nitrophenol and dibenzofuran in Figure 2.
Figure 2 - 4-Nitrophenol retention time shift relative to dibenzofuran as the GC column activity increases with use.
Dibenzofuran is relatively inert for a heterocycle and has a very stable relative retention time (RRT) when compared to the PAH internal standard (Figure 3) while 4-nitrophenol is unusually mobile. Maybe the 4-nitrophenol RRT is highly sensitive to elution temperature (it happens, but that topic warrants its own article). The EZGC models in Figure 4 show that when all variables are constant except column length, the resolution of 4-nitrophenol and dibenzofuran degrades, but the elution order does not change. Column activity is a component of the selectivity term (α) in the resolution equation. Activity in the column will act as a second retention mechanism (adsorption), and has a greater effect on retention at lower temperatures and the elution temperatures of all the compounds decrease as lengths of column are removed during maintenance. Stronger interactions with an increasing number of active sites could explain the relative movement of 4-nitrophenol.
Figure 3 - Dibenzofuran and 4-nitrophenol retention time changes relative to Acenaphthene-D10 retention time over the lifetime of a GC column
Figure 4 - EZGC model of 4-nitrophenol and dibenzofuran resolution before and after 10 meters of column is removed (using the same run conditions used to collect the data for figures 2 and 3.
Compounding the problem is the EPA’s recommendation to use m/z = 139 as the quant ion for 4-nitrophenol. You might have noticed the data gap where 4-nitrophenol crosses over dibenzofuran in Figure 3, or how the 4-nitrophenol peak disappears in the extraction ion chromatogram shown in Figure 2C. The gap in the gray 4-nitrophenol RRT trace in Figure 3 is due to the 4-nitrophenol peak coeluting with dibenzofuran, which also has a significant response at m/z = 139 (highlighted by the red trace in Figure 5A). Changing the quant ion to m/z = 65 significantly reduces the possible bias due to coelution and prevents losing the 4-nitrophenol quant ion peak in dibenzofuran (Figure 5B).
Figure 5 - Extracted ion chromatograms of 4-nitrophenol and dibenzofuran showing possible interferences in red when the quant ion is 139 (5A) and 65 (5B)
So where did the 4-nitrophenol go? Referring back to Figure 2, if the 4-nitrophenol peak is not in the expected retention time window, it is probably coeluting with dibenzofuran or eluting after dibenzofuran. It is important to note that this is not a column specific phenomenon; we see this on every 5-type column from every manufacturer.