A quick evaluation of some disadvantages to performing semivolatiles analysis by EPA Method 8270D with splitless injection

This blog is part of a series; the previous installment can be found here.

Figure 1 - The later eluting PAH peaks are displaying the characteristic exaggerated shark fin shape of significantly overloaded peaks when analyzing the 8270 MegaMix at 120 ng on column by splitless injection.

Semivolatile calibrations on the 30 m x 0.25 mm ID x 0.25 µm df column format often range from 1.0 to over 100 ng/µL; however, a 0.25 mm ID column usually experiences peak overload as the mass on column approaches 10 ng. Column overload presents initially as slight peak fronting, skewing the tailing factor of a normally Gaussian peak to < 1.0. As mass on column continues to increase throughout the calibration range, isobars that elute close together—such as benzo[b]fluoranthene and benzo[k]fluoranthene— quickly become unquantifiable. Given the geometric nature of the typical calibration scheme, calibrations acquired under splitless conditions can quickly transition beyond acceptable chromatographic performance. Figure 1 clearly highlights the exaggerated shark fin appearance of severely overloaded peaks resulting from the splitless analysis of 1 µL of 120 µg/mL 8270 calibration standard. Peak shapes were so poor across the board for the 120 µg/mL calibration standard under splitless conditions that I dropped the level from the calibration, which has a clear impact on calibration dynamic range, as shown in Table 1.

Table 1 – Evaluation of the less volatile range of an 8270 calibration. The solid blue boxes indicate a dropped calibration point while a ‘*’ indicates the point was used (for easy counting).

Furthermore, the resolution of closely eluting isomeric pairs is particularly at risk when the column is overloaded because fronting causes the separation to collapse. Section 11.6.1.4 of EPA Method 8270D states that structural isomers are sufficiently resolved and may be reported individually when the valley height (h_v in Figure 2) is less than 50% of the average peak height. Insufficient resolution requires reporting the isomers as a pair with combined results. The most common critical separation that requires reporting as separate values for each compound is the benzo[b]fluoranthene and benzo[k]fluoranthene isomeric pair. Conversely, the coelution of 3-methylphenol and 4-methylphenol is a good example of insufficient resolution requiring the reporting of a combined result.

Figure 2 - EPA resolution criteria indicates that h_v must be less than 50% of the average h_p for the two peaks.

Take a look at how the benzo fluoranthene resolution held up across the 9-point splitless injection calibration range of 0.10 to 120 ng on column (Figure 3). The three highest concentration calibration standards (120, 80, and 40 µg/mL) do not meet the 50% valley resolution criteria. The peak fronting and resulting overlap from column overload make it impossible to generate a linear calibration including these points. Looking at Table 1, you can see that we dropped several high points for all the closely eluting PAH isomeric pairs (phenanthrene and anthracene, fluoranthene and pyrene, benz[a]anthracene and chrysene, and benzo[b]fluoranthene and benzo[k]fluoranthene) as well as indeno[123-cd]pyrene and dibenz[ah]anthracene (each has a minor product ion that interferes with the quant ion of the other).

Figure 3 – This overly of extracted quant ion chromatograms illustrates how benzo[b]fluoranthene and benzo[k]fluoranthene resolution rapidly collapses as the column overloads.Figure 3 also highlights the retention time drift that occurs as mass on column increases. The peak apex of benzo[b]fluoranthene shifts more than 0.2 minutes across the calibration standards. Section 11.3.5 of EPA Method 8270D defines the maximum acceptable retention time drift across a calibration as 0.06 relative retention time (RRT) units (normalized by the internal standard retention time). The RRT drift for both benzo fluoranthene isomers was 0.01, clearly within method specification, but hardly optimal. While the target peaks are easy to identify during evaluation of a continuing calibration verification (CCV), wandering retention times combined with multiple non-target peaks from coextracted material will make target peak identification significantly more difficult during real sample analysis.