Atropisomers of methoxylated PCBs were separated using an Agilent 7890A gas chromatograph equipped with an electron capture detector (μ-ECD) using the following enantioselective columns: HP-Chiral-20B (20B) and Cyclosil-B (CB) from Agilent, Chirasil-Dex (CD) from Varian, BGB-172 (BGB) for BGB Analytic, and Chiral-Dex B-DM (BDM), Chiral-Dex B-PM (B-PM) and Chiral-Dex G-TA (GTA) columns from Supelco Analytical (see Table S3 for details). The compounds were first analyzed individually using the following temperature program: 50°C for 1 min, 10°C/min to 140°C, hold for 20 min, 1°C/min to column maximum temperature, hold for 20 min (22 (link)) to establish retention times and elution order as well as identify atropisomers of methoxylated PCBs separating on these columns for further optimization. The injector and detector were kept at 250°C. The flow was set to 1 mL/min.
Subsequently, the separation of methoxylated PCBs that partially separated or showed significant peak widening on a particular column was further optimized. Because the resolution of PCB atropisomers improves with decreasing analysis temperature (Figure S1) (18 (link), 23 ), temperature programs with a long isothermal hold at progressively lower temperatures were used: 50°C for 1 min, 10°C/min to X, 10°C to 225°C, hold for 10 min, where X is in the range from 140°C to 180°C. The flow in these analyses was 3 mL/min to achieve elution in a reasonable amount of time. The length of the temperature programs varied from 220 min at 180°C to 500 min at 140°C. In some cases an analysis time of 8 h was not sufficient to elute the methoxylated metabolite. These long analysis times were considered impractical for enantioselective analysis, even if improvement of separation was observed. Atropisomers of PCBs in the microsomal incubation mixtures (see below) were analyzed on the CD column using the following temperature program: 2°C/min from 100°C to 150°C, 0.2°C/min to 185°C, 15°C to 200°C (22 (link), 24 (link)). The conditions provided acceptable resolution (from 0.63 to 0.89) for all four studied congeners.