APPENDIX

We applied duplicated urea adduction on samples from Section 208-1263A-34X-1. However, gas chromatograms of nonadducted residues for the samples demonstrated that complete recovery was not achieved during our process (small peaks of targeted n-alkanes were observed). Urea adduction should have been repeated until no target peaks in the nonadducted residue were observed. In the case of this study, the amount of each sample extract was limited. Repeated urea adduction might have caused laboratory contamination and/or considerable loss of precious compounds; therefore, we stopped urea adduction after duplication of this process. As long as no isotopic fractionation during the urea adduction process were demonstrated, analytical results from the GC-IRMS system are valuable, even if complete recovery was not achieved. It is generally known that there is no carbon isotopic fractionation during the urea adduction process; however, only limited reports were accessible (Ellis and Fincannon, 1998). Therefore, we tested and vindicated the urea adduction technique employed in this study using standard long-chain alkanes.

We used 0.25 µM of pure, commercially produced C₂₈ and C₃₂ n-alkanes as standard materials for each urea adduction. The mixed standards were processed in the same manner as the procedure described in "Methods." We omitted repeated adduction on nonadducted residue obtained after the first process in order to test the effect of a single adduction. We performed three experiments independently. Adducted and nonadducted standards for each experiment were injected onto the GC-IRMS system, and we made duplicate analyses. We analyzed original n-C₂₈ and n-C₃₂, without any processes, three times. We compared carbon isotope values of initial standards, adducted standards, and nonadducted standards to each other.

Isotope values for initial standards were determined on the basis of triplicate analyses (Table AT1) to be –32.3 for n-C₂₈ and –27.4 for n-C₃₂. ¹³C values for adducted n-C₂₈ were 0, 0.1, and 0.5 deviated from the nonadducted values in Experiments A, B, and C, respectively. For n-C₃₂, Experiment B yielded identical values for the adducted and nonadducted n-alkane, whereas Experiment C showed 0.3 deviation in the adducted value from the nonadducted value. The averaged magnitude of these deviations is well within the range of instrumental standard deviation (±0.3). Average values of the three experiments for adducted n-C₂₈ and n-C₃₂ are –32.0 and –27.4, respectively. The adducted values are 0.3 and 0.1 deviated positively from initial standard but are still within the range of instrumental standard deviation.

Experiments using long-chain n-alkanes n-C₂₈ and n-C₃₂ could not show conclusive carbon isotopic fractionation between their adducted and nonadducted statuses. ¹³C values of urea-adducted n-alkanes also did not show any considerable fractionation from their untreated status and do not indicate that the urea adduction technique we employed in this study introduced carbon isotope fractionation during the processes. On the basis of this fact and previous knowledge for urea adduction technique (Ellis and Fincannon, 1998), we conclude that our urea adduction technique did not fractionate original carbon isotope values.