Extended internal standard method for quantitative ¹H NMR assisted by chromatography (EIC) for analyte overlapping...

We devised a novel extended internal standard method of quantitative ¹H NMR (qNMR) assisted by chromatography (EIC) that accurately quantifies ¹H signal areas of analytes, even when the chemical shifts of the impurity and analyte signals overlap completely. When impurity and analyte signals overlap in the ¹H NMR spectrum but can be separated in a chromatogram, the response ratio of the impurity and an internal standard (IS) can be obtained from the chromatogram. If the response ratio can be converted into the ¹H signal area ratio of the impurity and the IS, the ¹H signal area of the analyte can be evaluated accurately by mathematically correcting the contributions of the ¹H signal area of the impurity overlapping the analyte in the ¹H NMR spectrum. In this study, gas chromatography and liquid chromatography were used. We used 2-chlorophenol and 4-chlorophenol containing phenol as an impurity as examples in which impurity and analyte signals overlap to validate and demonstrate the EIC, respectively. Because the ¹H signals of 2-chlorophenol and phenol can be separated in specific alkaline solutions, 2-chlorophenol is suitable to validate the EIC by comparing analytical value obtained by the EIC with that by only qNMR under the alkaline condition. By the EIC, the purity of 2-chlorophenol was obtained with a relative expanded uncertainty (k = 2) of 0.24%. The purity matched that obtained under the alkaline condition. Furthermore, the EIC was also validated by evaluating the phenol content with the absolute calibration curve method by gas chromatography. Finally, we demonstrated that the EIC was possible to evaluate the purity of 4-chlorophenol, with a relative expanded uncertainty (k = 2) of 0.22%, which was not able to be separated from the ¹H signal of phenol under any condition.