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.

我们设计了一种新颖的扩展内标方法，借助色谱（EIC）辅助定量¹ H NMR（qNMR），即使杂质和分析物信号的化学位移完全重叠，也可以准确地定量分析物的¹ H信号区域。当杂质和分析物信号在¹ H NMR光谱中重叠但可以在色谱图中分离时，可以从色谱图中获得杂质与内标（IS）的响应比。如果响应比可以被转换成^1个杂质的H信号面积比和，则^1个被分析物的H信号区域可以精确地在数学上修正的贡献评估¹在¹ H NMR光谱中与分析物重叠的杂质的H信号面积。在这项研究中，使用了气相色谱和液相色谱。我们以含2-氯苯酚和含4-氯苯酚的苯酚为例，其中杂质和分析物信号重叠以分别验证和论证EIC。因为2-氯苯酚和苯酚的¹ H信号可以在特定的碱性溶液中分离，所以2-氯苯酚适合通过比较EIC与仅在碱性条件下通过qNMR获得的分析值来验证EIC。通过EIC，获得了2-氯苯酚的纯度，且不确定度相对增大（k = 2）的0.24％。纯度与在碱性条件下获得的纯度相匹配。此外，通过绝对色谱曲线法通过气相色谱法评估苯酚含量，也验证了EIC。最后，我们证明了EIC可以评估4-氯苯酚的纯度，相对扩展不确定度（k  = 2）为0.22％，在任何条件下都无法与苯酚的¹ H信号分离。