National Metrology Institute of Japan, National Institute of Advanced Industrial Science and Technology, 1-1-1, Umezono, Tsukuba, Ibaraki 305-8563, Japan; Graduate School of Natural Science and Technology, Kanazawa University, Kakumamachi, Kanazawa, Ishikawa 920-1192, Japan.
National Metrology Institute of Japan, National Institute of Advanced Industrial Science and Technology, 1-1-1, Umezono, Tsukuba, Ibaraki 305-8563, Japan.
Talanta. 2018 Jul 1;184:484-490. doi: 10.1016/j.talanta.2018.03.003. Epub 2018 Mar 12.
We devised a novel extended internal standard method of quantitative H NMR (qNMR) assisted by chromatography (EIC) that accurately quantifies H 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 H 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 H signal area ratio of the impurity and the IS, the H signal area of the analyte can be evaluated accurately by mathematically correcting the contributions of the H signal area of the impurity overlapping the analyte in the H 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 H 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 H signal of phenol under any condition.
我们设计了一种新颖的扩展内标法定量核磁共振波谱（qNMR）辅助色谱（EIC），即使杂质和分析物信号的化学位移完全重叠，也能准确地定量分析物的 H 信号面积。当杂质和分析物信号在核磁共振波谱中重叠但在色谱图中可以分离时，可以从色谱图中获得杂质和内标（IS）的响应比。如果响应比可以转化为杂质和 IS 的 H 信号面积比，则可以通过数学校正核磁共振波谱中与分析物重叠的杂质 H 信号面积的贡献来准确评估分析物的 H 信号面积。在这项研究中，使用了气相色谱法和液相色谱法。我们使用含苯酚的 2-氯苯酚和 4-氯苯酚作为杂质的例子，分别验证和证明了 EIC。因为在特定的碱性溶液中，2-氯苯酚和苯酚的 H 信号可以分离，所以 2-氯苯酚适合通过比较碱性条件下 EIC 和仅 qNMR 获得的分析值来验证 EIC。通过 EIC，在相对扩展不确定度（k = 2）为 0.24%的情况下，获得了 2-氯苯酚的纯度。该纯度与碱性条件下获得的纯度相匹配。此外，还通过气相色谱的绝对校准曲线法评估苯酚含量验证了 EIC。最后，我们证明 EIC 可以评估 4-氯苯酚的纯度，在相对扩展不确定度（k = 2）为 0.22%的情况下，在任何条件下都无法与苯酚的 H 信号分离。
