Vol.2 No.1 2009

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Research paper : Expansion of organic reference materials for the analysis of hazardous substances in food and the environment (T. Ihara et al.)−15−Synthesiology - English edition Vol.2 No.1 (2009) A measurement method that can be applied to the calibration of a wide range of working RMs must satisfy the following conditions:1) It must satisfy market demands regarding uncertainty, while also provide speed and simplicity of use.2) It must be highly versatile and applicable to a wide variety of chemical compounds (general organic compounds for the purposes of this study).Quantitative NMR is the most feasible candidate that can satisfy both conditions 1) and 2), although the answer is not yet fully established. Accordingly, in this study, we endeavored to establish quantitative NMR as a universal calibration technology for working RMs in organic compounds.4.2 Principles of quantitative NMRNMR is one of the main methods for determining the molecular structure of a chemical compound. It has an extensive track record in unraveling molecular structures, including the analysis of complex molecules such as proteins. Information obtained using NMR, such as chemical shift (the resonance peak position dependent on atomic bonding and the ambient environment) and spin-spin coupling (a split of the peak due to bonded nearby nuclei), provide hints about the chemical species and ambient environment of a molecule. In addition, the area ratio of various peaks, which resonates according to different chemical shifts, generally indicates the ratio of the number of atomic nuclei contributing to the peaks. As Figure 2 shows, the area ratio of 1H NMR signals can easily be used to confirm the relative number of protons for the resonances, which is vital for the qualitative analysis of organic compounds.Conventionally, this aspect of NMR was used exclusively to determine the chemical structure, solely by expressing the number of protons as a ratio in a molecule. However, the concept can be applied differently. If the molecular structure of an organic compound is already known and assignments of its 1H NMR spectrum has been set, the number of protons contributing to each resonance peak is known, and this information can be applied to the quantitative analysis of chemical compounds. Thus, when the 1H NMR measurement is performed by adding a reference chemical compound to a sample solution separately in an analyte (substance to be analyzed) solution, the spectra of the two chemical compounds overlay each other, as shown in Fig. 3. At this point, if the mass (weight), molecular weight, and purity of the added reference chemical compound (hereinafter, will be called the Primary Standard: PS) are known, the amount-of-substance (number of molecules) corresponding to peak I in Fig. 3 will also be known, and can be used as the criterion for finding the number of molecules in the analyte. To illustrate with a specific example, if the number of protons in PS (I) is the same as the number of protons in analyte (D) (the number is 6 for both), the ratio of the areas for peak I and peak D indicates the relative number of molecules. As such, the relationship can be expressed as follows:(Peak area I)/(Number of molecules of PS) = (Peak area of D)/(Number of molecules in analyte)Since the number of molecules in PS is already known, the CBAHBHCHAHCOHHA:HB:HC=2:1:21H2HChemical shift δ (ppm)7.07.5 8.0 8.5 2HOHAChemical shift δ (ppm)9876543210-1ABCDEI0.050.090.270.050.390.057.367.257.252.931.471.54AnalytePrimary standard○○○○○××××○○×××××○General applicabilityRapid analysisUncertainty(less than 1 %)Reference standardMain targetsubstanceOutline of analytical methodAnalytical methodNot requiredMetallic elementsAmount of electricity used in electrysis of specified substances is measured.CoulometryNot requiredInorganic saltsSettling quantity of specified substances in solution is measured.GravimetryNot requiredHigh purity organic compoundsRelationship between fraction melted and temperature around the melting point is measured.Primary direct methodPrimary ratio methodReference standards based on the principles of titration are required.Acid, base, elementsThe specified substance is measured using chemical reactions.TitrimetryRequired for each analyteTrace metalstrace organic compoundsMass spectrometry is performed using a stable isotope.Isotope dilution mass spectrometry△(Unknown value)A reference standard for 1H is required.Organic compoundsThe ratio of areas of 1H peaks with different chemical shifts is measured.Quantitative NMRFreezing point depression methodTable 1 Types of primary methods of measurement and their characteristics.Fig. 2 Qualitative analysis of chemical compounds using 1H NMR.Fig. 3 Quantitative analysis of chemical compounds using 1H NMR.

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