JP6485640B2 - Determination of nitrogen compounds - Google Patents
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Description
本発明は、窒素化合物の定量方法に属する。 The present invention belongs to a method for quantifying nitrogen compounds.
従来、溶液中の窒素の形態分析方法として、イオンクロマトグラフィー（ＩＣ）およびイオン電極を使用した方法が行われており、特許文献１では硝酸、亜硝酸の定量をイオンクロマトグラフィーで行い、アンモニアの定量をイオン電極で行っている（特許文献１の［００３４］）。 Conventionally, as a method for analyzing the form of nitrogen in a solution, an ion chromatography (IC) and a method using an ion electrode have been performed. In Patent Document 1, determination of nitric acid and nitrous acid was performed by ion chromatography, Quantification is performed with an ion electrode ([0034] of Patent Document 1).
ＩＣでは、イオン性の異なる窒素化合物を同時に測定することが原理上難しく、測定モードの異なる方法でイオン性の異なる窒素化合物を別々に分析する必要が出てくる。そうなると、少なくとも２回の測定が必要となり、効率が良くない。このようなこともあり、特許文献１では陰イオンの定量のみをイオンクロマトグラフィーで行い、陽イオンの定量はイオン電極で行っている。 In IC, it is difficult in principle to simultaneously measure nitrogen compounds having different ionic properties, and it becomes necessary to separately analyze nitrogen compounds having different ionic properties by methods having different measurement modes. In that case, at least two measurements are required, which is not efficient. For this reason, in Patent Document 1, only anion quantification is performed by ion chromatography, and cation quantification is performed by an ion electrode.
本発明の主な目的は、窒素化合物を効率良く定量可能な技術を提供することにある。 The main object of the present invention is to provide a technique capable of efficiently quantifying nitrogen compounds.
上記の知見に基づいて成された本発明の態様は、以下の通りである。
本発明の第１の態様は、
窒素化合物を含有する測定対象に対してＮを核とした核磁気共鳴分光法を用いた測定を行い、前記窒素化合物を定量する定量工程を有する、窒素化合物の定量方法である。
The embodiments of the present invention made based on the above findings are as follows.
The first aspect of the present invention is:
This is a method for quantifying a nitrogen compound, comprising measuring a measurement object containing a nitrogen compound using nuclear magnetic resonance spectroscopy with N as a nucleus and quantifying the nitrogen compound.
本発明の第２の態様は、第１の態様に記載の発明において、
前記定量工程においては、１４Ｎを核とした核磁気共鳴分光法を用いた測定を行う。
According to a second aspect of the present invention, in the invention according to the first aspect,
In the determination step, measurement is performed using nuclear magnetic resonance spectroscopy with 14 N as a nucleus.
本発明の第３の態様は、第２の態様に記載の発明において、
前記測定対象は複数の前記窒素化合物を含有し、
前記定量工程においては、前記核磁気共鳴分光法により得られた一つのスペクトルから複数の前記窒素化合物を一度に定量する。
According to a third aspect of the present invention, in the invention according to the second aspect,
The measurement object contains a plurality of the nitrogen compounds,
In the quantification step, a plurality of the nitrogen compounds are quantified at a time from one spectrum obtained by the nuclear magnetic resonance spectroscopy.
本発明の第４の態様は、第３の態様に記載の発明において、
前記測定対象は、イオン性が互いに異なる複数の前記窒素化合物を含有する。
According to a fourth aspect of the present invention, in the invention according to the third aspect,
The measurement object contains a plurality of the nitrogen compounds having different ionic properties.
本発明の第５の態様は、第１〜第４の態様のいずれかに記載の発明において、
前記測定対象はタンパク質を含有しない。
According to a fifth aspect of the present invention, in the invention according to any one of the first to fourth aspects,
The measurement object does not contain protein.
本発明の第６の態様は、第１〜第５の態様のいずれかに記載の発明において、
予め、濃度が既知の前記窒素化合物と、窒素を含有する基準物質とに対して前記核磁気共鳴分光法を用いた測定を行い、濃度が既知の前記窒素化合物のスペクトルのピークの面積と、前記基準物質のピークの面積とから、前記窒素化合物のピークの面積と前記窒素化合物の濃度との関係を得る定量準備工程をさらに有し、
前記関係に基づいて前記定量工程を行う。
According to a sixth aspect of the present invention, in the invention according to any one of the first to fifth aspects,
The nitrogen compound having a known concentration and a reference substance containing nitrogen are measured using the nuclear magnetic resonance spectroscopy, and the peak area of the spectrum of the nitrogen compound having a known concentration, From the area of the peak of the reference substance, further comprising a quantitative preparation step for obtaining the relationship between the area of the nitrogen compound peak and the concentration of the nitrogen compound,
The quantification step is performed based on the relationship.
本発明の第７の態様は、第６の態様に記載の発明において、
前記核磁気共鳴分光法を用いた測定においては各管が同心となる多重管を用い、当該多重管のうち一つの管には前記基準物質を配置し、別の管には濃度が既知の前記窒素化合物または前記測定対象を配置し、前記定量準備工程および前記定量工程を行う。
According to a seventh aspect of the present invention, in the invention according to the sixth aspect,
In the measurement using the nuclear magnetic resonance spectroscopy, multiple tubes in which each tube is concentric are used, one of the multiple tubes is arranged with the reference substance, and the other tube has a known concentration. A nitrogen compound or the measurement object is arranged, and the quantitative preparation step and the quantitative step are performed.
本発明によれば、窒素化合物を効率良く定量可能となる。 According to the present invention, nitrogen compounds can be quantified efficiently.
以下、本発明の実施の形態について、以下の順に説明する。
１．窒素化合物の定量方法
１−１．（定量）準備工程
１−２．定量工程
なお、本明細書において「〜」は所定の数値以上かつ所定の数値以下のことを指す。
また、特記の無い場合、スペクトルやピークとは、核磁気共鳴分光法を用いた測定における、縦軸を強度、横軸を化学シフト（ｐｐｍ）とした際のスペクトルやピークを指す。
Hereinafter, embodiments of the present invention will be described in the following order.
1. 1. Determination method of nitrogen compound 1-1. (Quantitative) Preparatory process 1-2. Quantitative Step In the present specification, “to” indicates a predetermined numerical value or more and a predetermined numerical value or less.
Unless otherwise specified, the spectrum and peak refer to a spectrum or peak in a measurement using nuclear magnetic resonance spectroscopy when the vertical axis is intensity and the horizontal axis is chemical shift (ppm).
＜１．窒素化合物の定量方法＞
本実施形態においては、窒素化合物を含有する測定対象に対して窒素Ｎ（好ましくは１４Ｎ）を核とした核磁気共鳴分光法（以降、単にＮＭＲと称する。）を用いた測定を行い、窒素化合物を定量する。以下、説明する。
<1. Determination of nitrogen compounds>
In this embodiment, measurement using a nuclear magnetic resonance spectroscopy (hereinafter simply referred to as NMR) using nitrogen N (preferably 14 N) as a nucleus is performed on a measurement object containing a nitrogen compound, and nitrogen is measured. Quantify the compound. This will be described below.
１−１．（定量）準備工程
まず、本工程においては、窒素化合物を定量する前準備を行う。具体的には、例えば、以下の工程を行う。
（ｉ）予め、測定対象となる窒素化合物と同じ組成であって濃度が既知の窒素化合物Ａと基準物質とに対して１４Ｎを核としてＮＭＲを用いた測定を行う。その際に、窒素化合物Ａのスペクトルのピークとは別の化学シフト（以降、ピーク位置と称する。）にて参照用のピークを明瞭に表出させることができる、窒素を含有する基準物質を採用する。そして、濃度が既知の窒素化合物Ａのスペクトルのピークの面積と、当該基準物質のピークの面積の値を得る。
（ii）別の濃度に設定した以外は窒素化合物Ａと同様とした窒素化合物Ｂと、先ほどの基準物質と組成や濃度等を同様とした基準物質とに対して１４Ｎを核としてＮＭＲを用いた測定を行い、濃度が既知の窒素化合物Ｂのスペクトルのピークの面積と、基準物質のピークの面積の値を得る。
（iii）適宜（ii）を繰り返し、当該窒素化合物のピークの面積と当該窒素化合物の濃度との関係を示す検量線を作成する。
なお、後述の定量工程に係る準備のことを定量準備工程と称し、それ以外の準備のことを含めて本工程では（定量）準備工程と称する。
1-1. (Quantitative) Preparatory Step First, in this step, a preliminary preparation for quantifying the nitrogen compound is performed. Specifically, for example, the following steps are performed.
(I) In advance, measurement is performed using NMR with 14 N as a nucleus for a nitrogen compound A and a reference material having the same composition as the nitrogen compound to be measured and a known concentration. At that time, a nitrogen-containing reference material that can clearly express a reference peak by a chemical shift (hereinafter referred to as a peak position) different from the peak of the spectrum of nitrogen compound A is adopted. To do. Then, a peak area value of the nitrogen compound A having a known concentration and a peak area value of the reference substance are obtained.
(Ii) NMR was used with 14 N as the nucleus for the nitrogen compound B, which was the same as the nitrogen compound A except that it was set at a different concentration, and the reference material having the same composition, concentration, etc. The peak area of the nitrogen compound B spectrum having a known concentration and the peak area value of the reference substance are obtained.
(Iii) Repeat (ii) as appropriate to create a calibration curve showing the relationship between the peak area of the nitrogen compound and the concentration of the nitrogen compound.
In addition, the preparation which concerns on the below-mentioned fixed_quantity | quantitative_assay process is called a fixed_quantity | quantitative_assay process, and this process including other preparations is called a (quantitative) preparation process.
ちなみに、上記の検量線の作成には、本出願人が公開する特開２００８−２２４２８４号公報に記載の手法を採用しても構わない。また、本工程において用いるＮＭＲの試料管は、同じく当該公報の図１（本願図１にて再掲）に記載の二重管式試料管を採用しても構わない。こうすることにより、諸々のクロマトグラフィー（例えばＩＣ）のように別化合物への溶解等の処理を加えない状態で溶液中の窒素化合物を効率良く測定することが可能となる。
なお、本実施形態においては、当該公報に記載の手法を採用した場合について述べ、特記の無い内容については、当該公報に記載の内容を適宜参照しても構わない。
Incidentally, the method described in Japanese Patent Application Laid-Open No. 2008-224284, which is disclosed by the present applicant, may be employed to create the calibration curve. In addition, as the NMR sample tube used in this step, the double tube type sample tube described in FIG. 1 of the publication (reprinted in FIG. 1 of the present application) may be adopted. By doing so, it becomes possible to efficiently measure the nitrogen compound in the solution without applying a treatment such as dissolution in another compound as in various chromatography (for example, IC).
In the present embodiment, the case where the method described in the publication is employed is described, and the contents described in the publication may be referred to as appropriate for the contents not specifically mentioned.
本実施形態においては、スペクトルのピークの面積は、ピークが出ていないピークの両端部分の間に基準となる直線を設定し、当該直線から上の部分すなわちピークの部分の強度の積分値として得ている。当該手法は、後述の実施例におけるＮＭＲ装置を使用すれば容易に実施可能である。 In this embodiment, the area of the peak of the spectrum is obtained as an integral value of the intensity of the upper part of the straight line, that is, the peak part, by setting a reference straight line between both end parts of the peak where no peak appears. ing. This method can be easily implemented by using an NMR apparatus in the examples described later.
なお、測定対象としては窒素化合物を含有していれば特に限定は無い。また、窒素化合物についても窒素Ｎの元素を含有していれば特に限定は無い。具体例としては、アンモニア（またはそのイオン、以降列挙する化合物も同様）、硝酸、ヒドラジン、尿素、チオ尿素など、またはそれらのいずれかの組み合わせが挙げられる。また、測定対象は液体であるのが通常であるが、液体以外であっても特に限定は無い。ただ、測定対象に含有される物質によっては本実施形態がもたらす効果をさらに増幅するが、その点については後掲の定量工程にて述べる。
また、基準物質についても、窒素化合物Ａのスペクトルのピークとは別のピーク位置にて参照用のピークを明瞭に表出させることができれば特に限定は無い。具体例としては、グリシン、アセトニトリル、ギ酸アンモニウムなどが挙げられる。
The measurement target is not particularly limited as long as it contains a nitrogen compound. Further, the nitrogen compound is not particularly limited as long as it contains an element of nitrogen N. Specific examples include ammonia (or its ions, the same for the compounds listed below), nitric acid, hydrazine, urea, thiourea, etc., or any combination thereof. Further, the measurement object is usually a liquid, but there is no particular limitation even if it is other than a liquid. However, the effect brought about by the present embodiment is further amplified depending on the substance contained in the measurement object, but this point will be described in the quantitative step described later.
The reference substance is not particularly limited as long as the reference peak can be clearly expressed at a peak position different from the peak of the spectrum of the nitrogen compound A. Specific examples include glycine, acetonitrile, ammonium formate, and the like.
上記の定量準備工程の内容はあくまで一具体例であり、最終的に未知の濃度の窒素化合物を定量可能ならば、上記の手法の代わりに公知の手法を適宜採用しても構わない。 The content of the above quantitative preparation step is merely a specific example, and a known method may be appropriately employed instead of the above method as long as a nitrogen compound having an unknown concentration can be finally determined.
１−２．定量工程
本工程においては、窒素化合物を含有する測定対象に対してＮを核としたＮＭＲを用いた測定を行い、窒素化合物を定量する。本実施形態においては、ＮＭＲを用いた測定を行う際に、測定対象が窒素化合物を含有することを鑑みてＮ（好ましくは１４Ｎ）を核とすることに大きな特徴がある。以下、核種１４Ｎを例に挙げて詳述する。
1-2. Quantification process In this process, the measurement which contains N as a nucleus is performed to the measuring object containing a nitrogen compound, and a nitrogen compound is quantified. In the present embodiment, when performing measurement using NMR, there is a great feature in that N (preferably 14 N) is a nucleus in view of the fact that the measurement object contains a nitrogen compound. Hereinafter, the nuclide 14 N will be described in detail as an example.
まず、１４Ｎを核としたＮＭＲを用いた測定を行う場合、諸々のクロマトグラフィーのように、測定で使用する溶媒に溶液を混合する必要がなくなる。そのため、溶液中に溶解している窒素化合物が溶媒と相互作用して形態変化を起こさせるおそれを無くすることができるし、ＧＣやＧＣＭＳのように溶液を高温に加熱して気化する必要もなくなる。 First, when performing measurement using NMR with 14 N as a nucleus, it is not necessary to mix the solution with the solvent used in the measurement as in various chromatography. Therefore, it is possible to eliminate the possibility that the nitrogen compound dissolved in the solution interacts with the solvent to cause a shape change, and it is not necessary to heat and evaporate the solution at a high temperature like GC or GCMS. .
また、仮に、１Ｈを核としてＮＭＲを用いてしまうと、窒素化合物が尿素、チオ尿素、ヒドラジンあるいはアンモニア等の溶液である場合、当該溶液の主成分である水がスペクトルのピークとして検出される。そうなると、尿素、チオ尿素、ヒドラジンあるいはアンモニア等に起因する１Ｈのピークなのか、溶液中の水に起因する１Ｈのピークなのか、判別が不可能となってしまう。
しかしながら、１４Ｎを核としたＮＭＲを用いる場合、上記のような水に起因するピークを表出させずに済む。そのため、窒素化合物に起因するピークを精度良く表出させることができ、ひいては上記の検量線により精度良く定量することが可能となる。
Also, if NMR is used with 1 H as a nucleus, when the nitrogen compound is a solution of urea, thiourea, hydrazine, or ammonia, water that is the main component of the solution is detected as a peak of the spectrum. . Then, it becomes impossible to determine whether the peak is 1 H due to urea, thiourea, hydrazine, ammonia, or the like, or 1 H peak due to water in the solution.
However, when NMR using 14 N as a nucleus is used, it is not necessary to reveal the peak due to water as described above. Therefore, it is possible to accurately express the peak due to the nitrogen compound, and as a result, it is possible to quantify with high accuracy using the calibration curve.
また、１４Ｎを核としたＮＭＲを用いることによる大きな利点がある。それは、１４Ｎの核自体の天然存在比が非常に高く、測定感度が非常に高くなることである。例えば、後述の実施例に示すＮＭＲ装置（ＦＴＮＭＲ：Ｆｏｕｒｉｅｒ Ｔｒａｎｓｆｅｒ ＮＭＲ、以下、特記の無い限りＮＭＲはＦＴＮＭＲを指す。）を使用すると、スペクトルの入手には数分程度で済む。なお、上記の各クロマトグラフィーでは、１回の測定に３０分程度要することを考えると、本実施形態がもたらす効果は顕著である。もちろん、本工程は、ＦＴＮＭＲ以外の方式、たとえばＣＷ（Ｃｏｎｔｉｎｕｏｕｓ Ｗａｖｅ）−ＮＭＲを採用しても差し支えない。 In addition, there is a great advantage by using NMR with 14 N as a nucleus. That is, the natural abundance ratio of the 14 N nucleus itself is very high, and the measurement sensitivity is very high. For example, using an NMR apparatus (FTNMR: Fourier Transfer NMR; hereinafter, NMR refers to FTNMR unless otherwise specified) shown in Examples described later, it takes only a few minutes to obtain a spectrum. In addition, in each of the above-mentioned chromatography, considering that it takes about 30 minutes for one measurement, the effect brought about by this embodiment is remarkable. Of course, a method other than FTNMR, for example, CW (Continuous Wave) -NMR may be employed in this step.
ちなみに、１４Ｎを核としたＮＭＲを用いる場合、１４Ｎ核自体が四極子核であるため、ＦＴＮＭＲの測定で検出されるピークは非常にブロードなピークになってしまう。つまり、測定感度が非常に高くなってしまい、ピークがブロードになる、すなわち分解能は低くなる。そのため、１４Ｎを核とした窒素化合物の構造解析を行おうとすると、多々の困難が生じる。
ところが、本工程においては上記のような困難があっても全く支障はない。なぜなら、本工程においては窒素化合物の構造解析を主として行うのではなく、窒素化合物の定量を行うことが主だからである。具体的に言うと、各々の窒素化合物に起因するピークが得られればよく、さらに言うと、逆に各ピークがブロードであればあるほどすなわち各ピークの面積が大きければ大きいほど、検量線を作成しやすくなり、ひいては定量する際の効率を良くすることができる。つまり、本発明者は、上記のようなブロードなピークとなるという特徴を逆に活かし、窒素化合物の定量化という手法を想到したのである。
Incidentally, when NMR using 14 N as a nucleus is used, since the 14 N nucleus itself is a quadrupole nucleus, the peak detected by the FTNMR measurement becomes a very broad peak. That is, the measurement sensitivity becomes very high, and the peak becomes broad, that is, the resolution becomes low. For this reason, when trying to analyze the structure of a nitrogen compound having 14 N as a nucleus, many difficulties arise.
However, in the present step, there is no problem even if the above-described difficulties are present. This is because, in this step, the structural analysis of the nitrogen compound is not mainly performed, but the determination of the nitrogen compound is mainly performed. More specifically, it is only necessary to obtain a peak due to each nitrogen compound. In other words, the more broad each peak, that is, the larger the area of each peak, the more the calibration curve is created. As a result, the efficiency at the time of quantification can be improved. In other words, the present inventor conceived a method of quantifying nitrogen compounds by taking advantage of the above feature of broad peaks.
ところで、１５Ｎを核としたＮＭＲを用いる場合も変形例として挙げられる。確かに、窒素を核種とすることにより、上記に挙げたように水に起因するピークを表出させないことは可能となる。ただ、１５Ｎ核を観測してＦＴＮＭＲを用いて定量する場合、１５Ｎは天然存在比が著しく低い。そのため、ピークは非常にシャープとなり窒素化合物のピーク位置を容易に判別できるものの、ピークの面積が小さくなる。そのため、１４Ｎを核とした場合の方が検量線を作成しやすくなる。その結果、本実施形態のように１４Ｎを核としたＮＭＲを用いるのが非常に好ましい。 By the way, a case where NMR using 15 N as a nucleus is used can be cited as a modification. Certainly, by using nitrogen as a nuclide, it is possible to prevent the peak due to water from appearing as mentioned above. However, when 15 N nuclei are observed and quantified using FTNMR, 15 N has a remarkably low natural abundance ratio. Therefore, the peak is very sharp and the peak position of the nitrogen compound can be easily discriminated, but the peak area is reduced. Therefore, it is easier to create a calibration curve when 14 N is used as a nucleus. As a result, it is very preferable to use NMR with 14 N as a nucleus as in this embodiment.
上記の内容以外にも、以下の効果が得られる。
例えば、クロマトグラフィーの一つであるＩＣでは、イオン性の異なる窒素化合物を同時に測定することが原理上難しく、少なくとも２回の測定が必要となる。
しかしながら、１４Ｎを核としたＮＭＲを用いる場合、イオン性が互いに異なる複数の窒素化合物のピークを各々別個に表出させることが可能となる。そうなると、１回の測定で、効率良く複数の窒素化合物を定量することが可能となる。測定対象が、イオン性が互いに異なる複数の窒素化合物を含有するならば、クロマトグラフィーの一つであるＩＣに比べ、より顕著な効果を奏する。
なお、ここで言う「イオン性が互いに異なる複数の窒素化合物」とは、窒素含有陰イオン、窒素含有陽イオン、および電気的に中性な窒素化合物の組み合わせのことを指す。
In addition to the above contents, the following effects can be obtained.
For example, in an IC which is one of chromatography, it is difficult in principle to simultaneously measure nitrogen compounds having different ionic properties, and at least two measurements are required.
However, when NMR using 14 N as a nucleus is used, peaks of a plurality of nitrogen compounds having different ionic properties can be expressed separately. Then, it becomes possible to quantify a plurality of nitrogen compounds efficiently by one measurement. If the object to be measured contains a plurality of nitrogen compounds having different ionic properties, a more remarkable effect can be obtained as compared with IC which is one of chromatography.
Note that “a plurality of nitrogen compounds having different ionic properties” as used herein refers to a combination of a nitrogen-containing anion, a nitrogen-containing cation, and an electrically neutral nitrogen compound.
上記の特徴を活かすのならば、測定対象は複数の窒素化合物を含有し、かつ、本工程においては、ＮＭＲにより得られた一つのスペクトルから複数の窒素化合物を一度に定量するのが好ましい。なお、各々の窒素化合物のピークの面積と窒素化合物の濃度との関係（例えば、複数の窒素化合物の各々に応じた検量線）を得ていれば、複数の窒素化合物を一度に定量することは可能である。 If the above feature is utilized, it is preferable that the measurement object contains a plurality of nitrogen compounds, and in this step, the plurality of nitrogen compounds are quantified at a time from one spectrum obtained by NMR. In addition, if the relationship between the peak area of each nitrogen compound and the concentration of the nitrogen compound (for example, a calibration curve corresponding to each of the plurality of nitrogen compounds) is obtained, it is possible to quantify a plurality of nitrogen compounds at a time. Is possible.
また、測定対象はタンパク質を含有しないのが好ましい。窒素が多数含有されているタンパク質が測定対象に全く存在しないならば、窒素化合物のピークを各々別個に明確に表出させることが確実に可能となる。 Moreover, it is preferable that a measuring object does not contain protein. If a protein containing a large amount of nitrogen does not exist at all in the measurement object, it is possible to reliably express the peaks of nitrogen compounds separately.
また、ＮＭＲを用いた測定においては上記のような二重管、さらに言うと各管が同心となる多重管（もちろん三重管や四重管などであっても構わない。）を用い、当該多重管のうち一つの管には基準物質を配置し、別の管には測定対象を配置するのが好ましい。例えば、多重管が二重管の場合、中心の管には基準物質を配置し、その外側の管には測定対象を配置するのが好ましい。その際、ＦＴＮＭＲ測定で磁場を固定するために必要な重水素化溶媒を中心の管に添加する。
なお、定量準備工程において検量線を得る際は、外側の管に、濃度が既知の窒素化合物ＡやＢを配置するのが好ましい。
ただ、本実施形態においては多重管を用いず、単なる試料管を用いても構わない。その場合、定量準備工程においては窒素化合物Ａを試料管に入れてＮＭＲにて測定した後、別の試料管に基準物質を入れて別途ＮＭＲにて測定することになる。定量工程でも同様であり、窒素化合物を含有する測定対象と基準物質とを別途ＮＭＲにて測定することになる。その一方、多重管を用いると、先に述べたように各窒素化合物に対して一度に測定することが可能となる。そのため、多重管を用いた上記手法の方が、窒素化合物をより効率良く定量することが可能となる。
Further, in the measurement using NMR, a double tube as described above, more specifically, a multiple tube in which each tube is concentric (of course, it may be a triple tube or a quadruple tube) is used. It is preferable that a reference substance is disposed in one of the tubes, and a measurement target is disposed in another tube. For example, when the multiple tube is a double tube, it is preferable that the reference material is disposed in the central tube and the measurement target is disposed in the outer tube. At that time, a deuterated solvent necessary for fixing the magnetic field by FTNMR measurement is added to the central tube.
In addition, when obtaining a calibration curve in the quantitative preparation step, it is preferable to place nitrogen compounds A and B having known concentrations in the outer tube.
However, in this embodiment, a simple sample tube may be used instead of the multiple tube. In that case, in the quantitative preparation step, nitrogen compound A is put in a sample tube and measured by NMR, then a reference substance is put in another sample tube and separately measured by NMR. The same applies to the determination step, and the measurement object containing the nitrogen compound and the reference substance are separately measured by NMR. On the other hand, when multiple tubes are used, it is possible to measure each nitrogen compound at once as described above. Therefore, the above method using multiple tubes can more efficiently quantify nitrogen compounds.
以上の結果、本実施形態によれば、窒素化合物を効率良く定量可能となる。 As a result, according to the present embodiment, the nitrogen compound can be quantified efficiently.
なお、上記の好ましい形態は、本工程以外にも先の定量準備工程で採用するのも好ましい。窒素化合物の種類に応じた検量線が効率良く得られるためである。そのため、上記のような一度の定量を、本工程と定量準備工程とで行うのが好ましいが、そのいずれかで行っても構わない。 In addition, it is also preferable to employ | adopt said preferable form in the previous quantitative preparation process besides this process. This is because a calibration curve corresponding to the type of nitrogen compound can be obtained efficiently. For this reason, it is preferable to perform the above-described single quantification in this step and the quantitative preparation step, but it may be performed in either of them.
以下、本実施例について説明する。なお、本発明の技術的範囲は以下の実施例に限定されるものではない。 Hereinafter, this embodiment will be described. The technical scope of the present invention is not limited to the following examples.
（実施例１）
１−１．（定量）準備工程
まず、ＦＴＮＭＲの試料管として、図１に示す二重管を用意した。二重管の外管は日本精密化学製Ｎ−５Ｐとし、内管は日本精密化学製Ｎ−５０２Ａとした。
そして、二重管の外管に、既知濃度のアンモニアを含有する溶液を添加した。その一方、二重管の内管に、ＦＴＮＭＲ測定時に必要な磁場固定用重水素化溶媒である重水（関東化学製、特級）、および、定量の基準物質（すなわち基準物質）としてグリシン（関東化学製、特級）を添加した。
その後、内管を外管に挿入し、１４Ｎを核としたＦＴＮＭＲ測定（装置としてはＢｒｕｋｅｒ Ｂｉｏｓｐｉｎ製ＡＶＡＮＣＥ４００型を使用）を行い、得られた既知濃度のアンモニアを含有する溶液におけるアンモニアのピーク面積を基準物質であるグリシンのピーク面積で除して規格化した。
Example 1
1-1. (Quantitative) Preparation Step First, a double tube shown in FIG. 1 was prepared as a sample tube for FTNMR. The outer tube of the double tube was N-5P manufactured by Nippon Seimitsu Chemical, and the inner tube was N-502A manufactured by Nippon Seimitsu Chemical.
Then, a solution containing ammonia of a known concentration was added to the outer tube of the double tube. On the other hand, in the inner tube of the double tube, heavy water (made by Kanto Chemical Co., special grade), which is a deuterated solvent for magnetic field fixation necessary for FTNMR measurement, and glycine (Kanto Chemical Co., Ltd.) as a quantitative reference material (ie, reference material) Manufactured, special grade).
After that, the inner tube was inserted into the outer tube, and FTNMR measurement with 14 N as the nucleus (using AVANCE 400 manufactured by Bruker Biospin as the apparatus) was performed, and the peak area of ammonia in the obtained solution containing ammonia of a known concentration was obtained. Was normalized by dividing by the peak area of glycine, which is a reference substance.
本実施例での定量準備工程の一つのスペクトルの結果を示したのが図２である。なお、１６０ｐｐｍ付近に表出したピークは、今回の実施例を行う際に添加していたヒドラジンに由来するものであり、本実施例に記載の内容とは直接の関係は無い。 FIG. 2 shows the result of one spectrum of the quantitative preparation step in this example. In addition, the peak expressed in the vicinity of 160 ppm is derived from hydrazine added at the time of carrying out this example, and has no direct relationship with the contents described in this example.
次に、濃度水準を変更した既知濃度のアンモニアを含有する溶液についても同様の測定を行った。本実施例において定量準備工程にてアンモニアの濃度に応じたアンモニアに起因するピーク面積（基準物質であるグリシンのピーク面積を１として規格化した値）を以下の表に示す。
１−２．定量工程
その後、二重管の内管の内容物をそのままにした上で、外管を、測定対象となる試料溶液を添加したものと交換したこと以外は上記の定量準備工程と同様の測定を行った。そして、本工程により得られたＮＭＲのスペクトルにおけるアンモニウムイオンのピーク面積を基準物質であるグリシンのピーク面積で規格化した値を検量線に代入し、試料溶液中のアンモニアの濃度を定量した。
本工程の結果、試料溶液中のアンモニアの濃度としては１０２１ｍｇ／ｌという値を効率良く得られた。
1-2. Quantitative process After that, the contents of the inner pipe of the double pipe were left as they were, and the same measurement as in the above quantitative preparation process was performed except that the outer pipe was replaced with one to which the sample solution to be measured was added. went. And the value which normalized the peak area of the ammonium ion in the NMR spectrum obtained by this process with the peak area of glycine which is a standard substance was substituted for the calibration curve, and the concentration of ammonia in the sample solution was quantified.
As a result of this step, a value of 1021 mg / l was efficiently obtained as the ammonia concentration in the sample solution.
（実施例２）
本実施例においては、測定対象に、イオン性が互いに異なる複数の窒素化合物（硝酸とアンモニア）を含有させた。そして基準物質としてはアセトニトリル（関東化学製）を用いた。それ以外は、実施例１と同様とした。
(Example 2)
In this example, the measurement object was made to contain a plurality of nitrogen compounds (nitric acid and ammonia) having different ionic properties. Acetonitrile (manufactured by Kanto Chemical) was used as a reference substance. Other than that was the same as Example 1.
本実施例での定量準備工程の一つのスペクトルの結果を示したのが図４である。図４が示すように、硝酸とアンモニアは、イオン性が互いに異なる複数の窒素化合物（硝酸イオン（ＮＯ３−）、アンモニウムイオン（ＮＨ４ ＋））となっており、硝酸とアンモニアに基準物質のアセトニトリルを含めた上で、これらは全てピークとして表出していた。
なお、図４においてＮＨ４ ＋のピーク位置がゼロよりも小さくなっているが、これは、測定対象に含有されるＨ＋のせいでピーク位置がシフトしているためであり、決して誤測定によるものではない。
FIG. 4 shows the result of one spectrum of the quantitative preparation step in this example. As shown in FIG. 4, nitric acid and ammonia are a plurality of nitrogen compounds (nitric acid ions (NO 3− ) and ammonium ions (NH 4 + )) having different ionic properties. These were all expressed as peaks, with the inclusion of acetonitrile.
In FIG. 4, the peak position of NH 4 + is smaller than zero, but this is because the peak position is shifted due to H + contained in the measurement object, which is never due to erroneous measurement. It is not a thing.
その結果、試料溶液中の硝酸の濃度としては２５ｍｇ／ｌ、アンモニアの濃度としては３３ｍｇ／ｌという値を効率良く得られた。 As a result, the nitric acid concentration in the sample solution was 25 mg / l, and the ammonia concentration was 33 mg / l.
（比較例）
高速液体クロマトグラフィー（ＨＰＬＣ）、ガスクロマトグラフィー（ＧＣ）、イオンクロマトグラフィー（ＩＣ）、ガスクロマトグラフ質量分析（ＧＣＭＳ）、液体クロマトグラフ質量分析（ＬＣＭＳ）、１Ｈを核にしたＦＴＮＭＲを用い、窒素化合物を含有する測定対象に対する定量を図ったが、いずれも上手くいかなかった。
例えば、ＨＰＬＣ、ＬＣＭＳ、ＩＣだと、溶離液との混合による形態変化が生じてしまった。特にＩＣでは、窒素含有陰イオンか窒素含有陽イオンのどちらかが検出可能であるが、同時に検出することはできなかった。また測定対象中の塩により測定が妨げられた。
また、ＧＣ、ＧＣＭＳだと、加熱による分解が生じた。特にＧＣＭＳでは分離カラムへの吸着が生じ、測定が妨げられた。
また、１Ｈを核にしたＦＴＮＭＲだと、水のピークがスペクトルに表出してしまい、測定が妨げられた。
(Comparative example)
High-performance liquid chromatography (HPLC), gas chromatography (GC), ion chromatography (IC), gas chromatography mass spectrometry (GCMS), liquid chromatography mass spectrometry (LCMS), FTNMR with 1 H as the nucleus, nitrogen Although quantification was attempted for a measurement object containing a compound, none of them was successful.
For example, in the case of HPLC, LCMS, and IC, morphological changes occurred due to mixing with the eluent. In particular, in IC, either a nitrogen-containing anion or a nitrogen-containing cation can be detected, but not at the same time. Moreover, the measurement was hindered by the salt in the measurement object.
In the case of GC and GCMS, decomposition by heating occurred. In particular, in GCMS, adsorption to a separation column occurred, and measurement was hindered.
In addition, in the case of FTNMR with 1 H as a nucleus, a water peak appeared in the spectrum, which hindered measurement.
Claims (5)
予め、濃度が既知の前記窒素化合物と、前記窒素化合物のスペクトルのピークとは別のピーク位置にて参照用のピークが表出する基準物質であるグリシンまたはギ酸アンモニウムと、に対して前記核磁気共鳴分光法を用いた測定を行い、濃度が既知の前記窒素化合物のスペクトルのピークの面積と、前記基準物質のピークの面積とから、前記窒素化合物のピークの面積と前記窒素化合物の濃度との関係を得る定量準備工程と、
を有し、
前記関係に基づいて前記定量工程を行う、窒素化合物の定量方法。 A quantitative step of quantifying the nitrogen compound by performing measurement using a nuclear magnetic resonance spectroscopy with 14 N as a nucleus on a measurement object containing the nitrogen compound ;
The nuclear magnetism is previously measured with respect to the nitrogen compound having a known concentration and glycine or ammonium formate, which is a standard substance in which a peak for reference appears at a peak position different from the peak of the spectrum of the nitrogen compound. The measurement is performed using resonance spectroscopy, and the peak area of the nitrogen compound and the concentration of the nitrogen compound are calculated from the peak area of the spectrum of the nitrogen compound with a known concentration and the peak area of the reference substance. A quantitative preparation process to obtain the relationship;
I have a,
A method for quantifying a nitrogen compound, wherein the quantification step is performed based on the relationship .
前記定量工程においては、前記核磁気共鳴分光法により得られた一つのスペクトルから複数の前記窒素化合物を一度に定量する、請求項１に記載の窒素化合物の定量方法。 The measurement object contains a plurality of the nitrogen compounds,
2. The method for quantifying a nitrogen compound according to claim 1 , wherein, in the quantification step, a plurality of the nitrogen compounds are quantified at a time from one spectrum obtained by the nuclear magnetic resonance spectroscopy.
In the measurement using the nuclear magnetic resonance spectroscopy, multiple tubes in which each tube is concentric are used, one of the multiple tubes is arranged with the reference substance, and the other tube has a known concentration. The method for quantifying a nitrogen compound according to any one of claims 1 to 4 , wherein a nitrogen compound or the measurement object is arranged to perform the quantification preparation step and the quantification step.
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