JP2010025644A - Coloration reagent of nitrate ions and method for detecting and quantifying nitrate ions using it - Google Patents
Coloration reagent of nitrate ions and method for detecting and quantifying nitrate ions using it Download PDFInfo
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- -1 nitrate ions Chemical class 0.000 title claims abstract description 94
- 229910002651 NO3 Inorganic materials 0.000 title claims abstract description 88
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 73
- 238000000034 method Methods 0.000 title claims abstract description 34
- FWEOQOXTVHGIFQ-UHFFFAOYSA-N 8-anilinonaphthalene-1-sulfonic acid Chemical compound C=12C(S(=O)(=O)O)=CC=CC2=CC=CC=1NC1=CC=CC=C1 FWEOQOXTVHGIFQ-UHFFFAOYSA-N 0.000 claims abstract description 80
- 238000006243 chemical reaction Methods 0.000 claims abstract description 42
- 239000000010 aprotic solvent Substances 0.000 claims abstract description 35
- 150000002790 naphthalenes Chemical class 0.000 claims abstract description 35
- VTRBOZNMGVDGHY-UHFFFAOYSA-N 6-(4-methylanilino)naphthalene-2-sulfonic acid Chemical compound C1=CC(C)=CC=C1NC1=CC=C(C=C(C=C2)S(O)(=O)=O)C2=C1 VTRBOZNMGVDGHY-UHFFFAOYSA-N 0.000 claims abstract description 27
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical group ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 120
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 claims description 31
- 238000002835 absorbance Methods 0.000 claims description 22
- 238000001514 detection method Methods 0.000 claims description 13
- 238000005259 measurement Methods 0.000 claims description 12
- 125000003277 amino group Chemical group 0.000 claims description 10
- 125000000542 sulfonic acid group Chemical group 0.000 claims description 10
- 239000000243 solution Substances 0.000 description 50
- 238000012360 testing method Methods 0.000 description 35
- 239000011259 mixed solution Substances 0.000 description 23
- 239000002904 solvent Substances 0.000 description 22
- 150000001450 anions Chemical class 0.000 description 19
- 239000000523 sample Substances 0.000 description 18
- DZLFLBLQUQXARW-UHFFFAOYSA-N tetrabutylammonium Chemical compound CCCC[N+](CCCC)(CCCC)CCCC DZLFLBLQUQXARW-UHFFFAOYSA-N 0.000 description 18
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 16
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 15
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 150000002500 ions Chemical class 0.000 description 12
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 12
- XEZNGIUYQVAUSS-UHFFFAOYSA-N 18-crown-6 Chemical compound C1COCCOCCOCCOCCOCCO1 XEZNGIUYQVAUSS-UHFFFAOYSA-N 0.000 description 11
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 10
- 238000004040 coloring Methods 0.000 description 10
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 9
- 238000010521 absorption reaction Methods 0.000 description 9
- 150000001768 cations Chemical class 0.000 description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 6
- 235000010333 potassium nitrate Nutrition 0.000 description 6
- 239000004323 potassium nitrate Substances 0.000 description 6
- 238000011002 quantification Methods 0.000 description 6
- 238000001228 spectrum Methods 0.000 description 6
- 238000010586 diagram Methods 0.000 description 5
- ABBQHOQBGMUPJH-UHFFFAOYSA-M Sodium salicylate Chemical compound [Na+].OC1=CC=CC=C1C([O-])=O ABBQHOQBGMUPJH-UHFFFAOYSA-M 0.000 description 4
- RRKTZKIUPZVBMF-IBTVXLQLSA-N brucine Chemical compound O([C@@H]1[C@H]([C@H]2C3)[C@@H]4N(C(C1)=O)C=1C=C(C(=CC=11)OC)OC)CC=C2CN2[C@@H]3[C@]41CC2 RRKTZKIUPZVBMF-IBTVXLQLSA-N 0.000 description 4
- RRKTZKIUPZVBMF-UHFFFAOYSA-N brucine Natural products C1=2C=C(OC)C(OC)=CC=2N(C(C2)=O)C3C(C4C5)C2OCC=C4CN2C5C31CC2 RRKTZKIUPZVBMF-UHFFFAOYSA-N 0.000 description 4
- 238000004737 colorimetric analysis Methods 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-M dihydrogenphosphate Chemical compound OP(O)([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-M 0.000 description 4
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 4
- 239000012488 sample solution Substances 0.000 description 4
- 229960004025 sodium salicylate Drugs 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- KRHYYFGTRYWZRS-UHFFFAOYSA-M Fluoride anion Chemical compound [F-] KRHYYFGTRYWZRS-UHFFFAOYSA-M 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-M hydrogensulfate Chemical compound OS([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-M 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 159000000001 potassium salts Chemical class 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 230000000007 visual effect Effects 0.000 description 3
- HVBSAKJJOYLTQU-UHFFFAOYSA-N 4-aminobenzenesulfonic acid Chemical compound NC1=CC=C(S(O)(=O)=O)C=C1 HVBSAKJJOYLTQU-UHFFFAOYSA-N 0.000 description 2
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 2
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- 238000011481 absorbance measurement Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 150000008049 diazo compounds Chemical class 0.000 description 2
- 239000012954 diazonium Substances 0.000 description 2
- 150000001989 diazonium salts Chemical class 0.000 description 2
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 2
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 2
- 229910017604 nitric acid Inorganic materials 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 229910001414 potassium ion Inorganic materials 0.000 description 2
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 2
- QMGVPVSNSZLJIA-FVWCLLPLSA-N strychnine Chemical compound O([C@H]1CC(N([C@H]2[C@H]1[C@H]1C3)C=4C5=CC=CC=4)=O)CC=C1CN1[C@@H]3[C@]25CC1 QMGVPVSNSZLJIA-FVWCLLPLSA-N 0.000 description 2
- 0 *c1cccc2c1c(Nc1ccccc1)ccc2 Chemical compound *c1cccc2c1c(Nc1ccccc1)ccc2 0.000 description 1
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- 235000005979 Citrus limon Nutrition 0.000 description 1
- 244000131522 Citrus pyriformis Species 0.000 description 1
- QMGVPVSNSZLJIA-UHFFFAOYSA-N Nux Vomica Natural products C1C2C3C4N(C=5C6=CC=CC=5)C(=O)CC3OCC=C2CN2C1C46CC2 QMGVPVSNSZLJIA-UHFFFAOYSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 description 1
- 241001279009 Strychnos toxifera Species 0.000 description 1
- RDXARWSSOJYNLI-UHFFFAOYSA-N [P].[K] Chemical compound [P].[K] RDXARWSSOJYNLI-UHFFFAOYSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 235000013405 beer Nutrition 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 239000013626 chemical specie Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 150000003983 crown ethers Chemical class 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 125000001624 naphthyl group Chemical group 0.000 description 1
- 238000006396 nitration reaction Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- VLTRZXGMWDSKGL-UHFFFAOYSA-M perchlorate Chemical compound [O-]Cl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-M 0.000 description 1
- 229940085991 phosphate ion Drugs 0.000 description 1
- 239000003586 protic polar solvent Substances 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 229960004889 salicylic acid Drugs 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000012453 solvate Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 229960005453 strychnine Drugs 0.000 description 1
- 229950000244 sulfanilic acid Drugs 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
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- 238000002834 transmittance Methods 0.000 description 1
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- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
Description
本発明は、硝酸イオン呈色試薬に関し、詳細には、硝酸イオンを含む溶液を簡便に呈色できる硝酸イオンの呈色試薬、並びにこれを用いて硝酸イオンを検出する方法及び硝酸イオンの定量方法に関する。 TECHNICAL FIELD The present invention relates to a nitrate ion coloring reagent, and more specifically, a nitrate ion coloring reagent that can easily color a solution containing nitrate ions, a method for detecting nitrate ions using the same, and a nitrate ion quantification method About.
従来、アニオンを検出或いは定量する方法として、イオンクロマトグラフ法、イオン選択電極法、比色法等が主に利用されている。イオンクロマトグラフ法は、試料溶液をイオン交換樹脂が充填されたカラムに流し、交換能の差によってアニオンを分離し、濃度測定を行う方法である。また、イオン選択電極法は試料溶液中にイオン選択電極を入れ、参照電極との間の電位差を測定することでアニオンの定量を行う方法である。 Conventionally, an ion chromatography method, an ion selective electrode method, a colorimetric method and the like are mainly used as a method for detecting or quantifying anions. The ion chromatograph method is a method in which a sample solution is passed through a column packed with an ion exchange resin, anions are separated based on a difference in exchange capacity, and concentration measurement is performed. The ion selective electrode method is a method in which an anion is quantified by placing an ion selective electrode in a sample solution and measuring a potential difference with a reference electrode.
比色法はアニオン呈色試薬と試料溶液を反応あるいは相互作用させ、吸収位置と吸光度の変化量から試料溶液中のイオン濃度を定量する方法である。この方法はイオン選択電極やイオンクロマトグラフ法に比べ、簡便で迅速な分析が行えるという利点を有する。更に、イオンクロマトグラフ法と比較すると分析に使用する装置が安価で、操作が簡便である。 The colorimetric method is a method of reacting or interacting with an anion coloring reagent and a sample solution, and quantifying the ion concentration in the sample solution from the amount of change in absorption position and absorbance. This method has an advantage that simple and quick analysis can be performed as compared with an ion selective electrode or an ion chromatographic method. Furthermore, compared with the ion chromatograph method, the apparatus used for analysis is inexpensive and the operation is simple.
現在、硝酸イオンを定量する呈色試薬としては、ブルシンやサリチル酸ナトリウムなどが使用されている。
ブルシン（２，３‐ジメトキシストリキニジン‐１０‐オン）は、硫酸の存在下で、硝酸イオンと反応すると黄色の物質を生成する。この物質の吸光度（４１０ｎｍ）を測定して硝酸イオンを定量する。ブルシンは、アルカロイドであるストリキニーネの類縁体であり毒性がある。また、ブルシンは、黄色の生成物の吸光度がＢｅｅｒの法則に従わないため、正確な定量が困難であるという欠点がある。
Currently, brucine, sodium salicylate, and the like are used as a color reagent for quantifying nitrate ions.
Brucine (2,3-dimethoxystriquinidin-10-one) produces a yellow substance when it reacts with nitrate ions in the presence of sulfuric acid. Nitrate ions are quantified by measuring the absorbance (410 nm) of this substance. Brucine is an analog of the alkaloid strychnine and is toxic. In addition, brucine has a disadvantage that the quantitative determination is difficult because the absorbance of the yellow product does not follow Beer's law.
サリチル酸ナトリウムを用いた硝酸イオンの定量は、サリチル酸のニトロ化を利用した方法で、Ｃａｔａｌｄｏ法（非特許文献１及び２参照）と呼ばれている。サリチル酸ナトリウムは濃硝酸の存在下、硝酸イオンによって、ニトロ化される。ニトロ化されたサリチル酸ナトリウムはアルカリ条件下でキノリド型となり４１０ｎｍ付近に吸収を持つ黄色を呈する。この呈色反応を利用し、４１０ｎｍの吸光度により硝酸イオンの定量を行う。この方法では濃硫酸を用いる必要がある。また，比較的妨害イオンが少なく操作が簡単ではあるが感度が劣る。
Nitrate ion quantification using sodium salicylate is a method that utilizes nitration of salicylic acid, and is called the Cataldo method (see Non-Patent
非特許文献３には、硝酸イオンと1,8-ANSを反応させて呈色させることが報告されている。この文献の記載によると、まず、硝酸イオンを還元して亜硝酸イオンとし、この亜硝酸イオンによってスルファニル酸をジアゾニウム塩とする。次に、このジアゾニウム塩と1,8-ANSをカップリングさせることにより紫色のジアゾ化合物が生成する。このジアゾ化合物の吸光度を測定することで硝酸イオンの定量を行う。この呈色反応は、硝酸イオン及び1,8-ANSの他に、還元剤を用いる。
即ち、非特許文献３に記載の定量方法では多段階の操作を要し、操作が複雑であるという問題を有していた。
That is, the quantitative method described in Non-Patent
本発明の課題は、高い精度を有するとともに、硝酸イオンを含む溶液を簡便に呈色することができる硝酸イオンの呈色試薬を提供することである。
本発明の他の課題は、本発明の呈色試薬を用いて、この呈色試薬が硝酸イオンと選択的に呈色反応を起こすことにより、溶液中の硝酸イオンを簡便に検出する方法及び硝酸イオンの定量方法を提供することである。
An object of the present invention is to provide a color reagent for nitrate ions that has high accuracy and can easily color a solution containing nitrate ions.
Another object of the present invention is to provide a method for easily detecting nitrate ions in a solution by using the color reagent of the present invention and causing the color reagent to selectively cause a color reaction with nitrate ions and nitric acid. It is to provide a method for quantitative determination of ions.
請求項１に係る発明は、少なくともアミノ基とスルホン酸基を含むナフタレン誘導体を含み、該ナフタレン誘導体と硝酸イオンの呈色反応により硝酸イオンを検出可能である呈色試薬に関する。
請求項２に係る発明は、前記ナフタレン誘導体が、下式（化１）で表される8-anilino-1-naphthalenesulfonic acid（1,8-ANS）であることを特徴とする請求項１に記載の呈色試薬に関する。
The invention according to
The invention according to
請求項５に係る発明は、非プロトン性溶媒がジクロロメタンであることを特徴とする請求項１乃至４いずれかに記載の呈色試薬に関する。
請求項６に係る発明は、下記段階（１）乃至（３）を含む硝酸イオンの検出方法に関する。
（１）少なくとも硝酸イオンを含む試料を準備する段階
（２）少なくともアミノ基とスルホン酸基を含むナフタレン誘導体を含む呈色試薬を準備する段階
（３）前記硝酸イオンを含む試料と前記ナフタレン誘導体を、非プロトン性溶媒中で呈色反応させる段階
請求項７に係る発明は、前記ナフタレン誘導体が、下式（化３）で表される8-anilino-1-naphthalenesulfonic acid（1,8-ANS）であることを特徴とする請求項６に記載の検出方法に関する。
The invention according to claim 5 relates to the color reagent according to any one of
The invention according to
(1) A step of preparing a sample containing at least nitrate ions (2) A step of preparing a color reagent containing a naphthalene derivative containing at least an amino group and a sulfonic acid group (3) A sample containing the nitrate ions and the naphthalene derivative The step of causing a color reaction in an aprotic solvent, wherein the naphthalene derivative is represented by the following formula (Chemical Formula 3): 8-anilino-1-naphthalenesulfonic acid (1,8-ANS) It is related with the detection method of
請求項１０に係る発明は、下記工程（１）乃至（５）を含む硝酸イオンの定量方法に関する。
（１）少なくとも硝酸イオンを含む試料を準備する工程
（２）少なくともアミノ基とスルホン酸基を含むナフタレン誘導体を含む呈色試薬を準備する工程
（３）前記硝酸イオンを含む試料と前記ナフタレン誘導体を、非プロトン性溶媒中で呈色反応させる工程
（４）工程３で得られた溶液の吸光度を測定する工程
（５）工程４で得た測定結果に基づき硝酸イオン濃度を算出する工程
請求項１１に係る発明は、前記ナフタレン誘導体が、下式（化５）で表される8-anilino-1-naphthalenesulfonic acid（1,8-ANS）であることを特徴とする請求項１０に記載の定量方法に関する。
The invention according to
(1) A step of preparing a sample containing at least nitrate ions (2) A step of preparing a color reagent containing a naphthalene derivative containing at least an amino group and a sulfonic acid group (3) A sample containing the nitrate ions and the naphthalene derivative A step of performing a color reaction in an aprotic solvent (4) A step of measuring the absorbance of the solution obtained in step 3 (5) A step of calculating a nitrate ion concentration based on the measurement result obtained in
本発明の硝酸イオンの呈色試薬は、硝酸イオンを高い精度で検出することができる。
本発明の硝酸イオンの検出方法は、本発明の呈色試薬を用いて行われる。本発明の呈色試薬はナフタレン誘導体を含み、このナフタレン誘導体は硝酸イオンと選択的に呈色反応を起こすことにより硝酸イオンを検出することができる。従って、本発明の硝酸イオンの検出方法は、呈色反応を起こすために中間体を生成する必要性がないから、簡便に硝酸イオンを検出及び定量することができるという利点を有する。
The nitrate ion coloring reagent of the present invention can detect nitrate ions with high accuracy.
The nitrate ion detection method of the present invention is carried out using the color reagent of the present invention. The color reagent of the present invention contains a naphthalene derivative, and this naphthalene derivative can detect nitrate ions by causing a color reaction selectively with nitrate ions. Therefore, the nitrate ion detection method of the present invention has an advantage that nitrate ions can be easily detected and quantified because there is no need to generate an intermediate in order to cause a color reaction.
以下、本発明の硝酸イオンの呈色試薬（以下、本呈色試薬という）について説明する。
本呈色試薬は、ナフタレン誘導体を含有し、該ナフタレン誘導体と硝酸イオンの呈色反応により、溶液中の硝酸イオンを検出可能とした硝酸イオンの呈色試薬である。
Hereinafter, the color reagent for nitrate ions of the present invention (hereinafter referred to as the present color reagent) will be described.
The present color reagent is a color reagent for nitrate ions, which contains a naphthalene derivative and makes it possible to detect nitrate ions in a solution by a color reaction between the naphthalene derivative and nitrate ions.
本呈色試薬に含まれるナフタレン誘導体は、少なくともアミノ基とスルホン酸基を含むナフタレン誘導体であり、好ましくは、下式（化７）で示される8-anilino-1-naphthalenesulfonic acid（以下、1,8-ANSという）、或いは下式（化８）で表される6-p-toluidino-2-naphthalenesulfonic acid（以下、2,6-TNSという）が用いられる。 The naphthalene derivative contained in the present color reagent is a naphthalene derivative containing at least an amino group and a sulfonic acid group, and preferably 8-anilino-1-naphthalenesulfonic acid (hereinafter referred to as 1, 1) represented by the following formula (Formula 7). 8-ANS) or 6-p-toluidino-2-naphthalenesulfonic acid (hereinafter referred to as 2,6-TNS) represented by the following formula (Formula 8) is used.
即ち、本呈色試薬は、1,8-ANS又は2,6-TNS（以下、両者合わせて1,8-ANS等という）と、硝酸イオンが呈色反応を起こすことにより、硝酸イオンの有無を検出可能とする硝酸イオンの呈色試薬である。
本呈色試薬に含まれる1,8-ANS等は如何なる形態であってもよい。例えば、本呈色試薬は、粉末状の1,8-ANS等からなる粉末状の呈色試薬であってもよく、或いは、1,8-ANS等をクロロホルム，ジクロロメタン，アセトン，ベンゼン，アセトニトリル等に溶かした液体状の呈色試薬であってもよい。
In other words, the present color reagent contains 1,8-ANS or 2,6-TNS (hereinafter referred to as 1,8-ANS together) and the presence of nitrate ions by causing a color reaction of nitrate ions. Is a color reagent for nitrate ions.
1,8-ANS and the like contained in the present color reagent may be in any form. For example, the present color reagent may be a powder color reagent composed of powdered 1,8-ANS or the like, or 1,8-ANS or the like may be chloroform, dichloromethane, acetone, benzene, acetonitrile or the like. It may be a liquid color reagent dissolved in the solution.
本呈色試薬を用いたとき、前記1,8-ANS等と硝酸イオンの呈色反応は、非プロトン性溶媒の存在下で起こる。この非プロトン性溶媒とは、水酸基、アミノ基、スルホン酸基等の酸性度の高い水素原子を含まない溶媒である。一方で、酸性度の高い水素原子を含むプロトン性溶媒では水素結合を介しアニオンを強く溶媒和し、アニオンの反応性を極端に低下させるため、好ましくない。
前記非プロトン性溶媒として、クロロホルムやジクロロメタンが挙げられるが、好ましくはジクロロメタンである。この理由は、ジクロロメタンの存在下で1,8-ANS等は、硝酸イオンと選択的に呈色反応し、この呈色（紫色又はオレンジ色）が、短時間内に起こり、また色が鮮明であるからである。
When the present color reagent is used, the color reaction between 1,8-ANS and the like and nitrate ions occurs in the presence of an aprotic solvent. This aprotic solvent is a solvent that does not contain a highly acidic hydrogen atom such as a hydroxyl group, an amino group, or a sulfonic acid group. On the other hand, a protic solvent containing a hydrogen atom with high acidity is not preferable because it strongly solvates an anion through a hydrogen bond and extremely decreases the reactivity of the anion.
Examples of the aprotic solvent include chloroform and dichloromethane, with dichloromethane being preferred. The reason for this is that 1,8-ANS, etc., reacts selectively with nitrate ions in the presence of dichloromethane, and this coloration (purple or orange) occurs within a short time, and the color is clear. Because there is.
硝酸イオンの検出方法に関しては後に詳説するが、本呈色試薬で硝酸イオンの検出を行うときは、前記非プロトン性溶媒と硝酸イオンを予め混合して硝酸イオン含有溶液を作製して、この溶液に本呈色試薬を添加することにより、1,8-ANS等と硝酸イオンとの呈色反応を起こしてもよい。 The method for detecting nitrate ions will be described in detail later. When nitrate ions are detected with the present color reagent, a solution containing nitrate ions is prepared by previously mixing the aprotic solvent and nitrate ions. A color reaction between 1,8-ANS or the like and nitrate ions may be caused by adding the present color reagent.
或いは、本呈色試薬は、1,8-ANS等と前記非プロトン性溶媒を含んでも良い。このように予め本呈色試薬に前記非プロトン性溶媒を含む場合は、1,8-ANSと非プロトン性溶媒の含有比（重量比）は、１：１０４ 〜１０６ とされる。この理由は、1,8-ANSの重量「１」に対し、非プロトン性溶媒が「１０４」未満の場合は1,8-ANSが高濃度となるため吸光度測定が難しくなるため、また「１０６」を超えると1,8-ANSが低濃度となるため目視による色変化が確認しにくいため、いずれの場合も望ましくないからである。また、2,6-TNSと非プロトン性溶媒の含有比（重量比）は、１：１０４ 〜１０６とされる。この理由は、2,6-TNSの重量「１」に対し、非プロトン性溶媒が「１０４」未満の場合は2,6-TNSが高濃度となるため吸光度測定が難しくなるため、また「１０６」を超えると2,6-TNSが低濃度となるため目視による色変化が確認しにくいため、いずれの場合も望ましくないからである。 Alternatively, the coloring reagent may contain 1,8-ANS and the like and the aprotic solvent. Thus, when the aprotic solvent is previously contained in the present color reagent, the content ratio (weight ratio) between 1,8-ANS and the aprotic solvent is 1:10 4 to 10 6 . The reason for this is that when the aprotic solvent is less than “10 4 ” with respect to the weight “1” of 1,8-ANS, the concentration of 1,8-ANS becomes high, making it difficult to measure the absorbance. 10 6 "by weight, l, 8-ANS since the hard color change by visual inspection for a low concentration is confirmed, it is not desirable in any case. The content ratio (weight ratio) between 2,6-TNS and the aprotic solvent is set to 1:10 4 to 10 6 . The reason for this is that when the aprotic solvent is less than “10 4 ” with respect to the weight “1” of 2,6-TNS, the concentration of 2,6-TNS becomes high and the absorbance measurement becomes difficult. If it exceeds 10 6 ”, 2,6-TNS has a low concentration, and it is difficult to confirm a color change by visual observation.
次に、本呈色試薬を用いた硝酸イオンの検出方法（以下、本検出方法という）について説明する。
本検出方法は、以下の３段階を含む方法である。
（１）少なくとも硝酸イオンを含む試料を準備する段階
（２）本呈色試薬を準備する段階
（３）前記硝酸イオンを含む試料と本呈色試薬を、非プロトン性溶媒の存在下で呈色反応させる段階
Next, a method for detecting nitrate ions using the present color reagent (hereinafter referred to as the present detection method) will be described.
This detection method is a method including the following three steps.
(1) Step of preparing a sample containing at least nitrate ions (2) Step of preparing the coloring reagent (3) Coloring the sample containing the nitrate ions and the coloring reagent in the presence of an aprotic solvent Reaction stage
段階（１）は、少なくとも硝酸イオンを含む試料を準備する段階である。即ち、本呈色試薬を用いて硝酸イオンを検出しようとする試料が段階（１）で準備される。
段階（２）において、本呈色試薬が準備される。前述のとおり、本呈色試薬は、ナフタレン誘導体を含有する試薬であり、このナフタレン誘導体は、少なくともアミノ基とスルホン酸基を含む。本呈色試薬は如何なる形態でもよく、例えば、粉末状である1,8-ANSのみで構成される粉末状の呈色試薬であってもよく、或いはクロロホルム、ジクロロメタン等に溶解した液体状の呈色試薬であってもよい。
段階（３）において、段階（１）で得た試料と段階（２）で得た本呈色試薬を、非プロトン性溶媒（例えば、ジクロロメタン）の存在下で混合する。
Step (1) is a step of preparing a sample containing at least nitrate ions. That is, a sample for detecting nitrate ions using the present color reagent is prepared in step (1).
In step (2), the present color reagent is prepared. As described above, the present color reagent is a reagent containing a naphthalene derivative, and this naphthalene derivative contains at least an amino group and a sulfonic acid group. The present color reagent may take any form, for example, may be a powder color reagent composed only of powdered 1,8-ANS, or a liquid color solution dissolved in chloroform, dichloromethane or the like. It may be a color reagent.
In step (3), the sample obtained in step (1) and the present color reagent obtained in step (2) are mixed in the presence of an aprotic solvent (eg, dichloromethane).
本検出方法においては、非プロトン性溶媒をどの段階で添加してもよい。例えば、段階（１）において、硝酸イオンと非プロトン性溶媒を含む試料を準備してもよいし、段階（２）において、非プロトン性溶媒を含んで本呈色試薬を調製してもよい。或いは、段階（３）において、非プロトン性溶媒を準備して、この非プロトン性溶媒の存在下で硝酸イオンと1,8-ANS等の呈色反応を起こさせてもよい。 In this detection method, an aprotic solvent may be added at any stage. For example, in step (1), a sample containing nitrate ions and an aprotic solvent may be prepared. In step (2), the present color reagent may be prepared containing an aprotic solvent. Alternatively, in step (3), an aprotic solvent may be prepared to cause a color reaction such as nitrate ions and 1,8-ANS in the presence of the aprotic solvent.
尚、前記段階（１）及び段階（２）は、いずれを先に行ってもよく、例えば、最初に、本呈色試薬を調製して、次に、少なくとも硝酸イオンを含む試料を準備してもよい。 Any one of the steps (1) and (2) may be performed first. For example, first, the present color reagent is prepared, and then a sample containing at least nitrate ions is prepared. Also good.
本検出方法によると、本呈色試薬に含まれる1,8-ANS等と、硝酸イオンが呈色反応を起こす。このことにより、前記混合液中の硝酸イオンの有無を確認することができる。 According to this detection method, nitrate ions and 1,8-ANS, etc., contained in this coloring reagent cause a color reaction. Thereby, the presence or absence of nitrate ions in the mixed solution can be confirmed.
次に、本呈色試薬を用いた硝酸イオンの定量方法について説明する。
本発明の呈色試薬を用いて硝酸イオンを定量する方法は以下の工程を含む。
（１）少なくとも硝酸イオンを含む試料を準備する工程
（２）本呈色試薬を準備する工程
（３）前記硝酸イオンを含む試料と本呈色試薬を、非プロトン性溶媒の存在下で呈色反応させる工程
（４）工程３で得られた溶液の吸光度を測定する工程
（５）工程４で得られた測定結果に基づき、硝酸イオン濃度を算出する工程
Next, a method for quantifying nitrate ions using the present color reagent will be described.
The method for quantifying nitrate ions using the color reagent of the present invention includes the following steps.
(1) Step of preparing a sample containing at least nitrate ions (2) Step of preparing the present color reagent (3) Coloring the sample containing the nitrate ions and the present color reagent in the presence of an aprotic solvent Step of reacting (4) Step of measuring the absorbance of the solution obtained in step 3 (5) Step of calculating the nitrate ion concentration based on the measurement result obtained in
工程（１）は、少なくとも硝酸イオンを含む試料を準備する工程である。即ち、本呈色試薬を用いて硝酸イオンを定量しようとする（即ち、未知濃度の硝酸イオンを含む）試料が工程（１）で準備される。
工程（２）において、本呈色試薬が準備される。前述のとおり、本呈色試薬は、ナフタレン誘導体を含有する試薬であり、このナフタレン誘導体は、少なくともアミノ基とスルホン酸基を含む。本呈色試薬は如何なる形態でもよく、例えば、粉末状である1,8-ANS等のみで構成される粉末状の呈色試薬であってもよく、或いは非プロトン性溶媒（例えば、ジクロロメタン）に溶解した液体状の呈色試薬であってもよい。
工程（３）において、工程（１）で準備した試料と工程（２）で得た本呈色試薬を、非プロトン性溶媒（例えば、ジクロロメタン）の存在下で混合する。この溶液は、好ましくは、およそ３０分静置される。
Step (1) is a step of preparing a sample containing at least nitrate ions. That is, a sample intended to quantify nitrate ions using the present color reagent (that is, containing nitrate ions of unknown concentration) is prepared in step (1).
In step (2), the present color reagent is prepared. As described above, the present color reagent is a reagent containing a naphthalene derivative, and this naphthalene derivative contains at least an amino group and a sulfonic acid group. The present color reagent may take any form, for example, may be a powder color reagent composed only of powdered 1,8-ANS or the like, or an aprotic solvent (for example, dichloromethane). It may be a dissolved liquid color reagent.
In the step (3), the sample prepared in the step (1) and the present color reagent obtained in the step (2) are mixed in the presence of an aprotic solvent (for example, dichloromethane). This solution is preferably allowed to stand for approximately 30 minutes.
工程（４）において、工程３で得られた溶液の吸光度を測定する。具体的には、例えば、工程３で得られたジクロロメタン溶液は、５６５ｎｍにおける吸光度が測定され、吸光度が０．３以下であることを確認する。もし、吸光度が０．３を超えた場合は、工程１で得られた溶液にジクロロメタンを添加して希釈し，工程３で得られたジクロロメタン溶液の５６５ｎｍにおける吸光度が０．３以下となるようにする。
工程（５）において、硝酸イオン濃度を算出する。例えば，工程２で調製した1,8-ANSのジクロロメタン溶液の濃度が2.5×10-5 mol/L溶液であり，この溶液を３mL使用したとすると，下式（数１）により、未知試料の濃度（Ｃ）が算出できる。ただし濃度の単位はｍｏｌ／Ｌである。
In step (4), the absorbance of the solution obtained in
In step (5), the nitrate ion concentration is calculated. For example, if the concentration of 1,8-ANS dichloromethane solution prepared in
本発明の硝酸イオンの定量方法において、呈色試薬として1,8-ANSを使用する場合は、前記工程（３）において、硝酸イオン/1,8-ANSを、モル比で0.001〜0.3、好ましくは、0.1〜0.3とする。この理由は、値が小さすぎると吸光度変化が小さく，逆に大きすぎると発色が安定しないためである。2,6-TNSを使用する場合は、前記工程（３）において、硝酸イオン/2,6-TNSを、モル比で0.001〜1.0、好ましくは、0.1〜0.8とする。この理由は、値が小さすぎると吸光度変化が小さく，逆に大きすぎると発色が安定しないためである。 In the nitrate ion quantification method of the present invention, when 1,8-ANS is used as a color reagent, nitrate ion / 1,8-ANS is preferably 0.001 to 0.3, preferably in molar ratio in the step (3). Is 0.1 to 0.3. The reason for this is that if the value is too small, the change in absorbance is small, whereas if the value is too large, the color development is not stable. When 2,6-TNS is used, in the step (3), nitrate ion / 2,6-TNS is set to 0.001 to 1.0, preferably 0.1 to 0.8 in molar ratio. The reason for this is that if the value is too small, the change in absorbance is small, whereas if the value is too large, the color development is not stable.
標準液との比色により、硝酸イオンの定量を行う。この比色は、例えば、色彩色差測定、濃度反射測定、吸光度測定、透過率測定の何れかの測定による測定値の対比、または目視による対比である。 Nitrate ions are quantified by colorimetry with the standard solution. This colorimetry is, for example, a comparison of measured values by any one of color-color difference measurement, density reflection measurement, absorbance measurement, and transmittance measurement, or visual comparison.
以下、本発明を、実施例を用いてより詳細に説明する。 Hereinafter, the present invention will be described in more detail with reference to examples.
（試験例１）最適溶媒の検索と硝酸イオンに対する選択的呈色の確認
（１−１）
8-anilino-1-naphthalenesulfonic acidと硝酸イオンが溶液中で呈色反応を起こすために最適な溶媒を検索するために、以下（表１）に記載の各種溶媒を用いて、これら溶媒中で、8-anilino-1-naphthalenesulfonic acid（1,8-ANS）と硝酸イオンを反応させてこの溶液が呈色されるか否かを確認した。
（１−２）
さらに、本呈色試薬が、硝酸イオンと選択的に呈色反応を起こすことを確認するために、以下（表２）のアニオンのテトラブチルアンモニウム（ＴＢＡ）塩に対して呈色反応が起こるか否かを確認した。表中、ｐＫｂはアニオンの塩基解離定数を示す。
（１−３）
上記（１−１）と（１−２）を確認するため、下記の方法で試験を行った。
表１に記載の各種溶媒と1,8-ANSの混合溶液（1,8-ANSの濃度1.000×10-4 mol/L）3 mLに、表２のアニオンのテトラブチルアンモニウム塩の各種溶媒溶液（各種テトラブチルアンモニウム塩濃度3.00×10-4 mol/L）を1mL加えて、得られた混合溶液を時間ごとに写真撮影した。即ち、1,8-ANSと各ＴＢＡ塩はモル濃度比１：１で混合された。混合溶液の写真を図１〜６に示す。
(Test Example 1) Search for optimum solvent and confirmation of selective coloration for nitrate ion (1-1)
In order to search for the most suitable solvent for 8-anilino-1-naphthalenesulfonic acid and nitrate ion to cause a color reaction in the solution, various solvents described below (Table 1) were used. It was confirmed whether 8-anilino-1-naphthalenesulfonic acid (1,8-ANS) and nitrate ion were reacted to color the solution.
(1-2)
Furthermore, in order to confirm that the present color reagent selectively causes a color reaction with nitrate ion, the color reaction occurs with respect to the tetrabutylammonium (TBA) salt of the anion shown below (Table 2). Confirmed no. In the table, pK b shows the nucleotide dissociation constant anions.
(1-3)
In order to confirm the above (1-1) and (1-2), the test was conducted by the following method.
Various solvent solutions of tetrabutylammonium salts of anions in Table 2 in 3 mL of mixed solutions of various solvents listed in Table 1 and 1,8-ANS (concentration of 1,8-ANS: 1.000 × 10 -4 mol / L) 1 mL of various tetrabutylammonium salt concentrations of 3.00 × 10 −4 mol / L was added, and the resulting mixed solution was photographed every hour. That is, 1,8-ANS and each TBA salt were mixed at a molar concentration ratio of 1: 1. The photograph of a mixed solution is shown in FIGS.
図１乃至６は、メタノール（図１）、エタノール（図２）、アセトニトリル（図３）、アセトン（図４）、クロロホルム（図５）、ジクロロメタン（図６）を溶媒とした溶液中で1,8-ANSと各ＴＢＡ塩を反応させた時の溶液の写真である。
図１が示すとおり、４８時間経過した時点において、メタノール中で、各種アニオンと1,8-ANSの間で呈色反応は確認されなかった。
図２が示すとおり、４８時間経過した時点において、エタノール中で、各種アニオンと1,8-ANSの間で呈色反応は確認されなかった。
図３が示すとおり、アセトニトリルを溶媒とした場合、２４時間後まで全ての溶液において呈色は確認できなかった。しかし４８時間後に硝酸イオンの溶液は明るい黄色、リン酸二水素イオンの溶液は薄いオレンジ色、硫酸イオンの溶液は薄い赤色を呈した。
図４が示すとおり、アセトンを溶媒とした場合、１２時間後にリン酸二水素イオン、硫酸イオン、フッ素イオンの溶液は薄いオレンジ色、硫酸水素イオンと臭素イオンの溶液は薄い水色、塩化物イオンの溶液は薄い黄色を呈した。これらの色は時間が経過するにつれて濃くなっていった（写真（４８時間後））。
図５が示すとおり、クロロホルムを溶媒とした場合、３０分後にピロリン酸イオン、リン酸二水素イオン、硫酸イオン、硫酸水素イオン、フッ素イオン、塩化物イオン、硝酸イオンを加えた溶液は薄い緑色の呈色が確認できた（写真（３０分後））。８４時間後には、アニオンを加えていない溶液と過塩素酸イオン以外の溶液が濃い緑色を呈した。
図６が示すとおり、ジクロロメタンを溶媒とした場合は、硝酸イオンの溶液のみに２分後から紫色の呈色が見られた（写真（２分後））。この紫色の呈色は徐々に黄色へと変化し、３０分後には鮮やかな黄色となった（写真（３０分後））。その後、６時間後まで変化は見られなかったが、２４時間後にはピロリン酸イオン、リン酸一水素イオン、硫酸水素イオン、フッ素イオン溶液に薄い紫色の呈色が見られた（写真（２４時間後））。
FIGS. 1 to 6 are diagrams showing solutions of methanol (FIG. 1), ethanol (FIG. 2), acetonitrile (FIG. 3), acetone (FIG. 4), chloroform (FIG. 5) and dichloromethane (FIG. 6) in a solvent. It is the photograph of the solution when 8-ANS and each TBA salt are made to react.
As shown in FIG. 1, when 48 hours passed, no color reaction was observed between various anions and 1,8-ANS in methanol.
As shown in FIG. 2, when 48 hours passed, no color reaction was observed between various anions and 1,8-ANS in ethanol.
As shown in FIG. 3, when acetonitrile was used as a solvent, coloration could not be confirmed in all solutions until 24 hours later. However, after 48 hours, the nitrate ion solution was bright yellow, the dihydrogen phosphate ion solution was light orange, and the sulfate ion solution was light red.
As shown in FIG. 4, when acetone is used as the solvent, the solution of dihydrogen phosphate ion, sulfate ion and fluoride ion is pale orange after 12 hours, and the solution of hydrogen sulfate ion and bromine ion is pale light blue, chloride ion The solution had a pale yellow color. These colors became darker over time (photo (after 48 hours)).
As shown in FIG. 5, when chloroform is used as a solvent, a solution containing pyrophosphate ion, dihydrogen phosphate ion, sulfate ion, hydrogen sulfate ion, fluorine ion, chloride ion, and nitrate ion is light green after 30 minutes. Coloration was confirmed (photograph (after 30 minutes)). After 84 hours, a solution containing no anion and a solution other than perchlorate ions showed a dark green color.
As shown in FIG. 6, when dichloromethane was used as the solvent, a purple color was observed only after 2 minutes in the nitrate ion solution (photograph (after 2 minutes)). The purple color gradually changed to yellow and became bright yellow after 30 minutes (photo (30 minutes later)). Thereafter, no change was observed until 6 hours later, but after 24 hours, a pale purple color was observed in the pyrophosphate ion, monohydrogen phosphate ion, hydrogen sulfate ion, and fluorine ion solution (photograph (24 hours rear)).
以上の結果から、非プロトン性溶媒中で1,8-ANSは、硝酸イオンと選択的に呈色反応を起こすことが分かった。また、この硝酸イオン選択的な呈色反応は、非プロトン性溶媒であるジクロロメタンで顕著に確認できた。 From the above results, it was found that 1,8-ANS selectively colored with nitrate ions in aprotic solvents. This nitrate ion selective color reaction was remarkably confirmed with dichloromethane, which is an aprotic solvent.
（試験例２）硝酸塩と1,8-ANSの混合溶液のUV-visスペクトルの時間変化
ジクロロメタンを溶媒とした場合の1,8-ANSと硝酸イオンの混合溶液の呈色について、ジクロロメタン中で1,8-ANSと硝酸イオンのＴＢＡ塩のモル濃度比が１：１となるように溶液を調製し、時間ごとにUV-visスペクトルを測定した。詳細には、ジクロロメタンと1,8-ANSの混合溶液（1,8-ANSの濃度2.000×10-4 mol/L）1.5 mLに、硝酸ＴＢＡ塩のジクロロメタン溶液（硝酸ＴＢＡ塩濃度2.000×10-4 mol/L）を1.5 mL加えて、得られた混合溶液を時間ごとにUV-visスペクトル（Perkin Elmer UV-VIS/NMR Lamnda 19）測定した。即ち、1,8-ANSと各ＴＢＡ塩はモル濃度比１：１で混合された。測定結果を図７に示す。
(Test Example 2) Temporal change in UV-vis spectrum of a mixed solution of nitrate and 1,8-ANS The coloration of a mixed solution of 1,8-ANS and nitrate ions in dichloromethane was used as a color solution in dichloromethane. The solution was prepared so that the molar concentration ratio of 8-ANS and the TBA salt of nitrate ion was 1: 1, and the UV-vis spectrum was measured every time. Specifically, 1.5 mL of a mixed solution of dichloromethane and 1,8-ANS (concentration of 1,8-ANS: 2.000 × 10 −4 mol / L) is added to a dichloromethane solution of nitrate TBA salt (concentration of TBA nitrate : 2.000 × 10 − 4 mol / L) was added, and the obtained mixed solution was subjected to UV-vis spectrum (Perkin Elmer UV-VIS / NMR Lamnda 19) measurement every hour. That is, 1,8-ANS and each TBA salt were mixed at a molar concentration ratio of 1: 1. The measurement results are shown in FIG.
1,8-ANS自体（硝酸塩を加えていない）のスペクトルは、吸収極大が２８０ｎｍにあり、４００ｎｍ以上の可視領域には吸収を持たない。硝酸塩を加えた直後、３６０ｎｍ付近と５７０ｎｍ付近に吸収が現れ、1,8-ANS自体のスペクトルとは全く異なるスペクトルへと変化した。その後５７０ｎｍ付近の吸収は減少し、２４時間後には３６０ｎｍ付近と４７０ｎｍ付近に吸収を持つスペクトルへと変化した。これらスペクトル変化から1,8-ANSとは異なる化学種が生成していることが分かる。
次に、この反応速度を知るため同様の条件で５７０ｎｍの吸光度の時間変化を測定した（図８）。その結果、５７０ｎｍの吸光度は約２分後に最大となり（反応ａ）、その後徐々に減少し３０分後には殆ど変化が見られなくなった（反応ｂ）。速度定数を算出するため、この反応を一次反応と仮定して時間に対して吸光度変化率の自然対数をプロットした（図９）。ただし、100秒後の吸光度をA0とした。その結果、反応ａの速度定数Kaは０．０４１９ｓ−１、反応ｂの速度定数Kbは０．０００８ｓ−１となった。反応ａはｂに比べ、約５０倍と早い速度で起こることがわかった。
The spectrum of 1,8-ANS itself (without adding nitrate) has an absorption maximum at 280 nm, and has no absorption in the visible region of 400 nm or more. Immediately after the addition of nitrate, absorption appeared at around 360 nm and around 570 nm, and the spectrum changed to a spectrum completely different from that of 1,8-ANS itself. Thereafter, the absorption near 570 nm decreased, and after 24 hours, it changed to a spectrum having absorption near 360 nm and 470 nm. These spectral changes indicate that chemical species different from 1,8-ANS are generated.
Next, in order to know this reaction rate, the time change of absorbance at 570 nm was measured under the same conditions (FIG. 8). As a result, the absorbance at 570 nm became maximum after about 2 minutes (reaction a), and then gradually decreased, and almost no change was observed after 30 minutes (reaction b). In order to calculate the rate constant, the natural logarithm of the rate of change in absorbance was plotted against time assuming that this reaction was a first order reaction (FIG. 9). However, the absorbance after 100 seconds was set to A 0. As a result, the rate constant K a of reaction a was 0.0419 s −1 , and the rate constant K b of reaction b was 0.0008 s −1 . It was found that reaction a occurred at a rate about 50 times faster than b.
（試験例３）TBANO3による1,8-ANSのジクロロメタン中でのUV-visスペクトルの変化と色調の変化の観察
1,8-ANSのジクロロメタン溶液（濃度2.5×10-5M）3mLに、アニオンとしてTBANO3のジクロロメタン溶液（濃度1.5×10-3M）10μLを加えた溶液を調製し（G/H=0.2）、UV-visスペクトルを２分毎に測定した。測定結果を図１０に示す。図１１は、565nmにおけるジクロロメタン中での1,8-ANSの吸光度の時間変化を示す。
(Test Example 3) Observation of changes in UV-vis spectrum and color tone of 1,8-ANS in dichloromethane using TBANO 3
Prepare a solution by adding 10 μL of TBANO 3 in dichloromethane (concentration 1.5 × 10 −3 M) as anion to 3 mL of 1,8-ANS in dichloromethane (concentration 2.5 × 10 −5 M) (G / H = 0.2 ), UV-vis spectrum was measured every 2 minutes. The measurement results are shown in FIG. FIG. 11 shows the time course of the absorbance of 1,8-ANS in dichloromethane at 565 nm.
（試験例４）TBANO3による1,8-ANSのジクロロメタン中での色調の変化の観察
以下の表に示す1,8-ANS溶液をサンプル瓶に3mLとったものを13本用意し、それぞれに各アニオン溶液を10μLずつ加えよく振り、溶液の色の変化を観察した。観察は、目視とデジタルカメラでの撮影によって行った。結果を図１２に示す。図中の番号は、以下の表のアニオンの番号を表す。
(Test Example 4) Observation of changes in color tone of 1,8-ANS in dichloromethane using TBANO 3 Prepare 13 13 mL of the 1,8-ANS solution shown in the table below in a sample bottle. 10 μL of each anion solution was added and shaken well, and the color change of the solution was observed. Observation was performed by visual observation and photographing with a digital camera. The results are shown in FIG. Numbers in the figure represent anion numbers in the following table.
図１２が示すとおり、観察開始から8分後、（2）TBANO3に色調の変化が見られ、60分にかけて青紫色になった。さらに、60分を境に紫が赤みを帯び始め、徐々に赤紫、ピンク系統の色へと変化した。 As shown in FIG. 12, 8 minutes after the start of observation, (2) TBANO 3 showed a change in color tone and turned blue-purple over 60 minutes. Furthermore, after 60 minutes, purple began to appear reddish and gradually changed to reddish purple and pink.
本試験例の如く、アニオン/1,8-ANS=0.2(モル比)にすると紫色に発色するには、およそ３０分かかり、その後3時間たっても変化しない。したがって、このモル比（アニオン/1,8-ANS=0.2）とすることにより、硝酸イオンの定量を行うことができる。 As in this test example, when anion / 1,8-ANS = 0.2 (molar ratio), it takes about 30 minutes to develop a purple color, and it does not change even after 3 hours. Therefore, nitrate ions can be quantified by using this molar ratio (anion / 1,8-ANS = 0.2).
（試験例５）ジクロロメタン中での各種アニオンのカリウム塩/18-crown-6/1,8-ANS混合溶液の色調変化
前記の呈色反応が、対カチオンであるＴＢＡと1,8-ANSとの呈色反応ではないことを確認するため以下の試験を行った。
対カチオンをカリウムとして、各種イオンのカリウム塩と1,8-ANSとの呈色反応を調べた。カリウム塩は有機溶媒に全く溶解しないが、クラウンエーテル（18-crown-6）が存在するとカリウムイオンが環内に包摂され、ＴＢＡ同様脂溶性のカチオンとなり、有機溶媒に可溶化する（図１３参照）。
各種アニオンのカリウム塩と18-crown-6の混合溶液を1,8-ANS溶液に加え、色調の変化を観察した。詳細には、ジクロロメタンと1,8-ANSの混合溶液（1,8-ANSの濃度2.00×10-4 mol/L）2mLに、以下の表（表４）に記載の濃度のカリウム塩（リン酸水素イオン、リン酸二水素イオン、硝酸イオン、塩化物イオン、臭素イオンのカリウム塩）/18-crown-6のジクロロメタン溶液2mLを加えて、得られた混合溶液を時間ごとに写真撮影して色調の変化を確認した。即ち、各種カリウム，18-crown-6、1,8-ANSのモル濃度比は１：１：１とした。観察結果を、図１４に示す。
(Test Example 5) Color tone change of mixed solution of potassium salt of various anions / 18-crown-6 / 1,8-ANS in dichloromethane The color reaction described above was performed by using TBA as a counter cation and 1,8-ANS. The following test was conducted to confirm that the color reaction was not.
The color reaction between potassium salts of various ions and 1,8-ANS was investigated using potassium as the counter cation. Potassium salts do not dissolve in organic solvents at all, but in the presence of crown ether (18-crown-6), potassium ions are included in the ring and become lipid-soluble cations like TBA, solubilized in organic solvents (see FIG. 13). ).
A mixed solution of potassium salts of various anions and 18-crown-6 was added to the 1,8-ANS solution, and changes in color tone were observed. More specifically, 2 mL of a mixed solution of dichloromethane and 1,8-ANS (concentration of 2.00 × 10 −4 mol / L of 1,8-ANS) was added to potassium salt (phosphorus) with the concentration shown in the following table (Table 4). 2 ml of dichloromethane solution of 18-crown-6), and the resulting mixed solution is photographed every hour. The potassium salt of oxyhydrogen ion, dihydrogen phosphate ion, nitrate ion, chloride ion, bromine ion) The change in color was confirmed. That is, the molar concentration ratio of various potassium, 18-crown-6, and 1,8-ANS was 1: 1: 1. The observation results are shown in FIG.
図１４が示すとおり、対カチオンをカリウムイオンに代えても、対カチオンがＴＢＡの場合と同様に硝酸イオンを加えた溶液のみが紫色を呈し、時間が経過すると黄色へと変化した。 As FIG. 14 shows, even when the counter cation was replaced with potassium ions, only the solution to which nitrate ions were added showed a purple color as in the case of TBA, and changed to yellow over time.
（試験例６）硝酸カリウム/18-crown-6−1,8-ANS混合溶液UV-visスペクトルの時間変化
対カチオンをカリウム塩とした場合のUV-visスペクトルの変化を確認するために、1,8-ANS溶液に硝酸カリウム/18-crown-6溶液を加え、時間ごとにUV-visスペクトルを測定した。詳細には、ジクロロメタンと1,8-ANSの混合溶液（1,8-ANSの濃度2.000×10-4mol/L）1.5mLに、硝酸カリウム塩、18-crown-6、及びジクロロメタンの混合溶液を1.5mL加えて、得られた混合溶液を時間ごとにUV-visスペクトル測定した。尚、硝酸カリウム塩の濃度は2.08×10-4mol/Lであり、18-crown-6の濃度は2.11×10-4mol/Lである。即ち、硝酸カリウム、18-crown-6ならびに1,8-ANSのモル濃度比は約１：１：１である。測定結果を図１５に示す。
図１５が示すとおり、硝酸ＴＢＡ塩を加えた場合と同様に、約２分後に５７０ｎｍ付近に吸収を持つスペクトルが現れ、その後５７０ｎｍの吸光度は時間経過とともに減少、２４時間後には４７０ｎｍ付近に吸収を持つスペクトルへと変化した。
(Test Example 6) Change in time of UV-vis spectrum of potassium nitrate / 18-crown-6-1,8-ANS mixed solution To confirm the change in UV-vis spectrum when the counter cation is a potassium salt, Potassium nitrate / 18-crown-6 solution was added to 8-ANS solution, and UV-vis spectrum was measured every hour. Specifically, a mixed solution of potassium nitrate, 18-crown-6, and dichloromethane was added to 1.5 mL of a mixed solution of dichloromethane and 1,8-ANS (concentration of 1,8-ANS: 2.000 × 10 -4 mol / L). 1.5 mL was added, and the obtained mixed solution was subjected to UV-vis spectrum measurement every hour. The concentration of potassium nitrate is 2.08 × 10 −4 mol / L, and the concentration of 18-crown-6 is 2.11 × 10 −4 mol / L. That is, the molar concentration ratio of potassium nitrate, 18-crown-6 and 1,8-ANS is about 1: 1: 1. The measurement results are shown in FIG.
As shown in FIG. 15, as in the case of adding the nitrate TBA salt, a spectrum having an absorption near 570 nm appears after about 2 minutes, and then the absorbance at 570 nm decreases with the passage of time, and after 24 hours, the absorption near 470 nm. It changed to the spectrum it has.
（試験例７）TBANO3による2,6-TNS(6-p-toluidino-2-naphthalenesulfonic acid)の各溶媒下での色調の変化の観察
2,6-TNSとTBANO3をそれぞれ約１ｍｇずつはかりとり、これらをサンプル瓶に入れた。この操作を繰り返し行い、同じサンプルを9個作った。9個のサンプルにそれぞれ（１）アセトニトリル、（２）クロロホルム、（３）ジクロロメタン、（４）ベンゼン、（５）DMSO、（６）メタノール、（７）エタノール、（８）ヘキサン、（９）アセトンを各5mL加え攪拌し、目視とデジタルカメラでの撮影で色調の変化を観察した。結果を図１６に示す。図中の番号は、前記溶媒に付した番号と対応する。
(Test Example 7) Observation of changes in color tone of 2,6-TNS (6-p-toluidino-2-naphthalenesulfonic acid) with TBANO 3 under each solvent
About 1 mg each of 2,6-TNS and TBANO 3 was weighed and placed in a sample bottle. This operation was repeated to make nine identical samples. Nine samples were each (1) acetonitrile, (2) chloroform, (3) dichloromethane, (4) benzene, (5) DMSO, (6) methanol, (7) ethanol, (8) hexane, (9) acetone. 5 mL of each was added and stirred, and the change in color tone was observed visually and photographed with a digital camera. The results are shown in FIG. The numbers in the figure correspond to the numbers assigned to the solvents.
図１６が示すとおり、（３）ジクロロメタンでは溶媒を加えてすぐ黄色（オレンジ色に近い黄色）に変化し、その後徐々に色味が強くなっていった。（４）ベンゼンでは溶媒を加えて5分ほどで黄色みを帯びてきた。（レモン色）（２）クロロホルムの場合、約１時間で着色し始め，2日ほどで赤に近いオレンジ色となった。（７）エタノールではさらに着色が遅く、1週間ほどで赤みを帯びてきた。（８）ヘキサンは2,6-TNSが溶けなかった。 As shown in FIG. 16, in (3) dichloromethane, the color changed to yellow (yellow close to orange) immediately after the solvent was added, and then the color gradually became stronger. (4) Benzene became yellowish in about 5 minutes after adding solvent. (Lemon color) (2) In the case of chloroform, it started to color in about 1 hour, and in about 2 days, it turned orange near red. (7) The coloration of ethanol was even slower and it turned red in about a week. (8) 2,6-TNS did not dissolve in hexane.
前記試験例のうち、試験例５と６それぞれにおける、色調の変化とUV-visスペクトルの変化から、対カチオンはジクロロメタン中の呈色反応に関わっていないことが明らかになった。即ち、前記の呈色反応は1,8-ANSと硝酸イオンとの反応によるものである。
さらに、試験例７により、2,6-TNSと硝酸イオンが選択的に呈色反応を起こすことが分かった。
以上のとおり、本発明の呈色試薬は、硝酸イオンと選択的に呈色反応を起こすことにより、硝酸イオンを検出することができる。
Among the test examples, the change in color tone and the change in UV-vis spectrum in each of Test Examples 5 and 6 revealed that the counter cation was not involved in the color reaction in dichloromethane. That is, the color reaction is due to the reaction between 1,8-ANS and nitrate ions.
Furthermore, from Test Example 7, it was found that 2,6-TNS and nitrate ions selectively cause a color reaction.
As described above, the color reagent of the present invention can detect nitrate ions by causing a color reaction selectively with nitrate ions.
Claims (13)
（１）少なくとも硝酸イオンを含む試料を準備する段階
（２）少なくともアミノ基とスルホン酸基を含むナフタレン誘導体を含む呈色試薬を準備する段階
（３）前記硝酸イオンを含む試料と前記ナフタレン誘導体を、非プロトン性溶媒中で呈色反応させる段階 A method for detecting nitrate ions comprising the following steps (1) to (3).
(1) A step of preparing a sample containing at least nitrate ions (2) A step of preparing a color reagent containing a naphthalene derivative containing at least an amino group and a sulfonic acid group (3) A sample containing the nitrate ions and the naphthalene derivative , Color reaction in aprotic solvent
（１）少なくとも硝酸イオンを含む試料を準備する工程
（２）少なくともアミノ基とスルホン酸基を含むナフタレン誘導体を含む呈色試薬を準備する工程
（３）前記硝酸イオンを含む試料と前記ナフタレン誘導体を、非プロトン性溶媒中で呈色反応させる工程
（４）工程３で得られた溶液の吸光度を測定する工程
（５）工程４で得た測定結果に基づき硝酸イオン濃度を算出する工程 A method for quantifying nitrate ions comprising the following steps (1) to (5).
(1) A step of preparing a sample containing at least nitrate ions (2) A step of preparing a color reagent containing a naphthalene derivative containing at least an amino group and a sulfonic acid group (3) A sample containing the nitrate ions and the naphthalene derivative Step of color reaction in aprotic solvent (4) Step of measuring absorbance of solution obtained in step 3 (5) Step of calculating nitrate ion concentration based on measurement result obtained in step 4
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| US11857562B2 (en) | 2010-07-06 | 2024-01-02 | Glaxosmithkline Biologicals Sa | Delivery of RNA to trigger multiple immune pathways |
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| US11690864B2 (en) | 2010-07-06 | 2023-07-04 | Glaxosmithkline Biologicals Sa | Delivery of RNA to trigger multiple immune pathways |
| US11690861B2 (en) | 2010-07-06 | 2023-07-04 | Glaxosmithkline Biologicals Sa | Delivery of RNA to trigger multiple immune pathways |
| US11690865B2 (en) | 2010-07-06 | 2023-07-04 | Glaxosmithkline Biologicals Sa | Delivery of RNA to trigger multiple immune pathways |
| US11690862B1 (en) | 2010-07-06 | 2023-07-04 | Glaxosmithkline Biologicals Sa | Delivery of RNA to trigger multiple immune pathways |
| US11690863B2 (en) | 2010-07-06 | 2023-07-04 | Glaxosmithkline Biologicals Sa | Delivery of RNA to trigger multiple immune pathways |
| US11696923B2 (en) | 2010-07-06 | 2023-07-11 | Glaxosmithkline Biologicals, Sa | Delivery of RNA to trigger multiple immune pathways |
| US11707482B2 (en) | 2010-07-06 | 2023-07-25 | Glaxosmithkline Biologicals Sa | Delivery of RNA to trigger multiple immune pathways |
| US11717529B2 (en) | 2010-07-06 | 2023-08-08 | Glaxosmithkline Biologicals Sa | Delivery of RNA to trigger multiple immune pathways |
| US11739334B2 (en) | 2010-07-06 | 2023-08-29 | Glaxosmithkline Biologicals Sa | Immunisation of large mammals with low doses of RNA |
| US11730754B2 (en) | 2010-07-06 | 2023-08-22 | Glaxosmithkline Biologicals Sa | Delivery of RNA to trigger multiple immune pathways |
| US11857681B2 (en) | 2010-07-06 | 2024-01-02 | Glaxosmithkline Biologicals Sa | Lipid formulations with RNA encoding immunogens |
| US11759475B2 (en) | 2010-07-06 | 2023-09-19 | Glaxosmithkline Biologicals Sa | Delivery of RNA to trigger multiple immune pathways |
| US11766401B2 (en) * | 2010-07-06 | 2023-09-26 | Glaxosmithkline Biologicals Sa | Methods of administering lipid formulations with immunogens |
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| US11839686B2 (en) * | 2010-07-06 | 2023-12-12 | Glaxosmithkline Biologicals Sa | Lipid formulations with viral immunogens |
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| US11850305B2 (en) | 2010-07-06 | 2023-12-26 | Glaxosmithkline Biologicals Sa | Method of making lipid formulations with RNA encoding immunogens |
| US11851660B2 (en) | 2010-07-06 | 2023-12-26 | Glaxosmithkline Biologicals Sa | Immunisation of large mammals with low doses of RNA |
| US20220125724A1 (en) * | 2010-07-06 | 2022-04-28 | Glaxosmithkline Biologicals Sa | Methods of administering lipid formulations with immunogens |
| US11913001B2 (en) | 2010-07-06 | 2024-02-27 | Glaxosmithkline Biologicals Sa | Immunisation of large mammals with low doses of RNA |
| US11865080B2 (en) | 2010-07-06 | 2024-01-09 | Glaxosmithkline Biologicals Sa | Delivery of RNA to trigger multiple immune pathways |
| US11883534B2 (en) | 2010-07-06 | 2024-01-30 | Glaxosmithkline Biologicals Sa | Immunisation with lipid formulations with RNA encoding immunogens |
| US11891608B2 (en) | 2010-07-06 | 2024-02-06 | Glaxosmithkline Biologicals Sa | Immunization of large mammals with low doses of RNA |
| US11905514B2 (en) | 2010-07-06 | 2024-02-20 | Glaxosmithkline Biological Sa | Immunisation of large mammals with low doses of RNA |
| US11759422B2 (en) | 2010-08-31 | 2023-09-19 | Glaxosmithkline Biologicals Sa | Pegylated liposomes for delivery of immunogen-encoding RNA |
| US11639370B2 (en) | 2010-10-11 | 2023-05-02 | Glaxosmithkline Biologicals Sa | Antigen delivery platforms |
| US11896636B2 (en) | 2011-07-06 | 2024-02-13 | Glaxosmithkline Biologicals Sa | Immunogenic combination compositions and uses thereof |
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