JP7477522B2 - Concentration measurement method, concentration measurement device, and program - Google Patents
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- 238000005259 measurement Methods 0.000 title claims description 50
- 238000000691 measurement method Methods 0.000 title description 18
- 239000000243 solution Substances 0.000 claims description 119
- 239000000126 substance Substances 0.000 claims description 112
- 239000003153 chemical reaction reagent Substances 0.000 claims description 75
- 238000006243 chemical reaction Methods 0.000 claims description 69
- 239000012088 reference solution Substances 0.000 claims description 47
- 238000000034 method Methods 0.000 claims description 32
- 239000013076 target substance Substances 0.000 claims description 32
- -1 bromate ion Chemical class 0.000 claims description 19
- SXDBWCPKPHAZSM-UHFFFAOYSA-M bromate Inorganic materials [O-]Br(=O)=O SXDBWCPKPHAZSM-UHFFFAOYSA-M 0.000 claims description 18
- 238000004458 analytical method Methods 0.000 claims description 15
- 238000004364 calculation method Methods 0.000 claims description 15
- 239000012491 analyte Substances 0.000 claims description 10
- ZEWQUBUPAILYHI-UHFFFAOYSA-N trifluoperazine Chemical group C1CN(C)CCN1CCCN1C2=CC(C(F)(F)F)=CC=C2SC2=CC=CC=C21 ZEWQUBUPAILYHI-UHFFFAOYSA-N 0.000 claims description 6
- 229960002324 trifluoperazine Drugs 0.000 claims description 6
- 238000002835 absorbance Methods 0.000 claims description 5
- 239000000523 sample Substances 0.000 description 127
- 230000035484 reaction time Effects 0.000 description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 238000002360 preparation method Methods 0.000 description 6
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 4
- 238000011088 calibration curve Methods 0.000 description 3
- 238000000611 regression analysis Methods 0.000 description 3
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 2
- OFBQJSOFQDEBGM-UHFFFAOYSA-N Pentane Chemical compound CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 239000013074 reference sample Substances 0.000 description 2
- 230000001052 transient effect Effects 0.000 description 2
- IVRMZWNICZWHMI-UHFFFAOYSA-N Azide Chemical compound [N-]=[N+]=[N-] IVRMZWNICZWHMI-UHFFFAOYSA-N 0.000 description 1
- QTANTQQOYSUMLC-UHFFFAOYSA-O Ethidium cation Chemical compound C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 QTANTQQOYSUMLC-UHFFFAOYSA-O 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 238000006385 ozonation reaction Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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Description
本発明は、濃度測定方法、濃度測定装置およびプログラムに関し、特には、測定対象物質と反応して蛍光強度や吸光度等のパラメータの大きさを変化させる試薬を用いた濃度測定方法、並びに、当該濃度測定方法に好適に使用し得る濃度測定装置およびプログラムに関するものである。 The present invention relates to a concentration measurement method, a concentration measurement device, and a program, and in particular to a concentration measurement method using a reagent that reacts with a substance to be measured to change the magnitude of a parameter such as fluorescence intensity or absorbance, as well as a concentration measurement device and a program that can be suitably used for the concentration measurement method.
従来、溶液サンプル中に含まれている測定対象物質の濃度を測定する方法として、測定対象物質と反応する試薬を使用し、測定対象物質と試薬との反応によって変化するパラメータの大きさを測定することにより溶液サンプル中の測定対象物質の濃度を求める方法が知られている。A conventional method for measuring the concentration of a target substance contained in a solution sample involves using a reagent that reacts with the target substance and determining the concentration of the target substance in the solution sample by measuring the magnitude of a parameter that changes due to the reaction between the target substance and the reagent.
具体的には、例えば、水中の臭素酸イオン濃度を測定することが可能な方法として、臭素酸イオン濃度に応じて蛍光強度の大きさが変化するトリフルオペラジン（ＴＦＰ）を使用した定量方法が提案されている（例えば、特許文献１，２参照）。当該定量方法では、試料水中の臭素酸イオンとＴＦＰとを反応させた際の蛍光強度の大きさを測定し、得られた蛍光強度の測定値と、予め作成しておいた検量線とを対比することにより試料水中の臭素酸イオン濃度を求める方法が提案されている。Specifically, for example, a method capable of measuring the concentration of bromate ions in water has been proposed that uses trifluoperazine (TFP), whose fluorescence intensity changes depending on the concentration of bromate ions (see, for example,
しかし、測定対象物質と反応する試薬を用いた上記従来の濃度測定方法には、検量線の作成が必要であり、作業が煩雑であるという問題があった。However, the conventional concentration measurement method, which uses a reagent that reacts with the substance to be measured, requires the creation of a calibration curve, which makes the process complicated.
また、測定対象物質と反応する試薬を用いた上記従来の濃度測定方法には、溶液サンプル中に含まれている測定対象物質以外の物質（共存物質）と、試薬とが反応してパラメータの大きさの変化に影響を与えることにより測定対象物質の濃度の定量精度が低下するという問題もあった。 In addition, the conventional concentration measurement method using a reagent that reacts with the substance to be measured had the problem that the reagent reacts with substances (coexisting substances) other than the substance to be measured contained in the solution sample, affecting changes in the magnitude of the parameter, thereby reducing the quantitative accuracy of the concentration of the substance to be measured.
そこで、本発明は、測定対象物質と反応してパラメータの大きさを変化させる試薬を用いて溶液サンプル中の測定対象物質の濃度を測定する方法について、測定対象物質の濃度を高い精度で簡便に測定することを可能にすることを目的とする。Therefore, the present invention aims to provide a method for measuring the concentration of a target substance in a solution sample using a reagent that reacts with the target substance to change the magnitude of a parameter, thereby enabling the concentration of the target substance to be measured easily and with high accuracy.
この発明は、上記課題を有利に解決することを目的とするものであり、本発明の濃度測定方法は、溶液サンプル中に含まれる測定対象物質の濃度を、前記測定対象物質と反応して溶液サンプルのパラメータの大きさを変化させる試薬を用いて測定する方法であって、濃度未知の測定対象物質を含む溶液サンプル、及び、前記溶液サンプルに対し既知の量の測定対象物質を添加した参照溶液サンプルに、前記試薬を添加し、前記パラメータの経時変化を測定する測定工程と、前記測定工程による測定結果に基づいて、前記溶液サンプル中及び前記参照溶液サンプル中の測定対象物質と前記試薬との見掛け上の反応終了時間以降の前記パラメータの大きさと時間との関係を表す近似式を作成する分析工程と、前記近似式に時間Ｔ（但し、Ｔ≧０）を代入して得られるパラメータの値と、前記既知の量との関係から前記溶液サンプル中の測定対象物質の濃度を求める算出工程とを含むとを含むことを特徴とする。The present invention aims to advantageously solve the above-mentioned problems, and the concentration measurement method of the present invention is a method for measuring the concentration of a substance to be measured contained in a solution sample using a reagent that reacts with the substance to be measured to change the magnitude of a parameter of the solution sample, and is characterized by including a measurement step of adding the reagent to a solution sample containing a substance to be measured of an unknown concentration and a reference solution sample to which a known amount of the substance to be measured has been added to the solution sample, and measuring the change in the parameter over time; an analysis step of creating an approximation equation that represents the relationship between the magnitude of the parameter and time after the apparent end time of the reaction between the substance to be measured and the reagent in the solution sample and the reference solution sample based on the measurement results from the measurement step; and a calculation step of calculating the concentration of the substance to be measured in the solution sample from the relationship between the value of the parameter obtained by substituting time T (where T ≧ 0) into the approximation equation and the known amount.
また、この発明は、上記課題を有利に解決することを目的とするものであり、本発明の濃度測定装置は、溶液サンプル中に含まれる測定対象物質の濃度を、前記測定対象物質と反応して溶液サンプルのパラメータの大きさを変化させる試薬を用いて測定する装置であって、濃度未知の測定対象物質を含む溶液サンプル、及び、前記溶液サンプルに対し既知の量の測定対象物質を添加した参照溶液サンプルについて、前記試薬を添加した状態で、前記パラメータの経時変化を測定する測定部と、前記測定部による測定結果に基づいて、前記溶液サンプル中及び前記参照溶液サンプル中の測定対象物質と前記試薬との見掛け上の反応終了時間以降の前記パラメータの大きさと時間との関係を表す近似式を作成する分析部と、前記近似式に時間Ｔ（但し、Ｔ≧０）を代入して得られるパラメータの値と、前記既知の量との関係から前記溶液サンプル中の測定対象物質の濃度を求める算出部とを備えることを特徴とする。The present invention also aims to advantageously solve the above-mentioned problems, and the concentration measuring device of the present invention is a device that measures the concentration of a substance to be measured contained in a solution sample using a reagent that reacts with the substance to be measured to change the magnitude of a parameter of the solution sample, and is characterized in that it includes a measurement unit that measures the change in the parameter over time for a solution sample containing a substance to be measured of unknown concentration and a reference solution sample to which a known amount of the substance to be measured has been added, with the reagent added; an analysis unit that creates an approximation equation that represents the relationship between the magnitude of the parameter and time after the apparent end time of the reaction between the substance to be measured and the reagent in the solution sample and the reference solution sample based on the measurement results by the measurement unit; and a calculation unit that calculates the concentration of the substance to be measured in the solution sample from the relationship between the value of the parameter obtained by substituting time T (where T ≧ 0) into the approximation equation and the known amount.
更に、この発明は、上記課題を有利に解決することを目的とするものであり、本発明のプログラムは、溶液サンプル中に含まれる測定対象物質の濃度を、前記測定対象物質と反応して溶液サンプルのパラメータの大きさを変化させる試薬を用いて測定する装置に、濃度未知の測定対象物質を含む溶液サンプル、及び、前記溶液サンプルに対し既知の量の測定対象物質を添加した参照溶液サンプルについて、前記試薬を添加した状態で、前記パラメータの経時変化を測定した結果を取得させるステップと、取得した前記結果に基づいて、前記溶液サンプル中及び前記参照溶液サンプル中の測定対象物質と前記試薬との見掛け上の反応終了時間以降の前記パラメータの大きさと時間との関係を表す近似式を作成させるステップと、前記近似式に時間Ｔ（但し、Ｔ≧０）を代入して得られるパラメータの値と、前記既知の量との関係から前記溶液サンプル中の測定対象物質の濃度を求めるステップとを実行させることを特徴とする。Furthermore, the present invention aims to advantageously solve the above-mentioned problems, and the program of the present invention is characterized in that it executes the steps of causing an apparatus that measures the concentration of a substance to be measured contained in a solution sample using a reagent that reacts with the substance to be measured to change the magnitude of a parameter of the solution sample, to obtain results of measuring the change in the parameter over time for a solution sample containing a substance to be measured of unknown concentration and a reference solution sample to which a known amount of the substance to be measured has been added, while the reagent has been added; creating an approximation equation that expresses the relationship between the magnitude of the parameter and time after the apparent end time of the reaction between the substance to be measured and the reagent in the solution sample and the reference solution sample, based on the obtained results; and determining the concentration of the substance to be measured in the solution sample from the relationship between the value of the parameter obtained by substituting time T (where T ≧ 0) into the approximation equation and the known amount.
本発明によれば、測定対象物質の濃度を高い精度で簡便に測定することができる。 According to the present invention, the concentration of the substance to be measured can be measured easily and with high accuracy.
（濃度測定方法）
本発明の濃度測定方法は、測定対象物質を含む溶液サンプル中の測定対象物質の濃度を、測定対象物質と反応して溶液サンプルのパラメータの大きさを変化させる試薬を用いて測定する方法である。具体的には、本発明の濃度測定方法は、例えば、測定対象物質として臭素酸イオン（ＢｒＯ3
-）を含む溶液サンプル中の臭素酸イオン濃度を、臭素酸イオンと反応して溶液サンプルの蛍光強度の大きさを変化させる試薬としてトリフルオペラジンを用いて測定する際に用いることができる。
なお、本発明の濃度測定方法を適用可能な測定対象物質、パラメータおよび試薬の組み合わせは、上述した一例に限定されるものではなく、例えば、パラメータは吸光度であってもよい。また、測定対象物質は、鉄イオン、ｐＨ、ＤＮＡであってもよい。更に、試薬は、ペナントロリン、ＦＩＴＣ（ｆｌｕｏｒｅｓｃｅｉｎ ｉｓｏｔｈｉｏｃｙａｎａｔｅ）、ＥＭＡ（Ｅｔｈｉｄｉｕｍ ＭｏｎｏＡｚａｉｄｅ）であってもよい。
(Concentration measurement method)
The concentration measurement method of the present invention is a method for measuring the concentration of a target substance in a solution sample containing the target substance by using a reagent that reacts with the target substance to change the magnitude of a parameter of the solution sample. Specifically, the concentration measurement method of the present invention can be used, for example, when measuring the concentration of bromate ions in a solution sample containing bromate ions ( BrO3- ) as a target substance by using trifluoperazine as a reagent that reacts with bromate ions to change the magnitude of the fluorescence intensity of the solution sample.
The combination of the measurement target substance, the parameter, and the reagent to which the concentration measurement method of the present invention can be applied is not limited to the above-mentioned example, and for example, the parameter may be absorbance. The measurement target substance may be iron ions, pH, or DNA. Furthermore, the reagent may be pentane, FITC (fluorescein isothiocyanate), or EMA (ethidium monoazide).
ここで、本発明の濃度測定方法は、以下の知見を見出してなされたものである。測定対象物質と反応する試薬を用いた濃度測定方法では、溶液サンプル中に含まれている測定対象物質以外の物質（以下、「共存物質」と称することがある。）も試薬と反応してパラメータの大きさの変化に影響を与え得る。そして、上述した臭素酸イオンの定量系などでは、測定対象物質と試薬との反応は比較的早期に見掛け上の反応終了（平衡）に到達するのに対し、共存物質と試薬との反応は測定対象物質と試薬との反応が平衡に到達しても終了せずにパラメータの大きさの変化に影響を与え続ける。 Here, the concentration measurement method of the present invention was made based on the following findings. In a concentration measurement method using a reagent that reacts with the substance to be measured, substances other than the substance to be measured contained in the solution sample (hereinafter sometimes referred to as "coexisting substances") can also react with the reagent and affect the change in the magnitude of the parameter. In the above-mentioned bromate ion quantitative system, the reaction between the substance to be measured and the reagent reaches an apparent end of the reaction (equilibrium) relatively quickly, whereas the reaction between the coexisting substance and the reagent does not end even when the reaction between the substance to be measured and the reagent reaches equilibrium, and continues to affect the change in the magnitude of the parameter.
具体的には、本発明の濃度測定方法は、一例として試薬との反応により変化するパラメータが蛍光強度である場合について図１に示すように、溶液サンプルの蛍光強度の変化の大きさは、測定対象物質と試薬との反応に起因した蛍光強度の変化の大きさと、共存物質と試薬との反応に起因した蛍光強度の変化の大きさとの和であるところ、測定対象物質と試薬との反応が比較的早期に見掛け上の反応終了（平衡）に到達する一方で共存物質と試薬との反応は終了しない系では、共存物質と試薬との反応に起因した蛍光強度の変化が測定対象物質と試薬との見掛け上の反応が終了する時間ＴEの前後において略一定である場合、測定対象物質と試薬との見掛け上の反応終了後の蛍光強度の変化は、共存物質と試薬との反応に起因した蛍光強度の変化と略等しくなるという新たな知見を利用し、以下のようにして測定対象物質の濃度を求めるものである。 Specifically, in the concentration measurement method of the present invention, as an example, in the case where the parameter that changes due to the reaction with a reagent is fluorescence intensity, as shown in FIG. 1 , the magnitude of change in fluorescence intensity of a solution sample is the sum of the magnitude of change in fluorescence intensity caused by the reaction between the substance to be measured and the reagent and the magnitude of change in fluorescence intensity caused by the reaction between the coexisting substance and the reagent; in a system in which the reaction between the substance to be measured and the reagent reaches an apparent end of the reaction (equilibrium) relatively early while the reaction between the coexisting substance and the reagent does not end, if the change in fluorescence intensity caused by the reaction between the coexisting substance and the reagent is approximately constant before and after the time T E at which the apparent reaction between the substance to be measured and the reagent ends, the change in fluorescence intensity after the apparent end of the reaction between the substance to be measured and the reagent will be approximately equal to the change in fluorescence intensity caused by the reaction between the coexisting substance and the reagent, and the concentration of the substance to be measured is determined as follows.
本発明の濃度測定方法は、上記の新たな知見を利用し、濃度未知の測定対象物質が含まれる溶液サンプルと、溶液サンプルに対し既知の量の測定対象物質を添加した参照溶液サンプルとを用いて、測定対象物質の濃度を測定するものである。上述したような系では、溶液サンプルと参照溶液サンプルとについて試薬と反応させた際の蛍光強度の大きさの経時変化を測定すると、図２に示すような挙動を示す。また、図２に示すグラフにおいて、測定対象物質と試薬との見掛け上の反応が終了する時間ＴEよりも反応時間が長い部分（図２では時間ＴEよりも右側に位置する部分）における参照溶液サンプルの蛍光強度と溶液サンプルの蛍光強度との差は、参照溶液の調製時に溶液サンプルに対して添加した測定対象物質の量（既知の量）に対応した大きさとなる。また、時間ＴEよりも反応時間が長い部分における溶液サンプルの蛍光強度と反応時間との関係を反応開始時（Ｔ＝０）まで外挿して得られる蛍光強度は、溶液サンプル中で共存物質が試薬と反応していない状態の蛍光強度（即ち、溶液サンプル中の測定対象物質の濃度に対応した蛍光強度）となる。更に、時間ＴEよりも反応時間が長い部分における参照溶液サンプルの蛍光強度と反応時間との関係を反応開始時（Ｔ＝０）まで外挿して得られる蛍光強度は、参照溶液サンプル中で共存物質が試薬と反応していない状態の蛍光強度（即ち、参照溶液サンプル中の測定対象物質の濃度（＝既知の量の測定対象物質を添加した分だけ溶液サンプルよりも高い濃度）に対応した蛍光強度）となる。そして、図２に示すグラフでは、以下に示す［関係１］や［関係２］等の関係が成立するため、検量線などを作成しなくても、溶液サンプル中の測定対象物質の濃度を求めることが可能になる。なお、下記の関係を用いる場合には、参照溶液サンプルの蛍光強度と溶液サンプルの蛍光強度との差を取る際に共存物質による影響をキャンセルすることができるので、測定対象物質の濃度を高い精度で求めることができる。
［関係１］
溶液サンプルについて時間ＴE以降の蛍光強度の大きさと反応時間との関係を表す第１の近似式を作成し、参照溶液サンプルについて時間ＴE以降の蛍光強度の大きさと反応時間との関係を表す第２の近似式を作成した場合、下記の関係式（１）が成立する。
Ｃ＝｛（Ｐ1’－Ｐ0’）／（Ｐ2’－Ｐ1’）｝×（ｍ／Ｍ） ・・・（１）
Ｃ：溶液サンプル中の測定対象物質の濃度
Ｐ0’：反応開始時（Ｔ＝０）の蛍光強度の値
Ｐ1’：反応開始時（Ｔ＝０）における第１の近似式の蛍光強度の値（第１の近似式の切片）
Ｐ2’：反応開始時（Ｔ＝０）における第２の近似式の蛍光強度の値（第２の近似式の切片）
ｍ：溶液サンプルに添加した測定対象物質の質量（既知の量）
Ｍ：参照溶液サンプルの質量
［関係２］
溶液サンプルについて時間ＴE以降の蛍光強度の大きさと反応時間との関係を表す第１の近似式を作成し、参照溶液サンプルについて時間ＴE以降の蛍光強度の大きさと反応時間との関係を表す第２の近似式を作成し、反応開始時（Ｔ＝０）の蛍光強度の値Ｐ0’を通って第１の近似式および第２の近似式と略平行な仮想線とを作成した場合、下記の関係式（２）が成立する。
Ｃ＝｛（Ｐ1－Ｐ0）／（Ｐ2－Ｐ1）｝×（ｍ／Ｍ） ・・・（２）
Ｃ：溶液サンプル中の測定対象物質の濃度
Ｐ0：反応時間Ｔにおける仮想線の蛍光強度の値
Ｐ1：反応時間Ｔにおける第１の近似式の蛍光強度の値
Ｐ2：反応時間Ｔにおける第２の近似式の蛍光強度の値
ｍ：溶液サンプルに添加した測定対象物質の質量（既知の量）
Ｍ：参照溶液サンプルの質量
The concentration measurement method of the present invention utilizes the above-mentioned new findings to measure the concentration of a target substance using a solution sample containing a target substance of unknown concentration and a reference solution sample to which a known amount of the target substance has been added to the solution sample. In the above-mentioned system, when the change in the magnitude of the fluorescence intensity over time when the solution sample and the reference solution sample are reacted with a reagent is measured, the behavior shown in FIG. 2 is shown. In addition, in the graph shown in FIG. 2, the difference between the fluorescence intensity of the reference solution sample and the fluorescence intensity of the solution sample in the part where the reaction time is longer than the time T E at which the apparent reaction between the target substance and the reagent ends (the part located to the right of time T E in FIG. 2) corresponds to the amount (known amount) of the target substance added to the solution sample when the reference solution is prepared. In addition, the fluorescence intensity obtained by extrapolating the relationship between the fluorescence intensity of the solution sample and the reaction time in the part where the reaction time is longer than time T E to the start of the reaction (T=0) is the fluorescence intensity in the state where the coexisting substance in the solution sample has not reacted with the reagent (i.e., the fluorescence intensity corresponding to the concentration of the target substance in the solution sample). Furthermore, the fluorescence intensity obtained by extrapolating the relationship between the fluorescence intensity of the reference solution sample and the reaction time in the portion where the reaction time is longer than the time T E to the start of the reaction (T=0) is the fluorescence intensity in the state where the coexisting substance in the reference solution sample has not reacted with the reagent (i.e., the fluorescence intensity corresponding to the concentration of the substance to be measured in the reference solution sample (= a concentration higher than that of the solution sample by the amount of the substance to be measured added in a known amount)). In the graph shown in FIG. 2, the following relationships such as [Relationship 1] and [Relationship 2] are established, so that it is possible to determine the concentration of the substance to be measured in the solution sample without creating a calibration curve or the like. Note that when the following relationship is used, the influence of the coexisting substance can be canceled when taking the difference between the fluorescence intensity of the reference solution sample and the fluorescence intensity of the solution sample, so that the concentration of the substance to be measured can be determined with high accuracy.
[Relationship 1]
When a first approximation equation expressing the relationship between the magnitude of fluorescence intensity after time T and the reaction time for a solution sample is created, and a second approximation equation expressing the relationship between the magnitude of fluorescence intensity after time T and the reaction time for a reference solution sample is created, the following relational equation (1) is established.
C = {(P 1 '-P 0 ')/(P 2 '-P 1 ')} × (m/M) ... (1)
C: concentration of the substance to be measured in the solution sample; P 0 ': fluorescence intensity value at the start of the reaction (T=0); P 1 ': fluorescence intensity value of the first approximation formula at the start of the reaction (T=0) (intercept of the first approximation formula);
P2 ': the fluorescence intensity value of the second approximation formula at the start of the reaction (T=0) (the intercept of the second approximation formula)
m: mass of the substance to be measured added to the solution sample (known amount)
M: mass of reference solution sample [relationship 2]
When a first approximation equation is created that expresses the relationship between the magnitude of fluorescence intensity after time T E and the reaction time for the solution sample, and a second approximation equation is created that expresses the relationship between the magnitude of fluorescence intensity after time T E and the reaction time for the reference solution sample, and a virtual line is created that passes through the fluorescence intensity value P 0 ' at the start of the reaction (T = 0) and is approximately parallel to the first approximation equation and the second approximation equation, the following relationship (2) holds:
C = {(P 1 -P 0 )/(P 2 -P 1 )} × (m/M) ... (2)
C: concentration of the substance to be measured in the solution sample P 0 : fluorescence intensity value of the imaginary line at reaction time T P 1 : fluorescence intensity value of the first approximation equation at reaction time T P 2 : fluorescence intensity value of the second approximation equation at reaction time T m: mass (known amount) of the substance to be measured added to the solution sample
M: mass of the reference solution sample
なお、本発明において、測定対象物質と試薬との見掛け上の反応が終了する時間は、例えば、（１）測定対象物質と試薬との反応速度定数を用いて理論的に算出する方法、（２）反応時間と蛍光強度との関係を示す曲線の微分値が初めてゼロまたは所定値以下になる時間を反応終了時間とする方法、（３）第１の近似式と第２の近似式とを作成した際に第１の近似式の傾きを第２の近似式の傾きで除した値が０．９以上１．１以下となる時間を反応終了時間とする方法、などの方法を用いて決定することができる。
中でも、使用し易さの観点からは上記（２）の方法が好ましい。
In the present invention, the time at which the apparent reaction between the substance to be measured and the reagent ends can be determined using, for example, (1) a method of theoretically calculating the time using the reaction rate constant between the substance to be measured and the reagent, (2) a method of determining the reaction end time as the time at which the differential value of the curve showing the relationship between reaction time and fluorescence intensity first becomes zero or a predetermined value or less, or (3) a method of determining the reaction end time as the time at which the value obtained by dividing the slope of the first approximation equation by the slope of the second approximation equation when the first approximation equation and the second approximation equation are created is 0.9 to 1.1.
Among these, the above method (2) is preferred from the viewpoint of ease of use.
ここで、図２では、溶液サンプル中の測定対象物質と試薬との見掛け上の反応が終了する時間Ｔ1と、参照溶液サンプル中の測定対象物質と試薬との見掛け上の反応が終了する時間Ｔ2とが一致する場合について示したが、本発明では、時間Ｔ1と時間Ｔ2とが一致しなくてもよい。但し、測定対象物質の濃度を更に簡便かつ高精度で求める観点からは、時間Ｔ1と時間Ｔ2とは一致させることが好ましい。
なお、時間Ｔ1と時間Ｔ2とは、特に限定されることなく、例えば反応温度などの測定対象物質と試薬との反応条件を同一にすることにより、一致させることができる。
2 shows a case where the time T1 at which the apparent reaction between the analyte in the solution sample and the reagent ends coincides with the time T2 at which the apparent reaction between the analyte in the reference solution sample and the reagent ends, but in the present invention, the time T1 and the time T2 do not have to coincide. However, from the viewpoint of determining the concentration of the analyte more simply and with higher accuracy, it is preferable that the time T1 and the time T2 are coincident.
It should be noted that time T1 and time T2 are not particularly limited, and can be made to coincide by making the reaction conditions between the substance to be measured and the reagent, such as the reaction temperature, the same.
また、本発明において、第１の近似式および第２の近似式は、特に限定されることなく、例えば最小二乗法などの既知の回帰分析方法（単回帰分析または重回帰分析）やカーブフィッティングを用いて求めることができる。 In addition, in the present invention, the first approximation formula and the second approximation formula are not particularly limited and can be obtained, for example, using a known regression analysis method such as the least squares method (simple regression analysis or multiple regression analysis) or curve fitting.
更に、本発明において、反応開始時（Ｔ＝０）の蛍光強度の値Ｐ0’は、特に限定されることなく、任意の方法で求めることができる。例えば、反応開始時（Ｔ＝０）の蛍光強度の値Ｐ0’は、溶液サンプルおよび参照溶液サンプルのそれぞれについて、測定対象物質と試薬との見掛け上の反応が終了する時間ＴEよりも反応時間が短い部分（図２では時間ＴEよりも左側に位置する部分）のデータをカーブフィッティングすることにより、求めることができる。
なお、溶液サンプルのデータを用いて算出した反応開始時（Ｔ＝０）の蛍光強度の値と、参照溶液サンプルのデータを用いて算出した反応開始時（Ｔ＝０）の蛍光強度の値とが相違する場合には、何れか一方の値を蛍光強度の値Ｐ0’として採用してもよいし、両者の算術平均値を蛍光強度の値Ｐ0’として採用してもよい。
Furthermore, in the present invention, the fluorescence intensity value P0 ' at the start of the reaction (T=0) can be determined by any method without any particular limitation. For example, the fluorescence intensity value P0 ' at the start of the reaction (T=0) can be determined by curve fitting the data for the part of the reaction time shorter than the time T at which the apparent reaction between the substance to be measured and the reagent ends (the part located to the left of time T in FIG. 2) for each of the solution sample and the reference solution sample.
In addition, if the fluorescence intensity value at the start of the reaction (T=0) calculated using the data from the solution sample differs from the fluorescence intensity value at the start of the reaction (T=0) calculated using the data from the reference solution sample, either one of the values may be adopted as the fluorescence intensity value P 0 ', or the arithmetic mean value of both may be adopted as the fluorescence intensity value P 0 '.
そして、本発明において、上述した［関係２］において用いる仮想線は、特に限定されることなく、（１）傾きが第１の近似式と等しく、反応開始時（Ｔ＝０）の蛍光強度の値がＰ0’となる直線、（２）傾きが第２の近似式と等しく、反応開始時（Ｔ＝０）の蛍光強度の値がＰ0’となる直線、（３）傾きが第１の近似式の傾きと第２の近似式の傾きとの算術平均値に等しく、反応開始時（Ｔ＝０）の蛍光強度の値がＰ0’となる直線、などとすることができる。 In the present invention, the virtual line used in the above-mentioned [Relationship 2] is not particularly limited, and can be (1) a straight line whose slope is equal to the first approximation formula and whose fluorescence intensity value at the start of the reaction (T=0) is P 0 ', (2) a straight line whose slope is equal to the second approximation formula and whose fluorescence intensity value at the start of the reaction (T=0) is P 0 ', or (3) a straight line whose slope is equal to the arithmetic average of the slopes of the first approximation formula and the second approximation formula and whose fluorescence intensity value at the start of the reaction (T=0) is P 0 ', etc.
なお、上述した［関係１］および［関係２］に関し、蛍光強度のバラツキの影響を低減して測定対象物質の濃度を更に高精度で求める観点からは、本発明では、上述した［関係１］を利用して測定対象物質の濃度を求めることが好ましい。 With regard to the above-mentioned [Relationship 1] and [Relationship 2], from the viewpoint of reducing the effect of variations in fluorescence intensity and determining the concentration of the substance to be measured with greater accuracy, in the present invention, it is preferable to determine the concentration of the substance to be measured using the above-mentioned [Relationship 1].
そして、上述した知見および着想を用いた本発明の濃度測定方法では、具体的には、濃度未知の測定対象物質を含む溶液サンプル、及び、前記溶液サンプルに対し既知の量の測定対象物質を添加した参照溶液サンプルに、前記試薬を添加し、前記パラメータの経時変化を測定する測定工程と、前記測定工程による測定結果に基づいて、前記溶液サンプル中及び前記参照溶液サンプル中の測定対象物質と前記試薬との見掛け上の反応終了時間以降の前記パラメータの大きさと時間との関係を表す近似式を作成する分析工程と、前記近似式に時間Ｔ（但し、Ｔ≧０）を代入して得られるパラメータの値と、前記既知の量との関係から前記溶液サンプル中の測定対象物質の濃度を求める算出工程とを実施して、溶液サンプル中の測定対象物質の濃度を高い精度で簡便に求めることができる。
より具体的には、本発明の濃度測定方法では、例えば、
（Ａ）濃度未知の測定対象物質を含む溶液サンプルと、溶液サンプルに対し既知の量の測定対象物質を添加した参照溶液サンプルとを準備するサンプル準備工程と、
（Ｂ）サンプル準備工程で準備した溶液サンプルに試薬を添加し、パラメータの経時変化を測定する第１の測定工程と、
（Ｃ）サンプル準備工程で準備した参照溶液サンプルに試薬を添加し、パラメータの経時変化を測定する第２の測定工程と、
（Ｄ）第１の測定工程の測定結果から、溶液サンプル中の測定対象物質と試薬との見掛け上の反応終了時間である時間Ｔ1を決定し、時間Ｔ1以降のパラメータの大きさと時間との関係を表す第１の近似式を作成する第１の分析工程と、
（Ｅ）第２の測定工程の測定結果から、参照溶液サンプル中の測定対象物質と試薬との見掛け上の反応終了時間である時間Ｔ2を決定し、時間Ｔ2以降のパラメータの大きさと時間との関係を表す第２の近似式を作成する第２の分析工程と、
（Ｆ）第１の近似式に時間Ｔ（但し、Ｔ≧０）を代入して得られるパラメータの値Ｐ1と、第２の近似式に時間Ｔ（但し、Ｔ≧０）を代入して得られるパラメータの値Ｐ2と、既知の量との関係から溶液サンプル中の測定対象物質の濃度を求める算出工程と、
を実施して、溶液サンプル中の測定対象物質の濃度を高い精度で簡便に求めることができる。
The concentration measurement method of the present invention, which uses the above-mentioned findings and ideas, specifically includes a measurement step of adding the reagent to a solution sample containing a substance to be measured of an unknown concentration, and a reference solution sample prepared by adding a known amount of the substance to be measured to the solution sample, and measuring the change in the parameter over time; an analysis step of creating an approximation equation expressing the relationship between the magnitude of the parameter and time after the apparent end time of the reaction between the substance to be measured and the reagent in the solution sample and the reference solution sample based on the measurement results from the measurement step; and a calculation step of determining the concentration of the substance to be measured in the solution sample from the relationship between the value of the parameter obtained by substituting time T (where T≧0) into the approximation equation and the known amount, thereby making it possible to easily determine the concentration of the substance to be measured in the solution sample with high accuracy.
More specifically, in the concentration measurement method of the present invention, for example,
(A) a sample preparation step of preparing a solution sample containing a target substance of unknown concentration and a reference solution sample obtained by adding a known amount of the target substance to the solution sample;
(B) a first measurement step of adding a reagent to the solution sample prepared in the sample preparation step and measuring a change in a parameter over time;
(C) a second measurement step of adding a reagent to the reference solution sample prepared in the sample preparation step and measuring the change in a parameter over time;
(D) a first analysis step of determining a time T1 , which is an apparent end time of the reaction between the substance to be measured in the solution sample and the reagent, from the measurement result of the first measurement step, and creating a first approximation equation that expresses the relationship between the magnitude of a parameter and time after time T1 ;
(E) a second analysis step of determining a time T2 , which is the apparent end time of the reaction between the substance to be measured in the reference solution sample and the reagent, from the measurement result of the second measurement step, and creating a second approximation equation that expresses the relationship between the magnitude of the parameter and time after time T2 ;
(F) a calculation step of determining the concentration of the substance to be measured in the solution sample from the relationship between a parameter value P1 obtained by substituting time T (where T ≧ 0) into the first approximation formula, a parameter value P2 obtained by substituting time T (where T ≧ 0) into the second approximation formula, and a known amount;
By carrying out the above steps, the concentration of the substance to be measured in the solution sample can be determined easily and with high accuracy.
なお、サンプル準備工程において溶液サンプルに対して添加する測定対象物質の量（既知の量）は、特に限定されることなく、例えば管理基準値の１／２、法定基準値の１／２または測定感度の単位の１０倍値（例：単位がｍｇ／Ｌの場合、１０ｍｇ／Ｌ）とすることができる。 The amount of the substance to be measured (known amount) added to the solution sample in the sample preparation process is not particularly limited, and can be, for example, 1/2 the control standard value, 1/2 the legal standard value, or 10 times the unit of measurement sensitivity (e.g., 10 mg/L if the unit is mg/L).
また、第１の測定工程および第２の測定工程におけるパラメータの大きさの測定は、試薬を添加した時間を反応開始時（Ｔ＝０）として、パラメータの種類に応じた測定装置（例えば、蛍光光度計や吸光度計）を用いて行うことができる。ここで、第１の測定工程および第２の測定工程の実施順は、特に限定されない。また、前述したとおり、測定対象物質の濃度をより高い精度で求める観点からは、第１の測定工程と、第２の測定工程とでは、測定対象物質と試薬との反応条件を同一にすることが好ましい。 In addition, the measurement of the magnitude of the parameter in the first measurement process and the second measurement process can be performed using a measurement device (e.g., a fluorometer or an absorbance meter) according to the type of parameter, with the time when the reagent is added being the start of the reaction (T = 0). Here, the order in which the first measurement process and the second measurement process are performed is not particularly limited. In addition, as described above, from the viewpoint of determining the concentration of the substance to be measured with higher accuracy, it is preferable that the reaction conditions between the substance to be measured and the reagent be the same in the first measurement process and the second measurement process.
そして、算出工程では、上述した［関係１］または［関係２］を用いて測定対象物質の濃度を求めることが好ましく、［関係１］を用いて測定対象物質の濃度を求めることがより好ましい。 In the calculation process, it is preferable to determine the concentration of the substance to be measured using the above-mentioned [Relationship 1] or [Relationship 2], and it is more preferable to determine the concentration of the substance to be measured using [Relationship 1].
（濃度測定装置）
本発明の濃度測定装置は、溶液サンプル中に含まれる測定対象物質の濃度を、測定対象物質と反応して溶液サンプルのパラメータの大きさを変化させる試薬を用いて測定する装置である。そして、本発明の濃度測定装置は、例えば、上述した本発明の濃度測定方法を用いて溶液サンプル中の測定対象物質の濃度を求める際に好適に用いることができる。
(Concentration measuring device)
The concentration measuring device of the present invention is a device for measuring the concentration of a substance to be measured contained in a solution sample by using a reagent that reacts with the substance to be measured to change the magnitude of a parameter of the solution sample. The concentration measuring device of the present invention can be suitably used, for example, when determining the concentration of the substance to be measured in the solution sample by using the concentration measuring method of the present invention described above.
ここで、本発明の濃度測定装置の一例は、測定部と、分析部と、算出部とを備え、任意に他の構成を更に有していてもよい。Here, an example of a concentration measuring device of the present invention includes a measurement unit, an analysis unit, and a calculation unit, and may optionally further include other configurations.
測定部は、濃度未知の測定対象物質を含む溶液サンプル、及び、溶液サンプルに対し既知の量の測定対象物質を添加した参照溶液サンプルについて、試薬を添加した状態で、パラメータの経時変化を測定する。具体的には、測定部は、パラメータの種類に応じた測定装置（例えば、蛍光光度計や吸光度計）を用いて構成することができる。
なお、溶液サンプルおよび参照溶液サンプルへの試薬の添加は、測定者がマニュアル操作で行ってもよいし、測定部が自動で行ってもよい。
The measurement unit measures the change over time of a parameter for a solution sample containing a target substance of unknown concentration and a reference solution sample in which a known amount of the target substance has been added to the solution sample, with a reagent added. Specifically, the measurement unit can be configured using a measurement device (e.g., a fluorometer or an absorbance meter) according to the type of parameter.
The addition of the reagent to the solution sample and the reference solution sample may be performed manually by the measurer, or automatically by the measurement unit.
分析部および算出部は、単一の、または、複数のコンピュータ等で構成することができる。
そして、分析部は、測定部による測定結果に基づいて、溶液サンプル中及び参照溶液サンプル中の測定対象物質と試薬との見掛け上の反応終了時間以降のパラメータの大きさと時間との関係を表す近似式を作成する。
また、算出部は、近似式に時間Ｔ（但し、Ｔ≧０）を代入して得られるパラメータの値と、前記既知の量との関係から溶液サンプル中の測定対象物質の濃度を求める。
The analysis unit and the calculation unit can be configured with a single or multiple computers, etc.
Then, based on the measurement results from the measurement section, the analysis section creates an approximation equation that represents the relationship between the magnitude of the parameter and time after the apparent end time of the reaction between the substance to be measured in the solution sample and the reference solution sample and the reagent.
The calculation unit also determines the concentration of the substance to be measured in the solution sample from the relationship between the parameter value obtained by substituting time T (where T≧0) into the approximation formula and the known amount.
なお、分析部における近似式の作成および算出部における濃度の算出は、それぞれ、本発明の濃度測定方法の分析工程および算出工程と同様の手法を用いて行うことができるので、ここでは説明を省略する。 The creation of the approximation equation in the analysis section and the calculation of the concentration in the calculation section can be performed using techniques similar to the analysis process and calculation process of the concentration measurement method of the present invention, respectively, so a description thereof will be omitted here.
（プログラム）
本発明のプログラムは、溶液サンプル中に含まれる測定対象物質の濃度を、測定対象物質と反応して溶液サンプルのパラメータの大きさを変化させる試薬を用いて測定する際に用いられる。そして、本発明のプログラムは、例えば、上述した本発明の濃度測定装置を、上述した本発明の濃度測定方法を用いて溶液サンプル中の測定対象物質の濃度を求める濃度測定装置として機能させる際に好適に用いることができる。
(program)
The program of the present invention is used when measuring the concentration of a substance to be measured contained in a solution sample using a reagent that reacts with the substance to be measured to change the magnitude of a parameter of the solution sample. The program of the present invention can be suitably used, for example, when causing the concentration measuring device of the present invention described above to function as a concentration measuring device that determines the concentration of a substance to be measured in a solution sample using the concentration measuring method of the present invention described above.
具体的には、本発明のプログラムの一例は、溶液サンプル中に含まれる測定対象物質の濃度を、測定対象物質と反応して溶液サンプルのパラメータの大きさを変化させる試薬を用いて測定する濃度測定装置に、濃度未知の測定対象物質を含む溶液サンプル、及び、溶液サンプルに対し既知の量の測定対象物質を添加した参照溶液サンプルについて、試薬を添加した状態で、パラメータの経時変化を測定した結果を取得させるステップと、取得した測定結果に基づいて、溶液サンプル中及び参照溶液サンプル中の測定対象物質と試薬との見掛け上の反応終了時間以降のパラメータの大きさと時間との関係を表す近似式を作成させるステップと、近似式に時間Ｔ（但し、Ｔ≧０）を代入して得られるパラメータの値と、既知の量との関係から溶液サンプル中の測定対象物質の濃度を求めるステップとを実行させる。
より具体的には、本発明のプログラムの一例は、例えば、濃度測定装置の動作を制御するコンピュータ等の制御装置を介して濃度測定装置に上述したステップを実行させる。
Specifically, one example of the program of the present invention executes the steps of causing a concentration measuring device, which measures the concentration of a substance to be measured in a solution sample using a reagent that reacts with the substance to be measured to change the magnitude of a parameter of the solution sample, to obtain results of measuring the change in parameter over time for a solution sample containing the substance to be measured of an unknown concentration and a reference solution sample in which a known amount of the substance to be measured has been added to the solution sample, with the reagent added; creating an approximation equation based on the obtained measurement results, which represents the relationship between the magnitude of the parameter and time after the apparent end time of the reaction between the substance to be measured and the reagent in the solution sample and the reference solution sample; and determining the concentration of the substance to be measured in the solution sample from the relationship between the value of the parameter obtained by substituting time T (where T≧0) into the approximation equation and the known amount.
More specifically, an example of the program of the present invention causes the concentration measuring device to execute the above-mentioned steps via a control device such as a computer that controls the operation of the concentration measuring device.
なお、このプログラムは、コンピュータが読み取り可能な記録媒体に記録されていてもよい。このような記録媒体を用いれば、プログラムをコンピュータにインストールすることが可能である。ここで、プログラムが記録された記録媒体は、非一過性の記録媒体であってもよい。非一過性の記録媒体は、特に限定されるものではないが、例えば、ＣＤ－ＲＯＭ、ＤＶＤ－ＲＯＭなどの記録媒体であってもよい。また、このプログラムは、ネットワークを介したダウンロードによって提供することもできる。 The program may be recorded on a computer-readable recording medium. By using such a recording medium, the program can be installed on a computer. Here, the recording medium on which the program is recorded may be a non-transient recording medium. The non-transient recording medium is not particularly limited, and may be, for example, a CD-ROM, DVD-ROM, or other recording medium. The program may also be provided by downloading over a network.
（実施例１）
測定対象物質として臭素酸イオンを含む溶液サンプルとして、オゾン処理後の配管から採取したオゾン処理水（Ａ）を準備した。
オゾン処理水（Ａ）７５ｍｌに対し、臭素酸イオン標準を濃度が１０μｇ／Ｌ足されるように０．７５μｇ添加して参照サンプル溶液を準備した（サンプル準備工程）。
そして、溶液サンプルおよび参照溶液サンプルのそれぞれについて、試薬としてトリフルオペラジンを濃度が３μｍｏｌ／Ｌとなるように添加し、蛍光測定が可能な試作装置を使用して蛍光強度の経時変化を測定した（第１の測定工程および第２の測定工程）。結果を図３（ａ）に示す。
得られたデータから、以下のようにして、測定対象物質と試薬との見掛け上の反応が終了する時間Ｔ1，Ｔ2と、第１の近似式および第２の近似式と、反応開始時（Ｔ＝０）の蛍光強度の値Ｐ0’とを求めた（第１の分析工程および第２の分析工程）。
・時間Ｔ1，Ｔ2：反応時間と蛍光強度との関係を示す曲線の微分値が初めて０．５（ａ．ｕ.／秒）以下になる時間（＝５００秒）とした
・第１の近似式および第２の近似式：最小二乗法により求めた
・反応開始時（Ｔ＝０）の蛍光強度の値Ｐ0’：溶液サンプルの、時間Ｔ＝２００秒から時間Ｔ1までのデータを使用し、カーブフィッティングにより反応開始時（Ｔ＝０）の蛍光強度の指数を求め、反応開始時（Ｔ＝０）の蛍光強度を求めてＰ0’とした
そして、下記の関係式（１）：
Ｃ＝｛（Ｐ1’－Ｐ0’）／（Ｐ2’－Ｐ1’）｝×（ｍ／Ｍ） ・・・（１）
Ｃ：溶液サンプル中の臭素酸イオンの濃度
Ｐ0’：反応開始時（Ｔ＝０）の蛍光強度の値
Ｐ1’：反応開始時（Ｔ＝０）における第１の近似式の蛍光強度の値
Ｐ2’：反応開始時（Ｔ＝０）における第２の近似式の蛍光強度の値
ｍ：溶液サンプルに添加した臭素酸イオンの質量（既知の量）
Ｍ：参照溶液サンプルの質量
を用いて溶液サンプル（オゾン処理水（Ａ））中の測定対象物質の濃度を求めたところ、２．０１μｇ／Ｌであった。因みに、Ｐ0’は２.４３８（ａ．ｕ.）であり、Ｐ1’は２.６３４（ａ．ｕ.）であり、Ｐ2’は３．７８３（ａ．ｕ.）であり、ｍ／Ｍは１２μｇ／Ｌであった。
同じオゾン処理水（Ａ）について、イオンクロマトグラフ（Thermofisher社製、商品名「Dionex」）を用いて臭素酸イオン濃度の定量を行ったところ、濃度は１．９２μｇ／Ｌであった。
Example 1
As a solution sample containing bromate ions as a measurement target substance, ozonated water (A) collected from a pipe after ozone treatment was prepared.
A reference sample solution was prepared by adding 0.75 μg of a bromate ion standard to 75 ml of ozone-treated water (A) so that the concentration was increased to 10 μg/L (sample preparation step).
Then, trifluoperazine was added as a reagent to each of the solution sample and the reference solution sample to a concentration of 3 μmol/L, and the change in fluorescence intensity over time was measured using a prototype device capable of measuring fluorescence (first measurement step and second measurement step). The results are shown in FIG. 3(a).
From the obtained data, the times T1 and T2 at which the apparent reaction between the substance to be measured and the reagent ends, the first and second approximation equations, and the fluorescence intensity value P0 ' at the start of the reaction (T = 0) were determined as follows (first analysis step and second analysis step).
Times T1 , T2 : The times (=500 seconds) at which the differential value of the curve showing the relationship between reaction time and fluorescence intensity first becomes 0.5 (a.u./sec) or less. First approximation formula and second approximation formula: Calculated by the least squares method. Value of fluorescence intensity at the start of the reaction (T=0) P0 ': Using data from time T=200 seconds to time T1 of the solution sample, the exponent of the fluorescence intensity at the start of the reaction (T=0) was calculated by curve fitting, and the fluorescence intensity at the start of the reaction (T=0) was calculated and designated as P0 '. The following relational formula (1):
C = {(P 1 '-P 0 ')/(P 2 '-P 1 ')} × (m/M) ... (1)
C: concentration of bromate ions in the solution sample P0 ': fluorescence intensity value at the start of the reaction (T = 0) P1 ': fluorescence intensity value of the first approximation equation at the start of the reaction (T = 0) P2 ': fluorescence intensity value of the second approximation equation at the start of the reaction (T = 0) m: mass of bromate ions added to the solution sample (known amount)
The concentration of the substance to be measured in the solution sample (ozonated water (A)) was calculated using the mass of the reference solution sample, M, and was found to be 2.01 μg/L. Incidentally, P 0 ' was 2.438 (a.u.), P 1 ' was 2.634 (a.u.), P 2 ' was 3.783 (a.u.), and m/M was 12 μg/L.
The bromate ion concentration of the same ozone-treated water (A) was quantified using an ion chromatograph (manufactured by Thermofisher, product name "Dionex"), and the concentration was found to be 1.92 μg/L.
（実施例２）
サンプル準備工程において、測定対象物質として臭素酸イオンを含む溶液サンプルとして、オゾン処理後の配管から採取したオゾン処理水（Ｂ）を準備し、オゾン処理水（Ｂ）７５ｍｌに対し、臭素酸イオン標準を濃度が１０μｇ／Ｌ足されるように０．７５μｇ添加して参照サンプル溶液を準備した。それ以外は実施例１と同様にして溶液サンプル（オゾン処理水（Ｂ））中の測定対象物質の濃度を求めたところ、１１．９８μｇ／Ｌであった。因みに、Ｐ0’は２.３８１（ａ．ｕ.）であり、Ｐ1’は３．５３２（ａ．ｕ.）であり、Ｐ2’は４．６８５（ａ．ｕ.）であり、ｍ／Ｍは１２μｇ／Ｌであった。また、蛍光強度の経時変化の測定結果は、図３（ｂ）に示す通りであった。
また、同じオゾン処理水（Ｂ）について、イオンクロマトグラフ（Thermofisher社製、商品名「Dionex」）を用いて臭素酸イオン濃度の定量を行ったところ、濃度は１１．８５μｇ／Ｌであった。
Example 2
In the sample preparation step, ozonated water (B) collected from the piping after ozonation was prepared as a solution sample containing bromate ions as the measurement target substance, and 0.75 μg of bromate ion standard was added to 75 ml of ozonated water (B) so that the concentration was increased to 10 μg/L to prepare a reference sample solution. The concentration of the measurement target substance in the solution sample (ozonated water (B)) was determined in the same manner as in Example 1, and was found to be 11.98 μg/L. Incidentally, P 0 ' was 2.381 (a.u.), P 1 ' was 3.532 (a.u.), P 2 ' was 4.685 (a.u.), and m/M was 12 μg/L. The measurement results of the change in fluorescence intensity over time were as shown in FIG. 3(b).
In addition, the bromate ion concentration of the same ozone-treated water (B) was quantified using an ion chromatograph (manufactured by Thermofisher, product name "Dionex"), and the concentration was found to be 11.85 μg/L.
実施例１，２より、本発明の方法では高い精度で臭素酸イオン濃度を測定できることが分かる。 From Examples 1 and 2, it can be seen that the method of the present invention can measure bromate ion concentration with high accuracy.
本発明によれば、測定対象物質の濃度を高い精度で簡便に測定することができる。 According to the present invention, the concentration of the substance to be measured can be measured easily and with high accuracy.
Claims (6)
濃度未知の測定対象物質を含む溶液サンプル、及び、前記溶液サンプルに対し既知の量の測定対象物質を添加した参照溶液サンプルに、前記試薬を添加し、前記パラメータの経時変化を測定する測定工程と、
前記測定工程による測定結果に基づいて、前記溶液サンプル中及び前記参照溶液サンプル中の測定対象物質と前記試薬との見掛け上の反応終了時間以降の前記パラメータの大きさと時間との関係を表す近似式を作成する分析工程と、
前記近似式に時間Ｔ（但し、Ｔ≧０）を代入して得られるパラメータの値と、前記既知の量との関係から前記溶液サンプル中の測定対象物質の濃度を求める算出工程と、
を含み、
前記分析工程では、前記溶液サンプル中の測定対象物質に対応する第１の近似式と、前記参照溶液サンプル中の測定対象物質に対応する第２の近似式とを作成し、
前記算出工程では、下記の関係式（１）：
Ｃ＝｛（Ｐ １ ’－Ｐ ０ ’）／（Ｐ ２ ’－Ｐ １ ’）｝×（ｍ／Ｍ） ・・・（１）
Ｃ：溶液サンプル中の測定対象物質の濃度
Ｐ ０ ’：反応開始時（Ｔ＝０）のパラメータの値
Ｐ １ ’：反応開始時（Ｔ＝０）における第１の近似式のパラメータの値
Ｐ ２ ’：反応開始時（Ｔ＝０）における第２の近似式のパラメータの値
ｍ：溶液サンプルに添加した測定対象物質の質量（既知の量）
Ｍ：参照溶液サンプルの質量
を用いて前記溶液サンプル中の測定対象物質の濃度を求める、濃度測定方法。 1. A method for measuring a concentration of a target substance contained in a solution sample by using a reagent that reacts with the target substance to change a magnitude of a parameter of the solution sample, comprising:
a measuring step of adding the reagent to a solution sample containing a substance to be measured at an unknown concentration and a reference solution sample obtained by adding a known amount of the substance to be measured to the solution sample, and measuring the change over time of the parameter;
an analysis step of creating an approximation equation representing the relationship between the magnitude of the parameter and time after the apparent completion time of the reaction between the substance to be measured in the solution sample and the reagent in the reference solution sample based on the measurement results from the measurement step;
a calculation step of calculating a concentration of the substance to be measured in the solution sample from a relationship between a parameter value obtained by substituting time T (where T≧0) into the approximation formula and the known amount;
Including,
In the analyzing step, a first approximation equation corresponding to the analyte in the solution sample and a second approximation equation corresponding to the analyte in the reference solution sample are prepared;
In the calculation step, the following relational expression (1):
C = {(P 1 '-P 0 ')/(P 2 '-P 1 ')} × (m/M) ... (1)
C: Concentration of the substance to be measured in the solution sample
P 0 ': Parameter value at the start of the reaction (T = 0)
P 1 ': the parameter value of the first approximation equation at the start of the reaction (T = 0)
P 2 ': the parameter value of the second approximation equation at the start of the reaction (T = 0)
m: mass of the substance to be measured added to the solution sample (known amount)
M: mass of the reference solution sample
and determining the concentration of a substance to be measured in the solution sample using the method .
前記パラメータが蛍光強度であり、
前記試薬がトリフルオペラジンである、請求項１又は２に記載の濃度測定方法。 the substance to be measured is bromate ion,
the parameter is fluorescence intensity,
The method for measuring a concentration according to claim 1 or 2 , wherein the reagent is trifluoperazine.
濃度未知の測定対象物質を含む溶液サンプル、及び、前記溶液サンプルに対し既知の量の測定対象物質を添加した参照溶液サンプルについて、前記試薬を添加した状態で、前記パラメータの経時変化を測定する測定部と、
前記測定部による測定結果に基づいて、前記溶液サンプル中及び前記参照溶液サンプル中の測定対象物質と前記試薬との見掛け上の反応終了時間以降の前記パラメータの大きさと時間との関係を表す近似式を作成する分析部と、
前記近似式に時間Ｔ（但し、Ｔ≧０）を代入して得られるパラメータの値と、前記既知の量との関係から前記溶液サンプル中の測定対象物質の濃度を求める算出部と、
を備え、
前記分析部では、前記溶液サンプル中の測定対象物質に対応する第１の近似式と、前記参照溶液サンプル中の測定対象物質に対応する第２の近似式とを作成し、
前記算出部では、下記の関係式（１）：
Ｃ＝｛（Ｐ １ ’－Ｐ ０ ’）／（Ｐ ２ ’－Ｐ １ ’）｝×（ｍ／Ｍ） ・・・（１）
Ｃ：溶液サンプル中の測定対象物質の濃度
Ｐ ０ ’：反応開始時（Ｔ＝０）のパラメータの値
Ｐ １ ’：反応開始時（Ｔ＝０）における第１の近似式のパラメータの値
Ｐ ２ ’：反応開始時（Ｔ＝０）における第２の近似式のパラメータの値
ｍ：溶液サンプルに添加した測定対象物質の質量（既知の量）
Ｍ：参照溶液サンプルの質量
を用いて前記溶液サンプル中の測定対象物質の濃度を求める、濃度測定装置。 1. An apparatus for measuring a concentration of a target substance contained in a solution sample by using a reagent that reacts with the target substance to change a magnitude of a parameter of the solution sample, comprising:
a measurement unit that measures the change over time of the parameter in a state where the reagent is added, for a solution sample containing a substance to be measured at an unknown concentration and a reference solution sample obtained by adding a known amount of the substance to be measured to the solution sample;
an analysis unit that creates an approximation equation that represents the relationship between the magnitude of the parameter and time after the apparent end time of the reaction between the measurement target substance in the solution sample and the reference solution sample and the reagent based on the measurement results by the measurement unit;
a calculation unit that calculates the concentration of the substance to be measured in the solution sample from the relationship between the parameter value obtained by substituting time T (where T≧0) into the approximation formula and the known amount;
Equipped with
the analysis unit creates a first approximation equation corresponding to the analyte in the solution sample and a second approximation equation corresponding to the analyte in the reference solution sample;
In the calculation unit, the following relational expression (1):
C = {(P 1 '-P 0 ')/(P 2 '-P 1 ')} × (m/M) ... (1)
C: Concentration of the substance to be measured in the solution sample
P 0 ': Parameter value at the start of the reaction (T = 0)
P 1 ': the parameter value of the first approximation equation at the start of the reaction (T = 0)
P 2 ': the parameter value of the second approximation equation at the start of the reaction (T = 0)
m: mass of the substance to be measured added to the solution sample (known amount)
M: mass of the reference solution sample
and determining the concentration of the substance to be measured in the solution sample using the concentration measuring device.
濃度未知の測定対象物質を含む溶液サンプル、及び、前記溶液サンプルに対し既知の量の測定対象物質を添加した参照溶液サンプルについて、前記試薬を添加した状態で、前記パラメータの経時変化を測定した結果を取得させるステップと、
取得した前記結果に基づいて、前記溶液サンプル中及び前記参照溶液サンプル中の測定対象物質と前記試薬との見掛け上の反応終了時間以降の前記パラメータの大きさと時間との関係を表す近似式を作成させるステップと、
前記近似式に時間Ｔ（但し、Ｔ≧０）を代入して得られるパラメータの値と、前記既知の量との関係から前記溶液サンプル中の測定対象物質の濃度を求めるステップと、
を実行させ、
前記近似式を作成させるステップでは、前記溶液サンプル中の測定対象物質に対応する第１の近似式と、前記参照溶液サンプル中の測定対象物質に対応する第２の近似式とを作成し、
前記濃度を求めるステップでは、下記の関係式（１）：
Ｃ＝｛（Ｐ １ ’－Ｐ ０ ’）／（Ｐ ２ ’－Ｐ １ ’）｝×（ｍ／Ｍ） ・・・（１）
Ｃ：溶液サンプル中の測定対象物質の濃度
Ｐ ０ ’：反応開始時（Ｔ＝０）のパラメータの値
Ｐ １ ’：反応開始時（Ｔ＝０）における第１の近似式のパラメータの値
Ｐ ２ ’：反応開始時（Ｔ＝０）における第２の近似式のパラメータの値
ｍ：溶液サンプルに添加した測定対象物質の質量（既知の量）
Ｍ：参照溶液サンプルの質量
を用いて前記溶液サンプル中の測定対象物質の濃度を求める、プログラム。 An apparatus for measuring the concentration of a substance to be measured contained in a solution sample by using a reagent that reacts with the substance to be measured to change the magnitude of a parameter of the solution sample,
a step of acquiring results of measuring the change over time of the parameter in a state where the reagent is added, for a solution sample containing a substance to be measured at an unknown concentration and a reference solution sample obtained by adding a known amount of the substance to be measured to the solution sample;
creating an approximation equation representing the relationship between the magnitude of the parameter and time after the apparent end time of the reaction between the analyte in the solution sample and the reagent in the reference solution sample based on the obtained result;
determining a concentration of the substance to be measured in the solution sample from a relationship between a parameter value obtained by substituting time T (where T≧0) into the approximation formula and the known amount;
Run the command ,
In the step of generating the approximation equation, a first approximation equation corresponding to the analyte in the solution sample and a second approximation equation corresponding to the analyte in the reference solution sample are generated,
In the step of determining the concentration, the following relational expression (1):
C = {(P 1 '-P 0 ')/(P 2 '-P 1 ')} × (m/M) ... (1)
C: Concentration of the substance to be measured in the solution sample
P 0 ': Parameter value at the start of the reaction (T = 0)
P 1 ': the parameter value of the first approximation equation at the start of the reaction (T = 0)
P 2 ': the parameter value of the second approximation equation at the start of the reaction (T = 0)
m: mass of the substance to be measured added to the solution sample (known amount)
M: mass of the reference solution sample
and determining the concentration of the substance to be measured in the solution sample using the above program.
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| WO2009116554A1 (en) | 2008-03-19 | 2009-09-24 | メタウォーター株式会社 | Method and apparatus for measurement of bromate ion |
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| WO2014174818A1 (en) | 2013-04-26 | 2014-10-30 | パナソニックＩｐマネジメント株式会社 | Quantitative analyzing method for oxidant and quantitative analyzing instrument for oxidant |
| JP2016057162A (en) | 2014-09-09 | 2016-04-21 | メタウォーター株式会社 | Bromate ion concentration measuring method and measuring system |
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| DE3609217A1 (en) * | 1986-03-19 | 1987-09-24 | Boehringer Mannheim Gmbh | METHOD AND REAGENT FOR DETERMINING A REACTION PARTNER OF AN IMMUNOLOGICAL REACTION |
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| WO2009116554A1 (en) | 2008-03-19 | 2009-09-24 | メタウォーター株式会社 | Method and apparatus for measurement of bromate ion |
| JP2010271095A (en) | 2009-05-20 | 2010-12-02 | Hitachi High-Technologies Corp | Automatic analysis device and analysis method |
| US20130130308A1 (en) | 2011-11-23 | 2013-05-23 | Envolure | Process for directly measuring multiple biodegradabilities |
| JP2014002007A (en) | 2012-06-18 | 2014-01-09 | Metawater Co Ltd | Bromate ion measuring method and measuring apparatus |
| WO2014174818A1 (en) | 2013-04-26 | 2014-10-30 | パナソニックＩｐマネジメント株式会社 | Quantitative analyzing method for oxidant and quantitative analyzing instrument for oxidant |
| JP2016057162A (en) | 2014-09-09 | 2016-04-21 | メタウォーター株式会社 | Bromate ion concentration measuring method and measuring system |
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