JP3011855B2 - Fluorescence analysis method of chloride ion and carrier for measuring chloride ion concentration used therein - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for the selective fluorescence analysis of chloride ions, and more particularly to a method for continuously detecting the concentration of chloride ions in a sample having a complicated composition.

[0002]

2. Description of the Related Art Chloride ions are widely distributed in environmental waters such as rivers and seawater as well as in living organisms, and are deeply related to ecosystems through pH balance and the like. Further, chloride ions also play an important role as counter anions of metal ions such as sodium ions and various organic cations. Therefore, the chloride ion concentration is an important index in a wide field such as clinical analysis and environmental conservation.
Further, metal chlorides such as hydrochloric acid and sodium chloride are industrially used in large quantities, and the analysis of chloride ions occupies an important position also from the viewpoint of process control and quality control.

That is, in a medical or industrial process, it is necessary to continuously monitor the chloride ion concentration and feed back the measurement result to constantly control the chloride ion concentration.

The chemical composition of biological samples such as serum to be analyzed, environmental water such as river water, reaction solutions in plants, and industrial wastewater is extremely complicated, and coexisting ions and organic substances greatly hinder analysis. Not a few. Therefore, there is a demand for a method for analyzing chloride ions that is extremely small due to coexisting substances. In particular, in the process of producing a high-purity reagent, such as chlorine or metal chloride, which is essential in the semiconductor industry or the like, highly accurate measurement of chloride ions in the presence of other halogen ions is indispensable.

Conventionally, chloride ions in sample water have been analyzed by a gravimetric method, a titration method or a colorimetric method. Although these analytical methods have high sensitivity and accuracy,
Complex operations such as sample sampling, pretreatment, and measurement are required. For example, in a mercury thiocyanate method, which is one of the colorimetric methods, a sample solution is pretreated, a mercury thiocyanate solution and an iron alum solution are added, and the absorbance is measured after a predetermined time. In any of the analysis methods, pretreatment of the sample solution is important, and particularly, separation from bromide ion, iodide ion, cyanate ion, or the like is required. Therefore, these analytical methods are in situ
Alternatively, it is difficult to apply to many fields that require continuous measurement online.

On the other hand, an electrochemical method for analyzing chloride ions using an ion electrode is a simple continuous measurement method and a general-purpose method for analyzing chloride ions. However, the ion electrode is not so high in mechanical strength and is easily broken, so that its operability is not always good in the analysis of an actual sample. In addition, there are many practical problems due to strong interference with the pH of sample water and coexisting ions, particularly with bromide and iodide ions.

Therefore, as a continuous measurement method of the chloride ion concentration with more excellent selectivity, an analysis method utilizing the fluorescence quenching of a fluorescent dye by chloride ions has been proposed. Reports on the quenching of fluorescent dyes by chloride ions are old,
Dating back to 1869 (Stokes GG Get
al. Chem. Soc. 22: 174-18518
69). After that, many reports have been made on a method of analyzing the chemical species by utilizing the fact that the fluorescence of the polycyclic aromatic compound is quenched by oxygen or sulfur dioxide. However, there have been few reports on the development and search for fluorescent dyes for measuring chloride ions based on fluorescence quenching, and as fluorescent dyes for analyzing halogen ions, 6-methoxy-1- (3-sulfosulfonic acid) has been used. Propyl) quinolinium or 3- (10-methylacridinium-9-
Yl) propionic acid tetrafluoroborate (Urbano)
E et al. Anal. Chem. 56: 427-
429, 1984).

In general, fluorescence quenching by halogen ions such as chloride ions is performed by a Stern-Bomer system (Stern
O et al. Phys. Z. 20: 183-19
0, 1919), and if the concentration of the halogen ion is equal, the fluorescent intensity of the fluorescent dye having a larger extinction constant is greatly reduced. In other words, a fluorescent dye having a larger extinction constant can measure halogen ions with higher sensitivity. Normally, the extinction constant of an organic fluorescent dye for chloride ions is as small as ¹ M ^-1 or less, but the extinction constant for chloride ions of the above two types of fluorescent dyes is as large as ¹ to 10 M ^-1, and the sensitivity of chloride ions is high. It is possible to measure the concentration.

However, a halogen ion having a larger atomic weight causes fluorescence quenching more strongly, so that bromide ion and iodide ion greatly hinder analysis of chloride ion based on fluorescence quenching.

Therefore, in order to enable measurement of the chloride ion concentration which is less affected by these interfering ions,
There has been a demand for a fluorescent dye which has a large quenching constant with respect to chloride ions and is less affected by quenching by bromide ions or iodide ions.

[0011]

An analysis method utilizing the fluorescence quenching of a fluorescent dye by chloride ions utilizes the fact that the fluorescence intensity from the fluorescent dye decreases with an increase in the chloride ion concentration, and the fluorescence intensity is reduced. The chloride ion concentration is measured indirectly from the intensity change. Therefore, many organic or inorganic fluorescent dyes that are quenched by chloride ions can be used. Further, by searching for a compound having a large quenching constant specifically for chloride ions from these fluorescent dyes, selective measurement of chloride ions becomes possible.

However, the fluorescent dyes reported so far for the purpose of measuring chloride ions are strongly quenched by coexisting halogen ions, especially iodide ions. It was difficult to measure ions. The present invention has found a fluorescent dye capable of selectively detecting chloride ions by fluorescence quenching even in the presence of an interfering species represented by iodide ions, and selectively using the fluorescent dye to selectively detect chloride ions. It is intended to provide a simple continuous analysis method.

[0013]

Means for Solving the Problems In the analysis of chloride ions based on fluorescence quenching, the present inventor searched for a fluorescent dye from the above viewpoint and found that 3,6-bis (dimethylamino)
The inventors have found that acridine and its derivatives are excellent fluorescent dyes, and have completed the present invention.

The present invention relates to a method for measuring the chloride ion concentration in a sample solution, which comprises mixing an aqueous solution of a dye represented by the following general formula (1) with a sample solution containing the chloride ion, The present invention relates to a method for measuring a chloride ion concentration, comprising a step of measuring a degree of disappearance of fluorescence due to contact with the chloride ion.

[0015]

Embedded image

The present invention also relates to a fluorescence analysis method for detecting the chloride ion concentration in a sample solution, which comprises converting an aqueous solution of a dye represented by the following general formula (2) into a sample solution containing the chloride ion: And measuring the extent of disappearance of fluorescence due to contact of the dye with the chloride ion.

[0017]

Embedded image (However, R represents an alkyl group having 1 to 30 carbon atoms, and X ⁻ represents a halogen ion.)
The formula (2) includes that the halogen ion is a bromine ion.

Further, the present invention provides a method for measuring the chloride ion concentration in a sample aqueous solution, wherein a carrier having a dye represented by the following general formula (2) fixed on its surface is brought into contact with a sample solution containing chloride ions. Measuring the degree of disappearance of fluorescence caused by contact of the dye with the chloride ion.

Embedded image (However, R represents an alkyl group having 1 to 30 carbon atoms, and X ⁻ represents a halogen ion.)

The present invention also provides a carrier for measuring a chloride ion concentration, on the surface of which a dye whose fluorescence is lost by contact with chloride ions in an aqueous solution is fixed, wherein the dye has the following general formula (2): The present invention relates to a carrier for measuring chloride ion concentration, which has a structure represented by the following formula:

Embedded image (However, R represents an alkyl group having 1 to 30 carbon atoms, and X ⁻ represents a halogen ion.)

[0020]

The fluorescence quenching by a halogen ion such as a chloride ion is based on the fact that the excitation energy of a photoexcited fluorescent dye is transferred to a halogen ion, and the fluorescent dye is relaxed to a ground state without emitting fluorescence.

Therefore, at the time of analysis, a predetermined concentration of the fluorescent dye is dissolved in the sample solution in advance, or if the fluorescent dye cannot be added in advance, the fluorescent dye is added to the sample solution immediately before the analysis so as to obtain the predetermined concentration. Then, the fluorescence intensity is measured. The fluorescence intensity of a fluorescent dye solution having a predetermined concentration in the absence of chloride ions is determined in advance, and the chloride ion concentration is determined from the respective fluorescence intensities in the presence or absence of chloride ions.

When it is difficult to directly add the fluorescent dye to the sample solution due to diffusion, outflow, toxicity, etc. of the fluorescent dye into the sample solution, the fluorescent dye is substituted with the substituent at the 10-position of the fluorescent dye. Is fixed to an appropriate carrier, and the carrier is brought into contact with a sample solution, and the chloride ion concentration is measured from the fluorescence intensity. If this method is used, the fluorescent dye can be recovered together with the carrier after the analysis without consuming the fluorescent dye during the analysis.

For example, 3,6-bis (dimethylamino)
Since the fluorescence characteristics (fluorescence intensity, excitation wavelength, or fluorescence wavelength) of -10-alkylacridinium ion hardly depend on the length of the alkyl chain at the 10-position, one suitable alkyl group is used.
The fluorescent dye is physically immobilized on a carrier by introducing the compound at the 0-position and utilizing the hydrophobicity of the alkyl chain. The alkyl group introduced at the 10-position preferably has 1 to 30 carbon atoms.

3,6-bis (dimethylamino) -10-
The alkyl group at the 10-position of the alkyl acridinium ion is a halogen atom, a sulfonate group, an amino group, a styryl group, a nitro group, a hydroxyl group, a carboxyl group, a ketone group, a cyano group, a tertiary or higher substituted or unsubstituted alkyl group, Alternatively, it may contain an unsubstituted aryl group or a substituted or unsubstituted cycloalkyl group.

This alkyl group may be modified with a reactive group such as maleimide or succinimide.
The fluorescent dye modified with a reactive group can be chemically immobilized on a compound having a functional group such as -SH or on the surface of a carrier having such a functional group. Can also be provided.

Examples of the carrier on which the fluorescent dye is immobilized include a particulate carrier used for chromatography and the like, and a polymer compound or a gel compound used as a material for a functional film. For example, porous carriers, particulate carriers such as zeolite, silica gel, and alumina, ion exchange resins, polymer compounds such as polymethyl methacrylate, polyvinyl chloride, and urethane; and gel compounds such as polyvinyl alcohol and polyacrylamide.

It should be noted that 3,6-bis (dimethylamino)-
The 10-alkylacridinium ion is a cation, and as a counter ion, a counter ion is a counter ion such as a halide ion such as a chloride ion, a hydroxide ion, a thiocyanate ion, a borate ion or a perchlorate ion. Fluorescent dyes as ions can also be used in the analysis method of the present invention.

The fluorescence analysis method of the present invention is suitable for measuring chloride ions in raw materials, intermediate products and final products of chemical products. Further, the present invention can be applied to analysis of cooling water, wastewater, and the like in an industrial plant. Further, it can be used for measurement of chloride ions in environmental water such as groundwater or river water, or in biological samples such as blood or intracellular fluid. In particular, the sample solution is sent by a pump, a carrier on which a fluorescent dye is immobilized is placed in this flow, and the fluorescence intensity is measured, so that the chloride ion concentration can be continuously measured without consuming the fluorescent dye. Can be monitored.

The test sample is preferably an aqueous solution.
An organic solvent soluble in water such as ethanol or acetonitrile may be dissolved. Further, the pH of the sample solution is desirably around neutral, but 3,6-bis (dimethylamino)-
The fluorescence properties of 10-alkylacridinium ions are pH
Since there is almost no change in the range of 3 to 11, analysis is possible in a wide pH range. Furthermore, since the fluorescence characteristics of 3,6-bis (dimethylamino) -10-alkylacridinium ion hardly depend on the ionic strength of the sample solution, a sample having a wide range of ionic strength is used in the analysis method of the present invention. be able to. That is, due to the fluorescent characteristics of the fluorescent dye as described above, pretreatment such as pH adjustment and ionic strength adjustment of the sample is not required in the analysis operation, and it is possible to provide an in situ continuous analysis method for a wide range of measurement targets. Will be possible.

As an excitation light source for measuring the fluorescence of 3,6-bis (dimethylamino) acridine and its derivatives, a tungsten lamp, a xenon lamp, a light emitting diode, or the like, which is separated by a filter or a diffraction grating, can be used. The maximum excitation wavelength of 3,6-bis (dimethylamino) acridine and its derivatives is 4
Since it is near 90 nm, which coincides well with the oscillation wavelength (488 nm) of a commercially available argon ion laser, it is possible to use a laser as an excitation light source. In particular, when a sufficient amount of the fluorescent dye cannot be added to the sample, or when the amount of the fluorescent dye immobilized on the carrier is small, sufficient fluorescence intensity cannot be obtained by lamp excitation, which hinders analysis. In such a case, the fluorescence is measured with high sensitivity using a laser as the excitation light source, and the chloride ion concentration is determined with high accuracy. The fluorescence from the fluorescent dye is separated from the excitation light using a filter or a diffraction grating, and the intensity is measured using a photodiode, a photomultiplier or a CCD camera.

Further, the fluorescence analysis method using the dye according to the present invention is not limited to the measurement of the concentration of chloride ions, but also the chemical analysis having a stoichiometric relationship with the amount of chloride ions in a reaction involving chloride ions. It can also be used for species analysis. For example, in a halogen addition reaction or a dehalogenation reaction to an organic compound, the concentration of chloride ion is measured by the analysis method of the present invention, and the amount of the raw material organic compound and the amount of the reaction product can be obtained indirectly from the measured concentration. it can.

[0032]

EXAMPLES The present invention will now be described specifically with reference to examples, but these examples do not limit the scope of the present invention.

Example 1 <3,6-bis (dimethylamino) with chloride ion
Fluorescence quenching of -10-dodecylacridinium bromide> (1) 1 μM of 3,6-bis (dimethylamino) -10
-A 0.1 M potassium phosphate buffer (pH 7.0) containing dodecyl acridinium bromide (commercially available) was prepared. It was dissolved potassium chloride to a predetermined concentration (10 ^-4 to 10 ⁰ M) thereto.

(2) Excitation wavelength 490 nm, fluorescence wavelength 53
The fluorescence intensity of the above solution was measured at 0 nm, and the relative fluorescence intensity was determined assuming that the fluorescence intensity when the chloride ion concentration was 0 M was 1.

FIG. 1 shows the results. Assuming that F and F ₀ are the fluorescence intensity in the presence and absence of chloride ions, k is the extinction constant, and [Cl] is the chloride ion concentration, the Stern-Bomer equation is represented by the following equation.

[0036]

F / F ₀ = (1 + k [Cl]) ^{−1 The} extinction constant k for chloride ions was determined from FIG. 1 and the above equation to be 6.7 M ⁻¹ .

Comparative Example 1 <Fluorescence quenching of 6-methoxy-1- (3-sulfopropyl) quinolinium and 3- (10-methylacridinium-9-yl) propionic acid tetrafluoroborate by chloride ions (1) 0.1 M phosphorus containing 1 μM each of 6-methoxy-1- (3-sulfopropyl) quinolinium and 3- (10-methylacridinium-9-yl) propionic acid tetrafluoroborate Potassium acid buffer (pH 7.0)
Was prepared. Each of these solutions has a predetermined concentration (10 ^{-4 to} 10
⁰ M) was dissolved in potassium chloride.

(2) The fluorescence intensity of the above solution was measured at the excitation wavelength and the fluorescence wavelength shown in Table 1, and the relative fluorescence intensity was determined with the fluorescence intensity at 1 when the chloride ion concentration was 0 M as 1.

[0039]

[Table 1] When the quenching constant k was determined by the Stern-Bomer equation in the same manner as in Example 1, the case of 6-methoxy-1- (3-sulfopropyl) quinolinium was 7.6 M ⁻¹ , 3- (10
-Methylacridinium-9-yl) propionic acid was 9.3 M ^-1 . 3,6-Bis (dimethylamino) -10-dodecylacridinium was found to have a large extinction constant comparable to these fluorescent dyes.

Example 2 <3,6-bis (dimethylamino) with chloride ion
Acridine and 3,6-bis (dimethylamino) -1
Fluorescence quenching of 0-hexadecylacridinium bromide> (1) 1 μM 3,6-bis (dimethylamino) acrylic
Gin (commercially available), 1 μM 3,6-bis (dimethylamido
G) -10-hexadecylacridinium bromide (K
awabata Y et al. Anal. Che
m. 62: 2054-2055, 1990)
Each containing 0.1 M potassium phosphate buffer (p
H7.0) was prepared. A predetermined concentration (10
^-FourTo 10 ⁰ Dissolve potassium chloride to obtain M)
Was.

(2) Excitation wavelength 490 nm, fluorescence wavelength 53
The fluorescence intensity of the solution was measured at 0 nm, and the quenching constant for chloride ion was calculated. The quenching constants obtained are 7.3M ^-1 for 3,6-bis (dimethylamino) acridine and 3,6-bis (dimethylamino) -10.
6.4 for hexadecyl acridinium bromide
M ^-1 . It was confirmed that 3,6-bis (dimethylamino) acridine and its 10-position alkyl-substituted product had a large quenching constant for chloride ions.

Example 3 <Iodide ion interference in chloride ion fluorescence analysis> (1) 1 μM 3,6-bis (dimethylamino) -10
-Dodecylacridinium bromide, 1 μM 6-methoxy-1- (3-sulfopropyl) quinolinium, 1 μM
0.1 containing 0.1 M each of 3- (10-methylacridinium-9-yl) propionic acid tetrafluoroborate.
An M potassium phosphate buffer (pH 7.0) was prepared. Potassium iodide (1 mM) and potassium chloride were dissolved in each of these solutions to a predetermined concentration (10 ^{-4 to} 10 ⁰ ).

(2) The fluorescence intensity of the above solution was measured, and the relative fluorescence intensity was determined by setting the fluorescence intensity when the chloride ion concentration was 0 M to 1. Note that the excitation wavelength and the fluorescence wavelength for each dye in the fluorescence measurement are the same as in Comparative Example 1.

(3) At a predetermined chloride ion concentration,
The interference of iodide ions in the analysis of chloride ions was examined by subtracting the fluorescence intensity in the presence of iodide ions from the relative fluorescence intensity in the absence of iodide ions (FIG. 1).

FIG. 2 shows the obtained results. In the figure, the vertical axis represents an error from the relative fluorescence intensity (true fluorescence intensity) when no iodide ion is present, and the horizontal axis represents the chloride ion concentration.

From FIG. 2, it can be seen that 3,6-bis (dimethylamino) -10-dodecylacridinium has an extremely small interference with iodide ions as compared with the conventional fluorescent dye used for the analysis of chloride ions. It was confirmed that.

Further, 3,6-bis (dimethylamino) acridine and 3,6-bis (dimethylamino) -1
A similar experiment was performed using 0-hexadecylacridinium bromide as a fluorescent dye, and it was confirmed that the interference of iodide ions with these fluorescent dyes was extremely small.

[0048]

The fluorescent dye according to the present invention enables high-sensitivity detection of halogen ions as well as conventional fluorescent dyes, and also allows selective detection of chloride ions among halogen ions. is there.

Therefore, according to the analysis method of the present invention using such a fluorescent dye, continuous analysis of chloride ion is possible, and further, chloride ion in the presence of iodide ion, which has been a problem in the prior art, can be obtained. The analysis of ions can be performed accurately.

[Brief description of the drawings]

FIG. 1 is a graph showing the relationship between the fluorescence intensity and the chloride ion concentration of a solution according to Example 1.

FIG. 2 is a graph showing a relationship between an error from a true fluorescence intensity and a chloride ion concentration for a solution according to Example 4.

Claims (5)

(57) [Claims] 1. A method for measuring a chloride ion concentration in a sample solution, comprising mixing an aqueous solution of a dye represented by the following general formula (1) with a sample solution containing the chloride ion, A method for measuring chloride ion concentration, comprising a step of measuring a degree of disappearance of fluorescence due to contact with a chloride ion. Embedded image 2. A fluorescence analysis method for detecting a chloride ion concentration in a sample solution, comprising mixing an aqueous solution of a dye represented by the following general formula (2) with a sample solution containing the chloride ion: A fluorescence analysis method for chloride ions, comprising a step of measuring a degree of fluorescence disappearance due to contact of the dye with the chloride ions. Embedded image (However, R represents an alkyl group having 1 to 30 carbon atoms, and X ⁻ represents a halogen ion.) 3. The method according to claim 2, wherein the halogen ion in the formula (2) is a bromide ion. 4. A method for measuring a chloride ion concentration in a sample aqueous solution, comprising: contacting a carrier having a dye represented by the following general formula (2) fixed on a surface thereof with a sample solution containing chloride ions; A method for measuring chloride ion concentration, comprising a step of measuring a degree of disappearance of fluorescence caused by contact with the chloride ion. Embedded image (However, R represents an alkyl group having 1 to 30 carbon atoms, and X ⁻ represents a halogen ion.) 5. A carrier for measuring a chloride ion concentration having a dye immobilized on its surface, the dye having a fluorescence disappearance upon contact with chloride ions in an aqueous solution, wherein the dye has a structure represented by the following general formula (2): A carrier for measuring chloride ion concentration, comprising: Embedded image (However, R represents an alkyl group having 1 to 30 carbon atoms, and X ⁻ represents a halogen ion.)