JPH085628A - Fluorometry for chloride ion - Google Patents

Abstract

PURPOSE:To realize selective continuous analysis of chloride ion by employing a compound represented by a specific formula and its derivatives as a fluorescent pigment. CONSTITUTION:When the concentration of chloride ion in a sample is detected, 3.6-bis (dimethylamino) acridine and its derivatives are employed as a fluorescent pigment. At the time of analysis, the fluorescent pigment is previously dissolved into a sample solution at a predetermined concentration and the fluorescence concentration is measured. The fluorescence intensity of a fluorescent pigment solution is previously determined at a predetermined concentration when no chloride ion is present and then the concentration of chloride ion is determined based on the fluorescence intensity when the chloride is present and not present. When it is difficult to add the fluorescent pigment directly to the sample solution, a carrier fixed with the fluorescent pigment is brought into contact with the sample solution using a substituent at 10 position of the fluorescent pigment and the concentration is measured based on the fluorescence intensity. With such fluorescent pigment, pretreatment of sample, e.g. conditioning of pH or ion intensity, can be eliminated in the analytic operation thus realizing a selective continuous analysis.

Description

Detailed Description of the Invention

[0001]

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

[0002]

2. Description of the Related Art Chloride ions are widely distributed not only in living organisms but also in environmental water such as rivers and seawater, and are closely related to the ecosystem through pH balance and the like. The chloride ion also plays an important role as a counter anion for metal ions such as sodium ion and various organic cations. Therefore, chloride ion concentration is an important index in a wide range of fields such as clinical analysis and environmental protection.
In addition, a large amount of metal chlorides such as hydrochloric acid and sodium chloride are industrially used, and analysis of chloride ions occupies an important position in terms of process control and quality control.

That is, in medical or industrial processes, 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, industrial wastewater, etc. is extremely complicated, and coexisting ions and organic substances greatly interfere with the analysis. Not a few. Therefore, there is a demand for a method for analyzing chloride ions, which is less affected by coexisting substances. In particular, in the process of manufacturing high-purity reagents such as chlorine and metal chlorides, which are essential in the semiconductor industry, etc., highly accurate measurement of chloride ions in the presence of other halogen ions is essential.

Conventionally, chloride ions in sample test water have been analyzed by a gravimetric method, a titration method, a colorimetric method, or the like. Although these analytical methods have high sensitivity and accuracy,
Complex operations such as sample sampling, pretreatment, and measurement are required. For example, in the mercury thiocyanate method, which is one of the colorimetric methods, after pretreatment of a sample solution, a mercury thiocyanate solution and an iron alum solution are added, and the absorbance is measured after a predetermined time. In any analysis method, pretreatment of the sample solution is essential, and in particular, 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 mechanical strength of the ion electrode is not so high, and the ion electrode is easily damaged, so that its operability is not always good in the analysis of an actual sample. Further, the pH of the sample test water and the coexisting ions, especially bromide ion and iodide ion, are strongly interfered with, so that there are many problems in practical use.

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

Generally, fluorescence quenching by halogen ions such as chloride ions is carried out by the Stern-Boomer system (Stern
O et al. Phys. Z. 20: 183-19
0, 1919), the fluorescent intensity of a fluorescent dye having a larger extinction constant is greatly reduced if the concentration of halogen ions is equal. In other words, the higher the extinction constant of the fluorescent dye, the more sensitively the halogen ion can be measured. Usually, the quenching constants of organic fluorescent dyes for chloride ions are as small as ¹ M ^-1 or less, but the quenching constants of the above two types of fluorescent dyes for chloride ions are large at 1-10 M ^-1, which is highly sensitive to chloride ions. It is possible to measure the concentration.

However, since halogen ions having a large atomic weight cause fluorescence quenching to a greater extent, bromide ions and iodide ions are a major obstacle in the analysis of chloride ions based on fluorescence quenching.

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

[0011]

An analysis method utilizing the fluorescence quenching of a fluorescent dye by chloride ion utilizes the fact that the fluorescence intensity from the fluorescent dye decreases as the chloride ion concentration increases, and The chloride ion concentration is indirectly measured from the intensity change. Therefore, many organic or inorganic fluorescent dyes that are quenched by chloride ions can be used. Furthermore, by searching for compounds having a large quenching constant specifically for chloride ion from these fluorescent dyes, it becomes possible to selectively measure chloride ion.

However, fluorescent dyes which have been reported for the purpose of measuring chloride ions have been strongly quenched by coexisting halogen ions, particularly iodide ions, and a highly accurate chloride compound can be prepared for samples in which such ions coexist. It was difficult to measure the ions. The present invention has found a fluorescent dye capable of selectively detecting chloride ion by fluorescence quenching even in the presence of an interfering species typified by iodide ion, and selectively uses the fluorescent dye to selectively detect chloride ion. The purpose of the present invention is to provide a continuous analysis method.

[0013]

In the analysis of chloride ions based on fluorescence quenching, the present inventor conducted a search for fluorescent dyes from the above-mentioned viewpoint, and found 3,6-bis (dimethylamino)
We have found that acridine and its derivatives are excellent fluorescent dyes, and completed the present invention.

That is, the present invention provides a fluorescence analysis method for detecting the concentration of chloride ion in a sample using a fluorescent dye that quenches fluorescence by chloride ion, and the fluorescent dye is represented by the formula (1):

[0015]

[Chemical 3] The method for fluorescence analysis of chloride ion is characterized by using 3,6-bis (dimethylamino) acridine and its derivative represented by

Further, the fluorescence analysis method of the present invention uses the formula (2) as the fluorescent dye.

[0017]

[Chemical 4] (However, R is an alkyl substituent having 1 to 30 carbon atoms and I ⁻ represents a halogen ion.) Use of a halide of a 3,6-bis (dimethylamino) -10-alkylacridinium ion. Is included.

Further, in the fluorescence analysis method of the present invention, the fluorescent dye represented by the above formula (2) is immobilized on a carrier, and the carrier is brought into contact with a sample solution to remove chloride ion without consuming the fluorescent dye. This includes continuously detecting the concentration.

The fluorescence analysis method of the present invention includes that the halide represented by the above formula (2) is bromide.

[0020]

Fluorescence quenching by halogen ions such as chloride ions is based on the fact that the excitation energy of the photoexcited fluorescent dye is transferred to the halogen ions and the fluorescent dye relaxes to the ground state without emitting fluorescence.

Therefore, at the time of analysis, a fluorescent dye having a predetermined concentration is dissolved in the sample solution in advance, or when the fluorescent dye cannot be added in advance, the fluorescent dye is added to the sample solution immediately before the analysis so that the concentration becomes a predetermined concentration. Then, the fluorescence intensity is measured. The fluorescence intensity of the 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 used by using 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 to measure the chloride ion concentration from the fluorescence intensity. By using this method, the fluorescent dye can be collected 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 alkyl chain length at the 10-position, an appropriate alkyl group is
Introduced at the 0 position, the hydrophobicity of the alkyl chain is used to physically fix the fluorescent dye to the carrier. The alkyl group introduced at the 10-position is preferably one having 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, a substituted group Alternatively, it may contain an unsubstituted aryl group or a substituted or unsubstituted cycloalkyl group.

The alkyl group may be modified with a reactive group such as maleimide or succinimide.
A 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 these functional groups, and the carrier on which the fluorescent dye is chemically immobilized can be analyzed. You can also use it.

Examples of the carrier for immobilizing the fluorescent dye include a particulate carrier used for chromatography and the like, and a polymer compound or a gel compound as a material of the functional film. Examples thereof include particulate carriers such as porous glass, zeolite, silica gel and alumina, ion exchange resins, polymer compounds such as polymethylmethacrylate, 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 halogen ion such as bromide ion, anion such as chloride ion, hydroxide ion, thiocyanate ion, borate ion, and perchlorate ion are paired. A fluorescent dye used as an ion 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 of chemical products, intermediate products or final products. It can also be applied to analysis of cooling water, waste water, etc. in industrial plants. Further, it can be used for measurement of chloride ions in environmental water such as groundwater and river water, or biological samples such as blood and intracellular fluid. In particular, the sample solution is sent by a pump, a carrier on which a fluorescent dye is fixed is installed 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.

An aqueous solution is desirable as the test sample,
A water-soluble organic solvent such as ethanol or acetonitrile may be dissolved. The pH of the sample solution is preferably around neutral, but 3,6-bis (dimethylamino)-
The fluorescence characteristic of 10-alkylacridinium ion is 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 property of 3,6-bis (dimethylamino) -10-alkylacridinium ion hardly depends on the ionic strength of the sample solution, a sample having a wide range of ionic strength is used for the analysis method of the present invention. be able to. That is, due to the above-mentioned fluorescence characteristics of the fluorescent dye, pretreatment such as pH adjustment and ionic strength adjustment of the sample is unnecessary in the analysis operation, and it is possible to provide an in situ continuous analysis method for a wide range of measurement targets. It will be possible.

As an excitation light source for measuring the fluorescence of 3,6-bis (dimethylamino) acridine and its derivative, a tungsten lamp, a xenon lamp or a light emitting diode which is dispersed 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 around 90 nm, which is in good agreement with the oscillation wavelength (488 nm) of a commercially available argon ion laser, it is possible to use a laser as the excitation light source. In particular, when the fluorescent dye cannot be added to the sample in a sufficient amount, 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, fluorescence is measured with high sensitivity using a laser as an excitation light source, and the chloride ion concentration is obtained from this accurately. The fluorescence from the fluorescent dye is separated from the excitation light by using a filter or a diffraction grating, and its intensity is measured by a photodiode, a photomultiplier tube 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 ion, but a chemical which has a stoichiometric relationship with the amount of chloride ion in a reaction involving chloride ion. It can also be used for species analysis. For example, in the halogen addition reaction or dehalogenation reaction to an organic compound, the concentration of chloride ion can be measured by the analysis method of the present invention, and the amount of the raw material organic compound or the amount of the reaction product can be indirectly determined from this. it can.

[0032]

The present invention will be described in detail below 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-dodecyl acridinium bromide> (1) 1 μM 3,6-bis (dimethylamino) -10
A 0.1 M potassium phosphate buffer solution (pH 7.0) containing dodecyl acridinium bromide (commercially available product) 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 by setting the fluorescence intensity when the chloride ion concentration was 0 M to 1.

The results are shown in FIG. When F and F ₀ are fluorescence intensities in the presence and absence of chloride ions, k is an extinction constant, and [Cl] is a chloride ion concentration, the Stern-Boomer equation is represented by the following equation.

[0036]

## EQU1 ## F / F ₀ = (1 + k [Cl]) ^{-1 When the} extinction constant k for chloride ion was determined from FIG. 1 and the above equation, it was 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 with chloride ion (1) 0.1 M phosphorus containing 1 μM 6-methoxy-1- (3-sulfopropyl) quinolinium and 3- (10-methylacridinium-9-yl) propionic acid tetrafluoroborate, respectively Phosphate buffer (pH 7.0)
Was prepared. Each of these solutions has a predetermined concentration (10 ^{-4 to} 10
Potassium chloride was dissolved so that the concentration became ⁰ M).

(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 when the chloride ion concentration was 0M as 1.

[0039]

[Table 1] When the extinction constant k was determined by the Stern-Boomer equation in the same manner as in Example 1, it was 7.6M ⁻¹ , 3- (10 in the case of 6-methoxy-1- (3-sulfopropyl) quinolinium.
In the case of -methylacridinium-9-yl) propionic acid, it was 9.3 M ^-1 . It was found that 3,6-bis (dimethylamino) -10-dodecyl acridinium has 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 of 3,6-bis (dimethylamino) acryi
Gin (commercially available), 1 μM 3,6-bis (dimethylamido)
No) -10-hexadecyl acridinium bromide (K
awabata Y et al. Anal. Che
m. 62: 2054-2055, 1990)
0.1M potassium phosphate buffer solution (p
H7.0) was prepared. A predetermined concentration (10
^-FourThrough 10 ⁰ M) to dissolve potassium chloride so that
It was

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

Example 3 <Interference of iodide ion in fluorescence analysis of chloride ion> (1) 1 μM 3,6-bis (dimethylamino) -10
-Dodecyl acridinium bromide, 1 μM 6-methoxy-1- (3-sulfopropyl) quinolinium, 1 μ
0.1 containing M 3- (10-methylacridinium-9-yl) propionic acid tetrafluoroborate, respectively.
An M potassium phosphate buffer solution (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. 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 ion in the analysis of chloride ion was investigated by subtracting the fluorescence intensity in the presence of iodide ion from the relative fluorescence intensity in the absence of iodide ion (FIG. 1).

The obtained results are shown in FIG. In the figure, the vertical axis represents the error from the relative fluorescence intensity (true fluorescence intensity) in the absence of iodide ions, and the horizontal axis represents the chloride ion concentration.

As shown in FIG. 2, 3,6-bis (dimethylamino) -10-dodecylacridinium has extremely small interference with iodide ions as compared with the fluorescent dye used in the conventional chloride ion analysis. It was confirmed.

Further, 3,6-bis (dimethylamino) acridine and 3,6-bis (dimethylamino) -1
Similar experiments were carried out 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]

EFFECTS OF THE INVENTION The fluorescent dye according to the present invention enables highly sensitive detection of halogen ions as well as conventional fluorescent dyes, and also allows chloride ions to be selectively detected 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 in the presence of iodide ion, which has been a problem in the prior art, is present. The ions can be analyzed accurately.

[Brief description of drawings]

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

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

Claims (4)

[Claims] 1. In a fluorescence analysis method for detecting the concentration of chloride ion in a sample using a fluorescent dye that quenches fluorescence by chloride ion, the fluorescent dye is represented by the formula (1): 3. A method for fluorescence analysis of chloride ion, which comprises using 3,6-bis (dimethylamino) acridine and a derivative thereof represented by: 2. A fluorescent dye of formula (2): (However, R is an alkyl substituent having 1 to 30 carbon atoms and I ⁻ represents a halogen ion.) A halide of a 3,6-bis (dimethylamino) -10-alkylacridinium ion represented by the formula is used. Item 2. The fluorescence analysis method according to Item 1. 3. The fluorescent dye represented by the formula (2) is immobilized on a carrier, and the carrier is brought into contact with a sample solution to continuously detect the concentration of chloride ion without consuming the fluorescent dye. Item 3. The fluorescence analysis method according to Item 2. 4. The fluorescence analysis method according to claim 2, wherein the halide represented by the formula (2) is bromide.