JP2001124750A - Measuring method for alkylphenols - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method in which alkylphenols (an alkylphenol having a 4 to 12 alkyl group or bisphenol A), in an environment, suspected as endocrine disrupting chemicals can be measured with high sensitivity. SOLUTION: This measuring method, for alkylphenols, in which a problem is solved is provided with a process (1) in which the alkylphenols in a water sample are reacted with a fluorescence labeling agent by an ultrasonic treatment under an alkali condition and under the existence of a phase transfer catalyst and in which fluorescence-labeled alkylphenols are generated, a process (2) in which an uncreacted fluorescence labeling agent and a by-product in the previous process are removed, and a process (3) in which the fluorescence-labeld alkylphenols are separated by reversed phase liquid chromatography so as to be chemiluminescence-detected.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring highly sensitive alkylphenols (such as alkylphenols having an alkyl group having 4 to 12 carbon atoms and bisphenol A) which are suspected as environmental hormones. About.

[0002]

2. Description of the Related Art Alkylphenols in the environment suspected as environmental hormones (such as alkylphenols having an alkyl group having 4 to 12 carbon atoms and bisphenol A)
For example, in the case of a water sample, the sample is 1 L (liter)
, And the extract was concentrated 1000-fold, followed by the GC-MS method. However, GC-
The lower limit of detection by the MS method was about 10 to 100 ppb. In addition, the measurement requires a large amount of water sample and also requires complicated operations such as extraction and concentration.

On the other hand, 2,4-dimethylphenol, 3-
As a method for measuring an alkylphenol such as methyl-4-chlorophenol, the alkylphenol is
Under alkaline conditions, in the presence of a phase transfer catalyst, react with dansyl chloride while stirring with a vortex mixer to produce dansylated alkylphenols, remove unreacted dansyl chloride, and then reverse-phase liquid chromatography the dansylated alkylphenols. A method for detecting chemiluminescence after separation by J.I.
Chromatogr. , 553 (1991) 345-.
356). However, in this method, alkylphenol having an alkyl group having a large number of carbon atoms, such as nonylphenol, and bisphenol A, which are suspected as environmental hormones, have a problem in quantitativeness due to the low rate of dansylation, resulting in high sensitivity. Could not be measured.

[0004]

SUMMARY OF THE INVENTION The present invention provides a method for measuring alkylphenols (alkylphenols having an alkyl group having 4 to 12 carbon atoms or bisphenol A) in the environment suspected as environmental hormones with high sensitivity. The task is to provide

[0005]

The object of the present invention is to provide (1)
Reacting alkylphenols in a water sample with a fluorescent labeling agent under ultrasonic treatment under alkaline conditions and in the presence of a phase transfer catalyst to produce fluorescently labeled alkylphenols; ( 2) removing the unreacted fluorescent labeling agent and the by-product of the previous step;
And (3) a step of separating the above-mentioned fluorescently labeled alkylphenols by reversed-phase liquid chromatography and then detecting chemiluminescence.

That is, according to the present invention, (1) an aqueous solution of an alkylphenol is made alkaline by adding an aqueous alkali solution (eg, an aqueous NaOH solution), and then an aqueous solution of a phase transfer catalyst (eg, tetrabutylammonium bromide) and a fluorescent labeling agent ( A methylene chloride solution of dansyl chloride or the like is added, and a derivatization reaction is carried out by sonication at room temperature for about 10 minutes. (2) After the reaction, the methylene chloride layer is placed on a mini column (an NH ₂ mini column is placed under a silica gel mini column). Excess dansyl chloride and by-product sulfonate were removed, and (3) the resulting reaction product (dansyl derivative) was separated by reversed-phase HPLC. Mixing and reacting with a solution in which TDPO (peroxalate) and hydrogen peroxide are mixed in advance, and detecting the generated luminescence; Method for measuring Le kill phenols.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Examples of the alkylphenols measured by the method of the present invention include alkylphenol having an alkyl group having 4 to 12 (preferably 4 to 9) carbon atoms and / or bisphenol A.

The alkyl group having 4 to 12 carbon atoms includes n-butyl, t-butyl, n-pentyl and n-butyl.
-Hexyl group, n-heptyl group, n-octyl group, nonyl group, n-dodecyl group and the like. Examples of the alkylphenol having an alkyl group having 4 to 12 carbon atoms include pn-butylphenol, pt-butylphenol, pn-pentylphenol, and pn
-Hexylphenol, pn-heptylphenol,
Specific examples include pn-octylphenol and p-nonylphenol.

In the method of the present invention, it is preferable that the sample containing the alkylphenols is subjected to measurement as a water sample. The water sample may be one that can be normally collected from the environment (river water, lake water, seawater, groundwater, rainwater, etc.). When eluting from a polymer such as polycarbonate or polysulfone is targeted, a water sample in which an alkylphenol is eluted from the polymer with hot water or an aqueous alkaline solution is used.

[0010] These water samples may be concentrated or diluted if necessary, but usually 1 to 10 ml of the sample can be directly used for measurement without concentration. In the method of the present invention, since measurement can be easily performed with a small amount of sample,
For example, the water sample may be 1 ml. The concentration of the alkylphenols in the water sample is not particularly limited as long as the concentration can be measured by concentration or dilution of the water sample.

Examples of the fluorescent labeling agent used in the method of the present invention include dansyl chloride and bansyl chloride, and dansyl chloride is preferred. When dansyl chloride is used, dansylated alkylphenols are formed as fluorescently labeled alkylphenols, and sulfonates such as alkali salts of dansyl hydroxide (alkali metal salts of dansyl hydroxide) are formed as by-products. .

The procedure of the measuring method of the present invention will be described below. (1) In the first step, the alkyl phenols in the water sample were reacted with a fluorescent labeling agent under ultrasonic treatment under an alkaline condition and in the presence of a phase transfer catalyst to perform fluorescent labeling. An operation of generating alkylphenols (fluorescent labeling of alkylphenols) is performed.

In the above operation (1) (fluorescence labeling), the method under alkaline conditions is to adjust the pH of the water sample to 10
The method is not particularly limited as long as it can be adjusted to about 12. That is, the alkali species, alkali concentration, alkali addition amount, and the like are not particularly limited as long as the pH can be maintained in the above range and the operation is not complicated. Usually, an aqueous solution of an alkali metal hydroxide (such as sodium hydroxide or potassium hydroxide) or an alkali metal carbonate (such as sodium carbonate or potassium carbonate) is used. In the method of the present invention, for simple measurement with a small amount of a sample, for example, 0.1% is added to 1 ml of a neutral water sample.
What is necessary is just to add 20 microliters of 1N sodium hydroxide aqueous solution.

As the phase transfer catalyst, a tetraalkylammonium halide is preferably used. The alkyl group is preferably an alkyl group having 1 to 8 carbon atoms such as a methyl group, an ethyl group, a butyl group, a hexyl group, an octyl group, and the halide is preferably a chloride, a bromide or the like. Specifically, as a phase transfer catalyst. Examples thereof include tetrabutylammonium chloride, tetrabutylammonium bromide, and tetrahexylammonium bromide. Of these, tetrabutylammonium bromide and tetrahexylammonium bromide are preferable.

[0015] The phase transfer catalyst is 0.01 to
It is preferred to use 1% by weight. In the method of the present invention, for example, 100 μl of a 1% by weight aqueous solution of tetrabutylammonium bromide may be added to 1 ml of a water sample in order to easily measure a small amount of the sample. The phase transfer catalyst is usually added to a water sample as an aqueous solution (for example, a 1% by weight aqueous solution).

The fluorescent labeling agent is used in an amount of 0.0
Preferably, it is used in a proportion of from 0.01 to 0.01% by weight. In the method of the present invention, for simple measurement with a small amount of sample, for example, 1 ml of a fluorescent labeling agent / methylene chloride solution (fluorescent labeling agent concentration: 20 ppm) may be added to 1 ml of a water sample. In addition, it is preferable that fluorescent labeling is usually added to a water sample in an organic solvent solution (for example, a 20 ppm organic solvent solution). As the organic solvent, aliphatic halogenated hydrocarbons and the like are preferable, and among them, methylene chloride and chloroform are preferable.

In the method of the present invention, the fluorescent labeling of the alkylphenols is carried out by reacting the alkylphenols with a fluorescent labeling agent under the alkaline conditions under a phase transfer catalyst under ultrasonic treatment. At this time, the reaction temperature may be room temperature (15-30 ° C.), and the reaction time may be about 5-20 minutes. In the method of the present invention, in order to easily measure a small amount of a sample, for example, as described above, a water sample to which an alkali and a phase transfer catalyst have been added may be subjected to ultrasonic treatment at room temperature for about 10 minutes. By this ultrasonic treatment, the yield of the fluorescent labeling is significantly improved, and the alkylphenols in the water sample can be measured with high sensitivity.

In the measuring method of the present invention, the following steps
(2) Removal of unreacted fluorescent labeling agent coexisting with the generated fluorescently labeled alkylphenol and by-products of the previous step, for example, sulfonate (salt of dansyl hydroxide and alkali) Then, in the next step, (3) an operation of separating the fluorescently labeled alkylphenols by reversed-phase liquid chromatography and then detecting chemiluminescence is performed.

That is, the operations (2) and (3) are as follows:
Separate the organic phase (methylene chloride phase) from the fluorescent labeling reaction solution (consisting of an aqueous phase and an organic phase), extract the aqueous phase with methylene chloride. Subjecting to a mini-column (normal-phase mini-column) for removing the fluorescent labeling agent and by-products of the previous step, concentrating the fluorescently labeled alkylphenol-containing fraction passing through the mini column to remove methylene chloride,
Add exactly 1 ml of acetonitrile to this residue,
This acetonitrile solution is used to perform a series of operations in which fluorescent-labeled alkylphenols are separated by reverse phase chromatography and then chemiluminescence is detected.

The above-mentioned operation may be carried out by a usual method.
The operation is preferably performed twice, for example, using a silica gel mini column (Sep-Pak silica (manufactured by Waters)) and an NH ₂ mini column (Sep-Pak Pl).
us NH ₂ (manufactured by Waters) is preferably connected using a mini-column (normal-phase mini-column). When performing each operation on the above scale using 1 ml of a water sample for measurement, if the mini-column is washed with 5 to 8 ml of methylene chloride, the fluorescent-labeled alkylphenol-containing fraction becomes the fraction that has passed through the column. Obtainable. Is preferably performed under a nitrogen stream.

The operation is generally the reverse phase chromatography (C ₁₈ type, such as C ₈ based columns) and chemiluminescent detection (peroxyoxalate (tdpo etc.) - hydrogen peroxide, etc.)
For example, a high performance liquid chromatography apparatus (LC-9) equipped with a reversed-phase column and a chemiluminescence detector
It is preferable to carry out the measurement online using Model 00 (manufactured by JASCO Corporation). This analysis illustrates the conditions in the examples below. Although the sample amount in this analysis is not particularly limited, it is preferable to use 5 to 10 μl of the acetonitrile solution in consideration of measurement sensitivity and injection reproducibility.

In this analysis, when a standard solution (a water sample) of an alkylphenol compound prepared separately was subjected to each of the above operations to determine a measured value (reaction yield), the average value was obtained in a sample concentration range of 0.5 to 5 ppb. A measured value (reaction rate) of about 80% was obtained, and the reactivity was sufficiently high. Further, when the area count number in the high performance liquid chromatography analysis was plotted against the concentration of the alkylphenols, a high correlation (linearity) was observed between the concentration of the alkylphenols and the area count number, and the lower limit of detection was p. -0.05 pp with nonylphenol
b. The regression equation obtained from this straight line can be used as a calibration curve when analyzing a water sample collected from the environment.

According to the measuring method of the present invention, alkylphenols can be obtained by, for example, (1) subjecting alkylphenols in a water sample to a fluorescent labeling agent and an ultrasonic wave under alkaline conditions and in the presence of a phase transfer catalyst. A reaction device for producing a fluorescently labeled alkylphenol by reacting under the treatment; (2) a removing device for removing unreacted fluorescent labeling agent and a by-product of the previous step; And a detection device for detecting chemiluminescence after separating the fluorescent-labeled alkylphenols by reversed-phase liquid chromatography.

The reaction apparatus is not particularly limited as long as it can fluorescently label alkylphenols under ultrasonic treatment. For example, there is an apparatus in which an ultrasonic cleaning apparatus is combined with a glass reactor (such as an ampule tube). The removal device is not particularly limited as long as it can remove unreacted substances and by-products of fluorescent labeling. For example, there is a liquid chromatography apparatus provided with the normal phase column. As the detection device, for example, a high-performance liquid chromatography device (type LC-900; manufactured by JASCO Corporation) equipped with the above-described reversed-phase column and a chemiluminescence detector is preferably exemplified. This conceptual diagram is shown in FIG.

[0025]

Next, the present invention will be described specifically with reference to examples and comparative examples. Example 1 1 ml of a water sample (p-nonylphenol concentration: 1.0 ppb) prepared by adding p-nonylphenol as an alkylphenol to pure water was put into a 10 ml ampoule tube, and 20 μl of a 0.1N sodium hydroxide aqueous solution was added thereto.
1, 100% of a 1% by weight aqueous solution of tetrabutylammonium bromide and 1 ml of a solution of dansyl chloride / methylene chloride (concentration of dansyl chloride: 20 ppm) were added, and the mixture was subjected to dansylation by treatment in an ultrasonic cleaning device at room temperature for 10 minutes.

After the treatment, the methylene chloride phase was separated, and the aqueous phase was further extracted twice with 1 ml of methylene chloride. The methylene chloride phase and the extract were combined, and a silica gel mini column (Sep-Pak silica; manufactured by Waters) was added to NH _2.
Mini column (Sep-PakPlus NH _2; Waters) was subjected to normal phase mini column linked and washed with methylene chloride 6 ml, to give the dansyl alkylphenols containing fraction that passes through the normal phase mini-column.

Next, this fraction was concentrated under a stream of nitrogen to remove methylene chloride, 1 ml of acetonitrile was accurately added to the residue and mixed, and 5 μl of the residue was equipped with a reverse phase column and a chemiluminescence detector. The analysis was performed using a high performance liquid chromatography apparatus (LC-900; manufactured by JASCO Corporation). FIG. 1 shows a conceptual diagram of this device. As a result, the measured value (reaction yield) was 76.9% of the test p-nonylphenol.
Was a value corresponding to This chromatogram is shown in FIG. 2 together with the chromatogram of the standard solution (p-nonylphenol concentration: 1.0 ppb).

The conditions for high performance liquid chromatography are as follows. Column: J'sphere ODS-H80 (φ4.6
mobile phase: acetonitrile / 50 mM imidazole aqueous solution (90/10 (v / v); adjusted to pH 7.0 with nitric acid) Column temperature: 40 ° C. Detection: chemiluminescence detection Reaction solution: ethyl acetate / acetonitrile (50 / 50 (v /
v); TDPO at 0.25 mM and hydrogen peroxide at 12.
Reaction solution flow rate: 1.4 ml / min Reaction temperature: 40 ° C. Injection volume: 5 μl

Example 2 The concentrations of p-nonylphenol were 0.1 ppb and 0.5 ppb.
1 ml of each of the samples changed to pb and 5.0 ppb was analyzed in the same manner as in Example 1. Table 1 shows the results together with the results of Example 1.

[0030]

[Table 1]

When the results of Examples 1 and 2 were plotted against the area counts at the time of analysis, a straight line shown in FIG. 3 was obtained. As a result, according to the method of the present invention, the lower limit of detection of p-nonylphenol was 0.05 ppb, indicating that p-nonylphenol can be measured with extremely high sensitivity.

Comparative Example 1 The analysis was carried out in the same manner as in Example 1 except that treatment was performed with a vortex mixer for 10 minutes instead of the ultrasonic cleaning apparatus. As a result, the measured value was 20 to 5 for the test p-nonylphenol.
The value was a value corresponding to 0%, and reproducibility was not obtained.

Example 3 As alkylphenols, pn-butylphenol, pn-pentylphenol, pn-hexylphenol, pn-heptylphenol, pn-octylphenol, p-nonylphenol (mixture), 1 m water sample containing 5 ppb of bisphenol A
1 was analyzed in the same manner as in Example 1. FIG. 4 shows the chromatogram. As a result, the measured values were obtained in Example 1.
It was the same value as the test concentration. The lower limit of detection of bisphenol A was 0.03 ppb, indicating that bisphenol A can be measured with high sensitivity as well.

[0034]

According to the measurement method of the present invention, alkylphenols (alkylphenols having alkyl groups having 4 to 12 carbon atoms or bisphenol A) in the environment which are suspected as environmental hormones are reduced to about 0.05 ppb to 5 ppb.
It can be measured with a high sensitivity of ppb. For example, when measuring nonylphenol, the sample concentration is 0.5 to
A measured value (reaction rate) of about 80% on average could be obtained in the range of 5 ppb, and the reactivity was sufficiently high. The lower limit of detection is 50 ng / L (0.0
5 ppb), which is equivalent to or higher than the target detection lower limit of 100 ng / L (0.1 ppb) in the GC / MS (SIM) method. In addition, when the stability under room temperature and refrigeration conditions was examined using a reaction solution having a sample concentration of 1 ppb, no change was observed even after one week. In the method of the present invention, it is not necessary to concentrate the water sample containing these alkylphenols, so that even a small amount of water sample can be measured with sufficiently high sensitivity. Furthermore, for example, if the water sample is concentrated 1000 times, it is possible to measure with a high sensitivity of about 0.05 to 5 ppt.

[Brief description of the drawings]

FIG. 1 is a conceptual diagram showing a high-performance liquid chromatography apparatus (one example) equipped with a reversed-phase column and a chemiluminescence detector. In the figure, P1 and P2 indicate pumps, and TDPO
Represents bis [2- (3,6,9-trioxadecanyloxygalbonyl) -4-nitrophenyl] oxalate.

FIG. 2 is a chromatogram showing a result of Example 1 and an analysis result of a standard solution.

FIG. 3 is a diagram in which the results of Examples 1 and 2 are plotted against the area count number at the time of analysis.

FIG. 4 is a chromatogram showing the results of Example 3. In the figure, t-C4, n-C8 and the like represent the carbon number of the alkyl group of the alkylphenol, and Bis-A represents bisphenol A.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 30/48 G01N 30/48 L 30/74 30/74 A 30/88 30/88 C (72) Invention Person Takashi Muranaka 5F, 1978 Kogushi, Obe, Ube City, Yamaguchi Prefecture F-term in UBI Scientific Analysis Center Co., Ltd. 4D017 AA01 BA04 DA03

Claims (6)

[Claims] (1) A fluorescently labeled alkylphenol is obtained by reacting an alkylphenol in a water sample with a fluorescent labeling agent under ultrasonic treatment under alkaline conditions and in the presence of a phase transfer catalyst. Generating a class,
(2) a step of removing unreacted fluorescent labeling agent and a by-product of the preceding step; and (3) chemiluminescence detection after separating the fluorescently labeled alkylphenols by reverse phase liquid chromatography. And a method for measuring alkylphenols. 2. An alkylphenol having 4 to 1 carbon atoms.
The method for measuring alkylphenols according to claim 1, wherein the alkylphenol is an alkylphenol having two alkyl groups and / or bisphenol A. 3. The method for measuring an alkylphenol according to claim 1, wherein the alkylphenol is an alkylphenol having an alkyl group having 4 to 9 carbon atoms. 4. The method according to claim 1, wherein the alkylphenol is p-nonylphenol. 5. The method for measuring an alkylphenol according to claim 1, wherein the fluorescent labeling agent is dansyl chloride. 6. A fluorescently labeled alkylphenol by reacting an alkylphenol in a water sample with a fluorescent labeling agent under ultrasonic treatment under alkaline conditions and in the presence of a phase transfer catalyst. (2) a removing device for removing unreacted fluorescent labeling agent and by-products of the previous step, and (3) reverse-phase liquid chromatography of the fluorescent-labeled alkylphenols And a detection device for detecting chemiluminescence after separation by the method described above.