JP2001114520A - Method for separating arsenic compounds by chemical form - Google Patents

Abstract

(57) [Summary] [PROBLEMS] To be able to quantify arsenic compounds at a trace concentration,
Provided is a method for separating arsenic compounds by chemical form, which can employ an element-specific device such as an inductively coupled plasma analyzer as a detecting means. SOLUTION: In the method for separating an arsenic compound from an arsenic compound-containing liquid by using high performance liquid chromatography, as a chromatographic filler, two or more ammonium groups or amino groups each having a functional group linked via an alkyl group are used. A method for separating an arsenic compound using a functional type strongly basic anion exchange resin.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for separating anionic arsenic compounds by high performance liquid chromatography.

[0002]

2. Description of the Related Art Arsenic is considered to be an essential constituent element of the human body, but is generally known as a harmful element. Its acute toxicity varies greatly depending on its chemical form. In inorganic arsenic compounds, trivalent (arsenic anhydride / arsenite) toxicity is much higher than that of pentavalent, and the toxicity of organic arsenic compound is , Considerably weaker than inorganic ones. In addition, studies have been conducted not only on acute toxicity but also on carcinogenicity. The natural content of arsenic compounds in terrestrial organisms is 0.1
０．２0.2 ppm, but can reach several tens of ppm in marine fish and seaweeds. This is not due to contamination but metabolized and concentrated pentavalent inorganic arsenic contained in seawater, and its chemical form is arsenobetaine in fish and shellfish and dimethyl arsenic in seaweed in combination with ribose. Mostly organic forms such as arsenosugar and the toxicity is weak.

Performing separation and quantification for each chemical form in this way is of great significance from the viewpoint of toxicity evaluation and also from the viewpoint of evaluating health risks such as carcinogenicity. As a method for analyzing water-soluble arsenic compounds contained in food and environmental water by chemical form, element-specific quantification is performed using high performance liquid chromatography as a separation means and an inductively coupled plasma emission spectrometer or an atomic absorption apparatus as a detection means. Methods are known. (New Food Analysis Method / Korin Publishing, Separation
of seven arsenic compound
s by high performance liq
uid chromatograph with o
n-line detection by hydro
gen-argon frame atomic ab
sorption spectrometry and
inductively coupled plas
ma mass spectrometry / J. o
fAnalytical Atomic Spectr
omtry, June 1992, vol. ,
629-634).

[0004] However, the lower limit of quantification of these conventional techniques is several hundred ppb, and it cannot be said that the requirements for trace analysis are sufficiently satisfied. The main reason that the lower limit of quantification is high is that high sensitivity cannot be obtained because the concentration of the eluent used for high-performance liquid chromatography is as high as 20 to 100 mM, and inductively coupled plasma mass spectrometry capable of higher sensitivity detection This is because it is difficult to use in combination with the device.

[0005]

In the conventional method for separating arsenic compounds by high performance liquid chromatography, the concentration of the eluent in the conventional method is high. It was practically impossible to perform analysis in combination with an analyzer or an atomic absorption device, and it was difficult to detect trace components. An object of the present invention is to provide a chromatographic separation method capable of detecting a trace amount of an arsenic compound component.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies on separation conditions such as a separation column and eluent composition used for high performance liquid chromatography in order to solve the above-mentioned problems, and as a result, a specific anion exchange resin The present inventors have found that trace components can be detected by using as a chromatography packing material, and have reached the present invention. That is, the gist of the present invention is characterized in that a bifunctional strong basic anion exchange resin having a functional group in which two ammonium groups or amino groups are linked via an alkyl group is used as a chromatography filler. A method for separating arsenic compounds by high performance liquid chromatography.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail. In the present invention, the compound to be separated is an arsenic compound which dissociates into anions represented by arsenous acid, arsenic acid, methanearsonic acid (also referred to as methylarsonic acid), cacodylic acid (dimethylarsinic acid) and the like. However, the present invention is not particularly limited to these. The origin of the sample to be separated includes food, pharmaceuticals, environmental water, soil, etc., but it is not necessary to be limited to these, and the injected sample is subjected to appropriate pretreatment according to its origin. Need to be In the present invention, a bifunctional strong basic anion exchange resin having a functional group in which two ammonium groups or amino groups are linked via an alkyl group is used as a chromatography packing material. As a specific example, the functional group of the bifunctional strong basic anion exchange resin is represented by the general formula (1)

[0008]

Embedded image -N ⁺ (R ¹ R ² ) -C _n H _2n -N ⁺ (R ³ R ⁴ R ⁵ ) (1)

(Wherein -C _n H _2n -is a linear alkyl group, and n is an integer of 1 to 12. In addition, R ¹ and R ^1
2 , R ³ , R ⁴ and R ⁵ each represent a hydrogen atom and a carbon atom of 1
To 12 alkyl groups or hydroxyalkyl groups. ), And represents -R ¹ R ² in the formula (1).
The one in which the N atom of N- is bonded to the base of the anion exchange resin. Among them, n is 1 to 8, furthermore 1 to 1
6, more preferably 2 to 4, and R ^{1 to} R ⁵ each preferably have 1 to 8, more preferably 1 to 6, and more preferably 1 to 2 carbon atoms.

The parent structure of the anion exchange resin is not particularly limited. For example, aromatic monovinyl compound-aromatic polyvinyl compound copolymer, (meth) acrylate-polyfunctional vinyl monomer copolymer, ( Examples thereof include a (meth) acrylamide-based compound-polyfunctional vinyl monomer copolymer, and a (meth) acrylate-aromatic-based polyvinyl compound copolymer. Among them, an aromatic monovinyl compound-aromatic polyvinyl compound copolymer, a methacrylate-polyfunctional vinyl monomer copolymer and the like are preferable. Examples of the aromatic monovinyl compound include styrene, vinyl toluene, vinyl naphthalene, and ethyl vinyl benzene. Examples of the aromatic polyvinyl compound include divinylbenzene, divinyltoluene, divinylxylene, divinylnaphthalene, and the like.
(Meth) acrylic acid esters include 2-hydroxyethyl (meth) acrylate, diethylene glycol (meth) acrylate, triethylene glycol (meth) acrylate, and octaethylene glycol (meth)
Acrylate, polyethylene glycol (meth) acrylate, methyl (meth) acrylate, ethyl (meth)
Acrylate, glycerol (meth) acrylate,
2,3-dihydroxypropyl (meth) acrylate,
Glycidyl (meth) acrylate and the like can be mentioned. Examples of the polyfunctional vinyl monomer include ethylene glycol di (meth) acrylate, diethylene glycol di (meth) acrylate, triethylene glycol di (meth) acrylate, and polyethylene glycol di (meth) acrylate.
Acrylate, glycerol di (meth) acrylate,
Trimethylolpropane tri (meth) acrylate, ethylenebis (meth) acrylamide, triethylenebis (meth) acrylamide, and the like. Examples of the (meth) acrylamide-based compound include (meth) acrylamide, methyl (meth) acrylamide, and ethyl (meth) acrylamide.

The anion exchange resin of the present invention has an ion exchange capacity of usually 10 to 100 μeq / g, preferably 20 to 100 μeq / g.
６０60 μeq / g, and the average particle size is usually 2-15 μeq.
m. The anion exchange resin is preferably porous and spherical. In the method for separating an arsenic compound of the present invention, a separation column packed with a strongly basic anion exchange resin as described above is used. As the size of the column, a column tube having an inner diameter of 2 to 10 mm and a length of 30 to 300 mm can be usually used, but can be freely selected from this range in consideration of peak separation performance and analysis time. The concentration of the arsenic compound in the arsenic compound-containing solution to be separated is usually 0.01 ppb.
５００500 ppm, preferably 0.01 pp.
b to 200 ppm, more preferably 0.01 ppb to 1
It is in the range of 00 ppm.

In the separation method of the present invention, a method based on ordinary high performance liquid chromatography is employed. That is, the arsenic compound-containing liquid is injected into the separation column,
The arsenic compound is separated and eluted by passing an eluent through the column. The flow rate of the eluent is usually
It is about 0.1 to 2.0 ml / min. In addition, the temperature of the separation column during the passage of the liquid is usually about 10 to 50 ° C. The concentration of the dissolved component is usually 0.1 to 15 in the eluent.
mM, preferably 0.5-5 mM aqueous solution. The pH of the solution is usually 3 to 12, preferably 8 to 12.

The following compounds can be used for eluent preparation. Examples of the phosphate include sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium hydrogen phosphate, and the like. Examples of the organic acid include citric acid, phthalic acid, trimesic acid, succinic acid, and p-toluene sulfone. Acids, maleic acid, acetic acid, formic acid, etc., as organic acid salts, for example, sodium citrate, lithium citrate,
Sodium maleate, lithium maleate, potassium hydrogen phthalate, sodium acetate, ammonium acetate, etc., as the hydroxyl compound, for example, sodium hydroxide, lithium hydroxide, etc., and as the amine compound, triethanolamine, tris ( Hydroxymethyl) aminomethane, diethanolamine, diethylethanolamine, methyldiethanolamine, etc .; borate as sodium borate; and inorganic acids as phosphoric acid, hydrochloric acid, sulfuric acid, nitric acid, perchloric acid, etc. For example, sodium carbonate, sodium hydrogen carbonate and the like can be used. Usually, an aqueous solution of a predetermined concentration is prepared from these alone or in combination of two or more, and it is necessary that the pH be within a predetermined range. For the purpose of preventing iron ions present in the sample from contaminating the column, a chelating agent represented by EDTA can be added to the eluate. In order to prevent the deterioration of column performance caused by irreversible adsorption of contaminants present in the sample to the column packing material, 30 vol / vol% of a polar organic solvent such as methanol / acetonitrile is used in the eluent. It can be added to the upper limit. Means for detecting the measurement sample components separated and eluted by the separation column include an electric conductivity meter, an inductively coupled plasma analyzer, an atomic absorption analyzer, an ultraviolet absorption meter, and the like. The present invention is particularly suitable for the case where an element-specific measurement method such as an inductively coupled plasma analyzer or an atomic absorption analyzer is employed as a detection means. That is, according to the method of the present invention, since a low concentration aqueous solution of 0.1 mM to 15 mM is used as an eluent for column separation, an improvement in the S / N ratio is expected in the detection, and it is difficult to apply a high concentration eluent. The use of the inductively coupled plasma mass spectrometer, which is said to be possible, enables more sensitive detection.

[0014]

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples unless it exceeds the gist thereof. (1) HPLC apparatus LC pump; LC-10AS column oven; CTO-6A detector; SPD-10AV auto injector; SIL-10A data processing; CLASS-LC10 (all trade names, manufactured by Shimadzu Corporation)

(2) Measurement Samples Arsenic acid, arsenic acid, cacodylic acid (obtained from Wako Pure Chemical Industries, Ltd.) and methanearsonic acid (obtained from Trichemical Laboratories) were weighed in a predetermined amount, and Milli-Q water was added. After dissolving and constant-volume, an aqueous solution having each concentration of 50 ppm was prepared and used as a measurement sample. (3) Chromatogram separation conditions Flow rate: 0.5 mL / min. Column temperature: 40 ° C Sample injection volume: 40 μL Detection: Indirect absorbance method at a wavelength of 260 nm

[0016] (4) a separation column MCI ^(R) GEL SCA05 ^{(manufactured} by Mitsubishi Chemical Corporation); column bifunctional type strongly basic anion exchange resin is filled. The characteristics of the ion exchange resin are shown below. Base resin structure: Copolymer of ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, glycidyl methacrylate Functional group: Pentamethyldiaminopropane Ion exchange capacity: 50 μeq / g Average particle size: 5 μm Physical structure: Porous Appearance: Spherical

[0017] MCI ^(R) GEL SCA04 ^{(manufactured} by Mitsubishi Chemical Corporation); were used for comparison, the column of monofunctional type quaternary ammonium type strong basic anion exchange resin is filled.
The characteristics of the ion exchange resin are shown below. Base resin structure: Copolymer of ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, glycidyl methacrylate Functional group: Methyl diethanolamine Ion exchange capacity: 30 μeq / g Average particle size: 5 μm Physical structure: Porous appearance; Spherical column size; Each column has an inner diameter of 4.6 mm and a length of 35 mm

(5) Eluent An aqueous solution obtained by mixing 1 mM potassium hydrogen phthalate and 1 mM NaOH to adjust the pH to 10.0. 1 mM aqueous solution of potassium hydrogen phthalate pH 4.2 Example 1 MCI ^(R) GEL S of the above (4) as a separation column
Using CA05 (manufactured by Mitsubishi Chemical), eluent (5)
FIG. 1 shows a chromatogram obtained by separating the measurement sample of the above (2) under the separation conditions of the above (3) using the aqueous solution of pH 10.0 in the above HPLC apparatus. The retention time of each component is as follows. Cacodylic acid: 1.886 (min,
The same applies hereinafter), arsenous acid: 2.845, methanearsonic acid:
4.477, arsenic acid: 6.977

As is apparent from the above, four arsenic compounds are well separated in a short time (within 10 minutes). The elution order of each component reflects the ion dissociation state at pH 10.0. That is, the retention of arsenic acid which dissociates into a divalent anion is the strongest, and the retention of methanearsonic acid in which a divalent anion and a monovalent anion coexist is the next strongest. 1
Cacodylic acid and arsenite dissociated into valent anions are weakly retained and elute quickly from the column. As described above, it is understood that the separation is mainly achieved in the anion exchange mode. From about 1.5 minutes and from 7 minutes to 13 minutes, the baseline of the chromatogram deviated to the negative side, which was a problem in the indirect absorbance method used in the examples, and was due to an element such as an inductively coupled plasma analyzer. If a specific method is adopted as the detection means, the phenomenon of swinging to the negative side can be quantified without any practical effect.

Comparative Example 1 The MCI ^(R) GEL S of the above (4) was used as a separation column.
Example 1 except that CA04 (manufactured by Mitsubishi Chemical) was used.
Separation was performed in the same manner as described above. The chromatogram is shown in FIG. The retention time of each component is as follows. Cacodylic acid: 1.980 (min, the same applies hereinafter), arsenous acid: 3.30
2, methanearsonic acid: 4.787, arsenic acid: 6.333 In this comparative example, although the four types of arsenic compounds seem to be well separated, each peak shows a double peak. This indicates that there is a problem in the separability and the quantitativeness.

Reference Example 1 Separation was carried out in the same manner as in Example 1 except that the aqueous solution of the above (5) having a pH of 4.2 was used as the eluent. The chromatogram is shown in FIG. The retention time of each component is as follows. Cacodylic acid: 1.438 (min, the same applies hereinafter),
Methanearsonic acid: 2.086, arsenic acid: 2.899, arsenous acid: 4.417 Even under these conditions, four types of arsenic compounds were successfully prepared in a short time (within 10 minutes) as in Example 1. ing. As shown in Table 1, when the degree of separation was compared between the two, the eluent pH 1
In Example 1 with 0.0, four components were almost separated by baseline, whereas in Reference Example 1, the separation ability of methanearsonic acid and arsenic acid was slightly inferior. This is because under the condition of pH 4.2, each arsenic compound has a small degree of dissociation into anions, and separation modes such as hydrophobic adsorption other than the anion exchange mode become dominant. Is considered to be reduced. Thus, it can be seen that it is advantageous to use an anion exchange resin under basic conditions in order to separate the anionic arsenic compound with high separation.

Reference Example 2 MCI ^(R) GEL S of the above (4) was used as a separation column.
Using CA04 (manufactured by Mitsubishi Chemical), the eluent was the above (5)
Separation was carried out in the same manner as in Example 1 except that an aqueous solution having a pH of 4.2 was used. The chromatogram is shown in FIG. The retention time of each component is as follows. Cacodylic acid: 1.413 (min, the same applies hereinafter), methanearsonic acid:
2.148, arsenic acid: 2.914, arsenous acid: 4.514 Separation in this reference example was equivalent to that in reference example 1.

As described above, the above (4) used in Reference Example 2
Is not suitable for use under basic conditions. On the other hand, Example 1 and Reference Example 1 found by this study
The separation column (4) used in the above showed good separation performance under both acidic and basic conditions. Further, since the concentration of the eluent is extremely low, an inductively coupled plasma analyzer or the like can be used as a detecting means, and thus a path for quantifying arsenic compounds at a trace concentration has been opened.

[0024]

Table 1 Peak separation rate Example 1 (FIG. 1: pH = 10.0) cacodylic acid / arsenous acid = 100% arsenous acid / methanearsonic acid = 97.5% methanearsonic acid / arsenic acid Comparative Example 1 (FIG. 2: pH = 10.0) Cacodylic acid / arsenous acid = 96.6% Arsenous acid / methanearsonic acid = 97.1% Methanearsonic acid / arsenic acid = 97. 3% Reference Example 1 (FIG. 3: pH = 4.2) Cacodylic acid / methanearsonic acid = 100% Methanearsonic acid / arsenic acid = 94.6% Arsenic acid / arsenous acid = 97.8% Reference example 2 ( Figure 4: pH = 4.2) Cacodylic acid / methanearsonic acid = 100% Methanearsonic acid / arsenic acid = 94.2% Arsenic acid / arsenous acid = 98.7% Note: Baseline separation = 100%

[0025]

According to the present invention, an element-specific device such as an inductively coupled plasma analyzer is detected because an extremely low concentration eluent is used for separating anionic arsenic compounds by high performance liquid chromatography. It can be adopted as a means, and it becomes possible to quantify a trace concentration of an arsenic compound. Further, since an acidic to basic eluent can be used in a single column, the pH of the eluent can be freely selected according to the target substance, and the separation can be easily controlled.

[Brief description of the drawings]

FIG. 1 shows a chromatogram obtained in Example 1.

FIG. 2 shows a chromatogram obtained in Example 2.

FIG. 3 shows a chromatogram obtained in Comparative Example 1.

FIG. 4 shows a chromatogram obtained in Comparative Example 2.

[Explanation of symbols]

 1 cacodylic acid 2 arsenous acid 3 methanearsonic acid 4 arsenic acid

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 30/88 G01N 30/88 B

Claims (8)

[Claims] 1. A method for separating an arsenic compound from an arsenic compound-containing liquid using high performance liquid chromatography, wherein the chromatography filler has a functional group in which two ammonium groups or amino groups are linked via an alkyl group. A method for separating an arsenic compound, comprising using a bifunctional strong basic anion exchange resin. 2. The functional group of the bifunctional strong basic anion exchange resin is represented by the general formula (1): -N ⁺ (R ¹ R ² ) -C _n H _2n -N ⁺ (R ³ R) ⁴ R ⁵ ) (1) (wherein -C _n H _2n -is a linear alkyl group;
Is an integer of 1 to 12. R ¹ , R ² , R ³ , R
⁴ and R ⁵ are a hydrogen atom, an alkyl group having 1 to 12 carbon atoms or a hydroxyalkyl group, respectively. ) And the expressed ones are, the process of separation according to claim 1 arsenic compound according in which -R ¹ R ² N-N atoms in the formula (1) is attached to the parent of the anion exchange resin. 3. The method for separating an arsenic compound according to claim 1, wherein the exchange capacity of the bifunctional strong basic anion exchange resin is 10 to 100 μeq / g. 4. The method for separating an arsenic compound according to claim 1, wherein the structure of the bifunctional strong basic anion exchange resin is porous and spherical. 5. The method for separating an arsenic compound according to claim 1, wherein the bifunctional strong basic anion exchange resin has an average particle size of 2 to 15 μm. 6. The concentration of the arsenic compound in the arsenic compound-containing solution is from 0.01 ppb to 500 pp for each arsenic compound alone.
The method for separating an arsenic compound according to any one of claims 1 to 5, which is pm. 7. An eluent used for eluting an arsenic compound adsorbed on a chromatographic packing material includes phosphate, organic acid, organic acid salt, hydroxide compound, amine compound,
An eluent containing a solution of one or a mixture of two or more selected from boric acid, borate, inorganic acid and carbonate and having a concentration of 0.1 to 15 mmol / L in total of the compounds is used. A method for separating an arsenic compound according to claim 1. 8. The eluent has a pH of 8 to 12.
The method for separating an arsenic compound according to the above.