JP3300499B2 - Analysis of beryllium by liquid chromatography - 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 quantifying trace amounts of toxic beryllium with ultra-high sensitivity by liquid chromatography.

[0002]

2. Description of the Related Art The distribution of beryllium (Be) in the natural world is relatively small, and is several ng / l (ie, ppt) in natural water.
It is an order. However, it is highly toxic and is listed as a toxic element that is extremely harmful to the human body. In particular, when absorbed as airborne particles, irritative symptoms appear strongly in the trachea, bronchi, lungs, etc., and the mortality from chronic poisoning is high. Recently, attention has also been paid to the carcinogenicity of beryllium.

[0003] Although beryllium is highly toxic,
In recent years, it has been widely used as a raw material for various industries such as alloy materials for space rockets, aircraft, nuclear reactors, and engines.
Under these circumstances, there is concern that beryllium may cause environmental pollution, particularly adverse effects on the working environment. In Japan, the allowable standard for indoor working environments is set at 0.002 mg / m ³ . For this reason, a highly sensitive analysis method for beryllium is required.

[0004] As a method for analyzing a trace amount of beryllium, a morin fluorescence method, a 2-methyloxin method, an aluminone colorimetric method and the like have been known for a long time. Recently, a fluorometric method by the FIA method (Watanabe, Takahashi, Aoki, "Analytical Chemistry" 41, 11)
(1992)), atomic absorption spectrometry (LCRobles, C. Garci)
a-Olalla, MTAlemany, AJAllert, Analys
t, 116, 735 (1991)), ICP emission spectroscopy (P. Schrael, Mikrochim. Acta, II, 355).
(1989)) has been reported as a highly sensitive quantitative method.

However, the conventional methods for analyzing beryllium described above are not satisfactory in terms of sensitivity,
Since the apparatus itself is expensive, development of a relatively simple analysis method using a high-sensitivity and ordinary analysis apparatus is required. In particular, since a trace amount of beryllium needs to be quantified, the detection limits of 2 ppb and 0.2 ppb, which are the detection limits of the above-mentioned atomic absorption spectrometry and ICP emission spectrometry, respectively.
It is desired to provide an ultra-sensitive analytical method exceeding the above.

On the other hand, liquid chromatography, in particular, high performance liquid chromatography (HPLC) has been widely used as a high-performance analytical method as a result of the recent advances in science and technology, which have dramatically improved analytical column technology and analytical instrument performance. It is used for Above all, reversed-phase partitioning (RP) -HPLC using a non-polar stationary phase and a polar mobile phase is an ion-pair (IP) -HPLC used in the biochemical-related analysis field.
By introducing the method described above, it is possible to analyze ionic species.

A method of quantifying trace metal ions using such RP-IP-HPLC has been studied (Hoshino, Yotsuyanagi, Aomura,
"Analytical Chemistry", 27, 315 (1978)), a sample ion is converted into a non-dissociable complex by a complexing reagent, and a sample solution containing this complex is separated and analyzed by liquid chromatography to obtain a ppb or sub-ppb level. The detection limit can be obtained. However, no optimized analytical method has been known for beryllium analysis, including selection of complexing reagents.

[0008]

SUMMARY OF THE INVENTION In view of the above circumstances, the present invention provides a method for analyzing beryllium, which can quantify trace amounts of beryllium with ultra-high sensitivity by a relatively simple method using liquid chromatography. The purpose is to provide.

[0009]

In order to achieve the above object, a method for analyzing beryllium provided by the present invention comprises converting a sample ion into a non-dissociable complex with a complexing reagent, and separating and analyzing a sample solution containing the complex. 1- (2,4-dihydroxy-1-phenylazo) as a complexing reagent for beryllium ion
It is characterized by using -8-hydroxy-3,6-disulfonaphthalene or 1- (2-hydroxy-4-diethylamino-1-phenylazo) -8-hydroxy-3,6-disulfonaphthalene.

[0010]

The effective radius of the beryllium atom is only 0.89.
Since it is much smaller than the {1.22} between {} and metallic lithium, as a result of repeated studies on a chelating reagent having a small chelating site as a complexing agent for beryllium, 1-
(2,4-dihydroxy-1-phenylazo) -8-hydroxy-3,6-disulfonaphthalene or 1- (2
-Hydroxy-4-diethylamino-1-phenylazo) -8-hydroxy-3,6-disulfonaphthalene has been found to be effective.

1- (2,4-dihydroxy-1-phenylazo) -8-hydroxy- used as a complexing reagent
3,6-Disulfonaphthalene is also called H-resorcinol and is represented by the following chemical formula.

[0012]

Embedded image

Also, 1- (2-hydroxy-4-diethylamino-1-phenylazo) -8-hydroxy-3,6
-Disulfonaphthalene is Beryllon III
Also, it is represented by a chemical formula shown in the following chemical formula 2.

[0014]

Embedded image

The above two types of complexing reagents form a complex by taking in beryllium ion (Be ²⁺ ) to form two 6-membered rings, and furthermore, because of their small chelate sites, the formation of metal ions other than beryllium. Many are considered not to form complexes. It can also be seen that the two reagents are very similar in structure and therefore similar in complexing behavior. The complexation reaction with beryllium ions by these complexing reagents is completed in a relatively short time, but at a low concentration, it is preferable to heat to, for example, about 100 ° C. in order to complete the reaction.

As a complexing reagent, 1- (2,4-dihydroxy-1-phenylazo) -8-hydroxy-3,6-
When disulfonaphthalene (hereinafter referred to as H-resorcinol) is used, a beryllium complex having the chemical formula shown in the following chemical formula 3 is obtained. Complex.

[0017]

Embedded image

[0018]

Embedded image

As shown in FIG. 4, the complexes having the chemical formulas (3) and (4) have peak wavelengths of absorbance of 450-5.
50 nm, and from the pH behavior shown in FIG.
It can be seen that the absorbance reaches a maximum value in the range of 10. Therefore, it is preferable that the complex formation of H-resorcinol and beryllium ion and the measurement of the absorbance are performed in the pH range of 7.5 to 10.

When 1- (2-hydroxy-4-diethylamino-1-phenylazo) -8-hydroxy-3,6-disulfonaphthalene (hereinafter referred to as berylon III) is used as a complexing reagent, A complex having the chemical formula shown in Chemical Formula 5 is formed. This complex also has an absorbance peak wavelength of 450 to 550 nm as shown in FIG. 6, and it can be seen from the pH behavior shown in FIG. 7 that it shows a substantially constant absorbance in the range of pH 6 to 12. Therefore, berylon I
The formation of the complex between II and beryllium ion and the measurement of the absorbance are preferably performed in the pH range of 6 to 12.

[0021]

Embedded image

After the complex of the beryllium ion is formed in advance by adding the complexing reagent to the sample solution in this way, the sample solution containing the complex is injected into a column, and the complex is separated and analyzed by liquid chromatography. With this, the concentration of beryllium can be determined. In particular, high-performance liquid chromatography (HPLC) is a high-performance analysis method, and in particular, reverse-phase partitioning (RP) -HPLC
This is a preferable method because detection of pb or sub-ppb levels is possible.

However, the conventional RP-HPLC (method B in FIG. 3), as shown in FIG.
After forming complexes ML and M'L of the complexing reagent L with
This is injected from the injector I into the column, and the complexing reagent L is added to the eluate in order to prevent the complexes ML and M'L from decomposing in the column. For this reason, the background of the complexing reagent L appears in the output of the absorbance detector D, and the peaks of the complexes ML and M′L are recorded thereon. Cannot be amplified, and it is difficult to detect with ultra-high sensitivity.

Therefore, as a result of further study, as a method most suitable for ultra-sensitive detection of beryllium, a kinetic discrimination (KD) -HPLC method in which the eluent does not contain the complexing reagent L (Method A in FIG. 3) ) Has been found to be preferred. That is, in this method, as shown in FIG.
The dissociation-active complex ML in M'L is positively dissociated in the column, and only the kinetically stable (dissociation-inactive) complex M'L is detected. Therefore, since the complexing reagent and the complex are separated from each other, there is no problem of overlapping of the spectra of the two, and detection with ultra-high sensitivity can be performed by an absorptiometer.

In the present invention, it was confirmed that each complex represented by Chemical Formula 3, Chemical Formula 4, or Chemical Formula 5 of beryllium with H-resorcinol or berylon III was a dissociation-inactive complex in the KD-HPLC method. However, in the case of the complexing reagent of the present invention, the peaks of the absorbance of the reagent and the complex tend to overlap, so that various separation conditions need to be optimized.

For example, by using methanol as an eluent for liquid chromatography, the peak of the complex can be made ahead of the peak of the complexing reagent. Further, the retention time and the peak height vary depending on the pH of the eluent. The longer the retention time, the better the separation, but the smaller the height of the peak. Therefore, the pH range for optimizing this relationship is preferably pH 8-9.

Further, the eluent preferably contains, in addition to the solvent, tetrabutylammonium bromide (TBAB) and / or ethylenediaminetetraacetic acid (EDTA). Tetrabutylammonium bromide is known as an ion-pairing agent in liquid phase chromatography, but in the method of the present invention, in particular, 5 × 10 ⁻³ to 2 × 10 ⁻² mol.
It was found that the effect was remarkable in the concentration range of / kg. Ethylenediaminetetraacetic acid has an effect of masking aluminum and iron eluted from the tube wall of the flow channel, and also has an effect of extremely improving the separation of the complex from the complexing reagent in the present invention.

The eluate from the column is guided to an absorbance detector as usual, and the concentration of beryllium contained in the sample solution is determined from the detected absorbance pattern.
At this time, as can be seen from FIGS. 4 and 6, the absorbance measurement wavelength was 450 to 5 for any complexing reagent.
It is preferably within the range of 50 nm.

[0029]

EXAMPLE A solution containing 1 × as a complexing reagent was ^added to a solution containing Be ^2+.
10 ⁻³ mol / dm ³ of H-resorcinol, ie 1-
1 ml of (2,4-dihydroxy-1-phenylazo) -8-hydroxy-3,6-disulfonaphthalene was added, and 5 ml of a 0.1 mol / dm ³ Tris buffer was added to adjust the pH to 8.5, followed by boiling. The complex was formed by heating in a water bath for 25 minutes and then cooled to room temperature.

100 μl of the sample solution containing the obtained complex
Was injected into an analytical column of a high performance liquid chromatograph.
The analytical column has Asahipak ODP- made by chemically bonding an octadecyl group to a vinyl alcohol copolymer.
50 (trade name of Asahi Kasei Corporation) was used.

Thereafter, an eluent containing no complexing reagent was flowed at a flow rate of 0.5 ml / min as a mobile phase to separate the complex. The eluent was methanol 38.1 as a solvent.
Wt%, tetrabutylammonium bromide 1.29 ×
10 ^-2 mol / kg, and ethylenediaminetetraacetic acid 1.
2.5 × 10 ^-2 mol including 0 × 10 ^-4 mol / kg
/ Kg Tris buffer adjusted to pH 8.8 was used.

The eluate obtained from the analytical column was led to an absorbance detector, and the absorbance was measured at a measurement wavelength of 500 nm. A typical example of the chromatogram output to the recorder of the detector is shown in FIG. As can be seen from the chromatogram in FIG. 1, the peak of Be according to the method of the present invention appeared before the peak of the complexing reagent L, and the resolution of the peak of Be was high, indicating a good peak shape.

The calibration curve shown in FIG. 2 was obtained from each chromatogram obtained by changing the concentration of Be ²⁺ contained in the sample solution and repeating the same operation as described above. This calibration curve has a Be ²⁺ concentration of 10 ⁻⁸ to 10 ⁻⁶ mol / dm ^3.
Shows a very good linearity with a correlation coefficient of 0.9999 in the range of, and the theoretical detection limit (S / N = 3) is 2.0 × 10 ⁻¹⁰ m
ol / dm ³ (1.8 ppt).

In the case where berylon III, ie, 1- (2-hydroxy-4-diethylamino-1-phenylazo) -8-hydroxy-3,6-disulfonaphthalene, was used as the complexing reagent, As in the case of the above, the Be ²⁺ concentration can be quantified with almost the same sensitivity. The detection limits of atomic absorption spectrometry and ICP emission spectrometry are 2 ppb and 0.2 ppb, respectively, and the conventional RP-H
Comparing with the same detection limit of PLC, it is understood that the method of the present invention is an unprecedented ultra-sensitive quantification method. In addition, if the pre-concentration is performed, which is an effective means for detecting an ultra-trace amount, it is considered that beryllium at a lower concentration can be reliably quantified.

[0035]

According to the present invention, beryllium, which is extremely toxic despite its widespread use as an industrial raw material, can be selectively separated and quantified with ultra-high sensitivity at the ppt level.

[Brief description of the drawings]

FIG. 1 is a typical example of a chromatogram obtained by the method of the present invention.

FIG. 2 is a calibration curve showing the relationship between Be ²⁺ concentration and absorbance according to the method of the present invention.

FIG. 3 KD-HPLC of high performance liquid chromatography
FIG. 5 is an explanatory diagram comparing the RP-HPLC method and the RP method.

FIG. 4 is a graph showing the pH behavior of the spectrum of the complex of the complexing reagent H-resorcinol and Be.

FIG. 5 is a graph showing the relationship between the absorbance at a fixed wavelength of the spectrum and the pH of the complex of the complexing reagent H-resorcinol and Be.

FIG. 6 is a graph showing the pH behavior of the spectrum of the complex of the complexing reagent berylon III and Be.

FIG. 7 is a graph showing the relationship between the absorbance at a fixed wavelength of the spectrum and the pH in the complex of the complexing reagents berylon III and Be.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁷ , DB name) G01N 30/00-30/96

Claims (8)

(57) [Claims] In a liquid chromatography for separating and analyzing a sample ion into a non-dissociable complex with a complexing reagent and separating and analyzing a sample solution containing this complex, 1- (2,4-dihydroxy- as a complexing reagent for beryllium ion is used. It is characterized by using 1-phenylazo) -8-hydroxy-3,6-disulfonaphthalene or 1- (2-hydroxy-4-diethylamino-1-phenylazo) -8-hydroxy-3,6-disulfonaphthalene. Beryllium analysis method. 2. A complex of a beryllium ion is previously formed by adding a complexing reagent to a sample solution, and a sample solution containing the complex is injected into a column, and then the complex is purified using an eluent containing no complexing reagent. The method for analyzing beryllium according to claim 1, wherein the elution is carried out and the light is detected by an absorption photometer. 3. A complexing reagent comprising: 1- (2,4-dihydroxy-1-phenylazo) -8-hydroxy-3,6
3. The method for analyzing beryllium according to claim 1, wherein a complex of beryllium ion is formed at pH 7.5 to 10 using disulfonaphthalene. 4. Use of 1- (2-hydroxy-4-diethylamino-1-phenylazo) -8-hydroxy-3,6-disulfonaphthalene as a complexing reagent at a pH of 6
The method for analyzing beryllium according to claim 1, wherein a complex of beryllium ions is formed in any one of (a) to (b). 5. The liquid chromatography according to claim 1, wherein the eluent contains methanol as a solvent.
Or the method for analyzing beryllium according to 2. 6. The method for analyzing beryllium according to claim 5, wherein the eluent contains tetrabutylammonium bromide and / or ethylenediaminetetraacetic acid in addition to the solvent. 7. The method for analyzing beryllium according to claim 5, wherein the pH of the eluent is in the range of 8 to 9. 8. The method for analyzing beryllium according to claim 2, wherein a wavelength within a range of 450 to 550 nm is used as a measurement wavelength of the absorbance in the absorptiometer.