JPH0783908A - Method for analyzing beryllium by liquid chromatography - Google Patents

Abstract

PURPOSE:To relatively simply determine a small amount of beryllium with an extremely high sensitivity by setting sample ion as a non-dissociation com plex by a specific complex reagent and then' isolating and analyzing the sample liquid containing the complex using liquid chromatography. CONSTITUTION:1-(2,4-dihydroxy-phenylazo)-8-hydroxy-3,6 disulfonaphthalene, or 1-(2-hydroxy-4-diethylamlno 1-phenylazo)-8-hydroxy-3,6-disulfonaphthalene is used as a complex reagent. These two types of complex reagents creates two 6-member rings incorporating beryllium ion and form a complex. The sample liquid containing the complex is injected into the column, the complex is isolated and analyzed by liquid chromatography, and then beryllium concentration is determined. It is desirable to used kinetics discrimination(KD) - HPLC as the HPLC(high performance liquid chromatography) most suited for ultra- high-sensitivity detection of beryllium and beryllium can be determined with the ultra high sensitivity of PPt level.

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 a trace amount of toxic beryllium by liquid chromatography with an extremely high sensitivity.

[0002]

2. Description of the Related Art Beryllium (Be) has a relatively small distribution in nature and has a few ng / l (ie ppt) in natural water.
Is the 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, irritation symptoms strongly appear in the trachea, bronchi, lungs, etc., and the mortality rate due to chronic poisoning is high. Recently, there has been much interest in the carcinogenicity of beryllium.

Although beryllium is highly toxic,
Recently, it has been widely used as various industrial raw materials such as space rockets, aircrafts, nuclear reactors, and alloy materials for engines.
Under such 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 . Therefore, a highly sensitive analysis method for beryllium is required.

As a method for analyzing a trace amount of beryllium, a morin fluorescence method, a 2-methyloxine method, an aluminone colorimetric method and the like have long been known. Recently, a fluorometric method by 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)) is reported as a highly sensitive quantitative method.

However, the above-mentioned conventional method for analyzing beryllium is not satisfactory in terms of sensitivity,
Since the device itself is expensive, it is required to develop a relatively simple analysis method with high sensitivity and using a normal analysis device. In particular, since it is necessary to quantify a very small amount of beryllium, the detection limits of 2 ppb and 0.2 ppb, which are the detection limits of the atomic absorption spectrometry and ICP emission spectrometry
It is desired to provide an ultra-sensitive analysis method that exceeds the limit.

On the other hand, liquid chromatography, especially high performance liquid chromatography (HPLC), has been widely used as a high-performance analytical method as a result of the dramatic improvement in the technology relating to analytical columns and the performance of analytical instruments due to the recent progress in science and technology. Is used for. Among them, reverse 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 analytical field.
By introducing the method described in (1), it is possible to analyze ionic species.

A method for quantifying trace metal ions using such RP-IP-HPLC has been studied (Hoshino, Yotsuyanagi, Aomura,
“Analytical Chemistry” 27, 315 (1978)), a sample ion is made into a non-dissociative complex with 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 is obtained. However, for the analysis of beryllium, an optimized analysis method including selection of a complexing reagent has not been known.

[0008]

In view of such conventional circumstances, the present invention is a method for analyzing beryllium capable of quantifying a very small amount of beryllium with ultrahigh 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 is such that a sample ion is made into a non-dissociative complex with a complexing reagent, and a sample solution containing this complex is separated and analyzed. 1- (2,4-dihydroxy-1-phenylazo) as a beryllium ion complexing reagent.
It is characterized in that -8-hydroxy-3,6-disulfonaphthalene or 1- (2-hydroxy-4-diethylamino-1-phenylazo) -8-hydroxy-3,6-disulfonaphthalene is used.

[0010]

[Function] The effective radius of the beryllium atom is only 0.89.
Since it is much smaller than 1.22Å of Å and metallic lithium, as a result of repeated studies on a chelating reagent having a small chelate site as a beryllium complexing reagent, 1-
(2,4-dihydroxy-1-phenylazo) -8-hydroxy-3,6-disulfonaphthalene, or 1- (2
-Hydroxy-4-diethylamino-1-phenylazo) -8-hydroxy-3,6-disulfonaphthalene was 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 chemical formula shown below.

[0012]

[Chemical 1]

Further, 1- (2-hydroxy-4-diethylamino-1-phenylazo) -8-hydroxy-3,6
-Disulfonaphthalene is Beryllon III
It is also referred to as a chemical formula and is represented by the chemical formula shown below.

[0014]

[Chemical 2]

The above-mentioned two kinds of complexing reagents form a complex by incorporating beryllium ion (Be ²⁺ ) to form two 6-membered rings, and since the chelate site is small, metal ions other than beryllium can be used. Most are believed not to form a complex. It is also observed that the two reagents are very similar in structure and therefore similar in complexing behavior. The complex formation reaction with beryllium ions by these complexing reagents is completed in a relatively short time, but it is preferable to heat to about 100 ° C., for example, in order to complete the reaction at a low concentration.

1- (2,4-dihydroxy-1-phenylazo) -8-hydroxy-3,6-as a complexing reagent
When disulfonaphthalene (hereinafter, referred to as H-resorcinol) is used, a beryllium complex having a chemical formula shown in the following chemical formula 3 is obtained, and this complex has a chemical formula shown in the chemical formula 4 in which an OH group is dissociated at pH 5 or higher. Becomes a complex of.

[0017]

[Chemical 3]

[0018]

[Chemical 4]

As shown in FIG. 4, the complex having the chemical formulas (3) and (4) has an absorbance peak wavelength of 450 to 5
It is at 50 nm, and the pH behavior shown in FIG.
It can be seen that the absorbance becomes maximum 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 range of pH 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 Formula 5 is formed. As shown in FIG. 6, the peak wavelength of the absorbance of this complex is still 450 to 550 nm, and it can be seen from the pH behavior shown in FIG. 7 that the absorbance is almost constant in the range of pH 6 to 12. Therefore, beryllon I
The complex formation between II and beryllium ion and the measurement of the absorbance are preferably carried out in the pH range of 6 to 12.

[0021]

[Chemical 5]

In this way, after the complexing reagent is added to the sample solution to form a beryllium ion complex in advance, the sample solution containing this complex is injected into the column, and the complex is separated and analyzed by liquid chromatography. Thus, the concentration of beryllium can be quantified. In particular, high performance liquid chromatography (HPLC) is a high-performance analytical method, and among them, reverse phase partition (RP) -HPLC is performed by an absorptiometric detector.
It is a desirable method because it can detect pb or sub-ppb levels.

However, according to the conventional RP-HPLC (method B in FIG. 3), as shown in FIG.
And the complex ML and M′L of the complexing reagent L are formed in advance,
This is injected from the injector I into the column, but the complexing reagent L is added to the eluent in order to prevent decomposition of the complexes ML and M′L in the column. Therefore, the background of the complexing reagent L appears in the output from the absorptiometric detector D, and the peaks of the complexes ML and M'L are recorded on the background, so that the spectrum of the complex spectrum should be large unless the spectrum shift during complex formation is large. However, there is a disadvantage that it is not possible to amplify only the peak of, and it is difficult to detect with high sensitivity.

As a result of further studies, as a method most suitable for the ultrasensitive detection of beryllium, a kinetic discrimination (KD) -HPLC method (method A in FIG. 3) containing no complexing reagent L in the eluent was used. ) Was found to be preferable. That is, in this method, as shown in FIG.
The complex ML that is active in dissociation reaction in M′L is positively dissociated in the column, and only the kinetically stable (dissociatively inactive) complex M′L is detected. Therefore, since the complexing reagent and the complex are separated, the overlapping of the spectra of the both does not pose a problem, and supersensitive detection is possible with an absorptiometric detector.

It was confirmed by the KD-HPLC method that each complex of H-resorcinol or beryllone III and beryllium shown in Chemical formula 3, Chemical formula 4 or Chemical formula 5 in the present invention was a dissociatively inactive complex. However, in the case of the complexing reagent according to the present invention, the absorbance peaks of this reagent and the complex are likely to overlap with each other, and therefore optimization is required for various separation conditions.

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

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

The eluate from the column is guided to an absorptiometric detector as usual, and the concentration of beryllium contained in the sample solution is determined from the pattern of the absorbance detected there.
At that time, as can be seen from FIGS. 4 and 6, the measurement wavelength of the absorbance is 450 to 5 in any of the complexing reagents.
A range of 50 nm is preferable.

[0029]

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

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

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

The eluate obtained from the analytical column was introduced into an absorptiometric 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 repeating the same operation as above while changing the concentration of Be ²⁺ contained in the sample solution. This calibration curve shows a Be ²⁺ concentration of 10 ⁻⁸ to 10 ⁻⁶ mol / dm ^3.
Shows a very good linearity with a correlation coefficient of 0.99999, and the theoretical detection limit (S / N = 3) is 2.0 × 10 ^-10 m
It was ol / dm ³ (1.8 ppt).

Also, when berylon III, that is, 1- (2-hydroxy-4-diethylamino-1-phenylazo) -8-hydroxy-3,6-disulfonaphthalene, was used as the complexing reagent, The Be ²⁺ concentration can be quantified with almost the same sensitivity as in the above case. The detection limits of atomic absorption spectrometry and ICP emission spectrometry are 2 ppb and 0.2 ppb, respectively.
Comparing with the fact that the detection limit of PLC is similar, it is understood that the method of the present invention is an unprecedented ultrasensitive quantitative method. In addition, it is considered that the lower concentration of beryllium can be reliably quantified by performing the first-stage concentration, which is an effective means for detecting an ultratrace amount.

[0035]

EFFECTS OF THE INVENTION According to the present invention, beryllium, which is extremely toxic despite its widespread use as various industrial raw materials, can be selectively separated and quantified with a super high sensitivity of ppt level.

[Brief description of 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
It is explanatory drawing which compared the method and RP-HPLC method.

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

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

Claims (8)

[Claims] 1. A liquid chromatograph for converting a sample ion into a non-dissociative complex with a complexing reagent and separating and analyzing a sample liquid containing the complex, 1- (2,4-dihydroxy-) as a complexing reagent for beryllium ion. 1-phenylazo) -8-hydroxy-3,6-disulfonaphthalene or 1- (2-hydroxy-4-diethylamino-1-phenylazo) -8-hydroxy-3,6-disulfonaphthalene is used Beryllium analysis method. 2. A complexing reagent is added to a sample solution to form a complex of beryllium ions in advance, a sample solution containing this complex is injected into a column, and then the complex is formed using an eluent containing no complexing reagent. The method for analyzing beryllium according to claim 1, which is eluted and detected by an absorptiometric detector. 3. 1- (2,4-dihydroxy-1-phenylazo) -8-hydroxy-3,6 as a complexing reagent
-A method for analyzing beryllium according to claim 1 or 2, characterized in that a complex of beryllium ions is formed at a pH of 7.5 to 10 using disulfonaphthalene. 4. 1- (2-Hydroxy-4-diethylamino-1-phenylazo) -8-hydroxy-3,6-disulfonaphthalene is used as a complexing reagent at pH 6
The method for analyzing beryllium according to claim 1 or 2, characterized in that a complex of beryllium ions is formed in a range of ~ 12. 5. The liquid chromatography 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 8-9. 8. The method for analyzing beryllium according to claim 2, wherein a wavelength in the range of 450 to 550 nm is used as the wavelength for measuring the absorbance in the absorptiometry detector.