JP3034654B2 - Continuous measurement of trace mercury - 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 continuously measuring trace amounts of mercury, which can continuously determine trace amounts of mercury.

[0002]

2. Description of the Related Art Conventionally, several methods have been used for measuring mercury, but in general, Japanese Industrial Standards (JIS K010
1 and K0102), the dithizone method (colorimetric method) and the atomic absorption method are widely used. The dithizone method is a method in which a water-insoluble complex formed by the reaction between mercury and dithizone is extracted with an organic solvent.
Is measured and analyzed. On the other hand, with atomic absorption spectrometry, stannous chloride is added to a sample solution containing mercury ions under acidity to reduce mercury ions, and metal mercury generated by aeration of this solution is vaporized.
Atomic absorption at 253.7 nm is measured and analyzed.

The above method has been widely used because it has many advantages over other methods in general, but it has been desired to improve it because the following problems remain. First, the dithizone method is not suitable for a continuous system because the operation is complicated and the required amount of the sample is relatively large. In addition, the sensitivity is lower than that of the atomic absorption method, and since there are relatively many interfering components such as silver and copper, errors tend to occur, which is not preferable as a method for measuring a trace amount of mercury. Furthermore, there is a danger in using harmful chemicals such as chloroform and hydroxylamine hydrochloride. On the other hand, the atomic absorption method is not suitable for a continuous system since it is based on a batch type operation requiring a relatively large amount of sample. In addition, there is a danger that a hazardous chemical such as tin chloride is used.

[0004] In addition, continuous measurement of mercury and automatic measurement using an autosampler have been attempted [Morita, H. et al.
et al. (1990) Analytical Science 6, 91 〜, Ping,
L. et al. (1990) Anal. Chem. 62 , 85-], because the gas flow is used for the measurement system, there is a problem that the apparatus becomes large-scale.

[0005] In many countries, mercury is still used for various purposes such as pesticides, and mercury pollution resulting from these uses is limited to water,
It affects each country in trace amounts through the atmosphere and crops. For example, it is said that rainwater contains about 0.0002 ppm of mercury and seawater contains about 0.0003 ppm of mercury. In Japan, the regulation value for wastewater discharge is 0.005 ppm
Therefore, if the concentration of mercury is about the regulated value, it can be measured by the above-mentioned conventional method.However, in order to use it as a monitor of mercury concentration, it is sufficient in terms of sensitivity and operability. I couldn't say that. Therefore, there has been a demand for establishing a method capable of continuously measuring even a very small amount of mercury with high sensitivity.

[0006]

SUMMARY OF THE INVENTION The present invention solves the above-mentioned problems of the prior art, and provides a method for continuously measuring a very small amount of mercury in which even a very small amount of mercury can be continuously measured with high sensitivity. The purpose is to:

[0007]

Means for Solving the Problems The present inventors have made intensive studies to achieve the above object, and as a result, added a predetermined amount of NADPH and a mercapto compound to a sample solution containing a trace amount of mercury, and added this solution to a porous material. By continuously contacting with a mercury ion reductase immobilized on a carrier, and measuring the amount of decrease in NADPH or increase in NADP ⁺ in the solution after the contact, so that the concentration of mercury ions in the sample solution can be continuously measured. They found that they could be quantified and invented this method.

That is, according to the present invention, a column is filled with a porous carrier on which mercury ion reductase is immobilized, for example, a high strength porous glass in which mercury ion reductase is immobilized by a diazo bond. Then, a solution containing a predetermined amount of NADPH (nicotinamide-adenine dinucleotide phosphate) and a mercapto compound is passed through a sample solution containing a trace amount of mercury, whereby divalent mercury ions (Hg ²⁺ ) in the solution are added. Is reduced to metallic mercury (Hg ⁰ ) by the catalytic action of mercury ion reductase, and the reaction to oxidize an equimolar amount of NADPH to NADP ⁺ proceeds continuously to reduce NADPH in the solution after passing through the column. by measuring the increase in the amount or NADP ^+, to quantify the mercury ion concentration in the sample solution is continuously It is intended to provide a continuous method of measuring trace amounts of mercury characterized by.

Further, according to the present invention, the porous carrier is
By immobilizing catalase together with mercury ion reductase, the measurement sensitivity can be further improved.

[0010] The substrate of the mercury ion reductase used in the present invention is not a free mercury ion in the solution but a mercaptide thereof. Therefore, in the present invention, a mercapto compound is added to the sample solution. In addition, experiments performed by the present inventors confirmed that the activity of the enzyme was highest when cysteamine was used, and that cysteine was second only to cysteine. The method for screening cells in which mercury ion reductase is present is described in Appl. Environ. Microbial. 54 , 2871-2.
873, (1988)].

In the present invention, the amount of decrease in NADPH is determined, for example, by the fluorescence (excitation wavelength 340 nm, emission wavelength) indicated by NADPH.
470 nm), but there is no problem using other suitable methods. When measuring organic mercury according to the method of the present invention, a known pretreatment may be applied to the sample solution.

[0012]

According to the present invention, a solution obtained by adding NADPH and a mercapto compound to a sample solution containing a trace amount of mercury is passed through a column packed with a porous carrier on which mercury ion reductase is immobilized. And the mercury ion reductase are continuously contacted, and the reaction shown in Chemical Formula 1 is advanced. In this reaction, the divalent mercury ion (Hg ²⁺ ) contained in the sample solution is reduced to metallic mercury (Hg ⁰ ) by the catalytic action of the mercury ion reductase, and at the same time, the reduced mercury ion Equimolar amount of NADP
H is oxidized to NADP ⁺ . Therefore, the mercury ion content can be quantified by measuring the decrease in NADPH or the increase in NADP ⁺ .

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In the present invention, the measurement sensitivity is further improved by immobilizing catalase together with mercury ion reductase on the porous carrier. This is because divalent mercury ions (Hg
²⁺ ) is reduced to metallic mercury (Hg ⁰ ), and a regenerative reaction occurs in which the produced metallic mercury is oxidized again by the catalytic action of the coexisting catalase to return to divalent mercury ions,
This is because a cycling reaction system in which oxidation and reduction of mercury ions are repeated is established. In this cycling reaction system, in one cycle, mercury ions contained in the sample solution and the equimolar amount of NADPH are reduced, and the equimolar amount of NADPH is reduced.
P ⁺ is generated. Therefore, in this cycling reaction, the amount of decrease in NADPH and the amount of increase in NADP ⁺ per unit time are constant, and when viewed over time, NADPH decreases linearly and NADP ⁺ increases linearly. Reaction occurs in the enzyme column. Therefore, the amount of decrease in NADPH per unit time or N
The increase in ADP ⁺ is greater than in the absence of catalase, and mercury can be quantified more sensitively.

The above-mentioned cycling reaction is an enzymatic cycling method [Lowry, O. et al.
H. et. Al. (1961) J. Biol. Chem. 236 , 2761-], and in addition to the reaction system of the substance A to be measured, a regeneration system for the substance A is provided to participate in the reaction repeatedly. By doing so, amplification is performed.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the scope of the present invention is not limited by the following examples.

[0016]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS One embodiment of the method for measuring trace mercury of the present invention will be described with reference to FIGS. In this example, continuous measurement of trace amounts of mercury was performed using the flow system shown in FIG. In addition, the enzyme column 4 in the above-mentioned flow system
Was manufactured as follows. Mercury ion reductase 6.0n
mole (0.35mg), 85mg of porous glass (pore size 1600
A, particle size 0.3 to 0.5 mm, a specific surface area of about 50 m ² / g, U.S. Patent
3,843,341) has a diazo bond [Mason, RD et al. (19
72) Biotechnol. And Bioeng. 14 , 637-], and 85 mg of porous glass or
The same amount of porous glass in which 7.2 mg of catalase was immobilized with a diazo bond in the same manner as described above was mixed, and packed into a column having an inner diameter of 3 mm and a length of 50 mm.

First, 500 mM Tris-sulfate buffer 3 (pH 7.5) containing 20 μM of NADPH was pumped by a pump 9.
At a flow rate of 0.2 ml / min, a 4 mM aqueous solution of cysteamine (sample solution 1) containing various concentrations of mercuric chloride (HgCl ₂ ) was fed into
It flowed at 0.8 ml / min for 2-3 minutes. At this time, the concentration of the sample solution 1 was adjusted by flowing pure water 2 into the flow path of the sample solution 1 through the valve 8. The mixed solution formed by merging the flow paths passes through the enzyme column 4 and reaches the flow cell 5. When the solution reached the flow cell 5, the solution was irradiated with excitation light 6, and the luminous intensity of fluorescence 7 (excitation wavelength 340 nm, fluorescence wavelength 470 nm) of NADPH contained in the solution was measured by Shimadzu Spectrofluorometer RF-5000.

From the fluorescence intensity measured as described above, N
When the amount of decrease in ADPH was determined, the difference between the amount of decrease in NADPH concentration and the concentration of mercuric chloride in the sample solution was determined as shown in FIG.
The relationship shown in Fig. 7 was obtained. As can be seen from FIG. 2, when immobilized alone without adding catalase to mercury ion reductase, mercuric chloride and NADPH react at a molar ratio of about 1: 1. When catalase was added to the ion reductase and immobilized (enzyme cycling method), the amount of NADPH reacted increased and the sensitivity was improved. Further, in the enzyme cycling method in the presence of catalase, it was confirmed that the sensitivity was further improved by reducing the flow rates of the sample and the NADPH solution.

In this example, Orange A Matrex gel (manufactured by Amicon) was used as a mercury ion reductase, and this was subjected to affinity chromatography [Fox, B. et al.
al. (1982) J. Biol. Chem. 257 , No. 5, 2498-]. Catalase was manufactured by Tokyo Kasei (derived from beef liver).

[0020]

[Comparative Example 1] Mercuric chloride was added at 0, 0.1, 0.2, 0.5,
Solutions containing 0.7 and 1.0 μM were prepared, and the measurement was carried out by the conventional techniques of atomic absorption spectrometry and the enzyme cycling method of the present invention. For the atomic absorption method, 0.5 ml of the above solution was placed in a 200 ml Erlenmeyer flask and subjected to pretreatment and measurement according to the method specified in Japanese Industrial Standards (JIS K0102). The measurement was performed in the same manner as described above. As a result, as shown in FIG. 3, there was almost no difference between the two measured values, and the quantification was accurate.

[0021]

According to the development of the present invention, the lower limit of detection of mercury ion concentration is 0.2 to 0.2 when catalase is not present.
In the presence of 0.3 μM (40-60 ppb Hg) and catalase, ultra-micro continuous analysis of 0.1 μM (20 ppb Hg) or less was possible. Further, since the method of the present invention can be measured without using harmful substances such as heavy metal salts and hydroxyamine hydrochloride as a reducing agent, as in the conventional method,
It is very favorable from the viewpoint of environmental protection and safety. Further, the mercury ion reductase in the method of the present invention,
Since it has high substrate specificity, it specifically reacts only with Hg2 +, and since it has high selectivity for other ions, there is little interference with other substances.

[Brief description of the drawings]

FIG. 1 is a conceptual diagram of a flow system in the method of the present invention used in an embodiment.

FIG. 2 is a graph showing the relationship between the decrease amount of the NADPH concentration and the HgCl ₂ concentration when measuring a trace amount of mercury according to the method of the present invention.

FIG. 3 is a graph showing a correlation between an atomic absorption method, which is one of the conventional methods for measuring trace mercury, and a measured value of trace mercury according to the method of the present invention.

[Explanation of symbols]

 1 ‥‥‥ Sample solution 2 ‥‥‥ Pure water 3 硫酸 Tris-sulfate buffer containing NADPH 4 ‥‥‥ Enzyme column 5 ‥‥‥ Flow cell 6 ‥‥‥ Excitation light 7 ‥‥‥ Fluorescence 8 ‥‥‥ Valve 9 ‥‥‥ pump

──────────────────────────────────────────────────の Continuing on the front page (72) Inventor Motohiro Uo 1-3-3-505, Kiyoshinmachi, Edogawa-ku, Tokyo (72) Inventor Ryoo Makishima 500-10 Shimohirooka, Tsukuba City, Ibaraki Prefecture (72) Inventor Karube Yasuo 1-3-16 Higashi-Arima, Miyamae-ku, Kawasaki-shi, Kanagawa Prefecture (58) Field surveyed (Int. Cl. ⁷ , DB name) C12Q 1/25-1/66 BIOSIS (DIALOG) CA (STN) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A sample solution containing a trace amount of mercury contains N
By adding ADPH and a mercapto compound and bringing them into contact with immobilized mercury ion reductase, divalent mercury ions (Hg ²⁺ ) in the solution can be converted to metallic mercury (Hg ⁰ ).
To an equivalent amount of NADPH to NA
The reaction of oxidizing to DP ⁺ is continuously progressed, and the amount of NADPH reduction or NADP ⁺ in the solution after passing through the column is reduced.
A continuous measurement method for a trace amount of mercury, wherein the concentration of mercury ions in a sample solution is continuously quantified by measuring an increase amount of the mercury. 2. The method according to claim 1, wherein a mercury ion reductase and catalase are present in the immobilized enzyme to amplify the reaction.