JPS6069552A - Method and reagent for colorimetric determination of copper - Google Patents

Abstract

PURPOSE:To enable ultra-microanalysis with high sensitivity by using a specific masking agent and coloring agent. CONSTITUTION:1-Hydroxyethane-1, 1-diphosphonic acid is used as a masking agent when iron and/or zinc exists and dimethylglyoxime is used as the masking material agent when nickel and/or cobalt coexists with or without combination use of a surface active agent. The 2-pyridylazo aminophenol deriv. expressed by the formula or the salt thereof is used as a coloring agent. The analysis is accomplished by dissolving suitably the masking agent, surface active agent and coloring agent in an aq. buffer soln. having required pH, measuring absorbancy and determining the concn. of copper in a sample. In the formula, X, Y denote H or halogen, R<1> denotes H or lower alkyl group and R<2>, R<3> denote a lower alkyl group, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel method for colorimetric determination of copper. More specifically, when iron and/or zinc are present, 1-hydroxyethane-1,1-diphosphonic acid is used as a masking agent, with or without a surfactant, and when nickel and/or cobalt are present. relates to a method for colorimetrically determining copper using dimethylglyoxime as a masking agent and a 2-pyridylazoaminophenol derivative or its salt as a coloring agent, and therefore to a reagent for the determination.

Copper is an element with a Clarke number of 55 ppm and is widely distributed in nature, and is indispensable as a monthly material for various industries.
, and is responsible for specific physiological activities. Copper, which is a heavy metal, is also subject to various standards from the perspective of environmental conservation. That is, it goes without saying that copper analysis is necessary for industrial, physiological, pathological, and other types of samples.

Copper analysis methods include the four-degree absorption method, typified by the bank proin method, and the 1,1,3-to-IJ cyanol-2-amino-J method.
- Fluorescence methods such as the propene method, luminol/hydrogen peroxide fluorescence method, and flame light or atomic absorption spectrometry using an ignition spectrum at 324.8 nm can process a large number of samples without requiring special equipment. As a method, the absorption 4 degree method is advantageous. Although a large number of colorimetric reagents for copper have been published, only a few are considered practical, such as diethyldithiocarbamate, cuproine, and dithizone. Among these, banproin, which has the highest sensitivity among the cuproin series, is used for precision analysis as a specific colorimetric reagent for copper, but even so, its molecular extinction coefficient is 12,860, which is over 12,860. Not sufficient for trace analysis, diethyldithiocarbamate (ε=15,890)
and dithizone (ε-28,600) are inferior in reagent stability, color stability, and specificity, and cannot be used for precise analysis.

Therefore, the present inventors developed a reagent for highly sensitive colorimetric determination of trace metals with a molecular extinction coefficient of 70 to 13.3xio'.
We focused on the compounds disclosed in JP-A No. 57-91975. These series of compounds are useful as highly sensitive colorimetric reagents for the determination of heavy metals, but due to their general non-specificity, pyridylazophenol compounds, like pyridylazonaphthol compounds, are sensitive to trace metals other than copper. It also has the disadvantage of being sensitive to

Previously, Kinoshi et al. reported that these series of compounds can be used in conjunction with one or more types of surfactants and conventionally known analytical masking agents to make them specific to zinc or nickel. He discovered that the invention could be used as a highly sensitive reagent, and filed a patent application for the invented technology (Japanese Patent Application No. 140095/1983).

This time, the present inventors have conducted intensive research on an ultra-trace analysis method that is specific to copper and has extremely high sensitivity.
Among the compounds disclosed in JP-A-57-9197'5, compounds that have never been used in conventional analysis methods are used as masking agents, with or without surfactants. We have completed a new and useful method of the present invention in which a group of compounds forming a part of the compound is used as a coloring agent.

The color former of the present invention has the general formula [1] or a halogen, R1 is hydrogen or a lower alkyl group, ' group, and ω-
The masking agent of the present invention is a 2-pyridylazoaminophenol derivative (representing a substituent selected from the group consisting of sulfohydroquine lower alkyl groups) or a salt thereof; Ethane
1,1-diphosphonic acid and dimethylglyoxime for nickel and/or cobalt. Of course, if it is known in advance that none of iron, zinc, nickel, and cobalt coexists, the combined use of these masking agents can be omitted.

In conventional analytical methods, citrate, condensed phosphate, sodium fluoride, nitrilotriacetic acid, tartrate, etc. are known as masking agents for iron, zinc, nickel, and cobalt, and the present coloring agent The masking agent of the present invention shows some effect when used in combination with a surfactant, and the effect is further increased by using a surfactant in combination with the masking agent of the present invention. It is far inferior in comparison. It was quite surprising that an existing compound, which had never been used as a masking agent in conventional analytical methods, showed perfect effects in the color quantitative determination of the present invention.

As a nonionic surfactant that is particularly effective in the present invention, T
ritonX-100 (polyoxyethylene octylphenyl ether; ROHM&)\-S Co., Ltd. trade name 1,
Br1j -35C polyoxyethylene lauryl ether; Kako Atlas ■trade name], Emulgen 120 [polyoxyethylene lauryl ether; Kako Atlas ■trade name], Tween 20 (polyoxyethylene sorbitan monolauryl ether; Kako Atlas ■trade name],
']"ween, 80 C polyoxyethylene sorbitan monooleyl ether; Kako Atlas ■Product name], Softanol 90 [Polyoxyethylene Alkyl Ether Gaku Kogyo (Kiku product name)], TritonX-4 0 5
[BO1) Oxyethylene 10-M & No. 1-S Co., Ltd. product name], Emulgen 1'47 (polyoxyethylene alkyl ether; Kako Atlas Co., Ltd. product name), Emulge/
920 [Polyoxyethylene lauryl ether truss■
Product name], Emulgen 950 (,I?l)
71-xyethylene nonylphenyl ethers ■Product name], Emulgen P P 29 0 N C
, 1-quinethylenenioxypropylene block d?
IJ Mama - Chemical Atlas ■Product name], Emmanuel 31
30 [Polyoxyethylene sorbita/monostearate; Kako Atlas ■Product name] etc. are used (・force 1 is not limited to these).

As anionic surfactants, &1-, Dunde Nodoching CMN [Polyethylene alkyl ether Na salt; Sanyo Chemical Industry ■Product name], Emar NC [Holyoxyethylene alkyl phenylether sulfate ester; Chemical Atlas ■Shangxing ” name], Al-Anon ACE [:
N-cocoyl-N-notyl-O-alanine Na salt; Yoken Fine Chemical ■Product name], Emar 20C [Polyoxy/ethylene alkyl sulfate Naq; Kako Atlas ■Product name], Nonipol S-4 0 (Polyethylene V-alkyl phenyl ether sulfate Ester Na salt; Sanyo Chemical Industry ■Product name], Sodium lauryl sulfate [Lauryl sulfate ester Na salt], Sandets L-BL [
Alkylbenzenesulfonic acid formalin condensate; Sanyo Kasei Kogyo ■trade name], Lebenol WX [polyoxyethylene alkyl sulfate Na salt; Kako Atlas ■trade name], Ultraphone W [bentadecylbenzimidazole sulfonic acid Na salt - Ciba Geigy Japan ■ Product name], Demol N [naphthalene sulfonic acid formalin condensate; Kako Atlas ■Product name), Softanol 308-25C secondary alkyl sulfate sodium salt; Nippon Shokubai Chemical Co., Ltd. ■Product name], Takanaka≠Imotsuma Yui 44015" 4 - Rehenau tv
W Z [Polyoxyethylene alkyl sulfate 1-
Na salt; Kako Atlas ■ trade name] and the like are used, but are not limited to these.

Examples of cationic surfactants include cation B13 [Lauryltrimethylammonium chloride; 1'' genuine oil ■trade name], Cortamine 86P [Stear 1J/l/trimethylammonium chloride; Kako Atlas ■tradename],
Levasoap NL [laurylpyridinium bromide; Miyoshi Yushi ■ trade name] and the like are used, but are not limited thereto.

Examples of amphoteric surfactants include A/Hitol 2413 [lauryl betaine; Kako Atlas ■ trade name] and A/Hitorl 86B [stearyl betaine; Kako Atlas ■ trade name].
etc., but are not limited to these.

Although the effective concentration of the surfactant of the present invention is not limited to eyebrows, it is preferably 0.01 to 200,000 p.
It is sufficient if it is clearly resolved within the range of H 4 to 12.

The color former of the present invention is as shown in general formula (1),
Among a series of compounds related to the compound group disclosed in JP-A-57-91975, those in which a carboxyl group is substituted at the ortho position to the azo group of the benzene ring and those in which an amine group is substituted can be used in the method of the present invention. is not necessarily sufficient, and this substituent should be a hydroxyl group. General formula [
When X and Y in [I] are halogens, synthesis is easiest when the halogen is bromine, followed by chlorine. The lower alkyl group in <it', 几2 and R3 practically has 1 to 4 carbon atoms. Also, general formula [
1] The salt of the color former compound of the present invention is an organic amine salt such as diethylamine salt or triethylamine salt, or an inorganic salt such as -ammonium salt, alkali metal salt or alkaline earth metal salt. The main purpose of using these salts is to make the color former of the present invention water-soluble. Among the color formers of the present invention, compounds disclosed in JP-A-57-91975, for example, A compound in which at least one of R2 or 3 has a sulfonic acid group is water-soluble even if it is not converted into a salt, and when using the method of the present invention in which a surfactant is used in combination, compounds with the general formula There is no need for the coloring agent itself to be water-soluble.

The method of the present invention involves dissolving an appropriate amount of the masking agent of the present invention, a surfactant, and a coloring agent in a buffered aqueous solution of the required pf, and adding the sample to be analyzed to the coloring reagent solution, that is, the reagent of the present invention. If so, remove the protein and add it for 5 minutes or so.
0 minutes, left for a while, and then tested at 560 nm using a reagent blind control.
The copper concentration in the sample can be calculated by measuring the absorbance of the sample and comparing it with the standard curve.

As such (the present invention has a molecular extinction coefficient of about 90 for copper,
It is extremely important to contribute to this industry by providing a method and reagent for ultra-fine analysis of copper, which has a high sensitivity of 0.000, a color specific to copper, and an extremely simple method. .

Examples are shown below.

Example 1 oIM carbonate buffer (pl-110,0) ] l,
Color former 2-(5-bromo-2-pyridylazo)-5-(
N-n-propyl-N-3-sulfopropylamino)phenol (hereinafter abbreviated as 5-Br-PAPS) 0.05 mM, l-hydroxyethane-1
, 1-diphosphonic acid (hereinafter referred to as !-1, EPA) sodium salt3. J9. Dimethylglyoxime (abbreviated as ``I11'IGO'') 2 t
17 Rum salt 300 mg, Bridge-3510,9
, and 10 p'i of cetyltrimethylammonium bromide were dissolved, which served as a coloring test solution.

Add 2.5 rnl of coloring reagent to 0.5 ml of a sample expected to contain 0.001 to 0.5 P'prn of copper,
After allowing it to stand for a minute, measure the absorbance at 560 nm using a reagent blind test. ! 1
'Get it.

According to the method of this example, a sample containing copper Y2O0μ9/cJI was allowed to coexist with iron, zinc, nickel, and cobalt, and the masking agent of the present invention was used in combination with the masking agent of the present invention. Table 1 shows the results of measurements made using the coloring agent of the present invention with and without the use of the coloring agent. In Table 1, iron, zinc, -chokel,
Cobalt and cobalt are each added in advance at 1000 μg/d I, and the dust generated by the metal chelate formed with the color former of the present invention is determined by the blue constant value (μ
I/dl), and the numerical value for copper is the absorbance.

Table 1 Formation degree of metal chelate by combination of color former, masking agent, and surfactant In Table 1, color former 5 B r P A P I-
I S 1 is 2-(5-bromo-2-pyridylazo)
-5-(N-n-propyl-N-2-hydroxy-
3-Sulfopropylamino)phenol'a? ,3,
5-Br-PAPS is 2-(3,5-dibromo-2-pyridylazo)-5-(N-n-propyl-N-3-sulfopropylamino)phenol'd
' , 315- B r -P A E S is 2-(
3,5-dibromo-2-pyridylazo)-5-(N-ethyl-N23-sulfopropylamino)phenol'&
, 3.5-B r -P A M E S is 2- (
3,5-dibromo-2-bi1Jdyl azo)-4-methyl-5-(N-ethyl-N-3-sulfopropylamine)
phenol, the masking agent a is HE P A f,
, ;, Same (b indicates MGO, and Levasorb NL (laurylpyridinium bromide) was used as the surfactant.

Example 2 A solution dissolved in 100 Tnl of 1N hydrochloric acid is used as a protein removal test solution. Also, 0.5M carbonate buffer (PH1O, 5) 800ral and coloring agent 5-B
r-PAPSo, 05mM, MGO2 sodium salt 30
0 m9, HEP A sodium salt 3.0 g, Softanol 90'fa0:5I, and cation BBIO,ji
+4 and make the total volume 11 with water and use it as a coloring test solution.

Add 5 ml of protein removal reagent solution to 0.5 ml of serum, stir, and centrifuge at 2.50 rpm for 10 minutes. Supernatant 0.5 m
1, add 2.5 ml of coloring test solution, leave it for 10 minutes, measure the absorbance at 5600 m using a reagent blind as a control, and calculate the concentration of copper in the serum by comparing it with the calibration curve (Figure 2). do.

Measured according to this example, conventional method (backproin method)
Table 2 shows the results compared with the measured values.

Incidentally, the backproin method was operated as follows. That is, 1.0 ail of IN-HCl to 2.0 ml of serum! "a = Added, stirred, soaked in a hot bath at 80-95℃ for 2 minutes, then added 1ml F4' of protein removal solution (20% chloracetic acid), stirred, centrifuged at 2.500 rpm for 10 minutes, supernatant 2m7'r
Add 0.025% L-ascorbic acid l〇- to this, add coloring solution I and omlyr consisting of 40g of crystalline sodium acetate, 15ml of Banc Brou/sodium sulfonate, and 100ml of water, mix, and adjust the absorbance at 4800m. , measured using a reagent blind test as a control, and a calibration curve (Figure 2)
Calculate the concentration of copper in serum in comparison to

Table 2 Comparison of copper measurements in serum between the method of the present invention and the pancproin method Y = 0.98 x+ 1.00 Example 3 0.1M carbonate buffer (i>Hl O, 0) 1 l, color former 5-Br-PAPSo, 05mM'a=, Table 3
As shown in Figure 2, the masking agent and surfactant are dissolved with or without coexistence, and the resulting coloring liquid 2.75
200 μg of copper containing 1000 μ97 dl each of iron, zinc, nickel, and cobalt was added to 25 ml of 7d + solution g, and after standing for 5 minutes, the absorbance at 55 Q nm was measured using a reagent blind control as a control. , quantitate copper by comparison with a standard curve.

The results are shown in Table 3. The degree of formation of metal chelates formed by coexisting metals and color formers on the wood surface is shown by the calculated value (μ, 9/d l) for each coexisting metal as copper, and the measured value for copper is shown by the absorbance. It is.

Table 3 5- B r -P A P S I) Formation degree of metal chelate under various conditions In Table 3, a and b are as in Table 1, and 2% surfactant was added. , However, the cloud mark is 1 t added, C is sandets) EM
N, d are Revenol WX, e is Softanol 90, f is Revanoop NL, g is Triton X-100,! 1 is bridge-35, l is cetyltrimethylammonium bromide,
J is amphitol 24B, and J is nirasan cation BB.

[Brief explanation of drawings]

Figure 1 shows the calibration curve for the conventional method (bank proin method) (the horizontal axis is the amount of Cu (μ, !i'/dl')' and the vertical axis is 480n
m indicates absorbance. ), and Figure 2 shows Kenpo (5-B
r-PAPS method) calibration curve (the horizontal axis is the amount of Cu (μ
g/old), and the vertical axis shows absorbance at 560 nm. ) is shown. Patent applicant Wako Pure Chemical Industries, Ltd. Figure 1 Cu l

Claims (4)

[Claims] (1) When iron and/or zinc coexist, 1-hydroxyethane-1,1-diphosphonic acid is used as a masking agent, and when nickel and/or conolte coexist, dimethylglyoxime is used as a masking agent. , general formula - R2 and R3 each represent the same or different types, hydrogen,
Copper colorimetry using a 2-pyridylazoaminophenol derivative (representing a substituent selected from the group consisting of a lower alkyl group, a sulfo-lower alkyl group, and an ω-sulfohydroxy lower alkyl group) or a salt thereof as a coloring agent. Quantification method. (2) When iron and/or zinc coexist, 1-hydroxyethane-1,]-diphosphonic acid is used as a masking agent; when nickel and/or cobalt coexist, dimethylglyoxime is used as a masking agent; or halogen, R1 is hydrogen or a lower alkyl group, 几2. 2-pyridylazoaminophenol (3) each represents the same or different type and represents a substituent selected from the group consisting of hydrogen, a lower alkyl group, a sulfo-lower alkyl group, and an ω-sulfo-hydroxy lower alkyl group. 1. A method for colorimetric determination of copper using a derivative or a salt thereof as a coloring agent, characterized in that a surfactant is used in combination. (3) A method for colorimetric determination of copper according to claim 2, which uses a nonionic surfactant, an anionic surfactant, a cationic surfactant, or an amphoteric surfactant as the surfactant. . (4) The method for colorimetric determination of copper according to claim 2, characterized in that the surfactants are a nonionic surfactant and a cationic surfactant.