JP2000171451A - Analytical method for protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method in which protein, in a trace amount, contained in an electrolytic solution or a plating solution can be quantitatively determined simply and with good accuracy. SOLUTION: A sample solution containing protein, such as an electrolytic solution, a plating solution or the like is passed through a column into which a hydrophobic adsorption resin is filled. The protein is adsorbed to the resin. Then, an eluent is made to flow to the column. The protein is eluted. An analytical reagent is added to an eluate. Then, the protein is quantitatively analyzed. Preferably, the protein is analyzed in a continuous flow. As the hydrophobic adsorption resin, a nonpolar resin or an intermediate polar resin is preferable.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for simply and accurately analyzing proteins in a solution, and more particularly to a method suitable for quantitative analysis of trace proteins such as glue and gelatin contained in an electrolytic solution or a plating solution.

[0002]

2. Description of the Related Art Additives are added to an electrolytic solution or a plating solution for various purposes such as glossing during electrodeposition, hardening of a plating layer, and smoothing of a surface. As an additive, glue is often used in electrolytic smelting and gelatin is often used in plating, but keeping its concentration within a certain range is extremely important for quality control. For example, glue is used for smoothing the electrodeposited surface, but when the concentration is excessive, there is a problem that polarization is remarkably increased and impurities such as bismuth are easily deposited.

[0003] However, generally known protein analysis methods are performed under conditions of weak acidity to weak alkalinity, and a strongly acidic sample such as an electrolytic solution or a plating solution having a pH of 1 or less is used. Few examples are applicable to solutions. Conventionally, proteins such as glue and gelatin contained in electrolytes and plating solutions have been measured by potentiometric titration, Kjeldahl distillation, etc., all of which require special equipment and complicated operations. Was. In the Kjeldahl distillation method, the measurement is performed after the glue is decomposed into ammonium nitrogen. However, the electrolyte often contains nitrogen compounds other than proteins, which makes accurate measurement difficult.

[0004] In addition, as a method for determining gelatin and glue in a strongly acidic solution, gelatin or glue is collected on a membrane filter and bound to a specific reagent (Amide Black 10B dye), and excess dye is washed. A method of eluting a dye to detect gelatin or the like (Japanese Patent Application Laid-Open No. 2-69660), or a method of drying a filter that has collected gelatin or the like and detecting by utilizing the reflectance (Japanese Patent Application Laid-Open No. 6-337247). ) Has been reported. However, these methods have a problem that the amount of collected glue is significantly affected by the pore size of the filter. Further, there is a problem that a filtration device is required for collecting the filter, the operation is complicated, and the analysis cost is increased because the filter is disposable.

[0005]

DISCLOSURE OF THE INVENTION The present invention solves the above-mentioned problems in the conventional protein measurement method, and provides an assay which can easily quantify proteins such as gelatin and glue in a solution even in a strongly acidic solution. It provides a method.

[0006]

The present invention uses a hydrophobic adsorbent resin having resistance to acids and organic solvents, and uses the resin to adsorb and elute proteins, which is more reliable than the conventional method using a filter. This completes a measurement method that is highly operable and easy to operate.

That is, the present invention relates to a protein analysis method having the following constitution. (1) A protein-containing sample solution is passed through a column filled with a hydrophobic adsorption resin to adsorb the protein to the resin, and then the eluent is passed through the column to elute the protein, and the eluate is analyzed. A protein analysis method comprising quantitatively analyzing a protein by adding a reagent. (2) The analysis method according to the above (1), wherein the hydrophobic adsorption resin is a nonpolar resin or an intermediate polar resin. (3) The analysis method according to the above (1) or (2), wherein the sample solution is a strongly acidic solution having a pH of 1 or less and uses an acid-resistant hydrophobic adsorption resin. (4) The analysis method according to (1), (2) or (3), wherein the sample solution is an electrolytic solution or a plating solution containing glue or gelatin. (5) A method for quantifying a protein in an electrolytic solution or a plating solution based on a flow analysis method in which addition, reaction, and analysis of a reagent are continuously performed while a sample solution flows through a pipeline. The protein contained in the liquid is adsorbed on the resin by passing through a column filled with a hydrophobic adsorption resin,
Next, an eluent is allowed to flow through the column to elute the protein, and while the eluate flows through the conduit, an analytical reagent is added and allowed to react, guided to the detection unit, and quantitatively analyzed for the protein.
The analysis method according to any one of (1) to (4). (6) In the flow analysis method of the above (5), a fixed amount of sample solution and eluent are alternately passed through a resin-packed column by switching a pipe line to continuously perform protein quantification. .

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION In the analysis method of the present invention, a protein-containing sample solution is passed through a column filled with a hydrophobic adsorption resin to adsorb the protein to the resin, and then the eluent is passed through the column. To elute the protein, and add an analytical reagent to the eluate to quantitatively analyze the protein.

The hydrophobic adsorbing resin used in the present invention is a resin having an adsorbing property to a protein, and the protein is adsorbed on the resin by mutual hydrophobic action of the protein and the resin. Generally, this hydrophobic adsorption resin is durable to acids and organic solvents, and can be used for relatively highly acidic sample liquids. By using a column filled with the hydrophobic adsorption resin, a means for adsorbing and eluting a protein can be easily formed, and its regeneration is also easy.

The hydrophobic adsorption resin is preferably a nonpolar resin or an intermediate polar resin. Examples of the non-polar resin include a styrene-divinylbenzene resin and the like, and examples of the intermediate polar resin include an ester resin such as an acylated ester resin. As shown in the examples below,
These non-polar resins and intermediate polar resins have higher protein recovery than ion exchange resins.

In the analysis method of the present invention, a protein-containing sample solution is passed through a column filled with the hydrophobic adsorption resin to adsorb the protein to the resin. Here, the column is preferably conditioned before the sample solution is passed through the resin. Conditioning enables stable adsorption of proteins and elution of unnecessary hydrophilic substances. Conditioning is performed, for example, by passing a small amount of acid, water, buffer, or the like through the column.

Specifically, when a large amount of metal ions or the like remains in the resin in the column, dilute sulfuric acid or the like is passed through the column before passing the sample liquid to remove the metal ions or the like. Elute and remove with dilute sulfuric acid. If a strongly acidic solution remains in the column, pass water through the column to wash the column, remove the acid inside, and adjust the pH before passing the sample solution. . When the inside of the column is alkaline, the inside of the column may be washed by passing a buffer solution or water for relaxing or neutralizing the alkali.

After the protein is adsorbed on the resin by passing the sample solution through, the protein is eluted from the resin, if necessary, washing is performed. For example, after a sample solution containing a large amount of metal ions together with a protein is passed through the column, dilute sulfuric acid or the like is passed through the column to remove residual metal ions before the protein is eluted. If it is too high, pour water to remove the acid in the column.
Prepare H. Thereafter, the eluent is passed through the column to elute the protein adsorbed on the resin.

As the eluent, an aqueous solution of an organic solvent can be used. Specifically, lower alcohols such as methanol and ethanol, and aqueous solutions such as acetonitrile can be used. The concentration of the organic solvent is preferably 20 to 50% by weight. If the concentration is too low, the protein is insufficiently eluted, while if the concentration is too high, the protein in the column or eluted precipitates, which hinders the reaction with the reagent.

[0015] A reagent is added to the eluate containing the eluted protein to quantitatively analyze the protein in the liquid. As the analysis method, a dye binding method, a biuret method, a fluorescence method, or the like can be used. The biuret method is a method in which a biuret reagent is added to react with a protein, and the protein is quantified based on the absorbance of the reaction solution. Fluorescence is a method in which a fluorescamine reagent or the like is added to react with a protein, and the protein is quantified from the fluorescence of the reaction solution.

Among them, the method using bicinchoninic acid (BCA) is suitable for detecting a trace amount of protein. This is a method utilizing reduction and coloring of copper by protein, and reacted with protein using a bicinchoninic acid reagent obtained by mixing a solution containing bicinchoninic acid (solution A) and a solution containing copper (solution B). Thereafter, a method of quantifying the protein concentration based on the absorbance of the reaction solution. This method is more sensitive than other analysis methods, and thus is suitable for quantifying a trace amount of protein contained in an electrolytic solution, a plating solution, or the like. Further, since the analysis is performed by the reaction of the sample liquid and the measurement of the absorbance thereof, it is suitable for continuous flow analysis.

The protein adsorption by the hydrophobic resin is due to the mutual hydrophobic action of the protein and the resin. However, when a polymer compound having a hydrophobic site is present in the liquid together with the protein, this is combined with the protein by the hydrophobic resin. It may be eluted by being adsorbed on the surface, which may be a hindrance to protein detection.In such cases, select a protein-specific detection method or adjust the eluent composition (type / concentration). This interference can be eliminated by selection and the like.

The analysis method of the present invention is a continuous flow analysis (FI
Analysis), and the protein contained in the electrolytic solution or the plating solution can be quantified based on the continuous flow analysis.
That is, in a flow analysis method in which addition, reaction, and analysis of a reagent are continuously performed while a sample solution flows through a pipeline, an electrolytic solution or a plating solution is used as a sample solution, and the sample solution is treated with a hydrophobic adsorption resin. Pass the liquid through the packed column to adsorb the protein contained in the liquid to the resin,
A protein is eluted by flowing an eluent through the column, and while the eluate flows through the conduit, an analytical reagent is added to cause a reaction to be led to a detection unit, where the protein is quantitatively analyzed.

In the above-mentioned flow analysis method, a pipe line for leading the sample liquid to the resin-packed column and leading to the outside of the system and a pipe line for leading the eluent to the resin-packed column and leading to the reagent addition section are alternately switched. Thus, a fixed amount of the sample solution and the eluent are alternately passed through the resin-packed column, and the protein can be continuously quantified.

[0020]

EXAMPLES The present invention will be specifically described below with reference to examples. Note that the present invention is not limited to these examples.

Example 1 As resins, four kinds of resins shown in Table 1 (two kinds of hydrophobic partition and adsorption resins:
(2 types of ion-exchange resins), and 2 g of each of these 2 g
mm column. After conditioning each column by washing it with a 0.1 M sulfuric acid solution in advance, a pseudo electrolyte containing 200 μg of gelatin (sulfuric acid: 1.5 M, copper: 40 g /
l, nickel: 20 g / l) was added to each column. After the sample solution was passed, 10 ml of a 0.1 M sulfuric acid solution was passed through the column to remove remaining metal ions, and 20 ml of water was further passed through the column to wash the inside to remove residual acid. Then
20 ml of a 50% aqueous solution of acetonitrile was passed through the column to elute the protein adsorbed on the resin. Water was added to 20 ml of the obtained eluate to make up to 50 ml. 1 ml of this was taken, 1 ml of bicinchoninic acid reagent was added, and the mixture was allowed to stand at room temperature for 2 hours to allow a sufficient reaction. Absorbance of this reaction solution
(Wavelength: 562 nm), the gelatin concentration was determined from a calibration curve created in advance, and the recovery was calculated. The results are shown in Table 1. As is clear from Table 1, the recovery rate of gelatin is much higher for the nonpolar resin and the intermediate polar resin than for the ion exchange resin. This indicates that the adsorption performance of the nonpolar resin and the intermediate polar resin is particularly excellent with respect to a trace amount of protein in the liquid.

[0022]

[Table 1]

Example 2 Using a column filled with two types of hydrophobic resins of Example 1, 1 ml of a 1.5 M sulfuric acid solution containing 5 mg of gelatin was added thereto to adsorb gelatin. Next, 5
Using four kinds of 0% methanol, 50% ethanol, 50% acetonitrile, and 100% methanol, gelatin was eluted by the same operation as in Example 1, and the recovery rate was determined. The results are shown in Table 2. As shown in Table 2, when the nonpolar resin (SM-2) and the intermediate polar resin (XAD-7) were used, the elution effect of 50% acetonitrile was the best, followed by 50% methanol and 50% ethanol. High dissolution effect. On the other hand, measurement was not possible with 100% methanol due to precipitation. From the above results, the eluent was 5
0% acetonitrile is preferred.

[0024]

[Table 2]

Example 3 A column packed with the nonpolar resin (SM-2) used in Example 1 was conditioned in advance with a 0.1 M sulfuric acid solution, and then a pseudo copper electrolytic solution containing 0 mg, 12.5 mg and 25 mg of gelatin respectively. 10 ml of a liquid (sulfuric acid: 1.5 M, copper: 40 g / l, nickel: 20 g / l) was added to the column. Next, the gelatin adsorbed on the resin was eluted in the same manner as in Example 1, and the obtained eluate was collected in a volumetric flask. After adding 15 ml of biuret reagent, the volume was adjusted to 50 ml with water. After leaving this at room temperature (25 ° C.) for 30 minutes, the absorbance (wavelength 540 nm) was measured, and the gelatin concentration was determined from a previously prepared calibration curve to calculate the recovery rate. The results are shown in Table 3. As is clear from Table 3, the recovery of gelatin was close to 100% at any concentration, and the correlation coefficient of the calibration curve was 0.99992, indicating high reliability.

[0026]

[Table 3]

Example 4 Using a column (inner diameter 8 mm) filled with a nonpolar resin (SM-2),
After conditioning with a 0.1 M sulfuric acid solution in advance, a copper electrolyte containing a predetermined amount of gelatin (sulfuric acid: 1.5 M, copper: 40 g /
l, nickel: 20 g / l) 50 ml was added to each column. After the sample solution was passed, 10 ml of a 0.1 M sulfuric acid solution was passed to remove the remaining metal ions, and then 20 ml of water was flown to wash the remaining acid. 20 ml of a 50% aqueous solution of acetonitrile was passed through this, water was added to the eluate to make 50 ml, 1 ml was collected, and 1 ml of bicinchoninic acid reagent was added.
The reaction was allowed to take place at room temperature for 2 hours. The absorbance (wavelength: 562 nm) of this reaction solution was measured, and the gelatin concentration was determined from a previously prepared calibration curve. On the other hand, 50
The same operation was performed for the sample to which 0 μg of glue was added,
The protein was adsorbed and eluted, and the recovery rate was determined.
The measurement was performed twice for gelatin and glue, and the results are shown in Table 4. As shown in Table 4, the concentration of gelatin in the electrolytic solution was about 38 ppm, and when 500 μg of glue was further added to the electrolytic solution, the detected protein concentration was proportional to the total amount of gelatin and glue, and the recovery rate of gelatin was It was about 90%, and accurate determination was possible for those containing gelatin and glue. The lower limit of quantification of gelatin in this example was about 10 ppm in terms of sample concentration.

[0028]

[Table 4]

Example 5 A column filled with a nonpolar resin (SM-2) and a reagent addition section were provided in a pipe through which a sample solution flows, and the pipe was formed into a coil-shaped reaction section, and the absorption was measured. In a flow analysis in which a measurement system leading to a meter was formed, dilute sulfuric acid (0.05 M) was used as a carrier liquid, and a copper electrolyte solution (sulfuric acid: 1.5 M, copper: 40 g / l, nickel: 20 g / ml) containing 25 μg / ml of gelatin was added. l) 1 ml was passed through a pipe and passed through the column. Then, about 4 ml of carrier liquid
After the flow, a 40% aqueous solution of acetonitrile was passed through the column, and the eluate was mixed with the eluate at a flow rate of 0.5 ml / min of the bicinchosic acid reagent while flowing through the line. Guide to the photometer and measure the absorbance of the reaction solution (wavelength 562
nm). The concentration of gelatin based on this absorbance was 24 μg / ml, which almost coincided with the amount added, and the measurement accuracy was close to 100%.

[0030]

[Table 5]

[0031]

According to the method of the present invention, the concentration of proteins such as gelatin and glue contained in a strongly acidic solution such as an electrolytic solution or a plating solution can be easily and accurately determined. Further, the analysis method of the present invention is suitable for continuous flow analysis, and the concentration of protein contained in an electrolytic solution or a plating solution can be continuously quantified by flow analysis.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Minoru Takeya 1-297 Kitabukuro-cho, Omiya-shi, Saitama F-term in Mitsubishi Materials Corporation General Research Laboratory 2G058 AA01 BA08 BA10 DA00 EA14 GA01 HA00

Claims (6)

[Claims] 1. A sample solution containing a protein is passed through a column filled with a hydrophobic adsorption resin to adsorb the protein to the resin, and then an eluent is passed through the column to elute the protein. A method for analyzing a protein, comprising quantitatively analyzing the protein by adding an analysis reagent to the protein. 2. The method according to claim 1, wherein the hydrophobic adsorption resin is a non-polar resin or an intermediate polar resin. 3. The analysis method according to claim 1, wherein the sample solution is a strongly acidic solution having a pH of 1 or less and uses an acid-resistant hydrophobic adsorption resin. 4. The analysis method according to claim 1, wherein the sample solution is an electrolytic solution or a plating solution containing glue or gelatin. 5. A method for quantifying a protein in an electrolytic solution or a plating solution based on a flow analysis method in which addition of a reagent, reaction and analysis are continuously performed while a sample solution flows through a pipe line. The liquid is passed through a column filled with a hydrophobic adsorption resin to adsorb the protein contained in the liquid onto the resin, and then the eluent is passed through the column to elute the protein. The analysis method according to any one of claims 1 to 4, wherein an analysis reagent is added during the flow, the reaction is performed, the reaction is led to the detection unit, and the protein is quantitatively analyzed. 6. The flow analysis method according to claim 5, wherein a fixed amount of a sample solution and an eluent are alternately passed through a resin-packed column by switching a pipe line to continuously perform protein quantification. .