JPS6229743B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for qualitatively and quantitatively determining carboxylic acids. Conventionally, the following methods are known as qualitative and quantitative methods for carboxylic acids.

(1) A method that measures the acidity exhibited by carboxylic acid in a sample, such as by observing the discoloration of a PH indicator, or by reacting with an alkaline reagent and observing the degree of neutralization.

(2) Convert the carboxylic acid in the sample into an active compound such as carboxylic acid chloride, carboxylic acid ester, or carboxylic acid anhydride, and convert these active compounds into hydroxamic acid by reacting hydroxylamine with the trivalent iron ion. React with iron to form iron hydroxamate, measure the red to reddish-purple color (qualitative), and measure the absorbance (quantitative).
method by doing.

(3) The carboxylic acid in the sample is combined with a colored or fluorescent compound in an aqueous or non-aqueous solvent to form a colored or fluorescent substance, and the coloration or fluorescence of these substances is A method based on analyzing degree as an indicator.

(4) A method in which carboxylic acid in a sample is converted to hydroxamic acid using a hydroxylamine salt and a carbodiimide compound in an aqueous or anhydrous organic solvent, and the hydroxamic acid is measured using a color reaction.

However, these conventional methods for qualitative and quantitative determination of carboxylic acids have the following problems.

(1) is a method that focuses on the acidity of carboxylic acids, but this method is common to acidic substances such as inorganic acids, sulfonic acids, and phenols.
This method is not specific to carboxylic acids and cannot be applied when these coexist.

Method (2) is the most common method, but the presence of water in the first step reaction, that is, the process leading to active derivatives such as carboxylic acid chloride, carboxylic acid ester, or carboxylic acid anhydride, can cause various problems. effect. Therefore, other measures must be taken for water-containing samples containing water-soluble carboxylic acids.

(3) is the same as (2), because the presence of water is an obstacle.
Besides having to be done in a non-aqueous solution,
Because the reagents used themselves have light absorption and fluorescence, prior to analysis, special measures must be taken to remove excess reagents or reduce reagent blanks, making operations complicated. Not only that, but there are limits to how blank values can be removed without performing a separation operation.

(4) is a result of adding a large amount of organic solvent because it is necessary to maintain the organic solvent concentration in the reaction system above a certain level.(i) Inorganic salts that are difficult to dissolve in organic solvents, such as phosphates, , sulfates, nitrates, halogen compounds, proteins, etc., are not removed in advance, otherwise these inorganic salts or proteins will precipitate during the operation. (ii) Since it is necessary to reduce the water content by adding an organic solvent several times the amount of the sample, a decrease in sensitivity due to dilution is unavoidable. Moreover, in order to avoid this, it is necessary to remove water in advance. (iii) As the hydroxylamine salt, it is necessary to use a salt that dissolves in a high concentration in an organic solvent, but these salts are usually oxidizing, such as perchlorates and nitrates, and hydroxylamine, which has a strong reducing power, cannot be used. Since these salts are explosive, there are difficulties in handling the reagents, and dissolving these explosive salts in organic solvents is dangerous.
(iv) As the carbodiimide compound, it is desirable to use dicyclohexylcarbodiimide, which is well soluble in organic solvents, but these carbodiimide compounds have a strong inflammatory effect on the skin, so they are dangerous to those engaged in experimental operations, and should not be used. In order to do this safely, it is necessary to acquire the skills. (v) Since an organic solvent is always required, not only are the reagents expensive, but also problems arise in waste liquid treatment when used in large quantities.

The method of the present invention eliminates the drawbacks of these conventional methods, and also enables the detection and quantification of carboxylic acids with high accuracy and with a safe and simple operation using low-risk and inexpensive reagents. It is something.

The method of the present invention will be explained in detail below.

In the method of the present invention, the carboxylic acid in the sample is converted to hydroxamic acid by treating a sample for which qualitative and quantitative determination of carboxylic acid is to be performed (hereinafter simply referred to as sample) and hydroxylamine salt with a water-soluble carbodiimide compound in an aqueous solution. The method is characterized in that the hydroxamic acid is measured using a color reaction. Examples of the hydroxylamine salts used include hydroxylamine hydrochloride, hydroxylamine sulfate, and water-soluble carbodiimide. Examples of the compound include 1-cyclohexyl-3-(2-morpholinoethyl)-
Carbodiimidemeth p-toluenesulfonate, 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride, and the like are used.

As the coloring reaction of hydroxamic acid, coloring due to trivalent iron ions is preferred. Qualitative determination can be performed by observing a color development phenomenon caused by a color reaction, while quantitative determination is performed by measuring and calculating the absorbance of this color development using a conventional measuring method.

In the above-mentioned hydroxamic acid production reaction, the pH can be adjusted more accurately by the presence of an inorganic salt buffer substance in the aqueous solution.

The method of the present invention has significant significance in that it enables the production reaction of hydroxamic acid in an aqueous solution, which is an advantage that cannot be achieved with the methods (2), (3), and (4) above.

In the method of the present invention, in order to produce hydroxamic acid, the organic solvent that conventionally needed to be present at a high concentration as in (4) above is no longer required, resulting in the following advantages. It will be done.

(i) Can be used directly for aqueous samples containing inorganic salts, proteins, enzymes, etc. In other words, if an organic solvent is used, a precipitate will form when added to the sample, which will interfere with absorbance measurement.
Since no precipitation occurs in the method of the present invention, there is no need for special operations such as removing inorganic salts, proteins, enzymes, etc. in advance or after precipitate formation, which makes operation in a fluid system extremely advantageous. do.

(ii) The PH of the solution, which is an important factor in the hydroxamic acid production reaction, can be accurately adjusted using an inorganic salt such as a phosphate, so the PH can be specified and the hydroxamic acid production reaction can be performed. By causing this, the variation in color development rate can be significantly reduced. This is not possible with non-aqueous solvent systems.

(iii) In the conventional method, the sample was diluted with an organic solvent, but since this is not necessary, sensitivity can be increased.

(iv) Since it is not necessary to dissolve the salt used as the hydroxylamine salt in an organic solvent at a high concentration, a normal non-explosive hydroxylamine salt can be used. Therefore, there is no need to use conventionally used dangerous reagents such as explosive hydroxylamine perchlorate or hydroxylamine nitrate, and as a result, safety is ensured in terms of reagent preparation and storage management. .

(v) Dicyclohexylcarbodiimide, which is widely used as a carbodiimide that is easily soluble in organic solvents, has a strong inflammatory effect and is difficult to wash off if it comes in contact with the skin because it is not water-soluble. Since it has little inflammatory effect and can be easily removed by washing with water, it is safe to operate and does not require special skills to use inflammatory reagents safely.

(vi) Processing of used reagents is extremely easy, as no expensive organic solvents are required, and no waste liquid containing highly concentrated organic solvents is produced after the operation. This means that the method of the present invention is extremely useful in cases where a large amount of reagents need to be consumed, such as in automatic analyzers that handle a large number of samples or in cases where this reaction is used in the detection section of liquid chromatography.

Thus, the method of the present invention can be applied to qualitative analysis of trace samples, detection of carboxylic acids on paper chromatograms or thin layer chromatography, absorption quantification of carboxylic acids using test tubes, automatic analyzers, flow systems, etc. food industry raw materials or products, industrial chemicals, or clinical chemistry test samples,
This method is particularly useful for detecting and quantifying carboxylic acids contained in environmental test materials, biochemical test materials, etc.

Examples are shown below.

Example 1 1.0 ml of a 0.24 M aqueous solution of hydroxylamine hydrochloride and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimidemeth P-toluenesulfonate (abbreviated as CMEC) in 1.0 ml of a 0.5-5 mM aqueous sodium acetate solution. Add 2.0 ml of a 0.06M aqueous solution of and react at 50°C for 15 minutes to generate hydroxamic acid. Next, 1.0 ml of a 0.1 M perchloric acid aqueous solution containing 1.0 M ferric perchlorate is added to this reaction solution to develop color, and the absorbance at 500 nm is measured to create a calibration curve. Next, add 1 ml of commercially available vinegar.
Dilute to 200 ml, add 1.0 ml of a 0.24M aqueous solution of hydroxylamine hydrochloride and 2.0 ml of a 0.06M aqueous solution of CMEC to 1 ml, and react at 50°C for 15 minutes to produce hydroxamic acid. A 0.1M perchloric acid aqueous solution containing 0.1M ferric perchlorate in this reaction solution.
Add 1.0 ml to develop color, measure the absorbance at 500 nm, and calculate the concentration of acetic acid in vinegar using the above calibration curve.

Example 2 Add 5μ of serum to 1.0ml of 0.5-5mM sodium acetate aqueous solution, then add 1/30M veronal buffer (PH7.4) or 1/30M phosphate buffer (PH7.4)
Add 1.5 ml of 0.6 M aqueous solution of hydroxylamine hydrochloride and 2.0 ml of 0.075 M aqueous solution of CMEC.
The reaction is carried out at 50°C for 15 minutes to generate hydroxamic acid. Next, 1.0 ml of a 0.1 perchloric acid aqueous solution containing 0.1M ferric perchlorate is added to this reaction solution to develop color, and the absorbance at 500 nm is measured to create a calibration curve. Next, acetylcholine perchlorate
1/30M veronal buffer with PH7.4 or 1/30M
Add 5μ of serum to 1ml of 25mM solution in phosphate buffer.
After adding and incubating at 37℃ for 1 hour,
0.5ml of 0.6M aqueous solution of hydroxylamine hydrochloride and
Add 2.0 ml of 0.075M aqueous solution of CMEC and react at 50°C for 15 minutes to generate hydroxamic acid. This reaction solution contains 0.1M ferric perchlorate.
Add 1.0ml of 0.1M perchloric acid aqueous solution to develop color for 500n.
The cholinesterase activity in the serum is measured by measuring the absorbance at m and quantifying the acid produced by the enzymatic reaction using the above-mentioned calibration curve.

Claims (1)

[Claims] 1. A sample for which qualitative and quantitative determination of carboxylic acid is to be performed is treated in an aqueous solution with a hydroxylamine salt and a water-soluble carbodiimide compound, the carboxylic acid in the sample is converted to hydroxamic acid, and the hydroxamic acid is converted to hydroxamic acid using a color reaction. A method for qualitatively or quantitatively measuring carboxylic acid.