JP2003075423A - Method for analyzing liberation sulfurous acid and total sulfurous acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To measure a sulfurous acid in wine with high selectivity and sensitivity by a simple method. SOLUTION: An inverse-phase column is used as a separation column 8, and an ion pair reagents are added to a mobile phase, thus mutually separating a stable hydroxymethanesulfone acid that is guided from a liberation sulfuric acid in wine and an acetoaldehyde combination sulfurous acid in wine to measure the liberation sulfurous acid. Additionally, with the total sulfurous acid in wine, the wine is subjected to alkali treatment, thus liberating acetoaldenyde in the wine and a sulfurous acid that is combined with sugar, and then guiding to a stable hydroxymethanesulfone acid for measurement as in the liberation sulfurous acid.

Description

Description: TECHNICAL FIELD [0001] The present invention relates to the field of analyzing sulfurous acid in wine such as food quality control. 2. Description of the Related Art As a quantitative analysis method of a sulfite compound in wine, the Rankine method and its applied method are frequently used. In this method, a sample is distilled under acidity, and the generated sulfurous acid gas is absorbed in an aqueous hydrogen peroxide solution to obtain sulfuric acid, and then titrated with sodium hydroxide. A method using a colorimetric method after distillation is also known. In the method using liquid chromatography, there is a method in which sulfurous acid is directly detected by an electric conductivity detector or an electrochemical detector. As a method for measuring sulfurous acid in the environment, sulfurous acid is reacted with formaldehyde to generate stable hydroxymethanesulfonic acid, which is then subjected to high performance liquid chromatography, and the eluate from the column is converted to orthophthalaldehyde and secondary phthalic acid. There is a post-column derivatization method in which a reaction is detected in the presence of one amine and detected. [0003] However, the aforementioned Rank
The ine method requires a distillation operation, requires skill and requires a large amount of sample, and is disadvantageous in that the operation is complicated. In addition, the high-performance liquid chromatography method using an electric conductivity detector has problems such as the influence of contaminants and low sensitivity. Furthermore, in the Rankine method or the high-performance liquid chromatography method using an electric conductivity detector or an electrochemical detector, since sulfurous acid is directly supplied to an analyzer, there is a possibility that a quantitative error may occur due to decomposition of sulfurous acid. The post-column derivatization method using high-performance liquid chromatography solves the above-mentioned problems, but when applied to the measurement of sulfurous acid in wine, the use of an ion-exchange column as a separation column requires free sulfurous acid and acetaldehyde. The problem is that the bound sulfurous acid cannot be separated and the free sulfurous acid concentration cannot be determined. Therefore,
An object of the present invention is to solve the above-mentioned problems, and to measure the sulfurous acid in wine with high selectivity and high sensitivity by a simple method. [0004] In order to solve the above-mentioned problems, the present invention applies the above-described post-column detection method by liquid chromatography, and uses a reversed-phase column as a separation column to form a mobile phase. Then, a stable hydroxymethanesulfonic acid derived from free sulfurous acid in wine and acetaldehyde-bound sulfurous acid in wine are mutually separated by adding an ion pair reagent to the mixture, and the free sulfurous acid is measured. In addition, the total sulfurous acid in wine is measured in the same manner as free sulfurous acid by treating the wine with alkali to release sulfurous acid bound to acetaldehyde and sugar in the wine, and then leading to stable hydroxymethanesulfonic acid. . [0005] The present invention is directed to a reverse phase chromatography using an ion pair reagent after reacting free sulfuric acid in wine and total sulfurous acid with formaldehyde to produce hydroxymethanesulfonic acid, which includes the above-mentioned measuring method. The present invention relates to a method for analyzing free sulfuric acid and total sulfurous acid in wine, wherein the sulfuric acid is separated and detected. Here, the formaldehyde used has a concentration of 1 to 100 mM, preferably 5 to 20 mM. Formaldehyde is used in a buffer solution, for example, citric acid (sodium), citric acid (potassium), etc., and by adding wine to the buffer solution, free sulfuric acid in the wine is converted to hydroxymethanesulfonic acid. This reaction is preferably performed, for example, at a temperature of 20 to 50 ° C. for a time of 1 to 10 minutes. When measuring total sulfurous acid, aldehyde,
Alkali treatment of bound sulfurous acid bound with ketones, sugars, etc. The alkali treatment is adjusted to pH 10 to 14, preferably pH 11 to 13, at 15 to 70 ° C, preferably 20 to 25 ° C, for 10 to 120 minutes, preferably 2 to 20 minutes.
By performing for 0 to 60 minutes, sulfurous acid can be released. The alkali is not particularly limited, and examples thereof include aqueous solutions of sodium hydroxide, potassium hydroxide, ammonium hydroxide and the like. As the reversed phase column, for example, a column filled with a filler in which a functional group is introduced into a silanol group of silica gel by a chemical bond is used. Various functional groups are known to be introduced. Examples thereof include an octadecyl group, an octyl group, an ethyl group, a methyl group, a phenyl group, and a cyano group (propionitrile group). Also,
As the ion pair reagent used for the mobile phase, for example, triethylamine or tributylamine having an ammonium ion as a counter ion can be used. The pH of the mobile phase is preferably from 2 to 7. The detection is carried out, for example, by reacting the eluate from the column in the presence of orthophthalaldehyde (OPA) and a primary amine. The OPA concentration is 0.1-100 m
M, preferably 1-20 mM. Ammonia can be used as the primary amine. The reaction temperature is 30 to 80 ° C, and the reaction time is 10 to 60 seconds. As the detector, for example, a fluorescence detector can be used. Embodiments of the present invention will be described with reference to the drawings. FIG. 1 shows a schematic diagram of an apparatus for performing the method of the present invention. In the figure, reference numeral 1 denotes a liquid sending pump for sending the mobile phase in the mobile phase reservoir 9. Reference numeral 3 denotes a sample introducer, for example, an auto injector can be used. Reference numeral 8 denotes a separation column, and a reactor 7 is disposed downstream of the separation column 8. The reaction reagent (OPA) in the reaction reagent reservoir 10 is sent to the reactor 7 by the liquid sending pump 2 and reacted with the eluate from the separation column 8. Further, the separation column 8 and the reactor 7 are housed in the thermostat 4. Reference numeral 5 denotes a fluorescence detector, and data from the detector 5 is processed by a data processor 6. In the above configuration, when a sample is analyzed, the mobile phase in the mobile phase reservoir 9 is first sent to the separation column 8 by the liquid sending pump 1 to be in a steady state, and then the sample (wine) is sent from the sample introduction device 3. ). In the wine in the sample, free sulfuric acid is converted to hydroxymethanesulfonic acid by pretreatment. In the separation column 8, hydroxymethanesulfonic acid and acetaldehyde-bound sulfurous acid are separated from each other and enter the reactor 7. In the reactor 7, hydroxymethanesulfonic acid reacts with OPA, which is a reaction reagent, to be converted into a fluorescent substance (isoindole derivative).
The fluorescent substance is detected by the fluorescence detector 5. (Experimental Example) The following experiment was conducted using the apparatus shown in FIG. (1) Pretreatment for measurement of free sulfurous acid 500 μl of wine was added to 50 ml of 10 mM citrate (sodium) buffer containing 10 mM formaldehyde, mixed vigorously, and allowed to stand for about 3 minutes to convert free sulfurous acid to hydroxymethanesulfonic acid. The filtered solution was injected by the sample introducer 3. (2) 50 mg of sodium hydroxide is added to 10 ml of pre-treated wine for the measurement of total sulfurous acid, and left to stand for 1 hour after mixing. 500 µl of the solution was added to 50 ml of 10 mM citrate (sodium) buffer containing 10 mM formaldehyde, mixed vigorously, and allowed to stand for about 3 minutes. A solution in which free sulfurous acid was converted to hydroxymethanesulfonic acid was filtered to obtain a sample introduction device. 3 and injected. (3) Blank adjustment The filtered wine (5 ml) is used as a blank. (4) Separation conditions Column: Shim-pack VP-ODS (inner diameter 4.6 mm, length 250 mm) Mobile phase: 20 mM tartaric acid and 5 mM ammonia and 25 mM triethylamine Flow rate: 1.0 ml / min Column temperature: 50 ° C. (5) Detection conditions Reaction reagent: mixture (mixing ratio A: B = 1: 4) A: 10 mM methanol solution of orthophthalaldehyde B: 200 mM boric acid (sodium) buffer (pH = 9.1) Flow rate: 0.5 ml / min Reaction temperature: 50 ° C. Detection: Ex = 320 nm, Em = 390 nm (6) Calculation of Sulfurous Acid Concentration The amount of free sulfurous acid is calculated by the peak amount of hydroxymethanesulfonic acid obtained by the procedure of (1) and the procedure of (3). It is calculated from the difference between the peak amounts of hydroxymethanesulfonic acid (formaldehyde-bound sulfurous acid) in the obtained blank.
The total sulfurous acid is calculated from the peak amount of hydroxymethanesulfonic acid obtained from the procedure (2). The chromatogram of wine analyzed according to the present invention is shown in FIG. FIG.
(a) is a chromatogram of free sulfite, and (b) is a chromatogram of total sulfite. In FIG. 2, peak a indicates hydroxymethanesulfonic acid derived from free sulfurous acid, b indicates acetaldehyde-bound sulfurous acid, and c indicates hydroxymethanesulfonic acid derived from total sulfurous acid. According to the present invention, it is possible to measure sulfurous acid in wine with a simple method with high selectivity and high sensitivity, and it is also possible to measure the sulfurous acid in wine with stable hydroxymethanesulfonic acid. And then subjected to high-performance liquid chromatography, which has higher quantitativeness than the conventional method. In addition, it is possible to perform quantitative analysis of both free and total sulfurous acid.

[Brief description of the drawings] FIG. 1 is a schematic diagram of an apparatus for performing the method of the present invention. Fig. 2 Chromatogram of wine [Explanation of symbols] 1, 2: liquid feed pump 3: Sample introducer 4: constant temperature bath 5: Fluorescence detector 6: Data processing machine 7: Reactor 8: Separation column

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Claims (1)

[Claim 1] Free sulfuric acid and total sulfurous acid in wine are reacted with formaldehyde to generate hydroxymethanesulfonic acid, which is then separated by reverse phase chromatography using an ion pair reagent. And analyzing the free and total sulfurous acid in wine.