JPH0424552A - Analyzing method for hydroquinone derivative - Google Patents

Abstract

PURPOSE:To make it possible to analyze a hydroquinone derivative which is readily subject to oxidation reaction by using a high-speed liquid chromatography device that is not in contact with metal, eluate from which dissolved oxygen is removed and filler whose content of impure metal is less in the liquid contact part of the eluate. CONSTITUTION:In order to avoid the contact between eluate wherein a sample is dissolved and a metal part, a glass-lined stainless pipe or a synthetic macromolecular material is utilized in a piping system, and solvent-resisting property and pressure resistance property are provided. For a pump head and the like, a material which is machined with resin is utilized. For a column, a column such as glass-lining coated column having excellent pressure resistance property and organic-solvent resisting property is used. A filler wherein the diameter of the particle is small is made to fill the column. Since the metal in the filler is made to be 10ppm or less, the filler such as highly pure silica gel is utilized. The filler containing a large amount of metal is cleaned with aqueous solution of inorganic acid or cleaned with chelate reagent and removed. Since the dissolved oxygen in the eluate is made to be 10ppm or less, the dissolved oxygen in the eluate is removed by pressure reduced degassing, ultrasonic degassing and the like.

Description

[Detailed description of the invention] <Industrial application field> The present invention relates to a method for analyzing hydroquinone derivatives.

More specifically, the present invention relates to a method for analyzing hydroquinone derivatives using high-performance liquid chromatography, which is characterized by using a measurement system in which the sample solution does not come into contact with metals.

<Prior Art and Problems> High performance liquid chromatography (IIPLC) is an important method as an analytical means, but it may involve decomposition or chemical changes of a sample during measurement. These decomposition reactions and chemical reactions include chemical reactions with the eluent, hydrolysis reactions,
Alternatively, oxidation reactions derived from dissolved oxygen in the solution N solution, oxidation reactions caused by metals in the eluent in the measurement system or from the measurement device, etc. can be mentioned. Such chemical reactions during measurement significantly impede the quantification of the analyte substance and cause a loss of accuracy in the analysis results, so they must be suppressed as much as possible.

In order to improve the reproducibility and accuracy of measurements, it is essential to remove factors that induce oxidation reactions when analyzing substances that are susceptible to oxidation reactions by HPLC, and the presence of oxygen and metal species in the measurement system is essential. Can be mentioned. Generally, in HPLC, stainless steel columns are widely used as analytical columns due to pressure resistance, organic solvent resistance, cost, etc., and pumps, piping, etc. are also made of metal in consideration of pressure resistance. For this reason, most of the eluent is in contact with the metal, so during measurement, the specimen comes into contact with the metal, and the sample undergoes an oxidation reaction due to the metal dissolved from the metal portion. Furthermore, the oxygen dissolved in the eluent causes an oxidation reaction and changes the sample substance. Medium- and low-pressure liquid chromatography devices designed to prevent the metal from coming into contact with the eluent are commercially available, but all of them lack pressure resistance because they are used to separate biopolymers such as proteins.

The present invention uses a high-performance liquid chromatography device that does not come into contact with metals in the eluent contact parts, piping, columns, and sample injection device, and an eluent from which dissolved oxygen has been removed, and further eliminates impurities. The present invention provides a method for analyzing hydroquinone derivatives that are susceptible to oxidation reactions by using a filler with a low metal content.

That is, the present invention provides a method for analyzing hydroquinone derivatives, which is characterized by using an eluent with dissolved oxygen of 1Q ppm or less, a packing material with a metal content of 10 ppm or less, and a liquid chromatography device in which the eluent does not come into contact with metals. It is related to.

The term "hydroquinone derivative" as used in the present invention refers to a compound that easily changes into a quinone structure substance through an oxidation reaction, such as hydroquinone, a substituted hydroquinone, or a compound that yields a derivative having a quinone structure as a result of an oxidation reaction. The hydroquinone derivative exchanges electrons with a metal ion to undergo a reaction as shown in formula (1), and is oxidized to a quinone derivative.

(R, -R4 are hydrogen or a substituent, aromatic condensed ring, X is oxygen or nitrogen with a substituent, X is a metal) For example, 1.2-
1 or 1,4-hydroquinone, hydroquinone having a substituent such as a methoxy group, a tertiary butyl group, or a methyl group, quinizarin, 1,4-dihydronaphthalene, and its derivatives, and hydroquinone analogs such as p-hydroxydiphenylamine. One example is the body.

In order to achieve the present invention, it is necessary to avoid contact between the eluent in which the sample is dissolved and the metal part, and to remove oxygen dissolved in the eluent.

In the measurement system, creating a measurement system in which the eluent does not come into contact with the metal is
Consideration must be given to the piping from the eluent to the pump, the solvent filter pump for particulate removal, the sample injection device (injector), the column, and the piping. For example, in the piping system, avoid contact with metal in everything from the eluent container to the liquid pump, from the liquid pump to the sample injection valve, to the sample solution loop, from the sample injection valve to the column inlet, and from the column outlet to the detector. There is a need.

Examples of materials that can be used to achieve this purpose include glass-lined stainless steel pipes, synthetic polymer materials such as tetrafluoroethylene (Teflon), and polyetheretherketone (PEEK). In particular, PEEK piping is solvent resistant and pressure resistant and is suitable for the present invention.

Liquid pumps and injectors whose pump heads and the like are processed with resin are commercially available and can be used in the present invention.

As for the column, materials other than stainless steel include glass, polycarbonate, Teflon inner-coated stainless steel, glass-lined stainless steel, PEEK, titanium, and the like. Glass columns and polycarbonate columns are used to separate biopolymers such as proteins that interact with metals. However, due to the low pressure resistance, the particle size of the packing material cannot be reduced, making it difficult to obtain a packed column with high theoretical plates. In addition, Teflon inner-coated columns, glass-lined columns, and PEEK columns have excellent pressure resistance and organic solvent resistance, so they can be filled with packing materials with small particle diameters, resulting in columns with high resolution. I can do it.

Since the metal contained in the filler is also considered to contribute to the oxidation reaction, it is necessary to use a filler with a low metal content. Therefore, the metal content in the filler must be less than 10 ppm. As the filler, high purity silica gel and polymer-based fillers can be used. Alternatively, even metal-rich fillers can be removed, for example, by washing with a dilute aqueous inorganic acid solution such as hydrochloric acid or sulfuric acid, or with a chelating reagent such as disodium ethylenediaminetetraacetate. .

Dissolved oxygen in the eluent is 10 ppm or less.

Removal of dissolved oxygen in the eluent can be achieved, for example, by vacuum degassing, ultrasonic degassing, helium bubbling, or membrane degassing. In particular, helium bubbling or online deaeration using a membrane deaerator are suitable for the present invention because they can remove dissolved oxygen in the eluent over a long period of time.

The present invention will be explained below using Examples, but the present invention is not limited to the Examples.

<Example> Commercially available glass-lined stainless steel column, inner diameter 4 mm
, Length 25cm (manufactured by Gascro Industries), PEEK
manufactured column, inner diameter 4.8mm, length 25c+a (S
(manufactured by AS), stainless steel column inner diameter 4.6sa+, length 25cm (manufactured by Sugiyama Shoji) with TSK packing material for reversed phase.
gel ODS-807M (5u tb.

(manufactured by Tosoh). The measurement system is a resin high-pressure liquid pump (CCPM) whose wetted parts are made of Teflon.

Tosoh) and injector (Rheodyne, 712)
5) A PEEK tube (manufactured by SAS) with an inner diameter of 0.25 mm was used for the piping, and a Teflon solvent filter was attached to pass the eluent. Furthermore, the eluent is M
12 hours before injection, helium was bled. 2,6-ditertiary-butylhydroquinone (DBIIQ) was used as a sample. Before injection, it was dissolved in acetonitrile, and 70 parts of acetonitrile and 30 parts of distilled water were added. An ultraviolet absorption photometer (UV-2151, LKB
The measurement was carried out using a Teflon cell manufactured by Teflon Co., Ltd., and a detection wavelength of 254 r+m). The measurement was carried out using the above-mentioned packed column while bubbling helium into the eluent during the measurement. The measurements were carried out three times, and the oxidants obtained in each measurement for the area of 2.6 ditertiary butylhydroquinone (DBBQ) and 2 methoxybenzoquinone (MBQ), the oxidant obtained by the peak column of the non-metal system, The area ratios are summarized in Table 1. In addition, the obtained chromatogram is
It is shown in Figure 1.

Dissolved oxygen in the eluent was measured using a dissolved oxygen system (manufactured by Benuchi). It was 7.4 ppm. In addition, the metal content in the filler is Fe, ippm or less, Na, 5ppm or less,
CLl % 1 ppm or less, Ti.

It was below ippm.

Rakuten Coagulation Low Comparative Example 1> A packed column was prepared in the same manner as in Example 1, except that a commercially available high-pressure liquid pump (CCPM, manufactured by Tosoh), a sample injection valve (7125, manufactured by Rheodyne), and a stainless steel pipe were used. The sample was measured using The results are summarized in Table 1.

Comparative Example 2> Example 1 except that an eluent with an oxygen concentration of 55 ppm was used.
Analyzed in the same way.

<Example 2> Using the analytical column prepared in Example 1, a membrane degasser (E
RC-3522 (manufactured by Tosoh) and an eluent degassed online for 24 hours, and then DBHQ analysis was performed. Table 2 shows the ratio of the area of the oxidant before and after passing the liquid to the area of the oxidant obtained in the peak column.

Further, a chromatogram is shown in FIG. 2.

Table 2 Peak area ratio of oxidized substances Before passing through Glass coated column after passing through liquid 1.02 1.0AP
EEK Column 1 1.03 Stainless Steel Column 1.39 3.28 Comparative Example 3> A peak column packed with a packing material with a high metal content and a peak column packed with a similar packing material were treated with 1mM ethylenediaminetetraacetic acid. 50 parts of disodium aqueous solution and 50 parts of acetonitrile for 24 hours, 0.5 1111/wi
Using a column washed with liquid at a flow rate of n, 90 parts of distilled water,
Add 10 parts of acetonitrile to the eluent and hydroquinone (IIQ
) was analyzed. The resulting chromatogram is shown in Figure 3.
It was shown to. Table 3 shows the peak areas of the oxidants and the results of metal analysis.

Table 3 Effect of metals in packing material BQ (%) Metal content (ppII) Na Pe ^I Cu Ti Metal-containing column 12.3 37 11 1>1>1> Washing column 0.2 5>1>1>
l>l><Effects of the Invention> According to the present invention, when a sample susceptible to oxidation reaction is analyzed by liquid chromatography, the measurement system is non-metallic, and the dissolved oxygen of the eluent is removed, thereby making it possible to It has become clear that decomposition can be suppressed.

[Brief explanation of the drawing]

Figure 1 was obtained in Example 1, and Figure 2 was obtained in Example 2 (
DBIIQ), where (A,) is the glass coated column, (B) is the PEEK column, and (C
) is a chromatogram of a stainless steel column; (a)
is the peak of D B +10, and (b) is the peak of DBBQ. FIG. 3 is a chromatogram of IIQ obtained with the column before (A) and after (B) washing with the EDTA solution in Comparative Example 3.

Claims (1)

[Claims] (1) A method for analyzing hydroquinone derivatives, which comprises using an eluent with dissolved oxygen of 10 ppm or less, a filler with a metal content of 10 ppm or less, and a liquid chromatography device in which the eluent does not come into contact with metals.