Quinone compound concentration detection method

Abstract

The invention discloses a method for detecting the concentration of quinone compounds, which comprises the following steps of: performing full-wave-band scanning by using an ultraviolet visible spectrophotometer under an anaerobic condition to obtain an ultraviolet visible light full-wave-band spectrogram L1 of quinone before electrochemical reduction and an ultraviolet visible light full-wave-band spectrogram L2 of quinone after electrochemical reduction, and determining the concentration of the quinone compounds according to the difference of characteristic absorption peaks in L1 and L2. Respectively selecting two test wavelengths with large difference, and recording the two test wavelengths as lambda 1 and lambda 2; establishing a concentration-absorbance straight line of the oxidation-state quinone compound (Q) under the wavelengths of lambda 1 and lambda 2, and calculating molar extinction coefficients epsilon Q, 1 and epsilon Q, 2 of the oxidation-state quinone compound (Q) under the wavelengths of lambda 1 and lambda 2 according to a formula 1; establishing a concentration-absorbance straight line of the reduction-state quinone compound (H2Q) under the wavelengths of lambda 1 and lambda 2, and calculating molar extinction coefficients epsilon H2Q1 and epsilon H2Q2 of the reduction-state quinone compound (H2Q) under the two wavelengths according to a formula 1; based on the molar extinction coefficient, the concentrations of quinone compounds in different oxidation and reduction states are tested by combining formulas 2 and 3. The determination method has the advantages of good linearity, good experiment repeatability, high determination result precision and high accuracy.

Description

Technical Field

[0001] The invention relates to the field of quinone compound detection, in particular to a method for detecting the concentration of quinone compounds. Background Art

[0002] Quinone compounds are an important class of organic matter, which are widely distributed in nature. Since quinone compounds can reversibly receive and release electrons, quinones in the environment can act as electron shuttles, mediate the electron transfer from microorganisms to the outside of the cell, participate in the extracellular respiration process of microorganisms, and can also act as terminal electron acceptors, thereby inhibiting the process of microorganisms producing methane. In addition, in the field of analytical chemistry, quinone compounds are also used as chemical probes to detect the redox capacity (i.e., electron supply capacity and electron acceptance capacity) of natural organic matter, iron-containing minerals, and clay sediments. Whether quinone compounds act as electron shuttles, terminal electron acceptors, or chemical probes, their characteristics are that the redox state of quinone compounds changes during the electron transfer process, that is, the change in the concentration of quinone compounds in different redox states. Therefore, it is of great environmental significance to establish a simple, fast, and accurate analytical method for quinone compounds.

[0003] At present, the test method of quinone compounds in different redox states is mainly electrochemical method, the principle of which is to completely reduce oxidized quinones to reduced quinones or completely oxidize reduced quinones to oxidized quinones under certain redox potential conditions, and obtain the concentration of quinone compounds in different redox states by integrating the current-time curve during the electrolysis process (i.e., chronoamperometry). Although the chronoamperometry can accurately test the concentration of quinone compounds in different redox states, its detection and analysis time cycle is long (tens of minutes or even hours), the operation is complicated, and it requires electrochemical related professional knowledge background, therefore, it is not conducive to being widely used in different environmental sciences and groundwater science and other related fields.

[0004] A Chinese patent application with publication number CN110579520A discloses a method for monitoring the electro-oxidation-reduction reaction process of quinone compounds, and discloses that infrared spectroscopy and electrochemistry are simultaneously monitored through a thin-layer electrolytic cell, and changes in reactants, intermediates, and products during the electro-oxidation-reduction process of quinone compounds are recorded in real time. S1: constructing a thin-layer electrolytic cell system: a platinum wire electrode is used as an auxiliary electrode (2), a platinum disk electrode with a diameter of 4 mm is used as a working electrode (3), and an Ag / AgCl electrode is used as a reference electrode (4), forming a three-electrode system, and arranged in a solution cavity (5) of the thin-layer electrolytic cell, and an infrared light window (6) is also arranged between the bottom of the solution cavity (5) and the inner wall of the cell body (1). This scheme uses infrared spectroscopy combined with electrochemistry to monitor the electro-redox reaction process of quinone compounds in real time through a thin-layer electrolytic cell. Although it is innovative, it has the following shortcomings: the equipment is complex and costly, the operation process is cumbersome, and the experimental conditions are high; the method is mainly suitable for the study of reaction mechanisms rather than rapid and quantitative detection; the detection cycle is long and it is difficult to meet the needs of rapid screening in complex systems. In particular, the ability to accurately quantify the final redox state concentration is limited, and the scope of application is restricted.

[0005] Quinone compounds often have characteristic molecular absorption spectra, so the concentration of quinone compounds is tested using UV-visible spectrophotometry. Compared with the electrochemical method, UV-visible spectrophotometry has the advantages of short test cycle (several seconds) and simple operation. The UV-visible spectrophotometry tests the sum of the absorbances of quinone compounds in different redox states at a specific wavelength (Formula 1), so the direct application of UV-visible spectrophotometry can accurately test the concentration of quinone compounds in fully oxidized or fully reduced states. For quinone compounds in the "intermediate" redox state, Formula 1 cannot be used to accurately calculate.

[0006]

[0007] Where A represents the total absorbance at a specific wavelength, C Q and Represent the concentrations of oxidized quinone compounds and reduced quinone compounds, εQ and εH respectively 2 Q represents the molar extinction coefficient of oxidized quinone compounds and reduced quinone compounds under specific wavelength conditions. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide a method for detecting the concentration of quinone compounds in view of the shortcomings of the prior art, which can detect the concentration of quinone compounds in an intermediate valence state between a fully oxidized state and a fully reduced state.

[0009] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0010] A method for detecting the concentration of quinone compounds comprises the following steps:

[0011] S1. dissolving an oxidized quinone compound in an electrolyte solution to obtain a solution A;

[0012] S2. Under anaerobic conditions, using a glassy carbon electrode as the working electrode, a platinum wire as the auxiliary electrode, and Ag / AgCl as the reference electrode, a portion of solution A was electrochemically reduced to obtain a reduced quinone solution B;

[0013] S3. Under anaerobic conditions, the solution A obtained in S1 and the reduced quinone solution B obtained in S2 are scanned in the full band using a UV-visible spectrophotometer to obtain the UV or / and visible light spectrum L1 of the quinone before electrochemical reduction and the UV or / and visible light spectrum L2 of the quinone after electrochemical reduction. According to the difference in characteristic absorption peaks in L1 and L2, two test wavelengths λ with larger differences are selected in L1 and L2, respectively. 1 and λ 2 ;

[0014] S4. Establish the oxidation state of quinone compounds (Q) at λ 1 and λ 2 The concentration-absorbance straight line under λ is calculated according to formula 1. 1 and λ 2 The molar extinction coefficient ε Q,1 and ε Q,2 ;

[0015]

[0016] Where A represents the total absorbance at a specific wavelength, e Q and Represent the concentrations of oxidized quinone compounds and reduced quinone compounds, ε Q and They represent the molar extinction coefficients of oxidized quinone compounds and reduced quinone compounds under specific wavelength conditions respectively;

[0017] S5. Establishment of reduced quinone compounds (H 2 Q) in λ 1 and λ 2 The concentration-absorbance straight line under λ is calculated according to formula 1. 1 and λ 2 The molar extinction coefficient ε H2Q1 and ε H2Q2 ;

[0018] S6. Based on ε Q , ε H2Q1 , ε H2Q2, combined with formula 2 and formula 3 to test the concentration of the quinone compound sample to be tested, the specific detection method is:

[0019] The quinone compound sample to be tested was acidified and diluted, and the UV-visible spectrophotometer was used to read the 1 and λ 2 The absorbance under , the concentration of the quinone compound sample to be tested is calculated according to Formula 2 and Formula 3;

[0020]

[0021] Among them A λ1 and A λ2 Represents λ 1 and λ 2 Total absorbance at wavelength, ε Q,1 and ε Q,2 Represent the oxidation state of quinone compounds in λ 1 and λ 2 Molar extinction coefficient under the conditions, ε H2Q,1 and ε H2Q,2 Represent the reduced quinone compounds in λ 1 and λ 2 The molar extinction coefficient under the conditions is: and C Q It represents the concentration of reduced quinone compounds and the concentration of oxidized quinone compounds in the quinone compound sample to be tested, and n is the dilution multiple of the quinone compound sample to be tested.

[0022] Oxidized quinones refer to the oxidized forms of quinone compounds. Quinones are usually organic compounds containing two carbonyl groups (C=O) connected to the same ring structure. Common forms of oxidized quinones include benzoquinone, naphthoquinone, and anthraquinone.

[0023] Reduced quinones are oxidized quinones converted to phenols through reduction reactions. These reduction products are usually reduced forms of quinones, which gain electrons or hydrogen atoms in the molecules and are converted into phenolic structures with strong reducing properties. For example, the reduction product of p-benzoquinone is p-phenol (hydroquinone), which has a phenolic hydroxyl group (-OH) instead of a carbonyl group (C=O).

[0024] In a preferred embodiment of the present invention, the electrolyte solution is a mixed solution of 0.05-0.2M KCl and 0.01-0.1M buffer, the pH is 6.0-8.0, and the volume ratio of the electrolyte solution to the oxidized quinone compound is 30-40mL:1-3mL. Preferably, the buffer is a MOPS solution.

[0025] In a preferred embodiment of the present invention, in S3, when L 1 The characteristic absorption peaks are not 2 Overlap, L 2 The characteristic absorption peaks are not 1 Overlap, take L 1 and L 2 The characteristic absorption peak is taken as λ 1 and λ 2 .

[0026] In a preferred embodiment of the present invention, in S3, when L 1 The characteristic absorption peak and L 2 Overlap, or L 2 The characteristic absorption peak and L 1 Overlap, in L 1 and L 2 The test wavelength with the largest difference in the ultraviolet-visible light absorbance of quinone before and after electrochemical reduction is selected as λ within the ±60nm wavelength range of the characteristic absorption peak of 1 and λ 2 .

[0027] In a preferred embodiment of the present invention, in S4, the oxidized quinone compound (Q) is established at λ 1 and λ 2 The concentration-absorbance linear method under the condition of λ is to take different concentrations of oxidized quinone compounds and measure the oxidized quinone compounds at λ 1 and λ 2 The concentration-absorbance line L 3 =ε Q,1 x and L 4 =ε Q,2 x.

[0028] In a preferred embodiment of the present invention, in S5, a reduced quinone compound (H 2 Q) in λ 1 and λ 2 The concentration-absorbance linear method under the condition of λ is to take different concentrations of reduced quinone compounds and measure the reduced quinone compounds at λ 1 and λ 2 The concentration-absorbance line L 5 =ε H2Q,1 x and L 6 =ε H2Q,2 x.

[0029] In a preferred embodiment of the present invention, S5 further includes measuring an electrochemical it curve using a reduced quinone compound and calculating a reduction rate b of the reduced quinone compound;

[0030] The current integral of the measured IT curve was used to calibrate the oxidized naphthoquinone (i.e., NQ) and reduced naphthoquinone (i.e., H) in the solution after electrochemical reduction. 2 NQ) concentration:

[0031]

[0032] Where F is the Faraday constant (96,485 s A mol -1 ), t 1 and t 2 (s) is the starting and ending point of the integrated current peak, I(t)(A) is the current function over time, C NQ1 (mol) is the concentration of oxidized naphthoquinone (i.e., NQ) in the solution before electrochemical reduction; Q2 (mol) is the concentration of oxidized naphthoquinone (i.e., NQ) in the solution after the electrochemical reduction; is the reduced naphthoquinone (i.e., H 2 N(Q) concentration; V(L) is the volume of the system during electrochemical reduction.

[0033] In a preferred embodiment of the present invention, when the quinone compound is 1,4-naphthoquinone (NQ), the NQ and H 2 The concentration of NQ can be calculated according to the following formula:

[0034]

[0035] Among them C NQ , The unit is mM, n is the sample dilution multiple, A 319nm and A 360nm Represent the total absorbance at a wavelength of 319 nm and a wavelength of 360 nm, respectively.

[0036] In a preferred embodiment of the present invention, when the quinone compound is 9,10-anthraquinone-2,6-disulfonic acid (AQDS), the AQDS and AH in the sample to be tested are 2 The concentration of DS can be calculated according to the following formula:

[0037]

[0038] Among them C AQDS , The unit is mM, n is the sample dilution multiple, A 387nm and A 325nm Represent the total absorbance at a wavelength of 387 nm and a wavelength of 325 nm, respectively.

[0039] A method for detecting the concentration of quinone compounds, when the quinone compound is 1,4-naphthoquinone (NQ), the concentration of NQ and H in the sample to be tested is 2 The concentration of NQ can be calculated according to the following formula:

[0040]

[0041] Among them C NQ , The unit is mM, n is the sample dilution multiple, A 319nm and A 360nm Represent the total absorbance at wavelengths of 319 nm and 360 nm, respectively;

[0042] When the quinone compound is 9,10-anthraquinone-2,6-disulfonic acid (AQDS), the AQDS and AH 2 The concentration of DS can be calculated according to the following formula:

[0043]

[0044] Among them C AQDS , The unit is mM, n is the sample dilution multiple, A 387nm and A 325nm Represent the total absorbance at a wavelength of 387 nm and a wavelength of 325 nm, respectively.

[0045] Preferably, 1-3 ml of 5-15 mM acid is added to the oxidized quinone compound and the reduced quinone compound respectively to acidify; preferably, the acid is H 2 SO 4 .

[0046] The quinone compound includes one of 1,4-naphthoquinone and 9,10-anthraquinone-2,6-disulfonic acid.

[0047] In S2, solution A is electrochemically reduced using chronoamperometry.

[0048] Dilute S6 within the absorbance range of the standard curve and acidify S6 by adding 1 to 3 ml of 5 to 15 mM acid.

[0049] In S3, under anaerobic conditions, solution A obtained in S1 and reduced quinone solution B obtained in S2 are fully scanned using a UV-visible spectrophotometer to obtain a UV-visible full-band spectrum L1 of quinone before electrochemical reduction and a UV-visible full-band spectrum L2 of quinone after electrochemical reduction.

[0050] Compared with the Chinese patent application with publication number CN110579520A, this application is based on dual-wavelength ultraviolet-visible spectrophotometry, which has significant advantages: it does not require complex electrochemical and infrared equipment, and can be completed with only an ultraviolet spectrophotometer, which is simple to operate and low in cost; through dual-wavelength characteristic absorption, a linear relationship standard curve is established to achieve simple and accurate quantitative analysis; the detection cycle is short, which is suitable for rapid screening of large batches of samples, and can cope with the determination of redox state concentration in complex systems, and has a wide range of practical application scenarios. In addition, the present invention combines electrochemical reduction data to further enhance the accuracy of concentration determination.

[0051] Compared with the prior art, the present invention has the following beneficial effects: the detection method for the concentration of quinone compounds in different redox states provided by the present invention requires low instrument cost, simple test process operation, few reagents required, short test cycle, and high detection efficiency; and the determination method has good linearity, good experimental repeatability, high precision of the determination results and high accuracy.

[0052] The method of the invention is simple, has a short analysis time, and has a low use cost. It has good application prospects in the field of action mechanism of quinone compounds participating in the electron transfer process and redox capacity analysis and testing, and makes up for the weaknesses of the existing technical means. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is the full-band UV-visible spectrum of quinone before and after electrochemical reduction in Example 1 of the present invention;

[0054] Figure 2 The electrochemical it curve of electrochemical reduction of 0.5 mM NQ in Example 1 of the present invention;

[0055] Figure 3 is the standard curve of NQ dual wavelength in Example 1 of the present invention;

[0056] Figure 4 is H in Example 1 of the present invention 2 Standard curve of NQ dual wavelength;

[0057] Figure 5 This is the full-band UV-visible spectrum of quinone before and after electrochemical reduction in Example 2 of the present invention;

[0058] Figure 6 The electrochemical it curve of electrochemical reduction of 0.5 mM AQDS in Example 2 of the present invention;

[0059] Figure 7 is the standard curve of AQDS dual wavelength in Example 2 of the present invention;

[0060] Figure 8 AH in Example 2 of the present invention2 Standard curve of DS at dual wavelengths. DETAILED DESCRIPTION

[0061] Example 1 Testing Method for Different Redox State Concentrations of 1,4-Naphthoquinone (NQ)

[0062] (1) Selection of test wavelength

[0063] Unless otherwise specified, all analyses and sample processing were performed at 25 ± 2 °C, 4% H 2 and 96% N 2 The results were carried out in an anaerobic glove box. Before placing the solution in the glove box, high-purity nitrogen (99.999%) was introduced for at least 30 minutes to eliminate the interference of oxygen in the solution. The glassware, plasticware, and chemicals used for method development were placed in the glove box transition chamber for vacuum degassing before being transferred to the glove box. The electrochemical analysis used a CHI1000C electrochemical workstation, a glassy carbon crucible as the working electrode and reaction vessel, an Ag / AgCl electrode filled with saturated KCl as the reference electrode, and a platinum wire electrode as the auxiliary electrode. The auxiliary electrode was separated from the working electrode chamber by a glass sand core. Each electrode was connected to the electrochemical workstation to construct a three-electrode system. The reaction vessel was placed on a stirrer and stirred continuously at 700 rpm with a 1 cm polytetrafluoroethylene-coated magnetic stirring bar. The It chronoamperometry was selected, and the voltage of the working electrode was set to -0.4 V (vs. SHE) to fully reduce 1,4-naphthoquinone. First, add 38 ml of a solution containing 0.1 M KCl and 0.01 M MOPS (pH 7.0) as the background solution, where MOPS acts as a pH buffer and KCl acts as an electrolyte. When the current signal of the background solution reaches stability (<3 μA), add 2 mL of 10 mM 1,4-naphthoquinone. When the current signal reaches stability again and remains basically unchanged, suspend the electrochemical test.

[0064] Take 3 mL of electrochemically reduced 1,4-naphthoquinone (i.e. H 2 NQ) was added to a quartz cuvette, covered with a rubber stopper, and wrapped with sealing glue to prevent it from being oxidized. It was taken out of the glove box and full-band scanned at a wavelength of λ = 200nm to λ = 800nm ​​using a UV-visible spectrophotometer (before scanning the sample, deionized water was used to correct the background). Then 3mL of 0.5mM 1,4-naphthoquinone (i.e., NQ) was taken and full-band scanned at a wavelength of λ = 200nm to λ = 800nm ​​using a UV-visible spectrophotometer. Figure 1 According to the difference in the characteristic absorption peaks before and after the reduction of 1,4-naphthoquinone, two test wavelengths with large differences were selected. According to the experimental results, NQ selected λ 1 =319nm and λ2 =360nm as the test wavelength.

[0065] 2. Calibration of the standard curve

[0066] According to step 1, naphthoquinone is fully reduced by an electrochemical method, and according to the IT curve measured in step 1 (such as Figure 2 The current integral is shown in Figure 2 to calibrate the electrochemical reduction of the oxidized naphthoquinone (i.e., NQ) and reduced naphthoquinone (i.e., H) in the solution. 2 NQ) concentration:

[0067]

[0068] Where F is the Faraday constant (96,485 s A mol -1 ), t 1 and t 2 (s) is the starting and ending point of the integrated current peak, I(t)(A) is the current function over time, C NQ1 (mol) is the concentration of oxidized naphthoquinone (i.e., NQ) in the solution before electrochemical reduction; Q2 (mol) is the concentration of oxidized naphthoquinone (i.e., NQ) in the solution after the electrochemical reduction; is the reduced naphthoquinone (i.e., H 2 N(Q) concentration; V(L) is the volume of the system during electrochemical reduction.

[0069] According to the current integral calibration of the IT curve, the reduction rate of naphthoquinone in the electrochemically reduced solution is 92.23%, and the reduced solution contains 0.4612 mM H 2 NQ, 0.0388 mM NQ.

[0070] Take 120 μL, 240 μL, 360 μL, 480 μL and 6000 μL of 0.5 mM oxidized naphthoquinone (i.e. 0.5 mM NQ) in 5 ml centrifuge tubes, add 1 ml of 10 mM H 2 SO 4 After acidification, use oxygen-free deionized water to make the volume 3mL. Then, the standard solution with NQ concentration gradient of 0.02mM, 0.04mM, 0.06mM, 0.08mM, and 0.1mM is obtained. 1 =319nm and λ 2 =360nm to read each absorbance, establish a linear relationship between the absorbance and the concentration of oxidized naphthoquinone at dual wavelengths, and obtain a linear standard curve (such as Figure 3 shown).

[0071] λ 1 =319nm A NQ=3.9718×C NQ R 2 =0.9997

[0072] λ 2 =360nm A NQ =3.6545×C NQ R 2 =0.9995

[0073] Among them C NQ The unit is mM.

[0074] Take 120 μL, 240 μL, 360 μL, 480 μL and 6000 μL of 0.5 mM electrochemically reduced naphthoquinone (i.e. 0.4612 mM H 2 NQ + 0.0388mM NQ) in a 5ml centrifuge tube, and add 1ml of 10mM H 2 SO 4 After acidification, use oxygen-free deionized water to make up to 3 mL. 2 The NQ concentration gradient is 0.0184mM, 0.0369mM, 0.0553mM, 0.0738mM, 0.0922mM; the NQ concentration gradient is 0.0016mM, 0.0031mM, 0.00047mM, 0.00062mM, 0.0078mM standard solution. 1 =319nm and λ 2 =360nm to read each absorbance, and calculate the theoretical absorbance of reduced naphthoquinone according to the following formula.

[0075]

[0076] The linear relationship between the absorbance and the reduced naphthoquinone concentration under dual wavelengths was established to obtain a linear standard curve, such as Figure 4 shown.

[0077] λ 1 =319nm R 2 =0.9999

[0078] λ 2 =360nm R 2 =0.9865

[0079] in The unit is mM.

[0080] 3. Sample concentration determination

[0081] Take the samples to be tested and add 1 ml of 10 mM H 2 SO4 After acidification, dilute with oxygen-free deionized water to the standard curve concentration range, respectively at λ 1 =319nm and λ 2 =360nm read the corresponding absorbance. It can be seen that:

[0082]

[0083] Simplifying the above two equations together yields:

[0084]

[0085] Substituting this into the first two equations, we can solve the equations to get:

[0086] Then the NQ and H in the sample 2 The concentration of NQ can be calculated according to the following formula.

[0087]

[0088] Among them C NQ , The unit is mM, and n is the sample dilution multiple.

[0089] Each quinone substance has a specific absorbance coefficient and absorbance-concentration relationship, so different standard curves need to be constructed for different quinone compounds.

[0090] Generally speaking, it is assumed that the concentration-absorbance curve formula of a quinone substance in different redox states can be expressed as:

[0091]

[0092] Further calculation yields:

[0093]

[0094] Each compound has its own specific absorption peak. Usually, the absorbance of a substance is strongest within a certain wavelength range. If the selected wavelength is far away from the absorption peak of the substance, the absorbance may be low, which will affect the sensitivity and accuracy of the measurement. The selection of 319nm and 360nm wavelengths is based on naphthoquinone (NQ and H 2 The absorbance characteristics of NQ) at these two wavelengths. These two wavelengths may be located in the strong absorption region and weak absorption region of the substance, respectively, so that substances in different chemical states can be distinguished.

[0095] Example 2 Testing Method for Different Redox State Concentrations of 9,10-Anthraquinone-2,6-Disulfonic Acid (AQDS)

[0096] (1) Selection of test wavelength

[0097] Unless otherwise specified, all analyses and sample processing were performed at 25 ± 2 °C, 4% H 2 and 96% N 2 The analysis was carried out in an anaerobic glove box. Before placing the solution in the glove box, high-purity nitrogen (99.999%) was introduced for at least 30 minutes to eliminate the interference of oxygen in the solution. The glassware, plasticware, and chemicals used for method development were placed in the glove box transition chamber for vacuum degassing before being transferred to the glove box. The electrochemical analysis used a CHI1000C electrochemical workstation, a glassy carbon crucible as the working electrode and reaction vessel, an Ag / AgCl electrode filled with saturated KCl as the reference electrode, and a platinum wire electrode as the auxiliary electrode. The auxiliary electrode was separated from the working electrode chamber by a glass sand core. Each electrode was connected to the electrochemical workstation to construct a three-electrode system. The reaction vessel was placed on a stirrer and stirred continuously at 700 rpm with a 1 cm polytetrafluoroethylene-coated magnetic stirring bar. The It chronoamperometry was selected, and the voltage of the working electrode was set to -0.4 V (vs. SHE) to fully reduce 9,10-anthraquinone-2,6-disulfonic acid (AQDS). First, add 38 ml of a solution containing 0.1 M KCl and 0.01 M MOPS (pH 7.0) as the background solution, where MOPS acts as a pH buffer and KCl acts as an electrolyte. When the current signal of the background solution reaches stability (<3 μA), add 2 mL of 10 mM 9,10-anthraquinone-2,6-disulfonic acid (AQDS). When the current signal reaches stability again and remains basically unchanged, suspend the electrochemical test.

[0098] Take 3 mL of electrochemically reduced 9,10-anthraquinone-2,6-disulfonic acid (AH 2 DS) was added to a quartz cuvette, covered with a rubber stopper, and wrapped with sealing glue to prevent it from being oxidized. It was taken out of the glove box and full-band scanned at a wavelength of λ = 200nm to λ = 800nm ​​using a UV-visible spectrophotometer (before scanning the sample, deionized water was used to correct the background). Then 3mL of 0.5mM 9,10-anthraquinone-2,6-disulfonic acid (AQDS) was taken and full-band scanned at a wavelength of λ = 200nm to λ = 800nm ​​using a UV-visible spectrophotometer, as shown in FIG. Figure 5 According to the difference in characteristic absorption peaks before and after reduction of 9,10-anthraquinone-2,6-disulfonic acid (AQDS), two test wavelengths with large differences were selected. According to the experimental results, AQDS selected λ 1 =325nm and λ 2 =387nm as the test wavelength.

[0099] (2) Calibration of standard curve

[0100] According to step (1), naphthoquinone is fully reduced by an electrochemical method, and the oxidation state of 9,10-anthraquinone-2,6-disulfonic acid (AQDS) and 9,10-anthraquinone-2,6-disulfonic acid (AH 2 DS) concentration.

[0101] Take 120 μL, 240 μL, 360 μL, 480 μL and 6000 μL of 0.5 mM oxidized 9,10-anthraquinone-2,6-disulfonic acid (i.e. 0.5 mM AQDS) in 5 ml centrifuge tubes, add 1 ml of 10 mM H 2 SO 4 After acidification, use oxygen-free deionized water to make the volume to 3 mL. The standard solution with AQDS concentration gradient of 0.02mM, 0.04mM, 0.06mM, 0.08mM, and 0.1mM is obtained. 1 =325nm and λ 2 =387 nm to read each absorbance, establish a linear relationship between the absorbance at dual wavelengths and the concentration of oxidized 9,10-anthraquinone-2,6-disulfonic acid, and obtain a linear standard curve.

[0102] like Figure 5-6 As shown, step (1) selects λ 1 =325nm and λ 2 =387nm as the test wavelength, the standard curve of the oxidized quinone AQDS in step (2) is as follows ( Figure 7 shown).

[0103] λ 1 =325nm A AQDS =5.7516×C AQDS R 2 =0.9995

[0104] λ 2 =387nm A AQDS =0.2022×C AQDS R 2 =0.9989

[0105] Take 120 μL, 240 μL, 360 μL, 480 μL and 6000 μL of 0.5 mM reduced 9,10-anthraquinone-2,6-disulfonic acid (i.e. 0.5 mM AH 2 DS) in a 5 ml centrifuge tube, add 1 ml of 10 mM H 2 SO 4 After acidification, use oxygen-free deionized water to make up to 3 mL. 2The DS concentration gradient is 0.02mM, 0.04mM, 0.06mM, 0.08mM, 0.1mM standard solution. 1 =325nm and λ 2 =387 nm to read each absorbance, establish a linear relationship between the absorbance at dual wavelengths and the concentration of reduced 9,10-anthraquinone-2,6-disulfonic acid, and obtain a linear standard curve.

[0106] In step (2), the reduced quinone AH 2 The standard curve of D is as follows ( Figure 8 shown).

[0107] λ 1 =325nm R 2 =0.998

[0108] λ 2 =387nm R 2 =0.9994

[0109] AQDS and AH in the sample in step (3) 2 The concentration of DS can be calculated according to the following formula.

[0110]

[0111] Among them C AQDS , The unit is mM, and n is the sample dilution multiple.

Claims

1. A method for detecting the concentration of quinone compounds, characterized in that The following steps are involved: S1. dissolving an oxidized quinone compound in an electrolyte solution to obtain a solution A; S2. Under anaerobic conditions, using a glassy carbon electrode as the working electrode, a platinum wire as the auxiliary electrode, and Ag / AgCl as the reference electrode, a portion of solution A was electrochemically reduced to obtain a reduced quinone solution B; S3. Under anaerobic conditions, the solution A obtained in S1 and the reduced quinone solution B obtained in S2 are subjected to full-band scanning using an ultraviolet-visible spectrophotometer to obtain an ultraviolet or / and visible light spectrum L1 of the quinone before electrochemical reduction and an ultraviolet or / and visible light spectrum L2 of the quinone after electrochemical reduction, and according to the difference in characteristic absorption peaks in L1 and L2, two test wavelengths λ1 and λ2 with large differences are selected in L1 and L2, respectively; S4. Establish the concentration-absorbance lines of the oxidized quinone compound (Q) at λ1 and λ2, respectively, and calculate its molar extinction coefficient ε at λ1 and λ2 according to formula 1 Q,1 and ε Q,2 ; Where A represents the total absorbance at a specific wavelength, C Q and C H2Q Represent the concentrations of oxidized quinone compounds and reduced quinone compounds, ε Q and They represent the molar extinction coefficients of oxidized quinone compounds and reduced quinone compounds under specific wavelength conditions respectively; S5. Establish the concentration-absorbance straight line of reduced quinone compound (H2Q) at λ1 and λ2, and calculate its molar extinction coefficient ε at λ1 and λ2 according to formula 1 H2Q1 and ε H2Q2 ; S6. Based on ε Q , ε H2Q1 , ε H2Q2 , combined with formula 2 and formula 3 to test the concentration of the quinone compound sample to be tested, the specific detection method is: Acidify and dilute the quinone compound sample to be tested, read the absorbance at λ1 and λ2 using an ultraviolet-visible spectrophotometer, and calculate the concentration of the quinone compound sample to be tested according to Formula 2 and Formula 3; Among them A λ1 and A λ2 Represents the total absorbance at wavelengths of λ1 and λ2, ε Q,1 and ε Q,2 represent the molar extinction coefficients of oxidized quinone compounds under λ1 and λ2 conditions, ε H2Q,1 and ε H2Q,2 represent the molar extinction coefficients of reduced quinone compounds under λ1 and λ2 conditions, respectively. and C Q It represents the concentration of reduced quinone compounds and the concentration of oxidized quinone compounds in the quinone compound sample to be tested, and n is the dilution multiple of the quinone compound sample to be tested.

2. The method for detecting the concentration of quinone compounds according to claim 1, characterized in that: The electrolyte solution is a mixed solution of 0.05-0.2M KCl and 0.01-0.1M buffer, with a pH of 6.0-8.0, and a volume ratio of the electrolyte solution to the oxidized quinone compound of 30-40mL:1-3mL.

3. The method for detecting the concentration of quinone compounds according to claim 1, characterized in that: In S3, when the characteristic absorption peak in L1 does not overlap with L2, and the characteristic absorption peak in L2 does not overlap with L1, the characteristic absorption peaks of L1 and L2 are taken as λ1 and λ2.

4. The method for detecting the concentration of quinone compounds according to claim 1, characterized in that: In S3, when the characteristic absorption peak in L1 overlaps with L2, or the characteristic absorption peak in L2 overlaps with L1, the test wavelengths with the largest difference in the ultraviolet-visible light absorbance of quinone before and after electrochemical reduction are selected as λ1 and λ2 within the wavelength range of ±60 nm of the characteristic absorption peaks of L1 and L2.

5. The method for detecting the concentration of quinone compounds according to any one of claims 1 to 4, characterized in that: In S4, the method for establishing the concentration-absorbance straight line of the oxidized quinone compound (Q) at λ1 and λ2 is to take different concentrations of the oxidized quinone compound and respectively measure the concentration-absorbance straight line L3=ε of the oxidized quinone compound at λ1 and λ2. Q,1 x and L4 = ε Q,2 x.

6. The method for detecting the concentration of quinone compounds according to any one of claims 1 to 4, characterized in that: In S5, the method for establishing the concentration-absorbance straight line of the reduced quinone compound (H2Q) at λ1 and λ2 is to take different concentrations of the reduced quinone compound and measure the concentration-absorbance straight line L5=ε at λ1 and λ2 respectively. H2Q,1 x and L6 = ε H2Q,2 x.

7. The method for detecting the concentration of quinone compounds according to claim 6, characterized in that: S5 also includes measuring an electrochemical it curve using a reduced quinone compound and calculating a reduction rate b of the reduced quinone compound; The concentrations of oxidized naphthoquinone (i.e., NQ) and reduced naphthoquinone (i.e., H2NQ) in the electrochemically reduced solution were calibrated according to the measured it curve current integral: Where F is the Faraday constant (96,485 s Amol -1 ), t1 and t2(s) are the starting and ending points of the integrated current peak, I(t)(A) is the current function over time, C NQ1 (mol) is the concentration of oxidized naphthoquinone (i.e., NQ) in the solution before electrochemical reduction; Q2 (mol) is the concentration of oxidized naphthoquinone (i.e., NQ) in the solution after the electrochemical reduction; is the concentration of reduced naphthoquinone (ie H2NQ) in the solution after the electrochemical reduction; V(L) is the volume of the system during the electrochemical reduction.

8. The method for detecting the concentration of quinone compounds according to claim 1, characterized in that: When the quinone compound is 1,4-naphthoquinone (NQ), the concentrations of NQ and H2NQ in the sample to be tested can be calculated according to the following formula: Among them C NQ , The unit is mM, n is the sample dilution multiple, A 319nm and A 360nm Represent the total absorbance at a wavelength of 319 nm and a wavelength of 360 nm, respectively.

9. The method for detecting the concentration of quinone compounds according to claim 1, characterized in that: When the quinone compound is 9,10-anthraquinone-2,6-disulfonic acid (AQDS), the concentrations of AQDS and AH2DS in the sample to be tested can be calculated according to the following formula: Among them C AQDS , The unit is mM, n is the sample dilution multiple, A 387nm and A 325nm Represent the total absorbance at a wavelength of 387 nm and a wavelength of 325 nm, respectively.

10. A method for detecting the concentration of quinone compounds, characterized in that: When the quinone compound is 1,4-naphthoquinone (NQ), the concentrations of NQ and H2NQ in the sample to be tested can be calculated according to the following formula: Among them C NQ , The unit is mM, n is the sample dilution multiple, A 319nm and A 360nm Represent the total absorbance at wavelengths of 319 nm and 360 nm, respectively; When the quinone compound is 9,10-anthraquinone-2,6-disulfonic acid (AQDS), the concentrations of AQDS and AH2DS in the sample to be tested can be calculated according to the following formula: Among them C AQDS , The unit is mM, n is the sample dilution multiple, A 387nm and A 325nm Represent the total absorbance at a wavelength of 387 nm and a wavelength of 325 nm, respectively.

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