A method for separating multiple target objects in CE-UV detection process and application

Abstract

The present application belongs to the technical field of detection, and relates to a method for separating multiple target objects in a CE-UV detection process and application, wherein the multiple target objects are two or more of SES-RO107, VS-RO107, HES-RO107, SES-RY201, VS-RY201 and HES-RY201, and the method for separating multiple target objects in a CE-UV detection process is as follows: controlling the type and concentration of an additive in a background electrolyte, a separation voltage, an injection time, the type and concentration of a buffer substance in the background electrolyte, and the pH value of the background electrolyte; and the application is as follows: when the concentration of multiple target objects is detected in a reactive dye color matching process, the method described above is adopted. The method is simple, and can separate reactive dyes with similar structures.

Description

Technical Field

[0001] This invention belongs to the field of detection technology and relates to a method and application for separating multiple target objects during CE-UV detection. Background Technology

[0002] Reactive dyes can dye a variety of natural fibers (such as cotton, linen, wool, and silk) as well as some synthetic fibers (such as polyester and nylon). When using reactive dyes to color fibers to a specific shade or deep color, two or more different reactive dyes are usually selected for color blending. Compared with dyeing with a single dye, this color blending dyeing process has many advantages, such as high color fastness and low cost. Therefore, dyeing and printing plants widely use color blending dyes for fiber dyeing.

[0003] In actual dyeing and color matching processes, interactions between different dyes can lead to poor color stability, affecting the final dyeing effect. Therefore, close attention must be paid to the compatibility between dyes used in color matching; only dyes with good compatibility will have good pairing properties. Simultaneously, the differences in the physical and chemical properties of the dyes must be clearly defined during color matching, particularly the differences in fiber affinity for the dye and the differences in dye diffusion within the fiber. Significant differences will result in large color variations between different batches. The smaller the differences, the better the compatibility. Only by using reactive dyes with good compatibility can dyeing stability and repeatability be guaranteed. To assess the compatibility between different dyes, the absorption rate of each dye during the color matching process needs to be monitored; therefore, accurate and effective monitoring methods need to be developed.

[0004] For common cellulosic fibers, reactive dyes require the addition of alkali during the dyeing process to ionize the hydroxyl groups on the fibers, thus providing a basis for subsequent covalent bond formation with the reactive dye. It is important to note that under alkaline conditions, the reactive groups on the reactive dye can also react with OH groups in the solution. - Irreversible hydrolysis occurs, forming hydrolyzed dyes that lose their reactivity and cannot form covalent bonds with fibers to achieve stable color fixation. However, the hydrolyzed dyes still adsorb onto the fibers and diffuse within them, affecting not only the dye fixation rate and the wash fastness of the fabric but also causing environmental pollution and complicated post-processing. Therefore, in the process of color-blocking dyeing, to more accurately monitor the dyeing and hydrolysis of each dye, it is necessary to precisely detect the concentration of each color-blocking dye and its hydrolyzed derivatives in the dye bath. This information can then be used to improve the dyeing process and increase dye utilization.

[0005] Because structurally similar reactive dyes share many similar physical and chemical properties, many commonly used analytical methods cannot accurately detect them during color mixing and dyeing processes. For example, Reactive Yellow 201 and Reactive Orange 107... Figure 1 The diagram shows the structures of two reactive dyes and their activated and hydrolyzed forms. SES-RO107 is Reactive Orange 107, VS-RO107 is the activated form of Reactive Orange 107, and HES-RO107 is the hydrolyzed form of Reactive Orange 107. SES-RY201 is Reactive Yellow 201, VS-RY201 is the activated form of Reactive Yellow 201, and HES-RY201 is the hydrolyzed form of Reactive Yellow 201. Figure 1 It can be observed that the two reactive dyes themselves have similar structures. Furthermore, since the structures of reactive dyes and their activated and hydrolyzed derivatives are also similar, it can be found that all six dye forms are structurally similar to each other. For example... Figure 2 As shown, this structural similarity results in their having the same maximum UV-Vis absorption wavelength. Reference 3 (Application of Raman Spectroscopy in the Study of Reactive Dyeing Behavior of Cellulose Fibers [D]. Donghua University) records that when these two original dye forms are mixed, it is difficult to use a UV-spectrophotometer for multi-wavelength identification, making it impossible to accurately qualitatively and quantitatively determine each dye form produced during the dyeing process. While Raman spectroscopy can perform qualitative and quantitative detection based on the position and intensity changes of characteristic peaks between different dyes, for structurally similar reactive dyes, their characteristic peaks highly overlap. Similarly, taking Reactive Yellow 201 and Reactive Orange 107 as examples, Reference 3 records that the Raman spectral characteristic peaks of the two dye aqueous solutions highly overlap, with no other obvious characteristic peaks between them. Therefore, Raman spectroscopy cannot be directly used for qualitative and quantitative analysis of the dyeing process of this type of dye.

[0006] Among chromatographic methods, high-performance liquid chromatography (HPLC) is a widely used separation and detection method. However, because a large amount of neutral salt needs to be added to the dye bath for staining promotion during the dyeing process, the dye matrix is ​​relatively complex. Direct injection analysis will affect the life of the chromatographic column, and dialysis desalting is usually required. The pretreatment operation is relatively cumbersome and the analysis cost is also high, making it difficult to use rapidly and on a large scale for routine detection. It still cannot accurately detect reactive dyes with similar structures.

[0007] Compared to high-performance liquid chromatography (HPLC), capillary electrophoresis is a more convenient and efficient detection method. However, currently reported analytical methods primarily analyze reactive dyes with different structures. Based on the separation mechanism of capillary electrophoresis, the composition of the background electrolyte is the most crucial factor in this analytical method. In the past, when separating reactive dyes with different structures, the background electrolyte typically consisted of a simple buffer solution, and in some cases, other agents were added to improve the separation efficiency between target analytes.

[0008] For example, in reference 4 (Capillary zone electrophoresis analysis of chlorotriazinylreactive dyes in dyebath effluent[J].1997,34(4):307-319.), a background electrolyte of 10 mmol / L K2HPO4 (pH=9.20) was used to analyze two reactive dyes with different structures, monochlorotriazine and bis-chlorotriazine, and their hydrolysis products in waste dye bath and wastewater, and good separation results were achieved.

[0009] Reference 5 (Identification of reactive dyes in spent dyebaths and wastewater by capillary electrophoresis–mass spectrometry[J]. Journal of Chromatography A, 2000, 886(1-2):271-282.) used capillary electrophoresis to analyze five reactive dyes (including three monovinyl sulfone reactive dyes, one divinyl sulfone reactive dye, and one heterodireactive dye) and their hydrolysis products. The structures of these dyes are quite different from each other, and the separation effect is good.

[0010] Reference 6 (Analysis of Reactive Dyestuffs and Their Hydrolysis by Capillary Electrophoresis[J]. Analytical Sciences, 2008.) used a background electrolyte of 10 mmol / L Na2B4O7 + 50.0 mmol / L SDS (pH = 9.3) to analyze eight reactive dyes with different structures and their activation and hydrolysis products, achieving good separation results.

[0011] However, none of these analytical methods can separate reactive dyes with similar structures and their hydrolysis products. Capillary electrophoresis separates components based on differences in mobility and partition. The mobility of the target analyte is related to factors such as the type of background electrolyte solution (BGE), pH, ionic strength, viscosity, and temperature. Reactive dyes with different structures exhibit significant differences in their physical and chemical properties. For reactive dyes with similar structures, their physical and chemical properties are similar. Therefore, capillary electrophoresis, which can be used to detect reactive dyes with different structures and their hydrolysis products, cannot be directly applied to the detection of various forms of dyes during color matching of structurally similar reactive dyes.

[0012] Therefore, it is of great significance to study a method and application for separating multiple target substances in the CE-UV detection process to solve the above problems. Summary of the Invention

[0013] The purpose of this invention is to solve the problems existing in the prior art and to provide a method and application for separating multiple target objects in the CE-UV detection process.

[0014] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0015] A method for separating multiple target analytes during CE-UV detection, wherein the multiple target analytes are two or more selected from SES-RO107, VS-RO107, HES-RO107, SES-RY201, VS-RY201 and HES-RY201, wherein SES-RO107 is Reactive Orange 107, VS-RO107 is the activated form of Reactive Orange 107, HES-RO107 is the hydrolyzed form of Reactive Orange 107, SES-RY201 is Reactive Yellow 201, VS-RY201 is the activated form of Reactive Yellow 201, and HES-RY201 is the hydrolyzed form of Reactive Yellow 201;

[0016] When separating multiple target analytes, the background electrolyte used is an additive containing cholic acid derivatives at a concentration of 15–60 mmol / L. The cholic acid derivatives are sodium cholate or sodium deoxycholate. The separation voltage is 12–18 kV and the injection time is 9–21 s.

[0017] The background electrolyte uses sodium borate as a buffer, with a concentration of 20–40 mmol / L and a pH of 8.3–9.

[0018] Alternatively, the background electrolyte may use disodium hydrogen phosphate as a buffer, with a concentration of 20–40 mmol / L and a pH of 7–8.

[0019] Alternatively, ammonium chloride can be used as a buffer in the background electrolyte, with a concentration of 20–40 mmol / L and a pH of 8–9.

[0020] Alternatively, the background electrolyte may use Tris as a buffer, with a Tris concentration of 20–100 mmol / L and a pH of 7.2–8.9.

[0021] When the pH value of the background electrolyte is less than 8.00, the inner surface of the capillary is coated; otherwise, the inner surface of the capillary is either uncoated or coated.

[0022] When the pH of the background electrolyte is less than 8.00, the EOF (electroosmotic flow) of an uncoated capillary increases rapidly as the pH of the background electrolyte increases. In this case, a capillary with a coating on the inner wall is required to ensure the stability of EOF and migration time. However, when the pH of the background electrolyte is greater than or equal to 8.00, the EOF does not increase significantly. Therefore, there are no requirements for the inner surface of the capillary. The inner surface of the capillary can be coated or uncoated.

[0023] This invention adjusts the formulation, pH value, separation voltage, and injection time of the background electrolyte to achieve baseline separation for SES-RO107, VS-RO107, HES-RO107, SES-RY201, VS-RY201, and HES-RY201 when using the CE-UV method. This is because bile acid derivatives, as surfactants, can form micelles in the background electrolyte when their concentration reaches or exceeds the critical micelle concentration (CMC). During the analysis, multiple target analytes interact with the micelles, resulting in sufficiently large differences in mobility among the target analytes. Consequently, the migration speed and migration time of the multiple target analytes also differ significantly, thus achieving the separation of multiple target analytes.

[0024] As a preferred technical solution:

[0025] The method described above for separating multiple target substances during CE-UV detection has the following conditions: the concentration of bile acid derivatives in the background electrolyte is 45.0 mmol / L; the separation voltage is 15 kV; and the injection time is 15 s.

[0026] The method described above for separating multiple target substances during CE-UV detection uses sodium borate as a buffer in the background electrolyte, with a sodium borate concentration of 30.0 mmol / L and a pH value of 8.60.

[0027] As described above, a method for separating multiple target analytes during CE-UV detection involves using sodium borate as a buffer in the background electrolyte. The pH of the background electrolyte is adjusted using a boric acid solution (concentration 0.30–0.80 mol / L). Buffer solutions typically consist of a conjugate acid-base pair; boric acid and sodium borate are indeed conjugate acid-base pairs, and are therefore commonly used in buffer solution preparation. Adjusting the pH of the background electrolyte with boric acid ensures that the pH is within the buffering range of borate, guaranteeing better buffering capacity and maintaining a stable pH. If hydrochloric acid is used to adjust the background electrolyte… While the pH of the background electrolyte can achieve the same results as boric acid and also has a certain buffering capacity, it introduces chloride ions into the background electrolyte, which may interfere with and affect the desired experimental results. The sodium borate-boric acid buffer system does not introduce additional ions, making the experiment more accurate and reliable. Similarly, when disodium hydrogen phosphate is used as the background electrolyte, the pH is adjusted with sodium dihydrogen phosphate solution; when ammonium chloride is used as the background electrolyte, the pH is adjusted with ammonium hydroxide solution; and when Tris is used as the background electrolyte, the pH is adjusted with HCl solution.

[0028] This invention also provides a method for detecting the concentration of multiple target substances during the dyeing process using reactive dyes. In the dyeing process using two reactive dyes, a sample of the dye solution is taken at time t, the pH is adjusted to neutral and diluted, and then detected using the CE-UV method. The peak area of ​​each target substance in the dye solution sample is measured, and the concentration of each target substance in the dye solution at time t during the dyeing process is calculated (here, the target substance concentration refers to the target substance concentration after the dye solution is diluted). Time t can be any one or more times during the dyeing process, and the two reactive dyes are SES-RO107 and SES-RY201.

[0029] The target substances are two or more of the following: SES-RO107, VS-RO107, HES-RO107, SES-RY201, VS-RY201, and HES-RY201.

[0030] When using the CE-UV method for detection, a method for separating multiple target objects during the CE-UV detection process, as described in any of the above items, is employed.

[0031] As a preferred technical solution:

[0032] The method described above for detecting the concentration of multiple target analytes during reactive dye color matching is described above, wherein the peak position of each target analyte is determined by the standard addition method;

[0033] The calculation uses a linear equation, and the steps to obtain the result are as follows:

[0034] (1) Prepare solutions containing SES-RO107, VS-RO107, HES-RO107, SES-RY201, VS-RY201 and HES-RY201, and dilute them to different concentrations to obtain a series of standard mixtures with a concentration range of 5-100 mg / L for each target analyte.

[0035] (2) A series of standard mixtures were detected by CE-UV method to obtain a series of electrophoretic spectra. When the CE-UV method was used for detection, a method for separating multiple target substances during the CE-UV detection process as described in any of the above items was adopted.

[0036] (3) Linear fitting was performed on the peak area of ​​each target in a series of electrophoresis spectra and the corresponding concentration to obtain a linear equation.

[0037] As described above, in a method for detecting the concentration of multiple target analytes during reactive dye color matching, in step (3), the correlation coefficient R between the peak area of ​​each target analyte and its corresponding concentration is... 2 All values ​​are greater than 0.99. The intra-day precision of peak area is 1.3%–3.9%, the inter-day precision of peak area is 1.5%–4.7%, the intra-day precision of migration time is 0.20%–0.30%, the inter-day precision of migration time is 0.56%–0.73%, the limit of detection is 0.9–3.2 mg / L, and the limit of quantitation is 3.1–10.9 mg / L.

[0038] The method described above for detecting the concentration of multiple target substances during reactive dye color matching involves obtaining the concentration of multiple target substances in the dye solution at time t during the dyeing process, and then calculating the absorption rate of each reactive dye at time t during the dyeing process.

[0039] The formula for calculating the absorbance of SES-RO107 at time t during the staining process is as follows:

[0040]

[0041] In the formula, C 0a C represents the concentration of SES-RO107 in the staining solution at the start of staining. ta总 It is the sum of the concentrations of SES-RO107, VS-RO107, and HES-RO107 in the staining solution at time t during the staining process;

[0042] The formula for calculating the absorbance of SES-RY201 at time t during the staining process is as follows:

[0043]

[0044] In the formula, C 0b C represents the concentration of SES-RY201 in the staining solution at the start of staining. tb总 It is the sum of the concentrations of SES-RY201, VS-RY201, and HES-RY201 in the staining solution at time t during the staining process.

[0045] Beneficial effects:

[0046] (1) A method for separating multiple target substances during CE-UV detection according to the present invention can separate reactive dyes with similar structures;

[0047] (2) When the method of separating multiple target substances in the CE-UV detection process of the present invention is applied to the color matching dyeing process of reactive dyes, the concentration of reactive dyes with similar structures can be detected separately. Attached Figure Description

[0048] Figure 1 Schematic diagrams of the structures of SES-RO107, VS-RO107, HES-RO107, SES-RY201, VS-RY201 and HES-RY201;

[0049] Figure 2 The UV absorption curves are for aqueous solutions of six dye forms.

[0050] Figure 3 This is a schematic diagram of the dyeing process using vinyl sulfone-based reactive dyes;

[0051] Figure 4 Electrophoretic spectra of the VS-RO107 preparation process;

[0052] Figure 5 Electrophoretic spectra of the VS-RY201 preparation process;

[0053] Figure 6 Electrophoretic spectra of the HES-RO107 preparation process;

[0054] Figure 7 Electrophoretic spectra of the HES-RY201 preparation process;

[0055] Figure 8 Electrophoretic spectra of six target compounds (SES-RO107, VS-RO107, HES-RO107, SES-RY201, VS-RY201 and HES-RY201) separated at different sodium borate concentrations.

[0056] Figure 9 Electrophoretic spectra of six target analytes separated at different concentrations of sodium cholate when the concentration of sodium borate in the background electrolyte is 30.0 mmol / L.

[0057] Figure 10 Electrophoretic spectra of six target analytes separated at different pH values ​​when the concentration of sodium borate in the background electrolyte was 30.0 mmol / L and the concentration of sodium cholate was 45 mmol / L.

[0058] Figure 11 Electrophoretic spectra of six target analytes separated at different separation voltages under optimal background electrolyte conditions;

[0059] Figure 12 Electrophoretic spectra of six target analytes separated at different injection times when the optimal concentrations of sodium borate, sodium cholate, pH, and separation voltage in the background electrolyte were found.

[0060] Figure 13 Electrophoretic spectra showing the separation of six target analytes at different concentrations of disodium hydrogen phosphate; where the concentration of disodium hydrogen phosphate in a is 20.0 mmol / L, in b is 30.0 mmol / L, and in c is 40.0 mmol / L.

[0061] Figure 14 Electrophoretic spectra of six target analytes separated at different concentrations of sodium cholate when the concentration of disodium hydrogen phosphate in the background electrolyte was 20.0 mmol / L; where the concentration of sodium cholate in a was 15.0 mmol / L, the concentration of sodium cholate in b was 30.0 mmol / L, and the concentration of sodium cholate in c was 60.0 mmol / L.

[0062] Figure 15 Electrophoretic spectra of six target analytes separated at different pH values ​​when the concentration of disodium hydrogen phosphate in the background electrolyte is 20.0 mmol / L and the concentration of sodium cholate is 30.0 mmol / L; where pH = 7.00 in a, pH = 7.50 in b, and pH = 8.00 in c.

[0063] Figure 16 Electrophoretic spectra showing the separation of six target analytes at different ammonium chloride concentrations; where the ammonium chloride concentration in a is 20.0 mmol / L, in b is 30.0 mmol / L, and in c is 40.0 mmol / L.

[0064] Figure 17 Electrophoretic spectra of six target analytes separated at different sodium cholate concentrations when the ammonium chloride concentration in the background electrolyte was 30.0 mmol / L; where sodium cholate concentration was 15.0 mmol / L in a, 30.0 mmol / L in b, and 60.0 mmol / L in c.

[0065] Figure 18 Electrophoretic spectra of six target analytes separated at different pH values ​​when the background electrolyte contains 30.0 mmol / L of both ammonium chloride and sodium cholate; where pH = 8.00 in a, pH = 8.50 in b, and pH = 9.00 in c.

[0066] Figure 19 Electrophoretic spectra of six target analytes separated at different Tris concentrations; where the Tris concentration in a is 20.0 mmol / L, the Tris concentration in b is 50.0 mmol / L, and the Tris concentration in c is 100.0 mmol / L.

[0067] Figure 20 The following are electrophoretic spectra of six target analytes separated at different sodium cholate concentrations when the Tris concentration in the background electrolyte is 50.0 mmol / L; where the sodium cholate concentration in a is 15.0 mmol / L, in b is 30.0 mmol / L, and in c is 60.0 mmol / L.

[0068] Figure 21 Electrophoretic spectra of six target analytes separated at different pH values ​​when the Tris concentration in the background electrolyte is 50.0 mmol / L and the sodium cholate concentration is 30.0 mmol / L; where pH = 7.2.00 in a, pH = 8.00 in b, and pH = 8.90 in c.

[0069] Figure 22 Electrophoretic spectra showing the effect of different types of buffer solutions on the separation effect;

[0070] Figure 23 Electrophoretic spectra showing the effect of different concentrations of sodium borate solution on the separation effect;

[0071] Figure 24 Electrophoretic spectra showing the effect of different concentrations of sodium deoxycholate on the separation effect;

[0072] Figure 25 This is a flowchart of the dyeing process;

[0073] Figure 26 Electrophoretic patterns of the dyeing process using two reactive dyes;

[0074] Figure 27 This is a graph showing the absorption rate of two reactive dyes during the dyeing process.

[0075] Figure 28 Electrophoretic patterns of SES-RO107 single staining process;

[0076] Figure 29 Electrophoretic patterns of SES-RY201 single staining process;

[0077] Figure 30 The absorption rate curve of SES-RO107 single dyeing process;

[0078] Figure 31 This is a graph showing the absorption rate of SES-RY201 during the single dyeing process. Detailed Implementation

[0079] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0080] To facilitate the representation of each target object, this invention refers to Reference 1 (Capillary electrophoretic analysis of the reactions of bifunctional reactive dyes under various conditions including a study of the analysis of the traditionally difficult to analyze phthalocyanine dyes[J]. Journal of Chromatography A, 1995, 706(1–2): 555-62.) and Reference 2 (The application of heterobifunctional reactive dyes to nylon6,6: process modifications to achieve high efficiencies[J]. Dyes and Pigments, 2001, 48(3): 245-51.) to label their names. After labeling, those skilled in the art can fully understand them. The English abbreviations of these target objects consist of two parts: "the corresponding form of name abbreviation + the name abbreviation of the reactive dye".

[0081] The two structurally similar reactive dyes used in this invention are both vinyl sulfone type reactive dyes. "SES" is an abbreviation for "β-sulfatoethyl sulfone," representing the original form of the vinyl sulfone type reactive dye, containing relatively stable reactive groups. In alkaline solution, it undergoes an elimination reaction to generate vinyl sulfone (VS) form. "VS" is an abbreviation for "vinyl sulfone," the activated form of the dye. It can react with hydroxyl groups on cellulose to complete the dyeing process. However, under dyeing conditions (usually high temperature and alkaline solution), it can also react with OH groups in the solution. - It undergoes a reaction and hydrolyzes, forming its hydrolyzed form; "HES" is an abbreviation for "β-hydroxyethyl sulfone," which is the hydrolyzed form of the dye. For example... Figure 3 As shown, the abbreviations for reactive dyes are: RO107 is CIReactive Orange 107, RY201 is CIReactive Yellow 201. Therefore, SES-RO107 is the original form of CIReactive Orange 107, and so on.

[0082] The instruments used in the methods and applications of this invention are mainly: a shaking water bath, a capillary electrophoresis apparatus, an ultraviolet detector, and a computer for data acquisition / processing.

[0083] In the following text, when confirming the peaks of each target analyte in the electrophoresis pattern, the migration time of each target analyte is different in different electrophoresis patterns due to the constantly changing test conditions. Therefore, the target analyte is determined by the standard addition method. Specifically, the concentrations of the other five analytes are kept constant, and only the concentration of one analyte is added. The analyte whose peak height and peak area increase is the analyte that has been spiked (this technique is existing technology).

[0084] The method for separating multiple target substances during CE-UV detection, as described in this invention, was obtained through a series of exploratory experiments, as follows:

[0085] (1) Preparation of the stock solution of the target substance;

[0086] SES-RO107 stock solution: Accurately weigh 25.0 mg of SES-RO107 and dissolve it in deionized water, then transfer it to a 25 mL volumetric flask to prepare a SES-RO107 stock solution with a concentration of 1.0 g / L.

[0087] The stock solution for SES-RY201 is prepared in a similar manner to that for SES-RO107, with the only difference being the dye.

[0088] Stock solution of VS-RO107: First, accurately weigh 10.0 mg of SES-RO107, then add SES-RO107 to 2 mL of 0.01 mol / L sodium hydroxide solution. React at room temperature for 10 min, then add 0.1 mol / L HCl solution to neutralize the solution to pH 7. Finally, transfer to a 10 mL volumetric flask and dilute to volume with deionized water to prepare a stock solution of 1.0 g / L VS-RO107. To verify the successful preparation of VS-RO107, samples were taken at 0, 1, 5, and 10 mins during the preparation process, neutralized, and analyzed. Electrophoresis patterns are shown below. Figure 4 As shown, after the addition of alkali, the original form of the reactive dye is rapidly converted into its reactive form, and after 10 minutes of reaction, the dye is completely converted into its reactive form.

[0089] The stock solution for VS-RY201 was prepared in a similar manner to that for VS-RO107, with the only difference being the dye. To verify the success of VS-RY201 preparation, samples were taken at 0, 1, 5, and 10 minutes during the preparation process, neutralized, and then analyzed. The electrophoretic patterns are shown below. Figure 5 As shown;

[0090] HES-RO107 stock solution: First, accurately weigh 50.0 mg of SES-RO107 and dissolve it in 20 mL of water. Then, add 4 mL of 0.1 mol / L sodium hydroxide solution and react in a water bath at 60°C for 20 min. After the reaction is complete, cool to room temperature and add 0.1 mol / L HCl solution dropwise to neutralize the solution to pH 7. Finally, transfer the solution to a 50 mL volumetric flask and add deionized water to make up to the final volume, thus preparing a 1.0 g / L HES-RO107 stock solution. To verify the success of the HES-RO107 preparation, samples were taken during the preparation process under the same conditions, neutralized and diluted, and then analyzed. Figure 6 As shown, after the addition of alkali, the original form of the reactive dye quickly disappeared and was completely converted into its reactive form; at the same time, the generated reactive form underwent a hydrolysis reaction under hot alkali conditions and was converted into its hydrolyzed form. After 20 minutes of reaction, it was completely converted into the hydrolyzed form.

[0091] The stock solution for HES-RY201: The preparation process is basically the same as that for HES-RO107, only the dye is different; the electrophoretic pattern of the preparation process is as follows. Figure 7 As shown;

[0092] (2) Exploration of analytical methods;

[0093] Equal volumes of the stock solutions of the six target analytes were mixed to obtain the test solution, and the conditions for separation of the six target analytes were investigated using CE-UV.

[0094] (2.1) Sodium cholate was used as an additive to the background electrolyte, and sodium borate was used as a buffer for the background electrolyte. The concentrations of sodium cholate and sodium borate, separation voltage, injection time, and pH of the background electrolyte were investigated. The details are as follows:

[0095] (2.11) Investigate the concentration of sodium borate;

[0096] Initially, a capillary tube with an inner diameter of 50 μm, a length of 50 cm, and an effective length of 40 cm was selected; the separation voltage was 15 kV; the injection pressure was 0.5 psi, and the injection time was 15 s; the concentration of sodium cholate was 50.0 mmol / L; the pH of the background electrolyte was 8.60, which was adjusted with 0.4 mol / L boric acid solution.

[0097] The concentration of sodium borate was adjusted within a certain range to obtain the separation results of six analytes;

[0098] like Figure 8 As shown, with the increase of sodium borate concentration, the migration time of HES-RO107 (corresponding to peak 1), HES-RY201 (corresponding to peak 2), VS-RO107 (corresponding to peak 3), VS-RY201 (corresponding to peak 4), SES-RO107 (corresponding to peak 5), and SES-RY201 (corresponding to peak 6) increases, and the resolution also increases. However, when the sodium borate concentration is too high, it will increase the working current and Joule heating, resulting in peak broadening. Therefore, the concentration of sodium borate should be controlled between 20 and 40 mmol / L, with 30.0 mmol / L being the optimal separation concentration.

[0099] (2.12) Investigate the concentration of sodium cholate;

[0100] Initially, a capillary tube with an inner diameter of 50 μm, a length of 50 cm, and an effective length of 40 cm was selected; the separation voltage was 15 kV; the injection pressure was 0.5 psi, and the injection time was 15 s; the concentration of sodium borate was 30.0 mmol / L; the pH of the background electrolyte was 8.60, which was adjusted with 0.4 mol / L boric acid solution.

[0101] The concentration of sodium cholate was adjusted within a certain range to obtain the separation results of six analytes;

[0102] like Figure 9 As shown, with the increase of sodium cholate concentration, the electroosmotic flow decreases, the migration time of each target increases, and the separation degree between each target also increases; therefore, the concentration of sodium cholate should be controlled between 15.0-60.0 mmol / L, among which, considering factors such as separation degree, migration time, and peak size, 45 mmol / L is the optimal separation concentration.

[0103] (2.13) Investigate the pH value of the background electrolyte;

[0104] Initially, a capillary tube with an inner diameter of 50 μm, a length of 50 cm, and an effective length of 40 cm was selected; the separation voltage was 15 kV; the injection pressure was 0.5 psi, the injection time was 15 s; the concentration of sodium borate was 30.0 mmol / L; and the concentration of sodium cholate was 45 mmol / L.

[0105] Within a certain range, the pH value of the background electrolyte was adjusted to obtain the separation of six analytes;

[0106] like Figure 10 As shown, within the pH range of 8.3–9, as pH increases, electroosmotic flow increases, the migration rate of each target analyte accelerates, and the separation degree slightly decreases. However, at pH 9.50, the addition of NaOH increases the ionic strength of the background electrolyte, leading to an increase in the working current and generating significant Joule heating, resulting in peak broadening and decreased sensitivity. Simultaneously, SES-RO107 and SES-RY201 are unstable under this pH condition, and their peaks almost disappear. Specifically, within the pH range of 8.3–9, adjustment was achieved using 0.4 mol / L boric acid, while at 9.50, adjustment was achieved using 0.1 mol / L NaOH. Therefore, a pH range of 8.3–9 is suitable, and after comprehensive comparison, the separation performance is optimal at pH 8.60.

[0107] (2.14) Investigate the separation voltage;

[0108] Initially, a capillary tube with an inner diameter of 50 μm, a length of 50 cm, and an effective length of 40 cm was selected; the injection pressure was 0.5 psi, and the injection time was 15 s; the concentration of sodium borate was 30.0 mmol / L; the concentration of sodium cholate was 45 mmol / L; the pH of the background electrolyte was 8.60, which was adjusted with 0.4 mol / L boric acid solution.

[0109] Within a certain range, the separation voltage was adjusted to obtain the separation results of six analytes;

[0110] like Figure 11 As shown, with the increase of voltage, the migration time and separation degree of each target object decrease. At the same time, excessively high voltage will result in a large operating current, generate more Joule heat, and have a large baseline noise. Therefore, 12V to 18KV is suitable, among which 15kV is the optimal separation voltage.

[0111] (2.15) Investigate the injection time;

[0112] Initially, a capillary tube with an inner diameter of 50 μm, a length of 50 cm, and an effective length of 40 cm was selected; the separation voltage was 15 kV; the injection pressure was 0.5 psi, and the injection time was 15 s; the concentration of sodium borate was 30.0 mmol / L; the concentration of sodium cholate was 45 mmol / L; the pH of the background electrolyte was 8.60, adjusted with 0.4 mol / L boric acid solution.

[0113] Within a certain range, the injection time was adjusted to obtain the separation results of the six analytes;

[0114] like Figure 12 As shown, with the increase of injection time, the peak shape increases, the baseline noise increases, and the resolution decreases slightly; therefore, 9s to 21s is suitable, with 15s being the optimal injection time.

[0115] Comprehensive analysis shows that when sodium cholate is used as an additive to the background electrolyte and sodium borate is used as a buffer, the optimal separation conditions are: sodium cholate concentration in the background electrolyte is 15–60 mmol / L, sodium borate concentration is 20–40 mmol / L, pH value is 8.3–9, separation voltage is 12–18 kV, and injection time is 9–21 s. The optimal separation conditions are: sodium cholate concentration in the background electrolyte is 45 mmol / L, sodium borate concentration is 30.0 mmol / L, pH value is 8.6, separation voltage is 15 kV, and injection time is 15 s.

[0116] (2.2) Sodium cholate was used as an additive to the background electrolyte, and disodium hydrogen phosphate was used as a buffer substance for the background electrolyte. The concentrations of sodium cholate, disodium hydrogen phosphate, and the pH value of the background electrolyte were investigated, as follows:

[0117] (2.21) Investigate the concentration of disodium hydrogen phosphate;

[0118] Initially, a capillary tube with an inner diameter of 50 μm, a length of 50 cm, and an effective length of 40 cm was selected. The separation voltage was 15 kV; the injection pressure was 0.5 psi; the injection time was 15 s; the concentration of sodium cholate was 60.0 mmol / L; and the pH was 7.00, adjusted with 0.10 mol / L sodium dihydrogen phosphate.

[0119] Within a certain range, the concentration of disodium hydrogen phosphate was adjusted to obtain the separation results of six analytes;

[0120] like Figure 13As shown, for 1-HES-RO107, 2-HES-RY201, 3-VS-RO107, 4-VS-RY201, 5-SES-RO107, and 6-SES-RY201, the migration time of each target analyte continuously increases with the increase of disodium hydrogen phosphate (as a buffer). Although the separation between the target analytes continuously increases, the peak shape deteriorates and the baseline noise is relatively large. Therefore, the concentration of disodium hydrogen phosphate is selected to be 20-40 mmol / L, and the optimal separation concentration is selected as 20.0 mmol / L.

[0121] (2.22) Investigate the concentration of sodium cholate;

[0122] Initially, a capillary tube with an inner diameter of 50 μm, a length of 50 cm, and an effective length of 40 cm was selected. The separation voltage was 15 kV; the injection pressure was 0.5 psi; the injection time was 15 s; the concentration of disodium hydrogen phosphate was 20.0 mmol / L; and the pH was 7.00, adjusted with 0.10 mol / L sodium dihydrogen phosphate.

[0123] Within a certain range, the concentration of sodium cholate was adjusted to obtain the separation results of the six analytes;

[0124] like Figure 14 As shown, with the continuous increase of sodium cholate concentration, the migration time and resolution of each target analyte continuously increase; however, when the concentration is too high, the peak shape deteriorates and the baseline noise increases; therefore, the concentration of sodium cholate is selected to be 15.0–60.0 mmol / L. Considering factors such as migration time, resolution, and peak shape, the optimal separation concentration is selected as 30.0 mmol / L.

[0125] (2.23) Investigate the pH value of the background electrolyte;

[0126] Initially, a capillary tube with an inner diameter of 50 μm, a length of 50 cm, and an effective length of 40 cm was selected. The separation voltage was 15 kV; the injection pressure was 0.5 psi; the injection time was 15 s; the concentration of sodium cholate was 30.0 mmol / L; and the concentration of disodium hydrogen phosphate was 20.0 mmol / L.

[0127] Within a certain range, the pH value of the background electrolyte was adjusted with 0.10 mol / L sodium dihydrogen phosphate to obtain the separation of six analytes;

[0128] like Figure 15As shown, with increasing pH, electroosmosis increases, the migration rate of each target increases, and the migration time continuously decreases, but the resolution also decreases slightly. At the same time, with increasing pH, due to the increased ionic strength of the background electrolyte, the baseline noise increases and the peak shape broadens, and the sensitivity of each target decreases. Therefore, a pH of 7-8 is suitable. Considering factors such as migration time, resolution, and peak shape, the separation is optimal at pH = 7.50.

[0129] Comprehensive analysis shows that when sodium cholate is used as an additive in the background electrolyte and disodium hydrogen phosphate is used as a buffer in the background electrolyte, the separation conditions are: the concentration of sodium dihydrogen phosphate in the background electrolyte is 20–40 mmol / L, the concentration of sodium cholate in the background electrolyte is 15.0–60.0 mmol / L, and the pH value of the background electrolyte is 7–8; the optimal separation conditions are: the concentration of sodium dihydrogen phosphate in the background electrolyte is 20.0 mmol / L, the concentration of sodium cholate in the background electrolyte is 30.0 mmol / L, and the pH value of the background electrolyte is 7.50.

[0130] (2.3) Sodium cholate was used as an additive to the background electrolyte, and ammonium chloride was used as a buffer substance for the background electrolyte. The concentrations of sodium cholate, ammonium chloride, and pH value of the background electrolyte were investigated, as follows:

[0131] (2.31) Investigate the concentration of ammonium chloride;

[0132] Initially, a capillary tube with an inner diameter of 50 μm, a length of 50 cm, and an effective length of 40 cm was selected. The separation voltage was 15 kV; the injection pressure was 0.5 psi; the injection time was 15 s; the concentration of sodium cholate was 30.0 mmol / L; and the pH was 8.50, adjusted with 0.20 mol / L ammonium hydroxide solution.

[0133] Within a certain range, the concentration of ammonium chloride was adjusted to obtain the separation results of the six analytes;

[0134] like Figure 16 As shown, for 1-HES-RO107, 2-HES-RY201, 3-VS-RO107, 4-VS-RY201, 5-SES-RO107, and 6-SES-RY201, the separation degree of each target analyte slightly increased with increasing ammonium chloride concentration, while the migration time also increased continuously. However, excessively high concentrations also caused peak broadening and decreased sensitivity. Therefore, a concentration of 20–40.0 mmol / L is suitable, and considering both separation degree and migration time, 30.0 mmol / L ammonium chloride was selected as the optimal separation concentration.

[0135] (2.32) Investigate the concentration of sodium cholate;

[0136] Initially, a capillary tube with an inner diameter of 50 μm, a length of 50 cm, and an effective length of 40 cm was selected. The separation voltage was 15 kV; the injection pressure was 0.5 psi; the injection time was 15 s; the concentration of ammonium chloride was 30.0 mmol / L; and the pH was 8.50, adjusted with 0.20 mol / L ammonium hydroxide solution.

[0137] Within a certain range, the concentration of sodium cholate was adjusted to obtain the separation of the six analytes;

[0138] like Figure 17 As shown, the separation degree of each target analyte increases slightly with increasing sodium cholate concentration; however, when the concentration is too high, the ionic strength of the background electrolyte increases, which not only makes the migration time of each target analyte too long, but also broadens the peaks of each target analyte, resulting in a decrease in sensitivity; therefore, a concentration of 15.0–60.0 mmol / L is suitable, and considering factors such as separation degree and migration time, 30.0 mmol / L is selected as the optimal separation degree.

[0139] (2.33) Investigate the pH value of the background electrolyte;

[0140] Initially, a capillary with an inner diameter of 50 μm, a length of 50 cm, and an effective length of 40 cm was selected. The separation voltage was 15 kV; the injection pressure was 0.5 psi; the injection time was 15 s; the concentration of ammonium chloride was 30.0 mmol / L; and the concentration of sodium cholate was 30.0 mmol / L.

[0141] Within a certain range, the pH value of the background electrolyte was adjusted using 0.20 mol / L ammonium hydroxide solution to obtain the separation of six analytes;

[0142] like Figure 18 As shown, with the increase of pH, electroosmosis increases, the migration speed of each target analyte continuously accelerates, the migration time continuously decreases, and the separation degree also decreases slightly; therefore, a pH of 8-9 is suitable, and after comprehensive comparison, the separation is best at pH=8.50.

[0143] Comprehensive analysis shows that when sodium cholate is used as an additive in the background electrolyte and ammonium chloride is used as a buffer in the background electrolyte, the separation conditions are: the concentration of ammonium chloride in the background electrolyte is 20–40.0 mmol / L, the concentration of sodium cholate in the background electrolyte is 15.0–60.0 mmol / L, and the pH value of the background electrolyte is 8–9; the optimal separation conditions are: the concentration of ammonium chloride in the background electrolyte is 30.0 mmol / L, the concentration of sodium cholate in the background electrolyte is 30.0 mmol / L, and the pH value of the background electrolyte is 8.50.

[0144] (2.4) Sodium cholate was used as an additive to the background electrolyte, and Tris was used as a buffer substance for the background electrolyte. The concentrations of sodium cholate, Tris, and the pH value of the background electrolyte were investigated, as follows:

[0145] (2.41) Investigate the concentration of Tris;

[0146] Initially, a capillary tube with an inner diameter of 50 μm, a length of 50 cm, and an effective length of 40 cm was selected. The separation voltage was 15 kV; the injection pressure was 0.5 psi; the injection time was 15 s; the concentration of sodium cholate was 30.0 mmol / L; and the pH was 8.00, adjusted with 0.10 mol / L hydrochloric acid.

[0147] Within a certain range, the concentration of Tris was adjusted to obtain the separation results of the six analytes;

[0148] like Figure 19 As shown, for 1-HES-RO107, 2-HES-RY201, 3-VS-RO107, 4-VS-RY201, 5-SES-RO107, and 6-SES-RY201, the migration time of each target analyte continuously increases with the increase of Tris concentration, and the resolution also increases. However, when the concentration is too high, more HCl needs to be added to adjust the pH, which increases the ionic strength of the background electrolyte, causing peak broadening and higher baseline noise. Therefore, a concentration of 20.0–100.0 mmol / L is suitable, and considering factors such as resolution and migration time, 50.0 mmol / L is selected as the optimal resolution.

[0149] (2.42) Investigate the concentration of sodium cholate;

[0150] Initially, a capillary with an inner diameter of 50 μm, a length of 50 cm, and an effective length of 40 cm was selected. The separation voltage was 15 kV; the injection pressure was 0.5 psi; the injection time was 15 s; the concentration of Tris was 50.0 mmol / L; and the pH was 8.00, adjusted with 0.10 mol / L hydrochloric acid.

[0151] Within a certain range, the concentration of sodium cholate was adjusted to obtain the separation of the six analytes;

[0152] like Figure 20As shown, the separation of each target analyte increases with increasing sodium cholate concentration, but the migration time also increases. At lower sodium cholate concentrations, both SES-RO107 and SES-RY201 exhibit peak broadening and delayed migration times. However, excessively high concentrations can lead to significant baseline noise and excessively long migration times. Therefore, a concentration range of 15.0–60.0 mmol / L is suitable, and considering factors such as separation and migration time, 30.0 mmol / L is selected as the optimal separation level.

[0153] (2.43) Investigate the pH value of the background electrolyte;

[0154] Initially, a capillary with an inner diameter of 50 μm, a length of 50 cm, and an effective length of 40 cm was selected. The separation voltage was 15 kV; the injection pressure was 0.5 psi; the injection time was 15 s; the concentration of Tris was 50.0 mmol / L; and the concentration of sodium cholate was 30.0 mmol / L.

[0155] Within a certain range, the pH value of the background electrolyte was adjusted with 0.10 mol / L hydrochloric acid to obtain the separation of six analytes;

[0156] like Figure 21 As shown, with increasing pH, electroosmosis increases, the migration time of each target analyte decreases, and the separation degree also decreases. Therefore, a pH of 7.2–8.9 is suitable. Considering factors such as migration time and separation degree, the separation is optimal at pH 8.00.

[0157] Comprehensive analysis shows that when sodium cholate is used as an additive to the background electrolyte and Tris is used as a buffer, the optimal separation conditions are: Tris concentration in the background electrolyte is 20.0–100.0 mmol / L, sodium cholate concentration is 15.0–60.0 mmol / L, and pH is 7.2–8.9. The optimal separation conditions are: Tris concentration is 50.0 mmol / L, sodium cholate concentration is 30.0 mmol / L, and pH is 8.00.

[0158] Furthermore, this invention further investigated the concentration of sodium cholate and found that when the concentration of sodium cholate was below its critical micelle concentration, the six target compounds could not be separated. The specific process is as follows:

[0159] (a) Select a capillary tube with an inner diameter of 50 μm, a length of 50 cm, and an effective length of 40 cm; the separation voltage is 15 kV; the injection pressure is 0.5 psi; and the injection time is 15 s. Figure 22The figure shows the electrophoretic spectra of the effect of different types of buffer solutions on the separation effect. In case a, the background electrolyte includes 10 mmol / L Na₂HPO₄ and 10 mmol / L NaH₂PO₄, with the pH adjusted to 6.93; in case b, the background electrolyte includes 20.0 mmol / L Na₂B₄O₇, with the pH adjusted to 9 using 0.4 mol / L boric acid solution; and in case c, the background electrolyte includes 20.0 mmol / L ammonium acetate, with the pH adjusted to 9 using 0.1 mol / L NaOH solution. The figure shows that when the background solution does not contain sodium cholate, the six target analytes cannot be separated.

[0160] (b) Select a capillary tube with an inner diameter of 50 μm, a length of 50 cm, and an effective length of 40 cm; the separation voltage is 15 kV; the injection pressure is 0.5 psi; and the injection time is 15 s. Figure 23 The image shows the electrophoretic spectra of the effect of different concentrations of sodium borate solution on the separation effect; the background electrolytes contained 20.0 mmol / L Na₂B₄O₇, 30.0 mmol / L Na₂B₄O₇, 40.0 mmol / L Na₂B₄O₇, and 50.0 mmol / L Na₂B₄O₇; the pH was adjusted to 9 with 0.4 mol / L boric acid solution. Figure 23 As can be seen, when the background electrolyte does not contain sodium cholate, the six target compounds cannot be separated;

[0161] Figure 22 and Figure 23 All studies used buffer solutions as background electrolytes without adding any other additives. Comprehensive analysis showed that even with changes in the concentration of buffer solutions such as phosphate, borate, and ammonium chloride, the six target analytes could not be separated when used alone. This also verifies that the CE method, which can be used to separate reactive dyes with different structures and their hydrolysis products, may not necessarily be directly applicable to the analysis of reactive dyes with similar structures.

[0162] Furthermore, this invention also investigated the use of sodium deoxycholate instead of sodium cholate, and found that the two have similar roles in the separation process, as detailed below:

[0163] A capillary tube with an inner diameter of 50 μm, a length of 50 cm, and an effective length of 40 cm was selected. The separation voltage was 15 kV; the injection pressure was 0.5 psi; the injection time was 15 s; the concentration of sodium borate in the background electrolyte was 30.0 mmol / L; and the pH was adjusted to 8.6 with 0.4 mol / L boric acid solution.

[0164] like Figure 24The image shows the electrophoretic spectra of the effect of different concentrations of sodium deoxycholate solution on the separation effect. When the concentration of sodium deoxycholate is in the range of 15–60.0 mmol / L, the migration time of each target analyte gradually increases with the increase of sodium deoxycholate solution concentration, but all six target analytes can be completely separated. However, without the addition of sodium deoxycholate (concentration of 0 mmol / L), the six target analytes cannot achieve baseline separation. This comparative result highlights the importance of sodium deoxycholate in the separation process. Therefore, sodium deoxycholate, also a derivative of cholic acid, plays a similar role to sodium cholate when added to the background electrolyte, and can play a key role in the separation of dyes with similar structures.

[0165] Based on the above research, this invention proposes a method for separating multiple target substances during CE-UV detection. The multiple target substances are two or more of SES-RO107, VS-RO107, HES-RO107, SES-RY201, VS-RY201, and HES-RY201. SES-RO107 is Reactive Orange 107, VS-RO107 is the activated form of Reactive Orange 107, HES-RO107 is the hydrolyzed form of Reactive Orange 107, SES-RY201 is Reactive Yellow 201, VS-RY201 is the activated form of Reactive Yellow 201, and HES-RY201 is the hydrolyzed form of Reactive Yellow 201.

[0166] The background electrolyte uses cholic acid derivatives as additives, with a concentration of 15–60 mmol / L. The cholic acid derivatives are sodium cholate or sodium deoxycholate. The separation voltage is 12–18 kV, and the injection time is 9–21 s.

[0167] The background electrolyte uses sodium borate as a buffer, with a concentration of 20–40 mmol / L and a pH of 8.3–9. The pH of the background electrolyte is adjusted using boric acid solution (concentration of 0.30–0.80 mol / L).

[0168] Alternatively, sodium dihydrogen phosphate can be used as a buffer in the background electrolyte, with a concentration of 20–40 mmol / L and a pH of 7–8; the pH of the background electrolyte can be adjusted using disodium hydrogen phosphate solution or sodium hydroxide solution.

[0169] Alternatively, ammonium chloride can be used as a buffer in the background electrolyte, with a concentration of 20–40 mmol / L and a pH of 8–9; the pH of the background electrolyte can be adjusted using ammonium hydroxide solution.

[0170] Alternatively, the background electrolyte uses Tris as a buffer, with a Tris concentration of 20–100 mmol / L and a pH of 7.2–8.9; the pH of the background electrolyte is adjusted using HCl solution.

[0171] When the pH value of the background electrolyte is less than 8.00, the inner surface of the capillary is coated; otherwise, the inner surface of the capillary is either uncoated or coated.

[0172] When sodium borate is used as the buffer in the background electrolyte, the pH of the background electrolyte is adjusted with boric acid solution; when disodium hydrogen phosphate is used as the buffer in the background electrolyte, the pH of the background electrolyte is adjusted with sodium dihydrogen phosphate solution or sodium hydroxide solution; when ammonium chloride is used as the buffer in the background electrolyte, the pH of the background electrolyte is adjusted with ammonium hydroxide solution; when Tris is used as the buffer in the background electrolyte, the pH of the background electrolyte is adjusted with HCl solution.

[0173] Based on the above research, this invention also proposes a method for detecting the concentration of multiple target substances during reactive dye color matching dyeing, the specific steps of which are as follows:

[0174] (1) Obtaining the linear equation;

[0175] (1.1) Prepare solutions containing SES-RO107, VS-RO107, HES-RO107, SES-RY201, VS-RY201 and HES-RY201, and dilute them to different concentrations to obtain a series of standard mixtures with a concentration range of 5-100 mg / L for each target analyte.

[0176] If sodium sulfate (neutral salt) is added during the reactive dye color matching process to promote staining, the same concentration of sodium sulfate (neutral salt) needs to be added to the standard mixture to eliminate the influence of sodium sulfate (neutral salt) on the analytical method. This is mainly because sodium sulfate, as a strong electrolyte, will increase the ionic strength of the solution after ionization, resulting in an increase in working current and excessive Joule heat, which leads to peak broadening and affects the peak area.

[0177] (1.2) A series of standard mixtures were detected by CE-UV method to obtain a series of electrophoretic spectra; wherein, when using CE-UV method for detection, a method described above for separating multiple target substances during CE-UV detection was adopted;

[0178] (1.3) Linear fitting was performed on the peak area and corresponding concentration of each target in a series of electrophoresis spectra to obtain a linear equation; the elution position of each target was determined by the standard addition method; the correlation coefficient R between the peak area and corresponding concentration of each target was calculated. 2All values ​​were greater than 0.99; the intra-day precision of peak area was 1.3%–3.9%; the inter-day precision of peak area was 1.5%–4.7%; the intra-day precision of migration time was 0.20%–0.30%; the inter-day precision of migration time was 0.56%–0.73%; the limit of detection was 0.9–3.2 mg / L; and the limit of quantitation was 3.1–10.9 mg / L.

[0179] (2) Test the concentration of the target substance;

[0180] In the dyeing process using two reactive dyes, a sample of the dye solution is taken at time t, the pH is adjusted to neutral and diluted, and then detected by the CE-UV method. The peak area of ​​each target substance in the dye solution sample is measured, and the concentration of each target substance in the dye solution at time t during the dyeing process is calculated. Time t can be any one or more times during the dyeing process.

[0181] Among them, the two reactive dyes are SES-RO107 and SES-RY201; the multiple target compounds are two or more of SES-RO107, VS-RO107, HES-RO107, SES-RY201, VS-RY201 and HES-RY201; the peak position of each target compound is determined by the standard addition method;

[0182] When using the CE-UV method for detection, a method described above is employed to separate multiple target substances during the CE-UV detection process.

[0183] (3) Calculate the absorption rate of each reactive dye at time t during the dyeing process;

[0184] The formula for calculating the absorbance of SES-RO107 at time t during the staining process is as follows:

[0185]

[0186] In the formula, C 0a C represents the concentration of SES-RO107 in the staining solution at the start of staining. ta总 It is the sum of the concentrations of SES-RO107, VS-RO107, and HES-RO107 in the staining solution at time t during the staining process;

[0187] The formula for calculating the absorbance of SES-RY201 at time t during the staining process is as follows:

[0188]

[0189] In the formula, C 0b C represents the concentration of SES-RY201 in the staining solution at the start of staining. tb总It is the sum of the concentrations of SES-RY201, VS-RY201, and HES-RY201 in the staining solution at time t during the staining process.

[0190] The method for detecting the concentration of multiple target substances during reactive dye color matching is described below with specific examples:

[0191] Dyeing processes such as Figure 25 As shown, the dye bath (100.0 mL in volume, composed of dye and deionized water, with a dye concentration of 2.0 g / L, and the dye being a mixture of Reactive Orange 107 and Reactive Yellow 201 in a 1:1 mass ratio) was placed in a shaking water bath. The initial temperature of the shaking water bath was set to 30°C. The fabric (plain cotton fabric, 5.0 g in mass) was immersed in the dye bath and dyed at 30°C for 30 min. At the 10th and 20th mins, 2 g of sodium sulfate was added to promote dyeing. After this dyeing stage, the temperature was increased at a rate of 1.5°C / min until it reached 60°C. The temperature was then kept constant, and 0.75 g of anhydrous sodium carbonate was added to the dye bath. When the dyeing continued for 40 min, another 0.75 g of anhydrous sodium carbonate was added to the conical flask. Dyeing was stopped at 60 min.

[0192] The testing steps are as follows:

[0193] (1) Obtaining the linear equation;

[0194] (1.1) Mix equal volumes of the stock solutions of the six target substances mentioned above to obtain a solution containing SES-RO107, VS-RO107, HES-RO107, SES-RY201, VS-RY201 and HES-RY201. Add sodium sulfate to the solution and dilute it to different concentrations to obtain a series of standard mixed solutions with a concentration range of 5 to 100 mg / L for each target substance (the concentration of sodium sulfate in the mixed solution before dilution is the same as the concentration of sodium sulfate in the dye solution during the actual dyeing process).

[0195] (1.2) A series of standard mixtures were detected using the CE-UV method to obtain a series of electrophoretic spectra. When using the CE-UV method, the background electrolyte used a bile acid derivative as an additive with a concentration of 45 mmol / L and sodium cholate as the bile acid derivative. The separation voltage was 15 kV and the injection time was 15 s. The background electrolyte used sodium borate as a buffer with a concentration of 30.0 mmol / L and a pH of 8.6. The pH of the background electrolyte was adjusted using boric acid solution (concentration of 0.40 mol / L).

[0196] (1.3) Linear fitting was performed on the peak area of ​​each target analyte in a series of electrophoresis spectra and the corresponding concentration to obtain a linear equation; the elution position of each target analyte was determined by the standard addition method;

[0197] As shown in Table 1, the correlation coefficient R between the concentration and the corresponding peak area of ​​the six target compounds in the concentration range of 5-100 mg / L is... 2 All values ​​were greater than 0.99, indicating that the analytical method has good linearity and can be used to analyze and detect dye solutions containing these target analytes. Intra-day precision was determined by injecting a mixed standard sample solution (containing 0.4 g / L sodium sulfate solution) at a concentration of 20 mg / L five times consecutively, while inter-day precision was determined by injecting samples over five consecutive days, and finally obtaining the relative standard deviation (RSD) values ​​of peak area and migration time. The limit of detection (LOD) was determined by continuously reducing the sample concentration until the sample peak height was approximately three times higher than the baseline noise. The limit of quantitation (LQD) was determined by continuously reducing the sample concentration until the sample peak height was approximately ten times higher than the baseline. Table 1 shows the intra-day precision of peak area, inter-day precision of peak area, intra-day precision of migration time, inter-day precision of migration time, limit of detection, and limit of quantitation for each target analyte.

[0198] Table 1 Linearity, Limit of Detection, and Reproducibility

[0199]

[0200]

[0201] (2) Test the concentration of the target substance;

[0202] In the dyeing process using two reactive dyes, a sample of the dye solution is taken at time t, the pH is adjusted to neutral and diluted, and then detected by the CE-UV method. The peak area of ​​each target substance in the dye solution sample is measured, and the concentration of each target substance in the dye solution at time t during the dyeing process is calculated. Time t can be any one or more times during the dyeing process.

[0203] Among them, the two reactive dyes are SES-RO107 and SES-RY201; the multiple target compounds are two or more of SES-RO107, VS-RO107, HES-RO107, SES-RY201, VS-RY201 and HES-RY201; the peak position of each target compound is determined by the standard addition method;

[0204] When using the CE-UV method for detection, the background electrolyte uses a bile acid derivative as an additive, with a concentration of 45 mmol / L. The bile acid derivative is sodium cholate. The separation voltage is 15 kV, and the injection time is 15 s. The background electrolyte uses sodium borate as a buffer, with a concentration of 30.0 mmol / L. The pH of the background electrolyte is 8.6. The pH of the background electrolyte is adjusted using boric acid solution (concentration 0.40 mol / L).

[0205] (3) Calculate the absorption rate of each reactive dye at time t during the dyeing process;

[0206] The formula for calculating the absorbance of SES-RO107 at time t during the staining process is as follows:

[0207]

[0208] In the formula, C 0a C represents the concentration of SES-RO107 in the staining solution at the start of staining. ta总 It is the sum of the concentrations of SES-RO107, VS-RO107, and HES-RO107 in the staining solution at time t during the staining process;

[0209] The formula for calculating the absorbance of SES-RY201 at time t during the staining process is as follows:

[0210]

[0211] In the formula, C 0b C represents the concentration of SES-RY201 in the staining solution at the start of staining. tb总 It is the sum of the concentrations of SES-RY201, VS-RY201, and HES-RY201 in the staining solution at time t during the staining process;

[0212] like Figure 26 The figure shows the electrophoretic patterns of the two reactive dyes during the dyeing process; Tables 2 and 3 show the concentrations of each target analyte and the calculated absorbance during the dyeing process of the two reactive dyes, respectively.

[0213] Table 2. Concentration and absorption rate of various forms of Reactive Orange 107 dyes during the dyeing process.

[0214]

[0215] Table 3. Concentration and absorption rate of various forms of Reactive Yellow 201 dyes during the blending process.

[0216]

[0217]

[0218] like Figure 27 As shown, the absorption rate curves of the two reactive dyes during the blending dyeing process are as follows: During the entire blending dyeing process, the absorption rate of both dyes continuously increases in the first 50 minutes, then decreases at 60 minutes. This indicates that during this period, a significant amount of dye is transferred from the fiber to the dye bath. Analysis of the data in Tables 2 and 3 reveals that at 50 minutes, the dye in the dye bath is completely converted to a hydrolyzed form. At 60 minutes, the concentration of the hydrolyzed dye increases. Analysis of the dyeing process reveals that this is due to the use of excessively high concentrations of alkali during the fixation stage. Under the influence of high fixation temperatures, all dyes in the dye bath undergo hydrolysis. Simultaneously, the excessive alkalinity of the dye bath causes a stripping effect on the dyed fabric sample, leading to the hydrolysis of some dyes already bound to the cellulose fibers, forming hydrolyzed dyes that diffuse into the dye bath. This increases the concentration of the hydrolyzed form in the dye bath, resulting in a decrease in the absorption rate at 60 minutes.

[0219] In addition, to verify the rationality of the analysis results of the color-matching dyeing process, two dyes were used separately for single-dye dyeing, and the dyeing process was the same as the color-matching dyeing process of the two reactive dyes. Sampling and analysis were carried out during the dyeing process.

[0220] When performing single staining process analysis, we not only used the CE-UV method of this invention to detect the samples, but also used the traditional ultraviolet-visible spectrophotometry (UV-Vis) to detect the samples; we compared the detection results of the two methods to verify the applicability and accuracy of capillary electrophoresis in the detection of staining process.

[0221] Electrophoretic patterns of individual staining processes for Active Orange 107 and Active Yellow 201 are shown below. Figure 28 , 29 As shown;

[0222] The UV-Vis method, based on the Lambert-Beer law and the principle of light additive properties, determines the dye concentration by measuring changes in the absorbance of the dye solution. The specific steps are as follows: First, the maximum absorption wavelength of SES-RO107 and SES-RY201 is determined to be 411 nm. Standard solutions of the two dyes with concentrations of 0, 10, 20, 40, 60, 80, and 100 mg / L are prepared, and the absorbance of these solutions is measured at 411 nm. A standard curve equation for the absorbance and concentration of each dye is then fitted. Next, the unknown sample is diluted to a suitable concentration, and its absorbance is measured at 411 nm. The absorbance rate can be calculated based on the change in absorbance. Simultaneously, the obtained absorbance is substituted into the respective standard curve equation to determine the concentration.

[0223] Tables 4 and 5 present the color absorption data of the two dyes during the single dyeing process determined by the CE-UV method, and Table 6 presents the color absorption of the two dyes determined by the UV-Vis method. Figure 30 and Figure 31 The absorbance curves determined by the two methods are shown;

[0224] Table 4. Concentrations and absorbance of various dye forms during the single dyeing process of Reactive Orange 107, as determined by CE-UV.

[0225]

[0226] Table 5. Concentrations and absorbance of various dye forms during single dyeing of Reactive Yellow 201 as determined by CE-UV.

[0227]

[0228]

[0229] Table 6. Absorption rates of Reactive Orange 107 and Reactive Yellow 201 during single dyeing determined by UV-Vis.

[0230]

[0231] From Tables 4-6 and Figures 30-31 It can be seen that the absorbance data measured by UV-Vis and CE-UV are basically consistent; at the same time, under the same dyeing process for color mixing, the absorbance of Reactive Orange 107 and Reactive Yellow 201 also showed a decrease at 60 min during the single dyeing process; through the analysis of the CE-UV method, it can be seen from Tables 4 and 5 that the dye is completely hydrolyzed in the dye bath at 50 min, and the degree of hydrolysis intensifies at 60 min, and the concentration of hydrolyzed dye in the dye bath increases, which is the same as the situation in the color mixing process;

[0232] While traditional UV-Vis methods can assess dye absorption by measuring changes in dye liquor absorbance, they cannot detect various forms of dye. This method only provides a rough evaluation of the dyeing process from a macroscopic perspective, failing to provide detailed descriptions and explanations of what happens during the dyeing process. More importantly, it cannot analyze the dyeing process of dyes with similar structures. In contrast, the CE-UV method of this invention can be applied to monitor the single dyeing and blending processes of reactive dyes with similar structures, accurately detecting multiple structurally similar activation and hydrolysis forms that appear during blending. Furthermore, this invention can effectively identify different situations during the dyeing process, thereby optimizing the dyeing process and improving dye utilization.

Claims

1. A method for separating multiple target substances during CE-UV detection, characterized in that, The target compounds are two or more of SES-RO107, VS-RO107, HES-RO107, SES-RY201, VS-RY201 and HES-RY201. Among them, SES-RO107 is Active Orange 107, VS-RO107 is the activated form of Active Orange 107, HES-RO107 is the hydrolyzed form of Active Orange 107, SES-RY201 is Active Yellow 201, VS-RY201 is the activated form of Active Yellow 201, and HES-RY201 is the hydrolyzed form of Active Yellow 201. The background electrolyte uses cholic acid derivatives as additives, with a concentration of 15-60 mmol / L. The cholic acid derivatives are sodium cholate or sodium deoxycholate. The separation voltage is 12-18 kV, and the injection time is 9-21 s. The background electrolyte uses sodium borate as a buffer, with a concentration of 20-40 mmol / L and a pH of 8.3-9. Alternatively, the background electrolyte can use disodium hydrogen phosphate as a buffer, with a concentration of 20-40 mmol / L and a pH of 7-8. Alternatively, ammonium chloride can be used as a buffer in the background electrolyte, with a concentration of 20-40 mmol / L and a pH of 8-9. Alternatively, the background electrolyte can use Tris as a buffer, with a Tris concentration of 20–100 mmol / L and a pH of 7.2–8.

9. When the pH value of the background electrolyte is less than 8.00, the inner surface of the capillary is coated; otherwise, the inner surface of the capillary is either uncoated or coated.

2. The method for separating multiple target substances during CE-UV detection according to claim 1, characterized in that, The concentration of bile acid derivatives in the background electrolyte was 45.0 mmol / L; the separation voltage was 15 kV; and the injection time was 15 s.

3. The method for separating multiple target substances during CE-UV detection according to claim 2, characterized in that, The background electrolyte uses sodium borate as a buffer, with a concentration of 30.0 mmol / L and a pH of 8.

60.

4. The method for separating multiple target objects during CE-UV detection according to claim 1, characterized in that, When sodium borate is used as a buffer in the background electrolyte, the pH of the background electrolyte is adjusted with boric acid solution. When disodium hydrogen phosphate is used as a buffer in the background electrolyte, the pH value of the background electrolyte is adjusted with sodium dihydrogen phosphate solution or sodium hydroxide solution; when ammonium chloride is used as a buffer in the background electrolyte, the pH value of the background electrolyte is adjusted with ammonium hydroxide solution. When Tris is used as a buffer in the background electrolyte, the pH of the background electrolyte is adjusted with HCl solution.

5. A method for detecting the concentration of multiple target substances during reactive dyeing, wherein during the dyeing process using two reactive dyes, a dye solution sample is taken at time t, the pH is adjusted to neutral and diluted, and then detected using the CE-UV method. The peak area of ​​each target substance in the dye solution sample is measured, and the concentration of each target substance in the dye solution at time t during the dyeing process is calculated. Time t can be any one or more times during the dyeing process. The two reactive dyes are SES-RO107 and SES-RY201; The target substances are two or more of the following: SES-RO107, VS-RO107, HES-RO107, SES-RY201, VS-RY201, and HES-RY201. When using the CE-UV method for detection, a method for separating multiple target objects during the CE-UV detection process as described in any one of claims 1 to 4 is employed.

6. The method for detecting the concentration of multiple target substances during reactive dye color matching dyeing according to claim 5, characterized in that, The peak position of each target analyte was determined using the standard addition method; The calculation uses a linear equation, and the steps to obtain the result are as follows: (1) Prepare a solution containing SES-RO107, VS-RO107, HES-RO107, SES-RY201, VS-RY201 and HES-RY201, and dilute it to different concentrations to obtain a series of standard mixtures with a concentration range of 5-100 mg / L for each target analyte. (2) A series of standard mixtures are detected by CE-UV method to obtain a series of electrophoretic spectra, wherein when the CE-UV method is used, a method for separating multiple target substances in the CE-UV detection process as described in any one of claims 1 to 4 is adopted; (3) Linear fitting is performed on the peak area of ​​each target substance in a series of electrophoresis spectra and the corresponding concentration to obtain a linear equation.

7. The method for detecting the concentration of multiple target substances during reactive dye color matching as described in claim 6, characterized in that, In step (3), the correlation coefficient R of the linear relationship between the peak area of ​​each target analyte and its corresponding concentration is... 2 All values ​​are greater than 0.

99. The intra-day precision of peak area is 1.3%–3.9%, the inter-day precision of peak area is 1.5%–4.7%, the intra-day precision of migration time is 0.20%–0.30%, the inter-day precision of migration time is 0.56%–0.73%, the limit of detection is 0.9–3.2 mg / L, and the limit of quantitation is 3.1–10.9 mg / L.

8. The method for detecting the concentration of multiple target substances during reactive dye color matching according to claim 5, characterized in that, After obtaining the concentrations of various target substances in the dye solution at time t during the dyeing process, the absorption rate of each reactive dye at time t during the dyeing process was also calculated. The formula for calculating the absorbance of SES-RO107 at time t during the staining process is as follows: Color absorption rate (%) = ; In the formula, This represents the concentration of SES-RO107 in the staining solution at the start of staining. It is the sum of the concentrations of SES-RO107, VS-RO107, and HES-RO107 in the staining solution at time t during the staining process; The formula for calculating the absorbance of SES-RY201 at time t during the staining process is as follows: Color absorption rate (%) = ; In the formula, This represents the concentration of SES-RY201 in the staining solution at the start of staining. It is the sum of the concentrations of SES-RY201, VS-RY201, and HES-RY201 in the staining solution at time t during the staining process.

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