Method for illumination-assisted in-situ cleaning of chromatographic stationary phase

Abstract

According to the novel illumination-assisted in-situ cleaning method for the chromatographic stationary phase, light-sensitive molecules modified on the surface of the stationary phase are driven to repeatedly generate conformational conversion through cyclic alternate illumination, so that disturbance or collision of a liquid layer on the surface of the stationary phase is realized, the mass transfer rate between the stationary phase and a mobile phase is increased, and the quality of the chromatographic stationary phase is improved. Pollutants are promoted to be separated from a chromatographic stationary phase, the effects of auxiliary cleaning and enhanced cleaning are achieved, the cleaning time is shortened, and the cleaning efficiency is improved. Besides, due to the assistance of light-driven molecular movement on cleaning, many pollutants can be cleaned under the conditions of less organic phases and more water, so that the use of high-toxicity and high-risk organic solvents can be reduced, the environmental protection property in the chromatographic stationary phase cleaning process is improved, and the cost is reduced.

Description

Technical Field

[0001] This application relates to the field of chromatographic stationary phase technology, and in particular to a method for light-assisted in-situ cleaning of chromatographic stationary phases. Background Technology

[0002] Liquid chromatography (LC) is a separation and analysis technique based on a liquid as the mobile phase. It is one of the most important and widely used techniques in modern analytical chemistry. Due to its high separation capability, high sensitivity, and wide applicability, LC has become an indispensable separation and analysis tool in fields such as chemistry, biology, medicine, environment, and food. The stationary phase is the core component that enables the separation function of LC. It is usually located inside the chromatographic column and is in direct contact with the sample and the mobile phase. With increasing usage time, the stationary phase gradually becomes contaminated or deteriorates due to sample residues, impurities in the mobile phase, buffer salt deposition, and contaminant adsorption. This leads to decreased column efficiency, poor peak shape, retention time drift, and even affects the accuracy and reproducibility of analytical results. The purpose of cleaning the stationary phase is to remove contaminants adsorbed on the surface or within the pores of the stationary phase, while protecting and maintaining its chemical structure and physical properties as much as possible.

[0003] Common methods for cleaning the stationary phase include multi-step solvent washing, such as washing the column in the order of water, methanol, isopropanol, chloroform, methanol, and water to remove contaminants from the stationary phase. However, this washing method suffers from problems such as low efficiency, large consumption of organic solvents, high cost, and environmental unfriendliness. Summary of the Invention

[0004] In view of the defects or deficiencies of the existing technology, the purpose of this application is to provide a new light-assisted in-situ cleaning method for chromatographic stationary phases. This method has high cleaning efficiency for chromatographic stationary phases, uses less organic solvent, is low in cost and environmentally friendly.

[0005] Specifically, this application provides a method for light-assisted in-situ cleaning of a chromatographic stationary phase, comprising the following steps: The mobile phase is used to wash the photosensitive molecule-modified chromatographic stationary phase to be cleaned; during the washing process, the chromatographic stationary phase is alternately irradiated with ultraviolet light and visible light, so that the contaminants on the chromatographic stationary phase are eluted by the mobile phase. The photosensitive molecule has the following structure:

[0006] R is selected from any one of H, -OH, -NH2, and -SO3Na.

[0007] Related techniques typically employ multi-step solvent rinsing to remove contaminants from the chromatographic stationary phase. However, this method is inefficient, consumes large amounts of organic solvents, is costly, and is environmentally unfriendly. To overcome at least one of these problems, this application proposes a novel light-assisted in-situ cleaning method for chromatographic stationary phases. This method uses cyclical alternating light irradiation to drive photosensitive molecules modified on the stationary phase surface to undergo repeated conformational changes, thereby agitating or colliding the liquid layer on the stationary phase surface. This accelerates the mass transfer rate between the stationary and mobile phases, promotes the removal of contaminants from the chromatographic stationary phase, achieves assisted cleaning, enhances cleaning, shortens cleaning time, and improves cleaning efficiency.

[0008] Furthermore, due to the assistance of light-driven molecular motion in cleaning, many pollutants can be cleaned with less organic phase and more water, thereby reducing the use of highly toxic and dangerous organic solvents, improving the environmental friendliness of the chromatographic stationary phase cleaning process and reducing costs.

[0009] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0010] Figure 1 This is a schematic diagram illustrating the principle of light-driven molecular motion according to an embodiment of the present invention.

[0011] Figure 2 Electron micrographs of the photosensitive molecule-modified chromatographic stationary phase prepared in Example 3: (a) 800x magnification, (b) 15,000x magnification.

[0012] Figure 3 XPS full-spectrum comparison analysis of the silica stationary phase (top) and the photosensitive molecule-modified chromatographic stationary phase prepared in Example 1 (bottom).

[0013] Figure 4 The following are images showing the effect of photo-assisted cleaning of the chromatographic stationary phase with different proportions of photosensitive molecules, using alternating light irradiation (4s blue light irradiation and 4s ultraviolet light irradiation) in an 8s cycle: (a) Cleaning effect of the chromatographic stationary phase in Comparative Example 1, (b) Cleaning effect of the chromatographic stationary phase in Example 1, (c) Cleaning effect of the chromatographic stationary phase in Example 2, (d) Cleaning effect of the chromatographic stationary phase in Example 3, (e) Cleaning effect of the chromatographic stationary phase in Example 4, and (f) Cleaning effect of the chromatographic stationary phase in Example 5.

[0014] Figure 5The following are chromatograms showing the effect of using different amounts of photosensitive molecules to light-assistedly clean contaminant v on the chromatographic stationary phase: (a) Chromatogram of the chromatographic stationary phase of Comparative Example 1, (b) Chromatogram of the chromatographic stationary phase of Example 1, (c) Chromatogram of the chromatographic stationary phase of Example 2, and (d) Chromatogram of the chromatographic stationary phase of Example 3. The compounds are: (i) thiourea, (ii) benzene, (iii) styrene, (iv) propenylbenzene, and (v) benzo[g,h,i]perylene. The chromatographic conditions are: column length 100 mm, column diameter 150 μm, packing particle size 5 μm, flow rate 5 μL / min, acetonitrile:water = 1:1 (volume ratio), detection wavelength 210 nm, and alternating illumination of the chromatographic stationary phase with an 8 s cycle (4 s of blue light irradiation followed by 4 s of ultraviolet light irradiation).

[0015] Figure 6 Chromatograms of carotene, a pollutant, were obtained by using a light-driven molecular motion-assisted cleaning chromatographic stationary phase. Chromatographic conditions: column length 100 mm, column diameter 150 μm, packing particle size 5 μm, flow rate 5 μL / min, acetonitrile:water = 9:1 (volume ratio), detection wavelength 430 nm, and alternating light irradiation of the stationary phase in 8-s cycles (4 s blue light irradiation: 4 s ultraviolet light irradiation). Detailed Implementation

[0016] The embodiments of this application are described in detail below. These embodiments are exemplary and intended to explain this application, and should not be construed as limiting this application.

[0017] In the description of this application, unless otherwise stated, "multiple" means two or more. "A variety" means two or more. In this document, the terms "comprising" or "including" are open-ended expressions, meaning they include the content specified in this application but do not exclude other aspects.

[0018] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0019] In this document, when the terms "optionally" or "optionally" appear before a technical feature, it indicates that the technical feature is not mandatory but can be selectively used depending on the actual situation. For example, in the description "a chromatographic stationary phase modified with hydroxyl groups, a photosensitive molecule as shown in Formula 1, and optional triethyl octadecylsilicate, reacted in a solvent at 70°C-90°C," "optional triethyl octadecylsilicate" indicates that triethyl octadecylsilicate can be used or not, and the choice can be made based on the actual situation.

[0020] Besides its excellent mechanical and chemical properties, silica also possesses high light transmittance, making it the most commonly used chromatographic stationary phase. However, with increasing usage time, the stationary phase gradually becomes contaminated or degraded due to sample residue, impurities in the mobile phase, buffer salt deposition, and contaminant adsorption. This leads to decreased column efficiency, poor peak shape, retention time drift, and even affects the accuracy and reproducibility of analytical results. Therefore, cleaning the chromatographic stationary phase is necessary. Related techniques typically employ multi-step solvent washing to remove contaminants from the stationary phase. However, this method is inefficient, uses large amounts of organic solvents, is costly, and is environmentally unfriendly.

[0021] This application proposes a method for assisting in cleaning the chromatographic stationary phase by utilizing light-driven molecular motion based on the motion phenomenon of azobenzene photosensitive molecules under light stimulation. Specifically, alternating ultraviolet and visible light irradiation is used to control the repeated cis-trans isomer tautomerism of photosensitive molecules (e.g., ...). Figure 1 As shown in the diagram, this invention utilizes the cis-trans isomer interconversion process to convert light energy into the kinetic energy of photosensitive molecules "twisting." During this "twisting" process, the photosensitive molecules collide with nearby contaminant molecules, thereby dislodging contaminants that were originally stably adsorbed on the surface of the chromatographic stationary phase through intermolecular forces. This allows the contaminants to enter the mobile phase and be carried out of the chromatographic column by the mobile phase flow. Compared with related technologies, this application employs an in-situ molecular perturbation design on the surface of the chromatographic stationary phase, eliminating the need for a large amount of organic solvent as the mobile phase. This reduces the dependence on a high proportion of organic solvent mobile phase during stationary phase cleaning, making cleaning more precise and efficient.

[0022] Specifically, this application provides a method for light-assisted in-situ cleaning of a chromatographic stationary phase, comprising the following steps: The mobile phase is used to wash the photosensitive molecule-modified chromatographic stationary phase to be cleaned; during the washing process, the chromatographic stationary phase is alternately irradiated with ultraviolet light and visible light, so that the contaminants on the chromatographic stationary phase are eluted by the mobile phase. The photosensitive molecule has the following structure:

[0023] R is selected from any one of H, OH, NH2, SO3Na.

[0024] Photosensitive molecules containing the above-mentioned structure can undergo cis-trans isomerization under different light stimuli. This application uses cyclical alternating light irradiation to drive photosensitive molecules modified on the surface of the chromatographic stationary phase to repeatedly undergo conformational transformation, thereby agitating or colliding the liquid layer on the surface of the chromatographic stationary phase, thus accelerating the mass transfer rate between the chromatographic stationary phase and the mobile phase, promoting the removal of pollutants from the chromatographic stationary phase, achieving auxiliary cleaning and enhanced cleaning effects, shortening cleaning time, and improving cleaning efficiency.

[0025] Furthermore, due to the assistance of light-driven molecular motion in cleaning, many pollutants can be cleaned with less organic phase and more water, thereby reducing the use of highly toxic and dangerous organic solvents, improving the environmental friendliness of the chromatographic stationary phase cleaning process and reducing costs.

[0026] The method described in this application has specific requirements for the stationary phase to be cleaned; it must be a chromatographic stationary phase modified with the aforementioned specific photosensitive molecules. As mentioned above, these photosensitive molecules can undergo cis-trans isomerization under different light stimuli, thus enabling efficient cleaning under light-assisted conditions. Therefore, before performing normal separation analysis, the chromatographic column should be filled with the chromatographic stationary phase modified with the aforementioned photosensitive molecules. During normal column use, without light irradiation, the photosensitive molecules are in a dormant state and will not affect the normal separation process of the chromatographic stationary phase. After the analysis is completed, the mobile phase is used to continue rinsing the stationary phase, while the column is alternately irradiated with ultraviolet and visible light. Under the action of alternating light irradiation, the photosensitive molecules on the stationary phase are activated by light and undergo repeated cis-trans isomerization with alternating light irradiation, thereby generating photosensitive molecular motion, which disturbs the liquid layer on the surface of the stationary phase, thereby reducing the adhesion of contaminants to the stationary phase. These contaminants are then carried out of the chromatographic column with the rinsing of the mobile phase, achieving light-driven molecular motion-assisted cleaning of the chromatographic stationary phase.

[0027] In some embodiments, the alternating light irradiation is performed in cycles of 6-30 seconds. For example, cycles can be 6s, 8s, 10s, 12s, 14s, 16s, 18s, 20s, 22s, 24s, 26s, 28s, or 30s. Taking a 6-second cycle as an example, irradiation with ultraviolet light for 3 seconds followed by irradiation with visible light constitutes one cycle. Repeating this alternating light irradiation cycle achieves cleaning. Periodic alternating light irradiation can disturb the liquid layer on the stationary phase surface, reducing the adhesion of contaminants to the stationary phase and thus efficiently removing contaminants. A cycle that is too short (less than 6s) means the photoinduction time is too short, resulting in insufficient proportion of molecules capable of isomerization at the interface, failing to effectively disturb the surface liquid layer. A cycle that is too long (greater than 30s) causes photosensitive molecules at the interface to maintain a single molecular configuration for an extended period, leading to a misalignment with the chromatographic separation time and resulting in "ineffective" cleaning.

[0028] In some embodiments, the total duration of the alternating illumination is 1-60 minutes, for example, 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 60 minutes. The entire alternating illumination process includes multiple cycles, thereby enabling efficient cleaning of contaminants by reducing their adhesion to the stationary phase through the movement of photosensitive molecules under light assistance. The total duration of the alternating illumination can be selected as needed; if the adhesion of contaminants to the stationary phase is low, they can be removed in a short time; conversely, the duration of the alternating illumination needs to be extended.

[0029] In some embodiments, the ratio of ultraviolet (UV) irradiation time to visible light irradiation time in one cycle can be (0.5-2):1, preferably (0.8-1.2):1. Taking a 6-second cycle as an example, the UV and visible light irradiation times are each 3 seconds; or, the UV irradiation time is 2 seconds and the visible light irradiation time is 4 seconds; or, the UV irradiation time is 4 seconds and the visible light irradiation time is 2 seconds. Most preferably, the ratio of UV irradiation time to visible light irradiation time is 1:1. Taking a 6-second cycle as an example, this further improves the cleaning efficiency of the chromatographic stationary phase compared to the group without light assistance.

[0030] In some embodiments, the wavelength of the ultraviolet light is in the range of 360nm-390nm. For example, the wavelength of the ultraviolet light is 360nm, 370nm, 380nm, or 390nm. The azobenzene photosensitive molecule of the present invention has maximum absorption in this wavelength range, and can obtain sufficient energy through illumination to overcome the energy barrier of the isomerization-tautomerization process, inducing the photosensitive molecule to change from one configuration to another, thereby realizing the movement of the photosensitive molecule.

[0031] In some embodiments, visible light includes blue light. The wavelength of blue light is in the range of 450 nm to 495 nm. The azobenzene photosensitive molecule of the present invention has maximum absorption in this wavelength range, and can acquire sufficient energy through illumination to overcome the energy barrier of the isomerization-tautomerization process, inducing the photosensitive molecule to change from one configuration to another, thereby realizing the movement of the photosensitive molecule. As a specific example, the wavelength of blue light may be 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, or 495 nm.

[0032] In some embodiments, the mobile phase includes acetonitrile, water, methanol, phosphate buffer solution, ammonium acetate buffer solution, or a mixture of two or more thereof. As a specific example, the mobile phase includes a mixture of acetonitrile and water. This application does not have specific requirements regarding the type of mobile phase; commonly used mobile phases can be used.

[0033] In some embodiments, the rinsing is initiated simultaneously with the alternating illumination. Alternatively, the alternating illumination is initiated and maintained after the rinsing has been performed for 1-5 minutes (e.g., 1 minute, 2 minutes, 3 minutes, 4 minutes, or 5 minutes). Thus, contaminants on the chromatographic stationary phase can be easily removed through the synergistic effect of mobile phase rinsing and alternating illumination.

[0034] In some embodiments, the photosensitive molecule-modified chromatographic stationary phase to be cleaned is packed in a chromatographic column, and the method is performed in the chromatographic column.

[0035] In some embodiments, the chromatographic stationary phase comprises silica.

[0036] In some embodiments, the photosensitive molecule-modified chromatographic stationary phase is prepared by a method comprising the following steps: The photosensitive molecule modified by the photosensitive molecule can be obtained by reacting the hydroxyl-modified chromatographic stationary phase, the photosensitive molecule shown in Formula 1, and optional triethyl octadecyl silicate in a solvent at 70℃-90℃.

[0037] Formula 1 Where R is selected from any one of H, OH, NH2, SO3Na; n is an integer from 1 to 4.

[0038] In some specific embodiments, n is 1 or 2.

[0039] In some specific embodiments, the photosensitive molecule shown in Formula 1 has the following structure: .

[0040] In some specific embodiments, the reaction temperature is 70°C, 75°C, 80°C, 85°C, or 90°C.

[0041] In some specific embodiments, the reaction time is 24h-48h, for example 24h, 28h, 32h, 36h, 40h, 44h or 48h.

[0042] In some specific embodiments, the solvent includes at least one of toluene and acetone.

[0043] In some specific embodiments, the mass ratio of the hydroxyl-modified chromatographic stationary phase to the total mass of the photosensitive molecule and octadecyltriethyl silicate shown in Formula 1 is (5-15):1, for example, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1 or 15:1.

[0044] In some specific embodiments, the molar ratio of the photosensitive molecule and triethyl octadecylsilicate shown in Formula 1 can be (5-100):(0-95), for example, 5:95, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, or 100:0. The molar ratio of the photosensitive molecule and triethyl octadecylsilicate shown in Formula 1 can be selected according to actual needs, and is not specifically limited herein.

[0045] In some specific embodiments, the reaction is carried out in a protective atmosphere, which includes nitrogen.

[0046] In some specific embodiments, the reaction is carried out in a closed environment.

[0047] In some specific embodiments, after the reaction is completed, the resulting photosensitive molecule-modified chromatographic stationary phase is repeatedly washed with a solvent. The solvent includes at least one of toluene and acetone.

[0048] In some embodiments, the hydroxyl-modified chromatographic stationary phase is prepared by a method comprising the following steps: The chromatographic stationary phase is subjected to alkalization and acidification treatments in sequence, washed with water until neutral, and then dried to obtain the hydroxyl-modified chromatographic stationary phase.

[0049] In some specific embodiments, after the alkalization treatment and before the acidification treatment, the preparation method further includes washing with water until neutral.

[0050] In some specific embodiments, the alkalization treatment time is 20 min to 50 min, for example, 20 min, 30 min, 40 min or 50 min.

[0051] In some specific embodiments, alkalization is performed using an alkaline solution (such as a 0.01 mol / L sodium hydroxide solution). Methods for alkalizing the chromatographic stationary phase are well known to those skilled in the art and will not be described in detail herein.

[0052] In some specific embodiments, the acidification treatment time is 5h-10h, for example, 5h, 6h, 7h, 8h, 9h or 10h. The acidification treatment can be carried out under reflux conditions.

[0053] In some specific embodiments, acidification is performed using an acidic solution (such as a 1 mol / L hydrochloric acid solution). Methods for acidifying the chromatographic stationary phase are well known to those skilled in the art and will not be described in detail herein.

[0054] In some specific embodiments, the drying temperature is 50°C-70°C, for example, 50°C, 55°C, 60°C, 65°C, or 70°C. The drying time is 1 hour-10 hours, for example, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, or 10 hours.

[0055] In some embodiments, the photosensitive molecule shown in Formula 1 is prepared by a method comprising the following steps: (1) The aminoazobenzene shown in Formula 2 undergoes a diazotization reaction to obtain the first intermediate; (2) The first intermediate is subjected to a diazo coupling reaction with phenol to obtain a second intermediate containing a phenolic hydroxyl group; (3) The second intermediate containing the phenolic hydroxyl group is converted into sodium phenolate; (4) The sodium phenoxide is reacted with C3-C6 haloolefins at 60℃-90℃ for 4h-24h, and the third intermediate is obtained after separation and purification. (5) In the presence of a catalyst, the third intermediate is reacted with triethoxysilane at 60℃-90℃ for 4h-24h to obtain the photosensitive molecule shown in Formula 1;

[0056] Formula 2 R is selected from any one of H, -OH, -NH2, and -SO3Na.

[0057] In some specific embodiments, the diazotization reaction of the aminoazobenzene shown in Formula 2 includes: dissolving the aminoazobenzene shown in Formula 2 in a solvent (such as a mixture of ethanol and water), adding NaNO2, and cooling to 0-5°C (e.g., 0°C or 5°C); adding an inorganic acid (such as hydrochloric acid), adjusting the pH to 0.5-1, and reacting for 1-4 hours (e.g., 1 hour, 2 hours, 3 hours, or 4 hours) to obtain the first intermediate. The molar ratio of the aminoazobenzene shown in Formula 2 to NaNO2 can be 1:(1.5-2), for example, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, or 1:2.

[0058] In some specific embodiments, the diazo coupling reaction between the first intermediate and phenol includes: dissolving phenol in sodium hydroxide solution, adding an appropriate amount of glacial acetic acid to adjust the pH to 8-12, cooling the solution to 0℃~5℃, adding the reaction solution containing the first intermediate obtained in step (1) dropwise to the resulting solution, adjusting the pH to 8-10 (e.g., 8, 9, or 10), heating to room temperature, and reacting for 1h-4h (e.g., 1h, 2h, 3h, or 4h) to obtain a second intermediate containing a phenolic hydroxyl group. The molar ratio of aminoazobenzene to phenol shown in Formula 2 can be 1:(1.1-3), for example, 1:1.1, 1:1.5, 1:2, 1:2.5, or 1:3. After the reaction is complete, water can be added to the resulting reaction solution to precipitate the product, and the pure product can be obtained by filtration and recrystallization. The filtration includes vacuum filtration. Recrystallization can be carried out in ethanol.

[0059] In some specific embodiments, converting the second intermediate containing phenolic hydroxyl groups into sodium phenolate includes: dissolving the second intermediate containing phenolic hydroxyl groups in a solvent (such as toluene), adding sufficient NaH under a protective atmosphere (such as nitrogen), and reacting at room temperature for 1-12 hours (e.g., 1 hour, 3 hours, 6 hours, 9 hours, or 12 hours) to obtain sodium phenolate.

[0060] In some specific embodiments, the method for preparing the third intermediate includes: adding a C3-C6 haloalkene to the sodium phenolate reaction solution obtained in step (3), heating to 60℃-90℃ (e.g., 60℃, 70℃, 80℃ or 90℃) and continuing the reaction for 4h-24h (e.g., 4h, 8h, 12h, 18h or 24h), and obtaining the third intermediate after separation and purification. The C3-C6 haloalkene includes at least one of bromopropene and bromobutene. The molar ratio of the second intermediate containing a phenolic hydroxyl group to the C3-C6 haloalkene can be 1:(1-5), for example, 1:1, 1:2, 1:3, 1:4 or 1:5. The separation and purification includes: adding a small amount of ethanol to the obtained reaction solution to quench the reaction, adjusting the pH to about 7, adding water to precipitate the product, filtering, drying, and then separating by column chromatography to obtain the pure product. The filtration includes vacuum filtration.

[0061] In some specific embodiments, the reaction of the third intermediate with triethoxysilane includes: adding 1.5-5 equivalents of triethoxysilane and an appropriate amount of catalyst to the third intermediate, and reacting at 60°C-90°C (e.g., 60°C, 70°C, 80°C, or 90°C) for 4-24 hours (e.g., 4 hours, 8 hours, 12 hours, 18 hours, or 24 hours) to obtain the photosensitive molecule shown in Formula 1. The catalyst includes a castor catalyst. The amount of catalyst can be adjusted as needed and is not specifically limited herein, as long as the catalytic effect is achieved. For example, the amount of catalyst can be 1 mg-50 mg, such as 1 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, or 50 mg. After the reaction is complete, column chromatography can be performed to obtain the photosensitive molecule shown in Formula 1.

[0062] In some specific embodiments, the synthetic route of the photosensitive molecule shown in Formula 1 is as follows: .

[0063] This invention utilizes a biazobenzene structure with sensitive light-responsive configurational conversion characteristics to perturb other molecules near the molecule during configurational conversion, thereby assisting in the cleaning of stubborn contaminants on the stationary phase surface. This enables rapid baseline leveling after chromatographic stationary phase analysis of complex matrix samples, effectively shortening the time for batch analysis of complex samples in the field of liquid chromatography, saving the amount of organic solvent used, and improving the service life of the chromatographic column.

[0064] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0065] Synthesis Example 1: Synthesis of the photosensitive molecule shown in Formula 1-1

[0066] Equation 1-1 1 mmol of 4-aminoazobenzene was dissolved in an aqueous acetone solution (acetone to water volume ratio 1:1), 1.5 mmol of NaNO2 was added, and the mixture was cooled to approximately 0°C. The pH was adjusted to 1 with the addition of hydrochloric acid, and the reaction was continued for 4 h to obtain a reaction solution containing the diazonium compound. Phenol was dissolved in a sodium hydroxide solution, and the pH was adjusted to 10 with the addition of glacial acetic acid. The solution was cooled to 5°C, and the reaction solution containing the diazonium compound was added dropwise to the resulting solution. The pH was then adjusted to 10, and the temperature was gradually raised to room temperature. The reaction was continued for 4 h, and the product was purified by column chromatography. 1 mmol of the diazonium compound was dissolved in toluene, and sufficient NaH was added under nitrogen protection. The reaction was carried out at room temperature for 6 h. After the reaction was complete, 1.2 mmol of bromopropene was added, and the temperature was raised to 60°C. The reaction was continued for 24 h, and the product was obtained by column chromatography. The product obtained in the previous step was added with 1.5 equivalents of triethoxysilane and 0.1 mL of caster catalyst, and the reaction was continued at 90 °C for 24 h. After separation and purification by column chromatography, the yellow photosensitive molecule shown in Formula 1-1 was obtained.

[0067] The photosensitive molecule shown in Formula 1-1 1 H NMR spectrum: 1 H NMR (300 MHz, CDCl3) δ 8.10 – 7.90(m, 8H), 7.58 – 7.47 (m, 3H), 7.02 (d, J = 9.1 Hz, 2H), 4.05 (t, J = 6.7 Hz,2H), 3.85 (q, J = 7.0 Hz, 6H), 2.04 – 1.89 (m, 2H), 1.25 (t, J = 7.0 Hz, 9H), 0.85 – 0.75 (m, 2H).

[0068] The photosensitive molecule shown in Formula 1-1 13 C NMR spectrum: 13 C NMR (75 MHz, CDCl3) δ 162.09,154.00, 153.29, 152.78, 147.04, 131.29, 129.16 (2C), 125.09 (2C), 123.79(2C), 123.43 (2C), 123.03 (2C), 114.83 (2C), 70.24, 58.49 (3C), 22.76, 18.34(3C), 6.52 ESI-MS high-resolution spectrum of the photosensitive molecule, m / z = 507.2423 ([M+H]) + ), 529.2243 ([M+Na] +The reference value for this molecular ion peak in the spectral library is: m / z = 507.2422 ([C 27 H 34 N4O4Si+H] + )), 529.2242= ([C 27 H 34 [N4O4Si+Na] + ).

[0069] Synthesis Example 2: Synthesis of Hydroxyl-Modified Chromatographic Stationary Phase At room temperature, commercially available spherical silica microspheres with a particle size of 5 μm were immersed in a 0.01 mol / L sodium hydroxide aqueous solution for 35 min to complete the surface activation treatment. The silica microspheres were then rinsed with ultrapure water until neutral, and refluxed at 1 mol / L hydrochloric acid aqueous solution at 100℃ for 8 hours to activate the hydroxyl groups on the silica surface. After washing the silica microspheres with water until the filtrate was neutral, they were dried in a vacuum drying oven at 60℃ for 4 h.

[0070] Synthesis of photosensitive molecule-modified chromatographic stationary phases Example 1 Take 2 g of the silica microspheres prepared in Synthesis Example 2 into an ampoule, add 2 mL of toluene and 200 mg of silane derivatizing agent (the silane derivatizing agent is a mixture of the photosensitive molecule prepared in Synthesis Example 1 and octadecyltriethoxysilane in a molar ratio of 5:95), replace the air with nitrogen, seal the ampoule, and heat to 80 °C. After reacting for 32 h, wash the product repeatedly with toluene to obtain the chromatographic stationary phase modified with the photosensitive molecule.

[0071] Its electron microscope image is as follows Figure 2 As shown in the image, the silica chromatographic stationary phase bonded with photosensitive molecules remains composed of uniformly sized spherical particles with a rough surface.

[0072] X-ray photoelectron spectroscopy (XPS) analysis was performed on the chromatographic stationary phase modified with photosensitive molecules, such as... Figure 3 As shown, the elemental composition of the chromatographic stationary phase modified with photosensitive molecules changed significantly. Compared with unmodified silica gel, the relative signal intensities of Si 2p and O 1s were significantly reduced, while the signal intensities of C 1s and N 1s were greatly enhanced. This systematic change in the relative content of elements directly confirms that the photosensitive molecules have been successfully modified onto the silica gel surface through chemical bonding.

[0073] Example 2 The photosensitive molecule-modified chromatographic stationary phase was prepared according to the method described in Example 1, except that the silane derivatizing agent was a mixture of the photosensitive molecule prepared in Example 1 and octadecyltriethoxysilane in a molar ratio of 10:90. The XPS chromatogram of the photosensitive molecule-modified chromatographic stationary phase prepared in this example is similar to... Figure 3similar.

[0074] Example 3 The photosensitive molecule-modified chromatographic stationary phase was prepared according to the method described in Example 1, except that the silane derivatizer was a mixture of the photosensitive molecule prepared in Example 1 and octadecyltriethoxysilane in a molar ratio of 20:80.

[0075] Example 4 The photosensitive molecule-modified chromatographic stationary phase was prepared according to the method described in Example 1, except that the silane derivatizer was a mixture of the photosensitive molecule prepared in Example 1 and octadecyltriethoxysilane in a molar ratio of 30:70.

[0076] Example 5 The photosensitive molecule-modified chromatographic stationary phase was prepared according to the method described in Example 1, except that the silane derivatizer was a mixture of the photosensitive molecule prepared in Example 1 and octadecyltriethoxysilane in a molar ratio of 50:50.

[0077] Example 6 The photosensitive molecule-modified chromatographic stationary phase was prepared according to the method described in Example 1, except that the silane derivatizer was a mixture of the photosensitive molecule prepared in Example 1 and octadecyltriethoxysilane in a molar ratio of 100:0.

[0078] Comparative Example 1 The chromatographic stationary phase was prepared according to the method described in Example 1, except that the silane derivatizer was a mixture of the photosensitive molecule prepared in Example 1 and octadecyltriethoxysilane in a molar ratio of 0:100.

[0079] In the following application examples, the wavelength of blue light used is 475nm, and the wavelength of ultraviolet light used is 380nm.

[0080] Application Example 1 Weigh 5.0 mg of Solvent Blue 36 powder into a 500 mL beaker, add 200 mL of 50% methanol-water solution, and sonicate to dissolve. Transfer the dissolved Solvent Blue 36 methanol-water solution to a 250 mL volumetric flask and add 50% methanol-water solution to the mark to obtain a Solvent Blue 36 stock solution with a concentration of 20 mg / mL. Weigh 10.0 mg of each of the following chromatographic stationary phases: Comparative Example 1, Example 1, Example 2, Example 3, Example 4, and Example 5, into six centrifuge tubes. Add 1 mL of 20 mg / mL Solvent Blue 36 stock solution to each centrifuge tube, seal, and incubate at 200 rpm for 12 h in a light-protected 20°C constant-temperature shaker. The centrifuge tubes were alternately irradiated with blue and ultraviolet light for 10 min, with each irradiation cycle lasting 8 s (4 s of blue light followed by 4 s of ultraviolet light). After irradiation, the tubes were centrifuged at 4000 rpm for 2 minutes. The supernatant was collected and stored, and the concentration of the remaining adsorbed dye was quantitatively analyzed using a UV-Vis spectrophotometer, i.e., the percentage of adsorbed dye (relative content). Subsequently, 1 mL of a 50% methanol-water solution was added to the centrifuge tubes for the first elution of the stationary phase containing the adsorbed dye. The process was as follows: After vortexing the solid-liquid mixture in the centrifuge tubes, the tubes were alternately irradiated with blue and ultraviolet light for 60 min (each irradiation cycle lasting 8 s, with 4 s of blue light followed by 4 s of ultraviolet light), and centrifuged at 4000 rpm for 2 minutes. After centrifugation, the supernatant was collected, and the dye content in the first elution was quantified using a UV-Vis spectrophotometer (i.e., the percentage of dye eluted in the first elution, relative content). Continue by adding 1 mL of 50% methanol-water solution to the centrifuge tube and performing a second wash following the "first elution" procedure described above. Measure and calculate the dye content in the second eluent (i.e., the percentage eluted in the second elution). Finally, subtract the percentage of remaining adsorption, the percentage eluted in the first elution, and the percentage eluted in the second elution from the total dye addition percentage (100%) to calculate the percentage of uneluted Solvent Blue 36 dye on the stationary phase packing. The control group experiment followed the same procedure as the experimental group, except that no light exposure was applied. All experiments in this application example were repeated three times. The experimental results are as follows: Figure 4 As shown.

[0081] Analysis of experimental results: like Figure 4As shown in (a), there was no difference in the first and second elution processes after the adsorption of dye on the stationary phase of Comparative Example 1 without bonded photosensitive molecules. The elution ratios for the first and second elutions were similar under light-assisted and non-light-assisted conditions, at 10.9% and 11.1% and 5.6% and 5.6%, respectively. In both cases, 5.1% to 5.2% of the dye failed to be eluted. This indicates that it is difficult to elute the dye from the stationary phase without assistance. The traditional method of cleaning compounds with strong retention on the stationary phase by using multiple solvents is inefficient.

[0082] like Figure 4 As shown in (b)-(f), the photosensitive molecule-modified stationary phases of Examples 1-5 exhibited significant differences in the elution ratios under light-assisted and non-light-assisted conditions during the first and second elutions. Under light-assisted conditions, the first elution ratio was 20.4%–26.3%, while without light-assisted conditions, it was 13.0%–16.0%, with light-assisted elution increasing the elution rate by 54.8%–64.9%. After the second elution, the residual dye ratio in the experimental group was 0%–1.3%, while the residual dye ratio in the control group was 8.3%–9.3%. This indicates that light-assisted elution significantly accelerates the dye elution process.

[0083] Experiments have shown that adding a certain proportion of photosensitive molecules to the chromatographic stationary phase under light-driven conditions can achieve faster cleaning of dyes adsorbed on it and reduce the amount of organic solvents used.

[0084] Application Example 2 The chromatographic stationary phases of Examples 1-3 and Comparative Example 1 were respectively packed into empty tubes of transparent capillary chromatographic columns. The general packing procedure for chromatographic stationary phase capillary packed columns is as follows: the above-mentioned chromatographic stationary phases are packed into commercially available silica capillary chromatographic column tubes using a column packer, the packing pressure is 5 MPa, the stationary phase packing length is 100 mm, and the packing rinsing solvent is pure methanol.

[0085] After filling, the chromatographic column is installed in the matching high-performance liquid chromatography instrument, which consists of a binary high-pressure pump, an injector, a chromatographic column, and an ultraviolet detector.

[0086] The general procedure for sample injection is as follows: Before injection, the signal baseline of the UV detector at 210 nm wavelength is flushed with a 50% acetonitrile-water (v / v) mixture at a flow rate of 5 μL / min until it is flat. Keeping the chromatographic conditions constant (detector wavelength 210 nm, flow rate 5 μL / min, 50% acetonitrile-water (v / v) as the mobile phase), 2 μL of a mixed standard sample of thiourea, benzene, styrene, propylbenzene, and benzo[g,h,i]perylene at a concentration of 5 ppm is added to the quantitative loop of the injector, and isocratic elution is performed for separation.

[0087] In this application example, experiments were conducted and compared on each chromatographic stationary phase under three cleaning modes: "no assisted cleaning," "assisted cleaning starting 5 minutes after injection," and "assisted cleaning under full light illumination." All three cleaning modes required solvent rinsing throughout the entire process. The rinsing solvent used in all three modes was the same: a 1:1 volume ratio acetonitrile-water solution.

[0088] Light-driven cleaning mode setting: The entire length of the capillary-packed column of the chromatographic stationary phase modified with photosensitive molecules is illuminated 360°. The alternating illumination process is carried out in 8-second cycles, with each cycle consisting of 4 seconds of blue light irradiation followed by 4 seconds of ultraviolet light irradiation.

[0089] The "no-assisted cleaning" process means that no light is applied during the entire chromatographic separation process; "Auxiliary cleaning begins 5 minutes after injection" means that circulating light will be started 5 minutes after injection, and the chromatographic column will also be in cleaning mode until the entire injection process, including the chromatographic cleaning process, is completed. The "full-process light-assisted cleaning" process means that the light is circulated immediately after sample injection until the entire chromatographic separation process is completed.

[0090] Figure 5 (a) Figure 5 (b) Figure 5 (c) Figure 5 The experimental procedure of (d) is basically the same, except that the amount of photosensitive molecules added in the stationary phase filler is different.

[0091] Analysis of experimental results: In the experiment of light-driven molecular motion-assisted cleaning of chromatographic stationary phase, hydrophobic benzo[g,h,i]perylene was used as a contaminant to contaminate the chromatographic column. The experiment compared the chromatographic retention time of the contaminant with and without light-assisted cleaning under the same chromatographic conditions. The cyclic light irradiation was carried out in 8-second cycles, including 4 seconds of blue light irradiation and 4 seconds of ultraviolet light irradiation.

[0092] like Figure 5 As shown in (a), after separating compounds i to iv, the stationary phase of Comparative Example 1 showed no difference in the removal of compound v under light-assisted and non-light-assisted conditions (the chromatographic retention time of compound v was 25.0 min for both).

[0093] like Figure 5 As shown in (b), the chromatographic retention time of compound v in Example 1 was 15.9 min under full-process light-assisted conditions, 16.7 min under light-assisted conditions starting 5 min after injection, and 19.3 min under no-light-assisted conditions. The removal time of compound v was shortened by 18% under light-assisted conditions compared to no-light-assisted conditions.

[0094] like Figure 5 As shown in (c), the chromatographic retention time of compound v in Example 2 was 18.5 min under full-process light-assisted conditions, 19.6 min under light-assisted conditions starting 5 min after injection, and 26.2 min under no-light-assisted conditions. The removal time of compound v was shortened by 29% under light-assisted conditions compared to no-light-assisted conditions.

[0095] like Figure 5 As shown in (d), the chromatographic retention time of compound v in Example 3 was 16.0 min under full-process light-assisted conditions, 17.5 min under light-assisted conditions starting 5 min after injection, and 21.0 min under no-light-assisted conditions. The removal time of compound v was shortened by 24% under light-assisted conditions compared to no-light-assisted conditions.

[0096] Experiments have shown that the method of the present invention can significantly accelerate the efficiency of pollutant cleaning without affecting the normal separation efficiency of the chromatographic column.

[0097] Application Example 3 This experiment used spinach juice rich in carotene as the actual sample to verify the effect of light-assisted cleaning.

[0098] The preparation process of spinach juice rich in carotene is as follows: Take 20 g of fresh spinach, crush and juice it, then filter the juice using a Buchner funnel. Place the juice in a separatory funnel and add 20 mL of n-hexane twice to extract the carotene and other weakly polar substances. Wash the obtained n-hexane extract three times with saturated saline solution. Concentrate the washed organic phase to dryness by nitrogen blowing, and redissolve the residue with 20 mL of acetonitrile. Subsequently, filter the acetonitrile solution through a 0.22 μm needle filter membrane to obtain the actual sample solution of the weakly polar pigments in spinach.

[0099] The photosensitive molecule-modified chromatographic stationary phase (photosensitive molecule and octadecyltriethoxysilane in a molar ratio of 10:90) prepared in Example 2 was used as the chromatographic stationary phase for the cleaning experiment of the obtained actual sample solution.

[0100] The general packing procedure for a chromatographic stationary phase capillary packed column is as follows: the above-mentioned chromatographic stationary phase is packed into a commercially available silica capillary chromatographic column using a column packer, the packing pressure is 5 MPa, the stationary phase packing length is 100 mm, and the packing rinsing solvent is pure methanol.

[0101] The parameter settings for light-assisted cleaning are the same as those for the cleaning mode in Application Example 2. In this application example, the three cleaning modes—"no assisted cleaning," "assisted cleaning starts 5 minutes after sample injection," and "full-process light-assisted cleaning"—are the same as in Application Example 2. The rinsing solvent used in all three cleaning modes is the same as in Application Example 2.

[0102] Experimental Results and Analysis: The chromatographic stationary phase of Example 2 was used for auxiliary cleaning of contaminants (carotene) during actual sample analysis, such as... Figure 6 As shown, the chromatographic retention time of the pollutant was 8.4 min under full-process light-assisted conditions, 10.8 min under light-assisted conditions starting 5 min after injection, and 13.6 min under no-light-assisted conditions. Light-assisted conditions reduced the carotene removal time by 38% compared to no-light-assisted conditions.

[0103] Experiments have shown that the method of the present invention can significantly improve the cleaning efficiency of pollutants.

[0104] Application Example 4 The experiment was conducted according to the method described in Application Example 1, except that the centrifuge tubes were alternately irradiated with blue light and ultraviolet light for 10 minutes during the first and second elution cycles. The specific procedure is as follows.

[0105] Weigh 5.0 mg of Solvent Blue 36 powder into a 500 mL beaker, add 200 mL of 50% methanol-water solution, and sonicate to dissolve. Transfer the dissolved Solvent Blue 36 methanol-water solution to a 250 mL volumetric flask and add 50% methanol-water solution to the mark to obtain a Solvent Blue 36 stock solution with a concentration of 20 mg / mL. Weigh 10.0 mg of the chromatographic stationary phase prepared in Comparative Example 1, the photosensitive molecule-modified chromatographic stationary phase prepared in Example 1, and the photosensitive molecule-modified chromatographic stationary phase prepared in Example 2 into three centrifuge tubes respectively. Add 1 mL of Solvent Blue 36 stock solution with a concentration of 20 mg / mL to each centrifuge tube, seal, and incubate at 200 rpm for 12 h in a light-protected 20°C constant temperature shaker. Irradiate the centrifuge tubes alternately with blue light and ultraviolet light for 10 min; the irradiation process is in 8-second cycles, with 4 seconds of blue light irradiation and 4 seconds of ultraviolet light irradiation. After irradiation, the sample was centrifuged at 4000 rpm for 2 minutes. The supernatant was collected and stored. The concentration of the remaining adsorbed dye was quantitatively analyzed using a UV-Vis spectrophotometer, i.e., the percentage of adsorbed dye (relative content). Subsequently, 1 mL of a 50% methanol-water solution was added to the centrifuge tube for the first elution of the stationary phase containing the adsorbed dye. The process was as follows: After vortexing the solid-liquid mixture in the centrifuge tube, the tube was alternately irradiated with blue light and UV light for 10 minutes (each irradiation cycle was 8 seconds, with 4 seconds of blue light irradiation followed by 4 seconds of UV light irradiation). The sample was then centrifuged at 4000 rpm for 2 minutes. After centrifugation, all the supernatant was collected, and the dye content in the first elution was quantified using a UV-Vis spectrophotometer (i.e., the percentage of dye eluted, relative content). 1 mL of a 50% methanol-water solution was added to the centrifuge tube for a second elution following the same "first elution" steps, and the dye content in the second elution was measured and calculated (i.e., the percentage of dye eluted). Finally, the percentage of uneluted Solvent Blue 36 dye on the stationary phase was calculated by successively subtracting the percentage of residual adsorption, the percentage of primary elution, and the percentage of secondary elution from the total dye addition percentage (100%). The control group experiment followed the same procedure as the experimental group, except that no light was applied. All experiments in this application example were repeated three times.

[0106] Analysis of experimental results: In Comparative Example 1, the stationary phase containing unbonded photosensitive molecules showed no significant difference in the first and second elution processes after dye adsorption. The elution ratios under light-assisted and non-light-assisted conditions were similar, at 10.8% and 11.1% and 5.6% and 5.6%, respectively. Furthermore, 5.0% to 5.1% of the dye remained uneluted in both cases. This indicates that the dye is difficult to elute from the stationary phase without assistance, and the traditional method of cleaning strongly retained compounds on the stationary phase using multiple solvents is inefficient.

[0107] In Examples 1-2, the photosensitive molecule-modified stationary phases showed significant differences in the elution ratios for the first and second elutions under light-assisted and non-light-assisted conditions. Under light-assisted conditions, the first elution ratios were 16.2% and 18.0%, respectively, while under non-light-assisted conditions, they were 13.0% and 14.2%, respectively. Light-assisted elution increased the elution amount by 24.6% and 11.1%, respectively. After the second elution, the remaining dye percentages in the experimental groups were 2.3% and 0.2%, respectively, while those in the control group were 8.3% and 8.4%, respectively. This indicates that light-assisted elution significantly accelerates the dye elution process.

[0108] Experiments have shown that adding a certain proportion of photosensitive molecules to the chromatographic stationary phase under light-driven conditions can achieve faster cleaning of dyes adsorbed on it and reduce the amount of organic solvents used.

[0109] Comparing the cleaning effect of Application Example 4 with that of Application Example 1, it can be seen that the illumination time has a significant impact on the cleaning effect. Therefore, the total alternating illumination time is preferably within the range of the present invention.

[0110] Application Example 5 The experiment was conducted according to the method described in Application Example 1, except that the irradiation process in the first and second elution cycles was in 30-second intervals, with 14 seconds of blue light irradiation and 16 seconds of ultraviolet light irradiation. The specific process is as follows.

[0111] Weigh 5.0 mg of Solvent Blue 36 powder into a 500 mL beaker, add 200 mL of 50% methanol-water solution, and sonicate to dissolve. Transfer the dissolved Solvent Blue 36 methanol-water solution to a 250 mL volumetric flask and add 50% methanol-water solution to the mark to obtain a Solvent Blue 36 stock solution with a concentration of 20 mg / mL. Weigh 10.0 mg of the chromatographic stationary phase prepared in Comparative Example 1, the photosensitive molecule-modified chromatographic stationary phase prepared in Example 1, and the photosensitive molecule-modified chromatographic stationary phase prepared in Example 2 into three centrifuge tubes respectively. Add 1 mL of Solvent Blue 36 stock solution with a concentration of 20 mg / mL to each centrifuge tube, seal, and incubate at 200 rpm for 12 h in a light-protected 20°C constant temperature shaker. Irradiate the centrifuge tubes alternately with blue light and ultraviolet light for 10 min; the irradiation process is in 8-second cycles, with 4 seconds of blue light irradiation and 4 seconds of ultraviolet light irradiation. After irradiation, the sample was centrifuged at 4000 rpm for 2 minutes. The supernatant was collected and stored. The concentration of the remaining adsorbed dye was quantitatively analyzed using a UV-Vis spectrophotometer, i.e., the percentage of adsorbed dye (relative content). Subsequently, 1 mL of a 50% methanol-water solution was added to the centrifuge tube for the first elution of the stationary phase containing the adsorbed dye. The process was as follows: After vortexing the solid-liquid mixture in the centrifuge tube, the tube was alternately irradiated with blue light and UV light for 60 minutes (each irradiation cycle was 30 seconds, with 14 seconds of blue light irradiation and 16 seconds of UV light irradiation). The sample was then centrifuged at 4000 rpm for 2 minutes. After centrifugation, all the supernatant was collected, and the dye content in the first elution was quantified using a UV-Vis spectrophotometer (i.e., the percentage of dye eluted in the first elution, relative content). Continue by adding 1 mL of 50% methanol-water solution to the centrifuge tube and performing a second wash following the "first elution" procedure described above. Measure and calculate the dye content in the second eluent (i.e., the percentage eluted in the second elution). Finally, subtract the percentage of remaining adsorption, the percentage eluted in the first elution, and the percentage eluted in the second elution from the total dye addition percentage (100%) to calculate the percentage of uneluted Solvent Blue 36 dye on the stationary phase packing. The control group experiment follows the same procedure as the experimental group, except that no light exposure is applied. All experiments in this application example are repeated three times.

[0112] Analysis of experimental results: In Comparative Example 1, the stationary phase containing unbonded photosensitive molecules showed no significant difference in the first and second elution processes after dye adsorption. The elution ratios under light-assisted and non-light-assisted conditions were similar, at 10.9% and 11.1% and 5.8% and 5.4%, respectively. Furthermore, 4.9% to 5.0% of the dye remained uneluted in both cases. This indicates that without assistance, the dye is difficult to elute from the stationary phase, and the traditional method of cleaning strongly retained compounds on the stationary phase using multiple solvents is inefficient.

[0113] In Examples 1-2, the photosensitive molecule-modified stationary phases showed significant differences in the elution ratios for the first and second elutions under light-assisted and non-light-assisted conditions. Under light-assisted conditions, the first elution ratios were 18.2% and 20.2%, respectively, while under non-light-assisted conditions, they were 13.4% and 14.3%, respectively. Light-assisted elution increased the elution amount by 35.8% and 41.3%, respectively. After the second elution, the remaining dye percentages in the experimental groups were 1.2% and 0%, respectively, while those in the control groups were 8.2% and 8.6%, respectively. This indicates that light-assisted elution significantly accelerates the dye elution process.

[0114] Application Comparative Example 1 The experiment was conducted according to the method described in Application Example 3, except that red light was used instead of blue light. The experimental results showed that the chromatographic retention time of the pollutants was 13.6 min in all three cleaning modes. It is evident that alternating between red and ultraviolet light cannot achieve conformational changes and molecular "twisting" of photosensitive molecules, and therefore cannot achieve light-assisted in-situ cleaning of the chromatographic stationary phase.

[0115] In summary, this invention utilizes the biazobenzene structure with sensitive photoresponsive configuration change characteristics to perturb other molecules near the molecule during the configuration change process, thereby achieving auxiliary cleaning of stubborn contaminants on the stationary phase surface and reducing the amount of organic solvent used. Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A method for light-assisted in-situ cleaning of a chromatographic stationary phase, characterized in that, Includes the following steps: The mobile phase was used to wash the chromatographic stationary phase modified with the photosensitive molecules to be cleaned. During the rinsing process, the chromatographic stationary phase is subjected to alternating ultraviolet and visible light irradiation, thereby eluting contaminants on the chromatographic stationary phase along with the rinsing of the mobile phase; The photosensitive molecule has the following structure: R is selected from any one of H, -OH, -NH2, and -SO3Na.

2. The method according to claim 1, characterized in that, The alternating light exposure has a cycle of 6s-30s; The total duration of the alternating illumination is 1 min to 60 min.

3. The method according to claim 2, characterized in that, In one cycle, the ratio of ultraviolet light irradiation time to visible light irradiation time is (0.5-2):1, preferably (0.8-1.2):1, and most preferably 1:

1.

4. The method according to claim 1, characterized in that, The wavelength of ultraviolet light is in the range of 360nm-390nm; Visible light includes blue light; the wavelength of blue light is in the range of 450nm-495nm.

5. The method according to claim 1, characterized in that, The rinsing is started simultaneously with the alternating light exposure; or, after the rinsing has been performed for 1-5 minutes, the alternating light exposure is started and the rinsing is continued.

6. The method according to claim 1, characterized in that, The photosensitive molecule-modified chromatographic stationary phase to be cleaned is packed in a chromatographic column, and the method is performed in the chromatographic column.

7. The method according to claim 1, characterized in that, The photosensitive molecule-modified chromatographic stationary phase is prepared by a method comprising the following steps: The photosensitive molecule modified by the photosensitive molecule can be obtained by reacting the hydroxyl-modified chromatographic stationary phase, the photosensitive molecule shown in Formula 1, and optional triethyl octadecyl silicate in a solvent at 70℃-90℃. Formula 1 Where R is selected from any one of H, OH, NH2, SO3Na; n is an integer from 1 to 4.

8. The method according to claim 7, characterized in that, The reaction time is 24-48 hours. And / or, the solvent includes at least one of toluene and acetone; And / or, the mass ratio of the hydroxyl-modified chromatographic stationary phase to the total mass of the photosensitive molecule and octadecyl triethyl silicate shown in Formula 1 is (5-15):1; And / or, the reaction is carried out in a protective atmosphere; And / or, the reaction is carried out in a closed environment; And / or, after the reaction is complete, the resulting photosensitive molecule-modified chromatographic stationary phase is repeatedly washed with a solvent.

9. The method according to claim 7, characterized in that, The hydroxyl-modified chromatographic stationary phase is prepared by a method comprising the following steps: The chromatographic stationary phase is subjected to alkalization and acidification treatments in sequence, washed with water until neutral, and then dried to obtain the hydroxyl-modified chromatographic stationary phase; Optionally, after the alkalization treatment and before the acidification treatment, the preparation method further includes: washing with water until neutral; The alkalization treatment time is 20-50 minutes; The acidification treatment time is 5h-10h; The drying temperature is 50℃-70℃, and the drying time is 1h-10h.

10. The method according to claim 7, characterized in that, The photosensitive molecule shown in Formula 1 is prepared by a method comprising the following steps: The aminoazobenzene shown in Formula 2 is subjected to a diazotization reaction to obtain the first intermediate; The first intermediate is subjected to a diazo coupling reaction with phenol to obtain a second intermediate containing a phenolic hydroxyl group; The second intermediate containing phenolic hydroxyl groups is converted into sodium phenolate; The sodium phenoxide was reacted with a C3-C6 haloalkene at 60℃-90℃ for 4h-24h, and the third intermediate was obtained after separation and purification. In the presence of a catalyst, the third intermediate is reacted with triethoxysilane at 60°C-90°C for 4-24 hours to obtain the photosensitive molecule shown in Formula 1. Formula 2 R is selected from any one of H, OH, NH2, SO3Na.

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