High-purity 2-hydroxyphenazine and preparation method thereof

Abstract

The invention discloses a high-purity 2-hydroxyphenazine compound and a purification method thereof.A large amount of 2-hydroxyphenazine compounds are successfully separated and purified from pseudomonas chlororaphis fermentation broth, and the purification method comprises the following steps that the pseudomonas chlororaphis fermentation broth is centrifuged to remove thalli, and a sample loading solution is obtained; carrying out dynamic adsorption on the loading solution by using a macroporous adsorption resin column until the loading solution is saturated; eluting impurities on the macroporous adsorption resin column by using a low-concentration ethanol water solution; desorbing by using a relatively high-concentration ethanol water solution as an eluent to obtain a desorption solution; and removing the eluent in the desorption solution to obtain a purified 2-hydroxyphenazine product. According to the method, the recovery rate reaches 93.01 + / -5.95%, and the method has the advantages of being easy to operate, environmentally friendly and free of pollution and has wide application value in the aspect of industrial large-scale production.

Description

Technical Field

[0001] The invention relates to the technical field, and in particular to high-purity 2-hydroxyphenazine and a preparation method thereof. Background Art

[0002] 2-Hydroxyphenazine has a good effect in antagonizing wheat take-all pathogen, sclerotinia pathogen and anthracnose pathogen, and has a good application prospect. In addition, 2-hydroxyphenazine has made certain progress in biosynthesis and can be naturally synthesized in Pseudomonas chlororaphis. At present, the traditional method for separating phenazine compounds is to use liquid phase extraction with organic solvents such as ethyl acetate and chloroform, but it has the disadvantages of complex separation process, long time consumption, and only suitable for small-scale preparation, which is not suitable for large-scale industrial production.

[0003] At present, the traditional separation method for obtaining 2-hydroxyphenazine is mainly liquid phase extraction using organic solvents such as ethyl acetate and chloroform. However, this traditional method has many serious defects. From a process point of view, its separation process is extremely complicated, involving multiple steps and delicate operations, requiring operators to have high professional skills and rich experience. In terms of time cost, the entire extraction process is time-consuming, and it often takes a lot of time from sample pretreatment to the final product. And this method is only suitable for small-scale preparation, and it is difficult to meet the needs of industrial large-scale production.

[0004] Against the backdrop of growing demand for industrial production, the limitations of traditional liquid phase extraction methods have become increasingly prominent. The extensive use of organic solvents not only significantly increases production costs, but also generates a large amount of organic solvent waste liquid. These waste liquids are difficult and costly to handle. If not handled properly, they will cause serious environmental pollution and violate the modern green production concept. At the same time, problems such as the volatilization of organic solvents pose a threat to the health of operators, requiring the provision of special protective equipment and sites.

[0005] As a high molecular polymer material that relies on van der Waals forces and hydrogen bonding forces to generate adsorption, macroporous adsorption resin has been widely used in the fields of pharmaceuticals and chemicals to separate polycyclic and heterocyclic compounds. It has unique physical and chemical properties and is expected to provide a new solution for the separation and purification of 2-hydroxyphenazine, overcome the many drawbacks of traditional organic solvent extraction methods, and meet the market's urgent demand for large-scale, efficient, and environmentally friendly production of 2-hydroxyphenazine. However, there are still many technical gaps and challenges in the application of macroporous adsorption resins to the separation and purification of 2-hydroxyphenazine, which urgently need further research and exploration. Summary of the invention

[0006] The present invention aims at the deficiencies in the prior art and provides a high-purity 2-hydroxyphenazine and a preparation method thereof.

[0007] The technical solution provided by the present invention is as follows:

[0008] S1, centrifuging the Pseudomonas chlororaphis fermentation broth and collecting the supernatant as the loading solution;

[0009] S2, using a macroporous adsorption resin column to dynamically adsorb the sample solution until saturation adsorption is achieved to capture the target compound 2-hydroxyphenazine;

[0010] S3, using a low concentration alcohol solution to elute the impurities on the macroporous adsorption resin column;

[0011] S4, using a high concentration alcohol solution as an eluent for desorption to obtain a desorption solution;

[0012] S5. Remove the eluent from the desorption solution, dry it, and obtain the purified 2-hydroxyphenazine.

[0013] The macroporous adsorption resin includes HZ-818 and / or HZ-801.

[0014] Preferably, the macroporous adsorption resin includes HZ-818.

[0015] The aspect ratio of the macroporous adsorption resin column is 80-100:1; the specific surface area of ​​the macroporous adsorption resin is 800-1200m 2 / g, particle size is 0.32~1.20mm.

[0016] The macroporous adsorption resin HZ-818 is milky white translucent spherical particles with a water content of 60-70 wt% and a wet true density of 1.00-1.10 g / mL.

[0017] The macroporous adsorption resin is pretreated before being loaded into the column, and the pretreatment step includes the following steps:

[0018] A. Soaking the macroporous adsorption resin in ethanol for 20-24 hours and washing with water in turn to obtain an alcohol-treated resin, wherein the concentration of ethanol is 95-98%, the rate of water washing is 1-2 BV / h, and the amount used is 2-5 BV;

[0019] B. soaking the alcohol-treated resin in an acid solution and washing it with water in sequence to obtain an acid-treated resin;

[0020] C. Soaking the acid-treated resin in an alkaline solution and washing with water in sequence;

[0021] D. The alkali-treated resin is subjected to ultrasonic treatment and then dried.

[0022] In step B, the acid solution is 0.4-0.8M HCl, and the immersion time is 4-6 hours; the water washing is carried out by distilled water at a rate of 1-2BV / h until the surface becomes neutral, and the amount used is 2-3BV;

[0023] And / or, in step C, the alkaline solution is 0.5-1M NaOH solution, and the immersion is 4-6 hours; the water washing is carried out by distilled water at a rate of 1-2BV / h until neutral, and the amount used is 2-3BV;

[0024] And / or, in step D, the alkali-treated resin is placed in 8-10 BV distilled water for ultrasonic treatment, the ultrasonic frequency is 25-50 kHz, the power is 100-162.5 W, and the ultrasonic time is 20-30 min.

[0025] The macroporous adsorption resin column is packed with the resin column by wet method, and the specific steps are as follows:

[0026] Soak the pretreated resin in 95-98% ethanol aqueous solution for 20-24h, stir evenly and pour into the chromatography column filled with 0.3-0.4BV 95-98% ethanol solution, release the solution at the same time and knock the column wall with an ear bulb to avoid uneven resin filling; when the liquid level drops to 1-2cm from the surface of the resin column, wash with distilled water to complete the wet filling of the resin column.

[0027] In step S2, during dynamic adsorption, the loading speed of the loading liquid is 0.8-2BV / h.

[0028] The low-concentration alcohol solution in step S3 is a low-concentration ethanol solution with a volume fraction of 40-50%, the flow rate of elution is 0.4-0.6 BV / h, and the amount of the low-concentration ethanol solution is 1-1.2 BV.

[0029] In step S4, the eluent is a high-concentration ethanol solution with a volume fraction of 75-95%; the desorption rate of the eluent is 0.6-1.5 BV / h, and the amount of the eluent is 4-5 BV.

[0030] In step S2, adsorption is performed at a temperature of 16-20°C.

[0031] In step S5, the drying step is: transferring the desorbed liquid to a rotary evaporator, sealing and reducing the pressure to 400-500 mmHg; heating to 80-85° C. until the volume is concentrated to 6-8% of the original desorbed liquid.

[0032] The present invention pre-treats the resin, including soaking in ethanol, washing with water, soaking in acid solution and alkaline solution and washing with water, and then ultrasonic treatment and drying, and each step cooperates with each other. Soaking in 95-98% ethanol for 20-24 hours can remove impurities in the resin and make it swell, and acid-base treatment further purifies the resin and adjusts its surface chemical properties. Ultrasonic treatment (frequency 25-50kHz, power 100-162.5W, duration 20-30min) can remove fine impurities inside the resin and make its structure more uniform and stable, enhance the adsorption performance and service life of the resin, and ensure stable function in the entire preparation process.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. Although macroporous adsorption resin has a certain adsorption capacity for 2-hydroxyphenazine, there are usually some compounds with similar structures in the fermentation broth that compete with 2-hydroxyphenazine for adsorption sites, affecting the selectivity of the resin; therefore, the present invention conducts a comparative study on a variety of macroporous adsorption resins; the macroporous adsorption resins HZ-818 and / or HZ-801 have better effects; preferably, the macroporous adsorption resin HZ-818 is used as an adsorbent to successfully separate and purify a large amount of 2-hydroxyphenazine compounds from the fermentation broth of Pseudomonas chlororaphis, and the maximum adsorption capacity of the HZ-818 resin for 2-hydroxyphenazine is as high as 6.8 mg / g, and it has excellent selective adsorption performance and stable physicochemical properties.

[0035] 2. Although the macroporous adsorption resin HZ-818 has a certain adsorption capacity for 2-hydroxyphenazine, in practical applications, the adsorption capacity cannot reach the ideal value due to various factors. For example, the pH value, temperature, ionic strength and other conditions of the solution may affect the adsorption performance of the resin. The present invention further optimizes and adjusts the process conditions such as temperature, sample flow rate, and eluent concentration, and its recovery rate reaches 93.01±5.95%. The purification method of the present invention has the advantages of simple operation, environmental protection and pollution-free, and has broad application value in industrial large-scale production.

[0036] 3. The loading speed of the sample solution is controlled at 0.8-2BV / h, which not only ensures that the target compound is fully contacted with the resin for adsorption, but also avoids the problem of incomplete adsorption caused by too fast a flow rate. A low-concentration ethanol solution with a volume fraction of 40-50% is used for elution at a flow rate of 0.4-0.6BV / h and a dosage of 1-1.2BV, which effectively removes impurities on the resin column and reduces the loss of the target compound. A high-concentration ethanol solution with a volume fraction of 75-85% is used as an eluent, and desorption is performed at a rate of 0.6-1.5BV / h and a dosage of 4-5BV, which ensures efficient desorption of the target compound. Combined with an operating temperature of 16-20°C, a stable and suitable reaction environment is provided for the entire process, so that each step is carried out in an orderly and efficient manner, and ultimately a recovery rate of up to 93.01±5.95% is achieved.

[0037] 4. Compared with the traditional organic solvent extraction separation method, the present invention avoids the generation of a large amount of organic solvent waste liquid and is more environmentally friendly. The entire preparation method has a clear operation flow, and the conditions of each step are clear and easy to control. It does not require complex and expensive equipment and operators with extremely high professional skills. For example, the desorption liquid is dried under specific conditions (sealed decompression to 400-500 mmHg, heated and concentrated at 80-85°C) by a rotary evaporator. The operation is simple and efficient, and has great advantages in industrial large-scale production. It can effectively reduce production costs and improve production efficiency and economic benefits.

[0038] 5. The present invention uses a relatively high concentration of ethanol as the eluent for desorption; compared with methanol, ethanol has a weaker polarity, but can provide a higher recovery rate at the same volume, further improving the purification efficiency and economic benefits. The present invention uses a relatively high concentration of ethanol as the eluent for desorption. Since the solubility of 2-hydroxyphenazine in aqueous solution under neutral conditions is less than 10 mg / L, according to the principle of "like dissolves like", the present invention uses an ethanol solution with a weaker polarity than methanol for elution. Compared with methanol, the use of high concentration ethanol as the eluent can provide a higher recovery rate at the same volume. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0040] Figure 1 is the adsorption equilibrium of 2-hydroxyphenazine by the resin at different temperatures;

[0041] Figure 2 The desorption of 2-hydroxyphenazine on the resin by ethanol solution with different volume fractions;

[0042] Figure 3 The residual 2-hydroxyphenazine in the effluent of the sample solution at different flow rates;

[0043] Figure 4 This is a comparison chart of the desorption effects of methanol and ethanol;

[0044] Figure 5 Comparison of HCl and NaOH soaking time in Example 6;

[0045] Figure 6 This is a comparison chart of the resin without ultrasonic treatment and with ultrasonic treatment during the pretreatment process. DETAILED DESCRIPTION

[0046] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0047] In the present invention, the specific surface area of ​​HZ-818 and / or HZ-801 used is 800-1200m 2 / g, particle size is 0.32~1.20mm.

[0048] Example 1 Determination of the Optimal Resin

[0049] (1) Seven macroporous adsorption resins, D001, D155, HP-20, HZD-2, HZ-801, HZ-818, and HZ-016, were used. Three portions of each resin were taken, each portion was accurately weighed 0.5 g, and soaked in 1 mL of 95% ethanol overnight;

[0050] (2) Filter and separate, wash the resin with deionized water to remove ethanol, and pour into 250 mL shake flasks respectively;

[0051] (3) Each portion of the resin was immersed in 25 mL of a 30 mg / L 2-hydroxyphenazine aqueous solution and mixed, the flask was sealed with a sealing film, and placed in a shaker at 28° C. and 200 rpm for 24 h, and samples were taken to detect the change in 2-hydroxyphenazine concentration;

[0052] (4) filtering and separating the resin and the 2-hydroxyphenazine aqueous solution, and washing the resin with deionized water for 1-2 times to remove the residual 2-hydroxyphenazine solution;

[0053] (5) Each portion of resin was immersed and mixed with 25 mL of 95% ethanol solution by volume, and the shake bottle was sealed with a sealing film and placed in a shaker at 28° C. and 200 rpm for 24 h. Samples were taken to detect changes in 2-hydroxyphenazine concentration. The test results are shown in Table 1.

[0054] Table 1 Separation effect of different macroporous resins

[0055] Macroporous resin types Manufacturer Final concentration of 2-hydroxyphenazine (mg / L) D001 Zhejiang Zhengguang Industrial Co., Ltd. 0.53 D155 Shanghai Nankai Resin Co., Ltd. 1.07 HZD-2 Shanghai Huazhen Technology Co., Ltd. 0.23 HZ-801 Zhengzhou Ainuo Chemical Technology Co., Ltd. 13.47 HZ-818 Shanghai Huazhen Technology Co., Ltd. 19.67 HP-20 Beijing Solable Technology Co., Ltd. 5.21 HZ-016 Greenlink Chemical Technology Co., Ltd. 0.17

[0056] As shown in Table 1, the macroporous resin HZ-818 has the best separation effect. Macroporous resin HZ-818 was selected for subsequent research.

[0057] Example 2 Determination of sample solution concentration and purification temperature

[0058] (1) Accurately weigh several portions of HZ-818 macroporous resin, 1.0 g each, and soak them in 2 mL of 95% ethanol overnight;

[0059] (2) Filter and separate, wash the resin with deionized water to remove ethanol, and pour into 250 mL shake flasks respectively;

[0060] (3) Each portion of resin was mixed with aqueous solutions of different concentrations prepared with pure 2-hydroxyphenazine, and the flasks were sealed with sealing film; the concentrations of 2-hydroxyphenazine were 17.5 mg / L, 40 mg / L, 80 mg / L, 140 mg / L, and 240 mg / L, respectively (as shown in Table 2).

[0061] Table 2

[0062] Test group number 2-Hydroxyphenazine concentration 2-1 17.5mg / L 2-2 40mg / L 2-3 80mg / L 2-4 140mg / L 2-5 240mg / L

[0063] (4) The mixed solution of each test group was placed in a shaker at 16°C, 28°C, and 45°C, respectively, and shaken at 200 rpm for 24 h. Samples were taken to detect the change in 2-hydroxyphenazine concentration, the change in the adsorption amount per unit mass of resin was calculated, and the adsorption isotherms produced by initial solutions of different concentrations at different temperatures were plotted.

[0064] The adsorption isotherms of different concentrations of initial solutions at different temperatures are shown in Figure 2. Figure 1 As shown, Figure 1 The data on the abscissa are the equilibrium concentrations of 2-hydroxyphenazine in the solution after resin adsorption.

[0065] Depend on Figure 1 It can be seen that under different sample solution concentrations, as the temperature decreases, the adsorption effect of HZ-818 resin on 2-hydroxyphenazine gradually increases. Among 16℃, 28℃ and 45℃, the adsorption effect of HZ-818 resin at 16℃ is the best. After fitting the isotherm curve with the Langmuir thermodynamic equation, it is found that the maximum adsorption capacity of HZ-818 resin is 6.8mg / g, so the maximum sample solution concentration is

[0066]

[0067] In the formula, ρ Resin is the wet true density of HZ-818 resin (1.00~1.10g / mL); V resin is the volume of resin involved in adsorption (mL); q m is the maximum adsorption capacity of HZ-818 for 2-hydroxyphenazine (6.8 mg / g); V A is the volume of the sample solution (L).

[0068] Example 3 Determination of eluent concentration

[0069] (1) Accurately weigh several portions of HZ-818 macroporous resin, 0.5 g each, and soak them in 1 mL of 95% ethanol overnight to ensure that the resin is fully infiltrated;

[0070] (2) Filter and separate, wash the resin with deionized water to remove ethanol, and pour into 250 mL shake flasks respectively;

[0071] (3) Each portion of resin was soaked and mixed with 25 mL of 2-hydroxyphenazine aqueous solution, the shake bottle was sealed with a sealing film, and placed in a shaker at 28°C and 200 rpm for 24 h. Samples were taken to detect the change in 2-hydroxyphenazine concentration to evaluate the adsorption effect of the resin;

[0072] (4) Washing: Filter and separate the resin and the 2-hydroxyphenazine aqueous solution, and wash the resin 1-2 times with deionized water to remove the residual 2-hydroxyphenazine solution;

[0073] (5) Elution: Add 25 mL of ethanol solution with volume fractions of 95%, 85%, 75%, 65%, 55%, 45%, 35%, and 25% to the resin, shake at 200 rpm at 28°C for 24 h, and take samples for testing to evaluate the elution effect of ethanol solutions of different concentrations. The results are shown in Figure 2 As shown;

[0074] Depend on Figure 2 It can be seen that when the eluent is 75-95% ethanol solution, the desorption effect of the eluent is the best. When the eluent is 75% ethanol solution, the desorption rate reaches 84.79±1.49%.

[0075] Example 4 Determination of sample loading flow rate

[0076] Test strain: Pseudomonas chlororaphis GP72 (CGMCC 1748CN 1932006A);

[0077] KB plate medium formula: 20g / L peptone, 15mL / L glycerol, 0.514g / L potassium dihydrogen phosphate, 0.732g / L magnesium sulfate, 20g / L agar;

[0078] KB liquid culture medium formula: 20g / L peptone, 15mL / L glycerol, 0.514g / L potassium dihydrogen phosphate, 0.732g / L magnesium sulfate.

[0079] The HZ-818 macroporous adsorption resin used in this embodiment is milky white translucent spherical particles. The height-to-diameter ratio of the macroporous adsorption resin column is preferably 100:1, and the specific surface area of ​​the macroporous resin is 900 m 2 / g, particle size is 1.20mm, moisture content is 60-70wt%, and wet true density is 1.00-1.10g / mL.

[0080] First, soak the HZ-818 macroporous resin in 95% ethanol for 24 hours, and wash the resin with distilled water at a rate of 1BV / h, with a dosage of 2BV; then soak it in 0.8M HCl solution for 6 hours, and wash it with distilled water at a rate of 2BV / h until it is neutral, with a dosage of 2BV. Finally, soak it in 1MNaOH solution for 6 hours, and wash it with distilled water at a rate of 2BV / h until it is neutral, with a dosage of 2BV; then place the alkali-treated resin in 8BV distilled water for ultrasonic treatment, with an ultrasonic frequency of 25kHz, a power of 162.5W, an ultrasonic duration of 0.5h, and dry it.

[0081] Soak the resin in 95% ethanol aqueous solution for 24 hours, stir evenly and pour it into a chromatography column filled with 0.3BV 95% ethanol aqueous solution. At the same time, open the chromatography column knob to release the solution and tap the column wall with an ear bulb to avoid uneven resin filling. When the liquid level drops to 1-2 cm from the surface of the resin column, wash with distilled water to complete the wet filling of the resin column.

[0082] (1) Fermentation broth preparation

[0083] The test strain Pseudomonas chlororaphis GP72 was inoculated on KB plate culture medium for activation once (28°C, 12h), and a single colony on the plate was picked and cultured in 5mL KB liquid culture medium overnight (28°C, 24h). The culture solution was inoculated and fermented at a concentration of 1 / 1000 for 4 days.

[0084] The Pseudomonas chlororaphis fermentation broth was centrifuged at 10,000 rpm for 10 min to remove the bacteria and retain the supernatant as the loading solution.

[0085] (2) 6 BV of the fermentation broth supernatant was passed through a chromatography column filled with HZ-818 macroporous resin at flow rates of 0.96 BV / h, 1.92 BV / h, and 6 BV / h, respectively. The effluent was collected and sampled at intervals of 1 BV to measure the concentration change. The results are shown in Table 1. Figure 3 shown.

[0086] Depend on Figure 3 It can be seen that when the loading flow rate is 0.96-1.92BV / h, HZ-818 resin can completely adsorb 2-hydroxyphenazine in the fermentation broth, but when the loading liquid flow rate is 1.92BV / h, 2-hydroxyphenazine residues appear in the effluent when the loading liquid volume increases to 4BV. At a loading flow rate of 0.96BV / h, the HZ-818 resin column can ensure complete adsorption of 2-hydroxyphenazine in 6BV fermentation broth.

[0087] Example 52 Purification of Hydroxyphenazine

[0088] 1. Treatment of Pseudomonas chlororaphis LX24 fermentation broth

[0089] The fermentation broth required for this experiment came from Pseudomonas chlororaphis GP72 (CGMCC 1748CN 1932006A); it was activated once on a KB plate (KB medium formula: 20 g / L peptone, 15 mL / L glycerol, 0.514 g / L potassium dihydrogen phosphate, 0.732 g / L magnesium sulfate) (28°C, 12 h), and a single colony on the plate was picked and cultured overnight in 5 mL KB liquid culture medium, and the culture medium was inoculated with a concentration of 1 / 1000 for fermentation.

[0090] 2. High-speed centrifugation for fermentation broth

[0091] The fermentation broth of Pseudomonas chlororaphis was centrifuged at 10,000 rpm for 10 min to remove the bacteria and retain the supernatant as the loading solution.

[0092] 3. Purification of 2-hydroxyphenazine compounds in fermentation broth by macroporous resin

[0093] 3.1. Resin column preparation

[0094] The HZ-818 macroporous adsorption resin used in this embodiment is milky white translucent spherical particles. The height-to-diameter ratio of the macroporous adsorption resin column is preferably 100:1, and the specific surface area of ​​the macroporous resin is 900 m 2 / g, particle size is 1.20mm, moisture content is 60-70wt%, and wet true density is 1.00-1.10g / mL.

[0095] Resin pretreatment: first soak HZ-818 macroporous resin in 95% ethanol for 24 hours, wash the resin with distilled water at a rate of 1BV / h, the amount is 2BV; then soak it in 0.8M HCl solution for 6 hours, wash it with distilled water at a rate of 2BV / h until it is neutral, the amount is 2BV. Finally, soak it in 1M NaOH solution for 6 hours, wash it with distilled water at a rate of 2BV / h until it is neutral, the amount is 2BV; then place the alkali-treated resin in 8BV distilled water for ultrasonic treatment, the ultrasonic frequency is 25kHz, the power is 162.5W, the ultrasonic time is 0.5h, and it is dried.

[0096] Wet-packing of resin column: Soak the pretreated resin in 95% ethanol aqueous solution for 24 hours, stir evenly and pour into the chromatography column filled with 0.3BV 95% ethanol aqueous solution, open the chromatography column knob to release the solution, and tap the column wall with an ear bulb to avoid uneven resin loading; when the liquid level drops to 1-2 cm from the surface of the resin column, wash with distilled water to complete the wet-packing of the resin column.

[0097] 3.2. Dynamic adsorption: The supernatant of the obtained fermentation broth was subjected to dynamic adsorption on a macroporous adsorption resin column (the temperature of the purification environment was 16°C) at a loading rate of 0.96 BV / h until saturated adsorption (when the 2-hydroxyphenazine concentration of the elution solution was 4% of the initial sample solution and the total volume of the elution solution was the dynamic processing capacity of the resin, the resin was defined to be in an adsorption equilibrium state).

[0098] 3.3. Elution: Use 40% ethanol aqueous solution to elute the impurities on the macroporous adsorption resin column. The elution rate is 0.6 BV / h and the amount of 40% ethanol aqueous solution is 1 BV.

[0099] 3.4. Desorption: Dynamic desorption was performed with 75% ethanol aqueous solution to obtain a desorption liquid. The desorption rate was 0.6 BV / h and the amount of 75% ethanol aqueous solution used was 5 BV.

[0100] 3.5. Concentration and drying: Finally, the collected desorption liquid was transferred to a rotary evaporator, sealed and decompressed to 400 mmHg, heated to 81°C in a water bath, the flask speed was 60 rpm, and the volume was concentrated to 6.67% of the original desorption liquid. The concentrated fermentation liquid was placed in a fume hood to dry, and the residual solid was collected to obtain pure 2-hydroxyphenazine. After HPLC inspection, the purity could reach up to 94.8%.

[0101] Example 6

[0102] This example compares the HCl and NaOH soaking times in the resin pretreatment in step 3.1 of Example 5, and the remaining steps are the same as in Example 5.

[0103] The results are as follows Figure 5 As shown in the figure, the recovery rate increased significantly with the extension of treatment time and eventually tended to be consistent, so the pretreatment method of acid soaking for 4 hours and alkali soaking for 6 hours was the best.

[0104] Comparative Example 1

[0105] The difference between this comparative example and Example 5 is that in step 3.4, 75% methanol aqueous solution is used for desorption.

[0106] Finally, the recovery rate of 75% methanol aqueous solution at the same volume is significantly lower than the recovery rate obtained by desorption using 75% ethanol aqueous solution in Example 5. Figure 4 shown.

[0107] Comparative Example 2

[0108] The difference between this comparative example and Example 5 is that the resin is not subjected to ultrasonic treatment during the pretreatment process. Figure 6As shown, it was found that the resin after ultrasonic treatment showed a faster adsorption rate and the adsorption capacity increased by 22.6%.

[0109] In summary, the present invention discloses a method for purifying 2-hydroxyphenazine compounds by using a macroporous adsorption resin, and successfully separates and purifies a large amount of 2-hydroxyphenazine compounds from a fermentation broth of Pseudomonas chlororaphis, comprising the following steps: centrifuging the fermentation broth of Pseudomonas chlororaphis to remove the bacterial cells, and obtaining a loading solution; using a macroporous adsorption resin column to dynamically adsorb the loading solution to saturated adsorption; using a low-concentration ethanol aqueous solution to elute impurities on the macroporous adsorption resin column; using a higher-concentration ethanol aqueous solution as an eluent for desorption, and obtaining a desorption solution; removing the eluent from the desorption solution, and obtaining a purified 2-hydroxyphenazine. The method of the present invention has a recovery rate of 93.01±5.95%, has the advantages of simple operation, environmental protection and no pollution, and has broad application value in industrial large-scale production.

[0110] It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention. In the absence of conflict, the embodiments of the present application and the features in the embodiments may be combined with each other at will.

Claims

1. A method for preparing high-purity 2-hydroxyphenazine, characterized in that: The following steps are involved: S1, centrifuging the Pseudomonas chlororaphis fermentation broth and collecting the supernatant as the loading solution; S2, using a macroporous adsorption resin column to dynamically adsorb the sample solution until saturation adsorption is achieved to capture the target compound 2-hydroxyphenazine; S3, using a low concentration alcohol solution to elute the impurities on the macroporous adsorption resin column; S4, using a high concentration alcohol solution as an eluent for desorption to obtain a desorption solution; S5. Remove the eluent from the desorption solution, dry it, and obtain the purified 2-hydroxyphenazine.

2. The preparation method according to claim 1, characterized in that: The macroporous adsorption resin includes HZ-818 and / or HZ-801.

3. The preparation method according to claim 2, characterized in that: Before the macroporous adsorption resin is filled into the column, a pretreatment is performed, and the pretreatment process includes the following steps: A. Soaking the macroporous adsorption resin in ethanol for 20-24 hours and washing with water in turn to obtain an alcohol-treated resin, wherein the concentration of ethanol is 95-98%, the rate of water washing is 1-2 BV / h, and the amount used is 2-5 BV; B. soaking the alcohol-treated resin in an acid solution and washing it with water in sequence to obtain an acid-treated resin; C. Soaking the acid-treated resin in an alkaline solution and washing with water in sequence; D. The alkali-treated resin is subjected to ultrasonic treatment and then dried.

4. The preparation method according to claim 3, characterized in that: In step B, the acid solution is 0.4-0.8M HCl, and the immersion time is 4-6 hours; the water washing is carried out by distilled water at a rate of 1-2BV / h until the solution becomes neutral, and the amount used is 2-3BV; And / or, in step C, the alkaline solution is 0.5-1M NaOH solution, and the immersion is 4-6 hours; the water washing is carried out by distilled water at a rate of 1-2BV / h until neutral, and the amount used is 2-3BV; And / or, in step D, the alkali-treated resin is placed in 8-10 BV water for ultrasonic treatment, the ultrasonic frequency is 25-50 kHz, the power is 100-162.5 W, and the ultrasonic time is 20-30 min.

5. The preparation method according to claim 1, characterized in that: In step S2, during dynamic adsorption, the loading speed of the loading liquid is 0.8-2BV / h.

6. The preparation method according to claim 1, characterized in that: The low-concentration alcohol solution in step S3 is a low-concentration ethanol solution with a volume fraction of 40-50%, the flow rate of elution is 0.4-0.6 BV / h, and the amount of the low-concentration ethanol solution is 1-1.2 BV.

7. The preparation method according to claim 1, characterized in that: In step S4, the eluent is a high-concentration ethanol solution with a volume fraction of 75-95%; the desorption rate of the eluent is 0.6-1.5 BV / h, and the amount of the eluent is 4-5 BV.

8. The preparation method according to claim 1, characterized in that: In step S2, adsorption is performed at a temperature of 16-20°C.

9. The preparation method according to claim 1, characterized in that: In step S5, the drying step is: transferring the desorbed liquid to a rotary evaporator, sealing and reducing the pressure to 400-500 mmHg; heating to 80-85° C. until the volume is concentrated to 6-8% of the original desorbed liquid.

10. High-purity 2-hydroxyphenazine prepared according to the preparation method according to any one of claims 1 to 9.

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