阅读说明：本技术 一种高纯度荧光素的纯化方法 (Purification method of high-purity fluorescein ) 是由 张兆元 高云兵 甄爱华 刘建洋 苗元旭 于 2021-08-20 设计创作，主要内容包括：本发明属于荧光素提纯技术领域,涉及一种高纯度荧光素的纯化方法。以粗品荧光素为原料,其特征在于,乙酰化反应后纯水打浆,重结晶,然后利用甲醇钠脱乙酰精制得到精品荧光素。本发明是利用制备荧光素衍生物的方法提纯荧光素的新工艺,由于荧光素二乙酸酯易于制备、易于还原,并且具有很好的结晶性,与杂质的性质差别比较大,因此利用荧光素钠制备荧光素二乙酸酯,再经皂化还原的提纯路线克服了现有技术中酸碱重复使用,以及杂质常与荧光素产生共沉淀的问题。(The invention belongs to the technical field of fluorescein purification, and relates to a method for purifying high-purity fluorescein. The method is characterized in that after acetylation reaction, pure water is pulped, recrystallization is carried out, and then sodium methoxide is used for deacetylation and refining to obtain the refined fluorescein. The invention relates to a new process for purifying fluorescein by using a method for preparing a fluorescein derivative, which is characterized in that fluorescein diacetate is easy to prepare and reduce, has good crystallinity and has larger property difference with impurities, so the purification route of preparing the fluorescein diacetate by using fluorescein sodium and saponifying and reducing overcomes the problems of acid-base reuse and impurity coprecipitation with fluorescein in the prior art.)

1. A method for purifying high-purity fluorescein takes crude fluorescein as a raw material, and is characterized in that after acetylation reaction, pure water is pulped, recrystallization is carried out, and then sodium methoxide is used for deacetylation and refining to obtain refined fluorescein.

2. The method for purifying high-purity fluorescein according to claim 1, which comprises the following steps:

(1) acetylation

Uniformly mixing the crude fluorescein with acetic acid or ethyl acetate, adding acetic anhydride and an acid-binding agent, reacting for 3-5 hours at 50-60 ℃, cooling to 20-30 ℃, crystallizing for 1-2 hours, filtering, pulping a filter cake by using pure water, and drying by blowing at 45-55 ℃ for 8 hours to obtain light yellow diacetyl fluorescein;

(2) recrystallization

Adding the light yellow diacetyl fluorescein into 5-6 times volume (m/V) of organic solvent, heating to 40-45 ℃, dissolving, carrying out hot filtration, adding 4-6 times volume (m/V) of precipitated solvent, cooling to 0-5 ℃, carrying out suction filtration, and then carrying out forced air drying at 45-55 ℃ for 7-8h to obtain white crystalline diacetyl fluorescein;

(3) saponification reaction

Adding methanol into white crystalline diacetyl fluorescein, wherein the amount of the methanol is 4-6L/kg diacetyl fluorescein, uniformly stirring, adding sodium methoxide until the color of the mixture turns to orange red, heating to 55-65 ℃, stirring for saponification reaction for 2-2.5h, adding acetic acid, continuously stirring for reaction for 3-3.5h, then cooling to 0-5 ℃, crystallizing for 1-1.5h, performing suction filtration, and drying by blowing at 50-55 ℃ for 7-8 h;

(4) adding ethanol according to the use amount of 4-6L/kg, heating to 65-75 deg.C, pulping for 30-40min, cooling to 20-25 deg.C, vacuum filtering, and air drying at 45-55 deg.C for 7-8 hr to obtain refined fluorescein.

3. The method for purifying high-purity fluorescein according to claim 2, wherein the ratio of crude fluorescein in step (1): acetic anhydride: the mass ratio of the acid-binding agent is 1 (0.2-0.4): (0.1-0.3), the adding amount of acetic acid or ethyl acetate is 4-6L/kg of crude fluorescein, and the acid-binding agent is any one of sodium carbonate, potassium carbonate and pyridine.

4. The method for purifying high-purity fluorescein as claimed in claim 2, wherein the organic solvent in step (2) is any one of dichloromethane, ethanol, ethyl acetate and acetone, and the precipitation solvent is petroleum ether or diethyl ether.

5. The method for purifying high-purity fluorescein according to claim 2, wherein the ratio of white crystalline diacetylfluorescein in step (3): sodium methoxide: the mass ratio of acetic acid is 1: (0.6-0.7): (0.55-0.6).

Technical Field

The invention belongs to the technical field of fluorescein purification, and relates to a method for purifying high-purity fluorescein.

Background

Fluorescein (fluorescein), also known as fluorescein or fluorescein red, is chemically named 3',6 ' -dihydroxyspiro [ isobenzofuran 1 (3H), 9 ' - (9H) -xanthene ] -3-one or 9- (o-carboxyphenyl) -6-hydroxy-3H-xanthene-3-one, first synthesized by von bayer in 1871. Fluorescein exists in three tautomeric structural forms: the structure of the interior cool type, the quinoid type and the zwitter-ion type is as follows:

the maximum absorption/emission wavelength of fluorescein was 492/571 nm (in water) and the fluorescence quantum yield was 0.92 (pH > 8). Because of the existence of oxygen bridge bond, two benzene rings are fixed on a plane, so that the molecule has a rigid coplanar structure and can generate strong fluorescence under the action of exciting light.

High-purity fluorescein sodium is widely applied to injection preparations or eye drop preparations for fundus radiography and is generally prepared from purified fluorescein. Fluorescein is usually obtained by condensation of resorcinol and phthalic anhydride, and the domestic conventional method for purifying fluorescein is generally obtained by dissolving with alkali, then performing acid precipitation, and repeating the steps for many times. The acidification fluorescein is amorphous crystal, so the surface area is large, and the adsorption to impurities is serious. Meanwhile, most of the impurities are acidic substances and are insoluble in an acidic aqueous solution, so the impurities often form coprecipitation with fluorescein. Therefore, it is difficult to obtain fluorescein with high purity by the conventional process.

Disclosure of Invention

The invention provides a novel method for purifying high-purity fluorescein, aiming at the problems in the traditional crude fluorescein refining process.

In order to achieve the purpose, the invention is realized by adopting the following technical scheme:

a purification method of high-purity fluorescein takes crude fluorescein prepared by co-heating phthalic anhydride and resorcinol as a raw material, the process is mature at present and widely applied to industrial production, the crude fluorescein can be directly purchased, and the method is not repeated. The crude fluorescein obtained by the process is subjected to acetylation reaction, then is pulped by pure water, then is recrystallized, and is refined by sodium methoxide deacetylation to obtain the refined fluorescein.

Preferably, the method comprises the following specific steps:

(1) acetylation

Uniformly mixing the crude fluorescein with acetic acid or ethyl acetate, adding acetic anhydride and an acid-binding agent, reacting for 3-5 hours at 50-60 ℃, cooling to 20-30 ℃, crystallizing for 1-2 hours, filtering, pulping a filter cake with pure water, and drying by air blast at 45-55 ℃ for 8 hours to obtain light yellow diacetyl fluorescein;

(2) recrystallization

Adding the light yellow diacetyl fluorescein into 5-6 times volume (m/V) of organic solvent, heating to 40-45 ℃, dissolving, carrying out hot filtration, adding 4-6 times volume (m/V) of precipitated solvent, cooling to 0-5 ℃, carrying out suction filtration, and then carrying out forced air drying at 45-55 ℃ for 8 hours to obtain white crystalline diacetyl fluorescein;

(3) saponification reaction

Adding white crystalline diacetyl fluorescein into methanol with the volume of 4-6 times (m/V), uniformly stirring, adding sodium methoxide till the color of the mixture becomes orange red, heating to 55-65 ℃, stirring for saponification reaction for 2-2.5h, adding acetic acid, continuously stirring for reaction for 3-3.5h, then cooling to 0-5 ℃, crystallizing for 1-1.5h, carrying out suction filtration, and drying for 8h at 50-55 ℃ by blowing;

(4) adding 4-6 times volume of (m/V) ethanol, heating to 65-75 deg.C, pulping for 30-40min, cooling to room temperature, vacuum filtering, and air drying at 45-55 deg.C for 8 hr to obtain refined fluorescein.

Preferably, in step (1), the crude fluorescein: acetic anhydride: the mass ratio of the acid-binding agent is 1 (2-4): (0.1-0.3), the adding amount of acetic acid is 4-6L/kg of crude fluorescein, and the acid-binding agent is any one of sodium carbonate, potassium carbonate and pyridine.

Preferably, in the step (2), the organic solvent is any one of dichloromethane, ethanol, ethyl acetate and acetone, and the precipitation solvent is petroleum ether or diethyl ether.

Preferably, in step (3), white crystalline diacetylfluorescein: sodium methoxide: the mass ratio of acetic acid is 1: (0.6-0.7): (0.55-0.6).

Compared with the prior art, the invention has the advantages and positive effects that:

the invention relates to a new process for purifying fluorescein by using a method for preparing a fluorescein derivative, which is characterized in that fluorescein diacetate is easy to prepare and reduce, has good crystallinity and has larger property difference with impurities, so the purification route of preparing the fluorescein diacetate by using fluorescein sodium and saponifying and reducing overcomes the problems of acid-base reuse and impurity coprecipitation with fluorescein in the prior art.

Drawings

FIG. 1 shows the analysis of the sample spot plate.

FIGS. 2 to 4 are liquid chromatography detection charts of the fine fluorescein prepared in examples 1 to 3.

FIGS. 5 to 7 are gas chromatography detection charts of the fine fluorescein prepared in examples 1 to 3.

Detailed Description

In order that the above objects, features and advantages of the present invention may be more clearly understood, the present invention will be further described with reference to specific embodiments. It should be noted that the embodiments and features of the embodiments of the present application may be combined with each other without conflict.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those specifically described herein, and thus the present invention is not limited to the specific embodiments of the present disclosure.

The products obtained in the following examples were measured by high performance liquid chromatography under the following conditions.

The instrument comprises the following steps: a high performance liquid chromatograph;

a chromatographic column: accucore ™ Vanqush-C18 + ultra-high performance liquid chromatography column (1.5 mu m), specification 150X 2.1mm, 1.5 mu m;

detection wavelength: 245 nm;

flow rate: 1.0 ml/min;

column temperature: 37 ℃;

mobile phase: taking potassium dihydrogen phosphate solution (0.32 g potassium dihydrogen phosphate, 1000ml water for dissolving, adjusting pH to 3.5 with phosphoric acid) as mobile phase A; acetonitrile is mobile phase B.

The gradient elution procedure is shown in table 1.

TABLE 1 gradient elution procedure

Solvent: mobile phase a-acetonitrile (30: 70).

The data processing adopts a self-contrast method.

The solvent residue of the product obtained in each example described below was measured by gas chromatography under the following conditions.

Maintaining at 40 deg.C for 8min, increasing to 120 deg.C at 8 deg.C/min, and maintaining for 10 min; nitrogen was used as a carrier gas, and the flow rate was 2.0 mL/min. When water is used as solvent, the headspace temperature is 70-85 deg.C, the headspace time is 30-60min, and the injection inlet temperature is 200 deg.C, such as FID detector, and the temperature is 250 deg.C. And taking the reference solution and the sample solution, continuously feeding the samples for at least 2 times respectively, and determining the peak area of the peak to be measured. Because the gas chromatography is a commonly used detection means for the residue of the fluorescein solvent, the detection condition of the gas chromatography is consistent with the conventional condition without special description.

Example 1

The crude fluorescein is prepared by co-heating phthalic anhydride and resorcinol and is used as a raw material, the purity of the raw material is not limited, and the industrially obtained conventional crude fluorescein is only used. Taking 25g of crude fluorescein and 125mL of glacial acetic acid in a 500mL three-mouth reaction bottle, uniformly stirring at 80-100 r/min, then sampling a point plate to obtain a reaction point shown in the figure 1, continuously adding 5g of pyridine (acid binding agent) and 10g of acetic anhydride into the reaction bottle, stirring at 50-60 ℃ for reacting for 3h at the rotation speed of 80-100 r/min, continuously sampling the point plate to obtain a product point shown in the figure 1, adding 2mL of petroleum ether into 1mL of ethyl acetate serving as a TLC (thin-layer chromatography) developer to generate diacetyl fluorescein Rf =0.8, obtaining the crude fluorescein Rf =0.5, and carrying out next treatment if the product point has no crude fluorescein.

Cooling to below 25 ℃ after complete reaction, stirring for crystallization for 1h, and performing suction filtration, wherein the stirring speed is 100-150 r/min, the filter cake is pulped once by 125ml of purified water, the pulping speed is 200 r/min, the pulping time is 10-30min, and the acetic anhydride, pyridine and acetic acid solvent residues which are easy to dissolve in water can be removed through the pulping of the purified water. And after the completion of the beating, drying by blowing at 50 ℃ for 8h to obtain 28g of orange diacetyl fluorescein. Dissolving and clearing with 5 times volume (140mL) of dichloromethane at 40 ℃, then carrying out heat filtration, adding 5 times volume of ether (700 mL) for precipitation, cooling to 0-5 ℃, stirring for 1h, carrying out suction filtration, wherein the stirring speed is 80-100 r/min, and carrying out forced air drying at 50 ℃ for 8h to obtain 25.3g of white diacetyl fluorescein.

Taking 25g of diacetyl fluorescein, adding 5 times of methanol (125 mL) in volume into a 500mL three-necked bottle, stirring and uniformly mixing at 100-150 r/min, adding 16.3g of sodium methoxide, changing the reaction liquid from yellow to red, and controlling the temperature to be 60 ℃ to continue reacting for 2 h; adding 14.5g of acetic acid, and continuing to react for 3 hours; cooling to 0-5 ℃, stirring, crystallizing for 1h, performing suction filtration for 0.5-1h, and performing forced air drying at 50 ℃ for 8h to obtain 18g of crude fluorescein. Adding the crude fluorescein into 5 times volume of anhydrous ethanol (90 mL), pulping at 70 deg.C at 100 rpm for 30min, cooling to room temperature, vacuum filtering for 0.5-1h, and forced air drying at 50 deg.C for 8h to obtain refined fluorescein 16.3 g.

The detection results of the related substances are shown in FIG. 2. Solvent residue detection is shown in figure 5.

From FIG. 2, it can be seen that the purity of the refined fluorescein reaches 99.91%. It can be seen from FIG. 5 that no organic solvent remains.

Example 2

This example is not particularly described, and corresponds to example 1.

Taking 100g of crude fluorescein and 500ml of glacial acetic acid, putting the mixture into a 1L three-mouth reaction bottle, uniformly stirring, adding 20g of pyridine and 40g of acetic anhydride, controlling the temperature to be 50-60 ℃, stirring and reacting for 3h, monitoring the reaction result by TLC, adding 2ml of petroleum ether into 1ml of ethyl acetate serving as a developing agent to generate diacetyl fluorescein Rf =0.8, and adding crude fluorescein Rf =0.5 serving as a reactant. And after the reaction is completed, cooling to below 25 ℃, stirring, crystallizing for 1h, carrying out suction filtration, pulping a filter cake with 500ml of purified water once, and carrying out forced air drying at 50 ℃ to obtain 112g of orange diacetyl fluorescein. Dissolving and clearing with 5 times volume of dichloromethane at 40 ℃, adding 5 times volume of ether for precipitation, cooling to 0-5 ℃, stirring for 1h, performing suction filtration, and performing forced air drying at 50 ℃ to obtain 101g of white diacetyl fluorescein.

Taking 100g of diacetyl fluorescein and 500ml of methanol in a 1L three-necked bottle, uniformly stirring, adding 65g of sodium methoxide, changing the reaction liquid from yellow to red, and stirring and reacting for 2 hours at the temperature of 60 ℃; adding 58g of acetic acid, and stirring for reaction for 3 hours; cooling to 0-5 ℃, stirring, crystallizing for 1h, performing suction filtration, and performing forced air drying at 50 ℃ to obtain 72g of crude fluorescein. And adding the crude fluorescein into 5 times of anhydrous ethanol, pulping at 70 ℃ for 30min, cooling to room temperature, filtering, and drying by blowing at 50 ℃ to obtain 65g of refined fluorescein.

The detection results of the related substances are shown in FIG. 3, and the detection of the solvent residue is shown in FIG. 6.

From FIG. 3, it can be seen that the purity of the refined fluorescein reaches 99.92%. It can be seen from FIG. 6 that no organic solvent remains.

Example 3

This example is not particularly described, and is consistent with example 1.

Taking 3kg of crude fluorescein and 15L of glacial acetic acid in a 50L glass reaction kettle, uniformly stirring, adding 600g of pyridine and 1.2kg of acetic anhydride, controlling the temperature to be 50-60 ℃, stirring for reacting for 3h, monitoring the reaction result by TLC, adding 2ml of petroleum ether into 1ml of ethyl acetate serving as a developing agent to generate diacetyl fluorescein Rf =0.8, and obtaining a reactant crude fluorescein Rf = 0.5. And after the reaction is completed, cooling to below 25 ℃, stirring, crystallizing for 1h, carrying out suction filtration, pulping a filter cake with 15L of purified water once, and carrying out forced air drying at 50 ℃ for 8h to obtain 3.40kg of orange diacetyl fluorescein. Dissolving and clearing with 5 times volume of dichloromethane at 40 ℃, adding 5 times volume of ether for precipitation, cooling to 0-5 ℃, stirring for 1h, performing suction filtration, and performing forced air drying at 50 ℃ to obtain 3.05kg of white diacetyl fluorescein.

Taking 3kg of diacetyl fluorescein and 15L of methanol in a 25L glass reaction kettle, stirring and uniformly mixing, adding 1.95kg of sodium methoxide, changing the reaction liquid from yellow to red, and stirring and reacting for 2 hours at the temperature of 60 ℃; adding 1.74kg of acetic acid, and stirring for reaction for 3 hours; cooling to 0-5 ℃, stirring, crystallizing for 1h, performing suction filtration, and performing forced air drying at 50 ℃ to obtain 2.17kg of crude fluorescein. Adding the crude fluorescein into 5 times of anhydrous ethanol, pulping at 70 deg.C for 30min, cooling to room temperature, vacuum filtering, and air drying at 50 deg.C to obtain 1.95kg of refined fluorescein.

The detection results of the related substances are shown in FIG. 4, and the detection of the solvent residue is shown in FIG. 7.

From FIG. 4, it can be seen that the purity of the refined fluorescein reaches 99.98%. It can be seen from fig. 7 that no organic solvent remains.

As can be seen from fig. 2 to 4 and 5 to 7, the purified luciferin obtained by the methods of examples 1 to 3 has very high purity and no solvent remains. The examples 1-3 are the amplification process from the pilot plant test to the pilot plant test step by step, no obvious abnormality occurs in the amplification process, the quality of the product is very stable, and the process is proved to be complete and suitable for large-scale industrial application. Example 4 this example differs from example 3 in that the glacial acetic acid is replaced by ethyl acetate and the remaining conditions are kept the same. 1.95kg of refined fluorescein is finally obtained, the purity of the refined fluorescein is up to 99.97 percent through detection, and no organic solvent is left.

Example 5

The crude fluorescein is prepared by co-heating phthalic anhydride and resorcinol and is used as a raw material, the purity of the raw material is not limited, and the industrially obtained conventional crude fluorescein is only used. Taking 25g of crude fluorescein and 100mL of ethyl acetate, putting the mixture into a 500mL three-mouth reaction bottle, uniformly stirring at 80-100 r/min, continuously adding 2.5g of pyridine (acid binding agent) and 5g of acetic anhydride into the reaction bottle, controlling the temperature to be 50-60 ℃, stirring and reacting for 5h, and rotating at 80-100 r/min. After the reaction is completed, the temperature is reduced to 20 ℃, the stirring crystallization is carried out for 2h, the suction filtration is carried out, the stirring speed is 100 plus materials and 150 r/min, the filter cake is pulped once by 150ml of purified water, the pulping speed is 200 r/min, the pulping time is 30min, and the acetic anhydride, pyridine and acetic acid solvent residues which are easy to dissolve in water can be removed through the pulping of the purified water. And after the beating is finished, drying by blowing at 50 ℃ for 8h to obtain the orange diacetyl fluorescein. Dissolving and clearing with 5 times volume of dichloromethane at 45 ℃, then carrying out heat filtration, adding 4 times volume of ether for precipitation, cooling to 0-5 ℃, stirring for 1.5h, carrying out suction filtration, wherein the stirring speed is 80-100 r/min, and carrying out forced air drying at 55 ℃ for 7h to obtain the white diacetyl fluorescein.

Taking the diacetyl fluorescein, adding methanol with 5 times volume (mass-volume ratio, the same below) into a 500mL three-necked flask, stirring and uniformly mixing at 100-; adding 15.2g of acetic acid, and continuing to react for 3.5 h; cooling to 0-5 ℃, stirring, crystallizing for 1.5h, performing suction filtration for 0.5-1h, and performing forced air drying at 55 ℃ for 7h to obtain a crude fluorescein. Adding the crude fluorescein into 5 times of anhydrous ethanol, pulping at 75 deg.C at 100 rpm for 40min, cooling to room temperature, vacuum filtering for 1h, and air drying at 45 deg.C for 7h to obtain refined fluorescein.

The purity of the refined fluorescein is up to 99.92% by detection, and the organic solvent has no residue.

Example 6

The crude fluorescein is prepared by co-heating phthalic anhydride and resorcinol and is used as a raw material, the purity of the raw material is not limited, and the industrially obtained conventional crude fluorescein is only used. Taking 25g of crude fluorescein and 1200mL of ethyl acetate, putting the mixture into a 500mL three-mouth reaction bottle, uniformly stirring at 90 r/min, continuously adding 7.5g of pyridine (acid binding agent) and 10g of acetic anhydride into the reaction bottle, controlling the temperature to be 60 ℃, stirring and reacting for 3h, and rotating speed to be 80-100 r/min. After the reaction is completed, the temperature is reduced to 22 ℃, the stirring crystallization is carried out for 1.5h, the suction filtration is carried out, the stirring speed is 100-150 r/min, the filter cake is pulped once by 200ml of purified water, the pulping speed is 200 r/min, the pulping time is 25min, and the acetic anhydride, pyridine and acetic acid solvent residues which are easy to dissolve in water can be removed through the pulping of the purified water. And after the beating is finished, drying by air blast at 45 ℃ for 7.5h to obtain the orange diacetyl fluorescein. Dissolving and clearing with 5 times volume of dichloromethane at 40 ℃, then carrying out heat filtration, adding 4 times volume of ether for precipitation, cooling to 0-5 ℃, stirring for 1.5h, carrying out suction filtration, wherein the stirring speed is 80-100 r/min, and carrying out forced air drying at 50 ℃ for 8h to obtain the white diacetyl fluorescein.

Taking diacetyl fluorescein, adding methanol with 5 times volume (mass-volume ratio, the same below) into a 500mL three-necked flask, stirring and uniformly mixing at 100-; adding 15.8g of acetic acid, and continuing to react for 3.5 h; cooling to 0-5 ℃, stirring, crystallizing for 1.0h, performing suction filtration for 0.5-1h, and performing forced air drying at 45 ℃ for 7.5h to obtain a crude product fluorescein. Adding the crude fluorescein into 6 times of anhydrous ethanol, pulping at 65 deg.C at 100 rpm for 30min, cooling to room temperature, vacuum filtering for 1h, and air drying at 55 deg.C for 8h to obtain refined fluorescein.

The purity of the refined fluorescein is up to 99.92% by detection, and the organic solvent has no residue.

The above description is only a preferred embodiment of the present invention, and not intended to limit the present invention in other forms, and any person skilled in the art may apply the above modifications or changes to the equivalent embodiments with equivalent changes, without departing from the technical spirit of the present invention, and any simple modification, equivalent change and change made to the above embodiments according to the technical spirit of the present invention still belong to the protection scope of the technical spirit of the present invention.