A crystal gel adsorption chromatography separation method for lactic acid

Abstract

The invention discloses a crystal gel adsorption chromatography separation method for lactic acid, wherein the method uses hydroxyethyl methacrylate as a monomer to polymerize a crystal gel matrix skeleton, obtains a crystal gel medium after chemical modification, dilutes a solution containing lactic acid with deionized water to obtain a sample solution, rinses the crystal gel medium with deionized water to wash away impurities that are not adsorbed, elutes with a 0.1-3 M salt solution, and collects an eluate containing lactic acid. The method is simple in process for separating lactic acid, easy to scale up, low in energy consumption, and free of secondary pollution, and the crystal gel medium can be reused, and is expected to be widely used in the separation and purification process of lactic acid.

Description

Technical Field

[0001] The invention belongs to the field of biochemical separation, and in particular relates to a crystal gel adsorption chromatography separation method of lactic acid. Background Art

[0002] As one of the three major organic acids, lactic acid is widely used in food, medicine, chemical industry and cosmetics. Lactic acid is an important raw material for synthesizing biodegradable material polylactic acid. Polylactic acid is expected to become an environmentally friendly material to replace petroleum-based plastics due to its biodegradability and biocompatibility. Therefore, the production of lactic acid has received widespread attention in recent years.

[0003] Lactic acid is usually synthesized by microbial fermentation. The fermentation raw materials are easy to obtain and cheap, and the lactic acid synthesis efficiency is high. However, the subsequent separation and purification is difficult. Crystallization, organic solvent extraction, membrane filtration, electrodialysis, etc. are commonly used in industry, which require a large amount of acid or alkali solution, high energy consumption, and produce a large amount of waste liquid. The separation efficiency is low, which affects the quality of downstream products.

[0004] Crystal gel matrix has broad application prospects in the field of bioseparation due to its excellent properties of ultra-large pores, high specific surface area, high permeability and low mass transfer resistance. However, there are no reports on the use of crystal gel matrix for adsorption of lactic acid after chemical modification. Summary of the invention

[0005] In view of the above technical problems existing in the prior art, the object of the present invention is to provide a crystal gel adsorption chromatography separation method for lactic acid.

[0006] The technical solution adopted by the present invention is:

[0007] The crystal gel adsorption chromatography separation method of lactic acid comprises the following steps:

[0008] S1: preparing a crystal gel chromatography column: the crystal gel medium filled in the crystal gel chromatography column is a crystal gel matrix skeleton formed by polymerization of hydroxyethyl methacrylate as a monomer, and further chemically modified with a monomer having a tertiary amino functional group to form a crystal gel;

[0009] S2: sample adsorption: using the lactic acid solution as the sample solution, the crystal gel chromatography column obtained in step S1 is loaded with the sample at a flow rate of 0.1-5 cm / min until the crystal gel chromatography column is saturated with adsorption;

[0010] S3: washing away impurities: washing the crystal gel medium in the crystal gel chromatography column with deionized water to wash away impurities that have not been adsorbed;

[0011] S4: Elution of lactic acid: elution with 0.1-2 M saline solution, and collection of the purified lactic acid-containing eluate;

[0012] S5 Crystal gel regeneration: Regenerate the crystal gel chromatography column with 1~2 M salt solution and a large amount of deionized water in sequence.

[0013] Furthermore, the specific preparation method of the crystal gel chromatography column in step S1 is:

[0014] 1) Dispersing hydroxyethyl methacrylate HEMA and polyethylene glycol diacrylate PEG-DA in ultrapure water at a mass ratio of 70-80:30-20 to prepare a monomer solution, wherein the total mass fraction of HEMA and PEG-DA in the monomer solution is 10-15%;

[0015] 2) Add initiator and accelerator to the monomer solution of step 1), continue to stir evenly, quickly pour the reaction solution into the microtube that has been pre-cooled at -5~-10℃, and set the cooling system temperature to -20~-30℃, and continue the low-temperature reaction for 24~48 hours to obtain the crystal gel matrix; take out the microtube, thaw it at room temperature, and rinse the composite crystal gel matrix with a large amount of pure water for use;

[0016] 3) The monomer with tertiary amino functional group is grafted onto the crystal gel matrix obtained in step 2) through a grafting reaction under the action of a catalyst to obtain an anionic crystal gel medium.

[0017] Furthermore, in step 2), the initiator and the accelerator are ammonium persulfate and tetramethylethylenediamine, respectively, and the amount of both is 0.5-1.5% of the total mass of HEMA and PEG-DA, preferably 1.0%.

[0018] Furthermore, in step 3), the monomer with a tertiary amino functional group in the grafting reaction is dimethylaminoethyl methacrylate.

[0019] Furthermore, in step 3), the specific process of the grafting reaction is: the catalyst solution is loaded and flowed through the prepared crystal gel matrix, so that the catalyst solution is fully in contact with the crystal gel matrix, and then the monomer solution with tertiary amino functional groups is loaded and flowed through the crystal gel matrix, so that the monomer with tertiary amino functional groups is fully in contact with the crystal gel matrix, and after reacting for a certain time at a certain temperature, the unreacted solution is rinsed with pure water to obtain an anionic crystal gel medium. The amount of the monomer solution with tertiary amino functional groups is 3 to 5 times the volume of the crystal gel matrix, and its monomer concentration is 1 to 2 M. The catalyst solution is 0.03 to 0.05 M Cu 3+ The amount of ion solution is 3 to 5 times the volume of the crystal gel matrix, the grafting reaction temperature is 45 to 60 ° C, and the reaction time is 1 to 3 hours.

[0020] Furthermore, the sample loading flow rate in step S2 is 1-4 cm / min.

[0021] Furthermore, the salt solutions described in step S4 and step S5 are both NaCl salt solutions.

[0022] Compared with the prior art, the beneficial technical effect of the present invention is that the lactic acid adsorption chromatography separation method provided by the present invention has simple process, no secondary pollution, low energy consumption and high separation efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the HPLC spectrum of the sample solution in Example 5 of the present application;

[0024] Figure 2 This is the HPLC spectrum of the eluent in Example 5 of the present application. Implementation

[0025] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto. Example

[0026] Hydroxyethyl methacrylate and polyethylene glycol diacrylate were added to pure water at a mass ratio of 77:23 to prepare a monomer solution, wherein the total mass concentration of the mixture of hydroxyethyl methacrylate and polyethylene glycol diacrylate was 10% (w / w). Initiator and accelerator were added to the well-stirred monomer solution, wherein the initiator and accelerator were ammonium persulfate and tetramethylethylenediamine, respectively, wherein the initiator and accelerator accounted for 1.0% (w / w) of the total amount of monomers (HEMA and PEG-DA), and the stirring was continued for 1 min. The reaction solution was quickly poured into a microtube that had been precooled at -10 °C, and the cooling system temperature was set to -20 °C, and the low-temperature reaction was continued for 24 h; the microtube was taken out, thawed at room temperature, and the crystal gel matrix was rinsed with a large amount of pure water. The grafting reaction was carried out on the crystal gel matrix. The catalyst solution was first loaded and flowed through the prepared crystal gel matrix, and then the monomer solution was loaded and flowed through the crystal gel matrix. The grafting monomer was dimethylaminoethyl methacrylate, and the amount of the monomer aqueous solution was 3 times that of the crystal gel matrix. The grafting reaction catalyst was 0.03 M Cu 3+ ion solution, the amount of catalyst used was 3 times the volume of the crystal gel matrix, the grafting reaction temperature was 45 °C, and the reaction time was 3 h. After the reaction, the crystal gel medium was rinsed with a large amount of pure water, and the diameter of the crystal gel medium was measured to be 1.1 cm, the length was 4.4 cm, and the porosity was 89%.

[0027] Use 100 mg / ml lactic acid solution as the loading liquid, load the sample at a flow rate of 1.0 cm / min, and load the sample to saturation; rinse the crystal gel column with pure water at a flow rate of 1.0 cm / min to wash away the impurities that are not adsorbed; elute with 11.7mL 1M NaCl salt solution at a flow rate of 1.0cm / min, and collect the eluate containing lactic acid. The entire experimental process was carried out under the monitoring of an ultraviolet detector with a wavelength of 220nm, and the collected eluate was measured for lactic acid concentration using an ultraviolet spectrophotometer at a wavelength of 210 nm, the same below. The detection showed that the lactic acid content of the eluate was 10.4 mg / mL, and the adsorption amount of the crystal gel was 29.6 mg / mL (wet crystal gel). Example

[0028] Hydroxyethyl methacrylate and polyethylene glycol diacrylate were added to pure water at a mass ratio of 77:23 to prepare a monomer solution, wherein the total mass concentration of the mixture of hydroxyethyl methacrylate and polyethylene glycol diacrylate was 10% (w / w). Initiator and accelerator were added to the well-stirred monomer solution, wherein the initiator and accelerator were ammonium persulfate and tetramethylethylenediamine, respectively, wherein the initiator and accelerator accounted for 1.0% (w / w) of the total amount of monomers (HEMA and PEG-DA), respectively, and stirring was continued for 1 min. The reaction solution was quickly poured into a microtube that had been precooled at -5 °C, and the cooling system temperature was set to -30 °C, and the low-temperature reaction was continued for 24 h; the microtube was taken out, thawed at room temperature, and the crystal gel matrix was rinsed with a large amount of pure water. The grafting reaction was carried out on the crystal gel matrix. The catalyst solution was first loaded and flowed through the prepared crystal gel matrix, and then the monomer solution was loaded and flowed through the crystal gel matrix. The grafting monomer was dimethylaminoethyl methacrylate, and the amount of the monomer aqueous solution was 5 times that of the crystal gel matrix. The grafting reaction catalyst was 0.03 M Cu 3+ ion solution, the amount of catalyst used was 5 times the volume of the crystal gel matrix, the grafting reaction temperature was 60 °C, and the reaction time was 1 h. After the reaction, the crystal gel medium was rinsed with a large amount of pure water, and the diameter of the crystal gel medium was measured to be 1.0 cm, the length was 4.6 cm, and the porosity was 87%.

[0029] Use 20 mg / ml lactic acid solution as the loading liquid, load the sample at a flow rate of 1.0 cm / min, and load the sample to saturation; rinse the crystal gel column with pure water at a flow rate of 1.0 cm / min to wash away the impurities that are not adsorbed; elute with 10.0 mL 1M NaCl salt solution at an elution flow rate of 1.0 cm / min, and collect the eluent containing lactic acid. The detection shows that the lactic acid content of the eluent is 5.3 mg / mL, and the adsorption amount of the crystal gel is 12.5 mg / mL (wet crystal gel). After repeated adsorption 5 times, the lactic acid content of the eluent is 4.9 mg / mL, and the adsorption amount of the crystal gel is 12.5 mg / mL (wet crystal gel). After repeated adsorption 10 times, the lactic acid content of the eluent is 5.1 mg / mL, and the adsorption amount of the crystal gel is 12.4 mg / mL (wet crystal gel). Example

[0030] Hydroxyethyl methacrylate and polyethylene glycol diacrylate were added to pure water at a mass ratio of 77:23 to prepare a monomer solution, wherein the total mass concentration of the mixture of hydroxyethyl methacrylate and polyethylene glycol diacrylate was 10% (w / w). Initiator and accelerator were added to the well-stirred monomer solution, wherein the initiator and accelerator were ammonium persulfate and tetramethylethylenediamine, respectively, wherein the initiator and accelerator accounted for 1.0% (w / w) of the total amount of monomers (HEMA and PEG-DA), respectively, and stirring was continued for 1 min. The reaction solution was quickly poured into a microtube that had been precooled at -5 °C, and the cooling system temperature was set to -20 °C, and the low-temperature reaction was continued for 48 h; the microtube was taken out, thawed at room temperature, and the crystal gel matrix was rinsed with a large amount of pure water. The grafting reaction was carried out on the crystal gel matrix. The catalyst solution was first loaded and flowed through the prepared crystal gel matrix, and then the monomer solution was loaded and flowed through the crystal gel matrix. The grafting monomer was dimethylaminoethyl methacrylate, and the amount of the monomer aqueous solution was 3 times that of the crystal gel matrix. The grafting reaction catalyst was 0.05 M Cu 3+ ion solution, the amount of catalyst used was 3 times the volume of the crystal gel matrix, the grafting reaction temperature was 45 °C, and the reaction time was 2 h. After the reaction, the crystal gel medium was rinsed with a large amount of pure water, and the diameter of the crystal gel medium was measured to be 1.1 cm, the length was 4.4 cm, and the porosity was 86%.

[0031] 100 mg / ml lactic acid solution was used as the loading liquid, and the sample was loaded at a flow rate of 4.0 cm / min until the sample was saturated; pure water was used to rinse the crystal gel column at a flow rate of 4.0 cm / min to wash away the impurities that were not adsorbed; 15.0 mL of 0.5 M NaCl salt solution was used for elution at a flow rate of 4.0 cm / min, and the eluate containing lactic acid was collected. The lactic acid content of the eluate was 6.8 mg / mL, and the adsorption amount of the crystal gel was 24.7 mg / mL (wet crystal gel). Example

[0032] Hydroxyethyl methacrylate and polyethylene glycol diacrylate were added to pure water in a mass ratio of 77:23 to prepare a monomer solution, wherein the total mass concentration of the mixture of hydroxyethyl methacrylate and polyethylene glycol diacrylate was 15% (w / w). Initiator and accelerator were added to the well-stirred monomer solution, wherein the initiator and accelerator were ammonium persulfate and tetramethylethylenediamine, respectively, wherein the initiator and accelerator accounted for 1.0% (w / w) of the total amount of monomers (HEMA and PEG-DA), respectively, and stirring was continued for 1 min. The reaction solution was quickly poured into a microtube that had been precooled at -10 °C, and the cooling system temperature was set to -20 °C, and the low-temperature reaction was continued for 24 h; the microtube was taken out, thawed at room temperature, and the crystal gel matrix was rinsed with a large amount of pure water. The grafting reaction was carried out on the crystal gel matrix. The catalyst solution was first loaded and flowed through the prepared crystal gel matrix, and then the monomer solution was loaded and flowed through the crystal gel matrix. The grafting monomer was dimethylaminoethyl methacrylate, and the amount of the monomer aqueous solution was 3 times that of the crystal gel matrix. The grafting reaction catalyst was 0.05 M Cu 3+ ion solution, the amount of catalyst used was 3 times the volume of the crystal gel matrix, the grafting reaction temperature was 45 °C, and the reaction time was 2 h. After the reaction, the crystal gel medium was rinsed with a large amount of pure water, and the diameter of the crystal gel medium was measured to be 1.0 cm, the length was 4.8 cm, and the porosity was 83%.

[0033] Dilute the solution containing lactic acid with pure water to obtain the loading solution, load the sample at a flow rate of 4.1 cm / min, and load the sample to saturation; rinse the crystal gel column with pure water at a flow rate of 4.1 cm / min to wash away the impurities that are not adsorbed; elute with 10.5 mL of 2 M NaCl salt solution at a flow rate of 4.1 cm / min, and collect the eluate containing lactic acid. The lactic acid content of the eluate was detected to be 6.7 mg / mL, and the adsorption amount of the crystal gel was 18.4 mg / mL (wet crystal gel). Example

[0034] Lactobacillus paracasei was cultured in broth medium for 48 h, and the obtained fermentation liquid was diluted 10 times with pure water (the lactic acid content was 6.6 mg / ml) to obtain the sample solution (the HPLC spectrum of the sample solution is shown in Figure 1 As shown), taking the crystal gel medium of Example 2 as an example, the sample was loaded at a flow rate of 1.0 cm / min until the sample was saturated; the crystal gel column was rinsed with pure water at a flow rate of 1.0 cm / min to wash away the impurities that were not adsorbed; 7.8 mL of 1 M NaCl salt solution was used for elution at a flow rate of 1.01 cm / min, and the eluate containing lactic acid was collected (the HPLC spectrum of the eluate containing lactic acid is shown in Figure 2 The lactic acid content of the eluent was detected to be 4.1 mg / mL, and the adsorption amount of the crystal gel was 7.9 mg / mL (wet crystal gel).

[0035] contrast Figure 1 and Figure 2 It can be seen that Figure 2 The impurity peaks in the liquid phase spectrum of the eluent are significantly reduced, indicating that the crystal gel column of the present invention can not only effectively adsorb lactic acid, but also can well separate lactic acid from impurities.

Claims

1. A method for separating lactic acid by crystal gel adsorption chromatography, characterized in that: The following steps are involved: S1 Preparation of crystal gel chromatography column: The crystal gel medium filled in the crystal gel chromatography column is a crystal gel matrix skeleton formed by polymerization of hydroxyethyl methacrylate as a monomer, and further a crystal gel formed by grafting reaction of a monomer with a tertiary amino functional group under the action of a catalyst; S2: sample adsorption: using the lactic acid solution as the sample solution, the crystal gel chromatography column obtained in step S1 is loaded with the sample at a flow rate of 0.1-5 cm / min until the crystal gel chromatography column is saturated with adsorption; S3: washing away impurities: washing the crystal gel medium in the crystal gel chromatography column with deionized water to wash away impurities that have not been adsorbed; S4: Elution of lactic acid: elution with 0.1-2 M saline solution, and collection of the purified lactic acid-containing eluate; S5 Crystal gel regeneration: regenerate the crystal gel chromatography column with 1~2 M salt solution and a large amount of deionized water in sequence; In step S1, the monomer with a tertiary amino functional group for the grafting reaction is dimethylaminoethyl methacrylate, and the specific process of the grafting reaction is: the catalyst solution is sampled and flowed through the prepared crystal gel matrix to make the catalyst solution fully contact with the crystal gel matrix, and then the monomer solution with a tertiary amino functional group is sampled and flowed through the crystal gel matrix to make the monomer with a tertiary amino functional group fully contact with the crystal gel matrix, at a certain temperature, after reacting for a certain time, the unreacted solution is rinsed with pure water to obtain an anionic crystal gel medium; the amount of the monomer solution with a tertiary amino functional group is 3 to 5 times the volume of the crystal gel matrix, and its monomer concentration is 1 to 2 M, and the catalyst solution is 0.03 to 0.05 M Cu 3+ The amount of ion solution is 3 to 5 times the volume of the crystal gel matrix, the grafting reaction temperature is 45 to 60 ° C, and the reaction time is 1 to 3 hours.

2. The method for separating lactic acid by crystal gel adsorption chromatography according to claim 1, characterized in that: The specific preparation method of the crystal gel chromatography column in step S1 is: 1) Dispersing hydroxyethyl methacrylate HEMA and polyethylene glycol diacrylate PEG-DA in ultrapure water at a mass ratio of 70-80:30-20 to prepare a monomer solution, wherein the total mass fraction of HEMA and PEG-DA in the monomer solution is 10-15%; 2) Add initiator and accelerator to the monomer solution of step 1), continue to stir evenly, quickly pour the reaction solution into the microtube that has been pre-cooled at -5~-10℃, and set the cooling system temperature to -20~-30℃, and continue the low-temperature reaction for 24~48 hours to obtain the crystal gel matrix; take out the microtube, thaw it at room temperature, and rinse the composite crystal gel matrix with a large amount of pure water for use; 3) The monomer with tertiary amino functional group is grafted onto the crystal gel matrix obtained in step 2) through a grafting reaction under the action of a catalyst to obtain an anionic crystal gel medium.

3. The method for separating lactic acid by crystal gel adsorption chromatography according to claim 2, characterized in that: In step 2), the initiator and accelerator are ammonium persulfate and tetramethylethylenediamine, respectively, and the amount of both is 0.5-1.5% of the total mass of HEMA and PEG-DA.

4. The method for separating lactic acid by crystal gel adsorption chromatography according to claim 3, characterized in that: In step 2), the initiator and accelerator are ammonium persulfate and tetramethylethylenediamine, respectively, and the amount of both is 1.0% of the total mass of HEMA and PEG-DA.

5. The method for separating lactic acid by crystal gel adsorption chromatography according to claim 1, characterized in that The sample loading flow rate in step S2 is 1-4 cm / min.

6. The method for separating lactic acid by crystal gel adsorption chromatography according to claim 1, characterized in that The salt solutions described in step S4 and step S5 are both NaCl salt solutions.

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