CN111606856B - Method for separating carnosine and histidine - Google Patents
Method for separating carnosine and histidine Download PDFInfo
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- CN111606856B CN111606856B CN202010513278.4A CN202010513278A CN111606856B CN 111606856 B CN111606856 B CN 111606856B CN 202010513278 A CN202010513278 A CN 202010513278A CN 111606856 B CN111606856 B CN 111606856B
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- C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
- C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
- C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine
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Abstract
The invention discloses a method for separating carnosine and histidine, which comprises the steps of adding a mixture containing the carnosine and the histidine into an aqueous two-phase system for extraction to realize the separation of the carnosine and the histidine; the double water phase system includes heavy phase as extractant and light phase as detergent, the heavy phase consists of inorganic salt and water, and the light phase consists of polymer and water or organic solvent and water; the inorganic salt is at least one of sulfate, phosphate, carbonate and chloride; the polymer is at least one of polyethylene glycol, polypropylene glycol, polyvinylpyrrolidone and ethylene oxide-propylene oxide copolymer; the organic solvent is at least one of lower alcohol with carbon number of 1-5, acetone and acetonitrile. The invention uses a double-aqueous phase system as a separation medium, realizes the high-efficiency separation of carnosine and histidine, has simple operation process and is easy to amplify.
Description
Technical Field
The invention relates to the technical field of chemical separation, in particular to a method for separating carnosine and histidine.
Background
Carnosine was found in the innervated tissues of spinal animals as early as 1900. Carnosine is known as beta-alanyl-L-histidine, an endogenous dipeptide composed of beta-alanine and L-histidine, can participate in regulating various physiological activities of human bodies, has excellent natural effects of resisting oxidation, free radicals, aging, nerves and blood vessels, relieving fatigue and the like, and is applied to the industries of medicines, health care, foods, cosmetics and the like.
Currently, most carnosine synthesis methods are to use beta-alanine or a beta-alanine precursor and L-histidine as starting materials, and synthesize the carnosine by protecting amino groups, activating carboxyl groups and selecting proper protective agents and activating agents; or directly synthesizing carnosine by changing proper conditions, salifying reaction and the like without protection and activation.
However, all of the conventional synthetic methods have a problem that separation is difficult, and particularly, carnosine and histidine are close in properties and are not easy to separate, so that it is difficult to obtain high-purity carnosine.
The existing literature has few reports on the separation of carnosine and histidine, and the literature reports that the separation of other amino acids and polypeptides mainly adopts an ion exchange method, the mixed solution is subjected to deacidification or neutralization until the pH value is reduced to be below the isoelectric point of the amino acids and polypeptides, then the salt is removed, most basic amino acids and part of neutral amino acids are adsorbed by cation exchange resin, and single amino acids and polypeptides are respectively obtained by gradient elution. However, the isoelectric point of histidine is 7.6, the isoelectric point of carnosine is 7.0, and the isoelectric points of histidine and carnosine are very close to each other, so that cross elution is serious, the purity and yield of carnosine are low, and the separation effect of the whole process is greatly reduced.
Therefore, no histidine and carnosine separation method suitable for industrial application is reported, and the technical progress of the carnosine and histidine separation technology is urgently needed.
Disclosure of Invention
Aiming at the defects in the field, the invention provides a method for separating carnosine and histidine, which takes a two-water-phase system as a separation medium to realize the high-efficiency separation of the carnosine and the histidine, and has simple operation process and easy amplification.
A method for separating carnosine and histidine comprises adding a mixture containing carnosine and histidine into an aqueous two-phase system for extraction to separate carnosine and histidine;
the aqueous two-phase system comprises a heavy phase serving as an extracting agent and a light phase serving as a washing agent, wherein the heavy phase consists of inorganic salt and water, and the light phase consists of a polymer and water or an organic solvent and water;
the inorganic salt is at least one of sulfate, phosphate, carbonate and chloride;
the polymer is at least one of polyethylene glycol, polypropylene glycol, polyvinylpyrrolidone and ethylene oxide-propylene oxide copolymer;
the organic solvent is at least one of low carbon alcohol with carbon number of 1-5, acetone and acetonitrile.
Compared with the traditional separation method, the double aqueous phase extraction technology can simplify the separation steps and reduce the production cost and energy consumption. In addition, the aqueous two-phase extraction also has the advantages of easy amplification, continuous operation, environmental protection and the like.
The aqueous two-phase system in the invention can be a two-phase system composed of polymer, inorganic salt and water, or a two-phase system composed of organic solvent, inorganic salt and water, specifically, the aqueous two-phase system can be composed of one of the polymer or organic solvent, one of the inorganic salt and water according to a proper proportion. The aqueous two-phase system is prepared from a polymer, inorganic salt and water, and when the polymer is liquid at normal temperature and normal pressure, the volume ratio of the polymer to the water in the aqueous two-phase system is preferably 0.2-2.5, and the mass ratio of the inorganic salt to the water is preferably 3-6; the double aqueous phase system is prepared from a polymer, inorganic salt and water, when the polymer is solid at normal temperature and normal pressure, the mass ratio of the polymer to the water in the double aqueous phase system is preferably 1-3:5, and the mass ratio of the inorganic salt to the water is preferably 3-6; when the aqueous two-phase system is prepared from an organic solvent, inorganic salt and water, the volume ratio of the organic solvent to the water in the aqueous two-phase system is preferably 0.2-2.5, and the mass ratio of the inorganic salt to the water is preferably 3-6. If the volume ratio of the light phase to the heavy phase is too high due to too high content of polymer or organic solvent in the aqueous two-phase system, less extractant is obtained. The content of inorganic salts (such as ammonium sulfate) in the aqueous two-phase system is too high, which causes salt precipitation in the system. If the content of the polymer, organic solvent, or inorganic salt (e.g., ammonium sulfate) in the aqueous two-phase system is too low, the aqueous two-phase system cannot be formed or the separation effect is not good.
The composition of the aqueous two-phase system is closely related to its separation performance. The system composition comprises the types and the occupied proportion of phase forming substances, the molecular weight of a polymer and the like, and all the factors can cause the capacity and the polarity of the light phase and the heavy phase to be associated with water molecules to be changed, so that the distribution coefficient of carnosine and histidine between the two phases is changed. Experiments show that when the two-phase system consisting of the polymer, the inorganic salt and the water or the two-phase system consisting of the organic solvent, the inorganic salt and the water is used as an extraction medium, higher distribution coefficient and selectivity coefficient can be obtained, and the separation of carnosine and histidine is facilitated.
Preferably, the molecular weight of the polymer is 400 to 10000. The polymer with too high molecular weight not only causes the difficulty of light and heavy two-phase delamination, but also causes the viscosity of the system to be too high, and increases the mass transfer resistance.
The pH of the aqueous two-phase system has a significant impact on its separation performance. Carnosine and histidine both belong to ampholytes and accept protons to present positive charges when the system pH is below the isoelectric point; when the pH is higher than the isoelectric point, protons bound to the pH are separated from the negative charges, so that the pH value of the solution influences the distribution ratio of carnosine and histidine between the two phases, and the charging properties of the carnosine and the histidine can be changed by adjusting the system pH, thereby influencing the separation effect of the carnosine and the histidine. Preferably, the pH of the aqueous two-phase system is 3 to 9. The pH adjustment can be carried out using an acid (e.g., hydrochloric acid, etc.) or a base (e.g., sodium hydroxide, etc.) which is commonly used in the art. Tests show that under the acidic condition (pH is more than or equal to 3 and less than 7), the aqueous two-phase system has higher separation selectivity on both carnosine and histidine.
Preferably, the temperature of the extraction is 15 to 40 ℃. The temperature is too low, the mass transfer rate between two phases is slow, the time required for the fractionation and extraction to reach the balance is long, and the production operation is not facilitated; meanwhile, the temperature is too high, the solvent is seriously volatilized, and carnosine and histidine are also possibly inactivated and denatured.
Preferably, the method for separating the carnosine and the histidine adopts a multistage fractional extraction device, the multistage fractional extraction device comprises an N-stage extraction section and an M-stage washing section, N, M are positive integers, and an extractant and the detergent in the multistage fractional extraction device are in countercurrent contact;
the extractant enters from the first stage of the extraction section, flows out from the Nth stage of the extraction section, enters into the Mth stage of the washing section, and finally flows out from the first stage of the washing section;
the detergent enters from the first stage of the washing section, flows out from the Mth stage of the washing section, enters into the Nth stage of the extraction section and finally flows out from the first stage of the extraction section;
the separation method specifically comprises the following steps:
(1) Dissolving a mixture containing carnosine and histidine with the light phase to obtain a raw material solution;
(2) Mixing the raw material liquid with a detergent flowing out of the Mth stage of the washing section, feeding the mixture into the Nth stage of the extraction section, flowing out of the first stage of the extraction section, collecting a raffinate rich in histidine, flowing out of the first stage of the washing section, collecting an extract rich in carnosine, and realizing the separation of the carnosine and the histidine.
The multistage fractional extraction device can adopt multistage fractional extraction equipment or devices commonly used in the field. According to the distribution coefficient of carnosine and histidine in the selected aqueous two-phase system, the flow ratio of the washing agent/the extracting agent and the flow ratio of the raw material liquid/the extracting agent are determined by comprehensively considering the aspects of product purity, recovery rate, treatment capacity and the like. Preferably, the flow ratio of the detergent/extractant is 0.1 to 10 and the flow ratio of the starting liquid/extractant is 0 to 5. More preferably, the flow ratio of the detergent/the extractant is in the range of 0.2 to 5, and the flow ratio of the raw material liquid/the extractant is in the range of 0 to 2.
When fractional extraction is carried out, the product recovery rate can be improved by increasing the number of stages of the extraction section; the number of washing stages is increased, so that higher product purity and higher recovery rate can be obtained.
Preferably, N is 3 to 30.
Preferably, M is 3 to 30.
More preferably, N, M are each independently selected from 3 to 30.
By using the optimized multistage fractional extraction separation method, the purity of carnosine in the extract can reach more than 98%, and the yield can reach more than 90%;
the purity of histidine in the raffinate can reach more than 98%, and the yield can reach more than 99%.
In the method for separating carnosine from histidine, the mass fraction of the carnosine in the mixture containing the carnosine and the histidine is preferably 1-88%.
Compared with the prior art, the invention has the main advantages that:
1. the invention adopts a double aqueous phase system as an extraction medium, and has higher selective separation capability on carnosine and histidine; the operation condition is mild, and the equipment is conventional; easy amplification, suitability for industrial production, small environmental pollution, high efficiency, simplicity and greenness.
2. The invention adopts the multi-stage fractionation extraction technology, the processing capacity is large, and the product purity and the yield can reach higher levels. According to different process conditions, the purity of carnosine can be controlled to be more than 95%, and the yield is more than 90%; the purity and yield of the histidine can be more than 90%.
Drawings
FIG. 1 is a schematic diagram of a multi-stage fractional extraction process according to the present invention.
Detailed Description
The invention is further described with reference to the following drawings and specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are conducted under conditions not specified, usually according to conventional conditions, or according to conditions recommended by the manufacturer.
The concentration of carnosine and histidine is quantitatively analyzed by High Performance Liquid Chromatography (HPLC), and the specific analysis conditions of the HPLC are as follows: the chromatographic column is a C18 column, the column temperature is 30 ℃, the mobile phase is sodium decane sulfonate buffer solution (1.22 g of sodium decane sulfonate and 11.41g of dipotassium hydrogen phosphate are dissolved in 1000mL of deionized water, and the pH is adjusted to 3.0 by phosphoric acid solution), and the acetonitrile =78 (v/v); the flow rate was 1.0mL/min, the detector was an ultraviolet detector, and the wavelength was 215nm.
The calculation method of the yield and the purity in the invention is as follows:
yield = mass of carnosine in product/mass of carnosine in starting material × 100%
Purity = mass of carnosine in product/total mass of carnosine and histidine in product × 100%
The flow of the method for separating carnosine and histidine in the following embodiments is shown in fig. 1, and a multi-stage fractional extraction device is adopted, wherein the multi-stage fractional extraction device comprises an N-stage extraction section and an M-stage washing section, N, M are positive integers, and an extractant and a washing agent are in countercurrent contact in the multi-stage fractional extraction device;
the extractant enters from the first stage of the extraction section, flows out from the Nth stage of the extraction section, enters into the Mth stage of the washing section, and finally flows out from the first stage of the washing section;
the detergent enters from the first stage of the washing section, flows out from the Mth stage of the washing section, enters into the Nth stage of the extraction section and finally flows out from the first stage of the extraction section;
the separation method specifically comprises the following steps:
(1) Dissolving a mixture containing carnosine and histidine with the light phase to obtain a raw material solution;
(2) Mixing the raw material liquid with a detergent flowing out of the Mth stage of the washing section, feeding the mixture into the Nth stage of the extraction section, flowing out of the first stage of the extraction section, collecting a raffinate rich in histidine, flowing out of the first stage of the washing section, collecting an extract rich in carnosine, and realizing the separation of the carnosine and the histidine.
Example 1
Preparing a double aqueous phase system consisting of polyethylene glycol 400, ammonium sulfate and water, wherein the volume ratio of the polyethylene glycol 400 to the water is 3, the mass ratio of the ammonium sulfate to the water is 1:2, pre-balancing, then taking out a light phase which is polyethylene glycol + aqueous phase and a heavy phase which is ammonium sulfate + aqueous phase, and taking out the light phase and the heavy phase for later use. A mixture of the raw material carnosine and histidine (carnosine/histidine =18.77% by mass) was dissolved in the light phase to prepare a raw material solution. And (2) performing fractional extraction at 25 ℃ by taking the heavy phase as an extracting agent and the light phase as a washing agent, wherein the flow ratio of the extracting agent to the raw material liquid to the washing agent is 2.6. The purity of carnosine in the extract is 98.37%, and the yield is 90.12%; the histidine purity in the raffinate was 98.18% with a yield of 99.72%.
Example 2
According to the weight ratio of polyethylene glycol 2000: ammonium sulfate: water =22.54, a two-aqueous phase system is prepared and pre-balanced according to the mass ratio of water = 22.54. A mixture of the raw material carnosine and histidine (carnosine/histidine =18.77% by mass) was dissolved in the light phase to prepare a raw material solution. And (2) carrying out fractional extraction at 25 ℃ by taking the heavy phase as an extracting agent, the light phase as a washing agent and the flow ratio of the extracting agent to the raw material liquid to the washing agent of 1. The purity of carnosine in the extract is 99.11%, and the yield is 91.69%; the purity of histidine in the raffinate was 98.46% with a yield of 99.85%.
Example 3
Preparing a double-aqueous-phase system consisting of polyethylene glycol 400, dipotassium hydrogen phosphate and water, wherein the volume ratio of the polyethylene glycol 400 to the water is 3. A mixture of carnosine and histidine (carnosine/histidine =18.77% by mass) was dissolved in the light phase to prepare a raw material solution. And (2) carrying out fractional extraction at 25 ℃ by taking the heavy phase as an extracting agent, the light phase as a washing agent and the flow ratio of the extracting agent to the raw material liquid to the washing agent of 1.01. The purity of carnosine in the extract liquor is 90.46 percent, and the yield is 89.24 percent; the histidine purity in the raffinate was 97.98% with a yield of 98.23%.
Example 4
Preparing a double aqueous phase system consisting of polyethylene glycol 400, ammonium sulfate and water, wherein the volume ratio of the polyethylene glycol 400 to the water is 1:5, the mass ratio of the ammonium sulfate to the water is 1:2, and adjusting the pH of the system to 4.2 by using concentrated hydrochloric acid. After pre-balancing, the light phase is polyethylene glycol + water phase, the heavy phase is ammonium sulfate + water phase, and the light phase and the heavy phase are taken out for later use. A mixture of carnosine and histidine (carnosine/histidine =18.77% by mass) was dissolved in the light phase to prepare a raw material solution. And (2) carrying out fractional extraction at 25 ℃ by taking the heavy phase as an extracting agent, the light phase as a washing agent and the flow ratio of the extracting agent to the raw material liquid to the washing liquid of 1. The purity of carnosine in the extraction liquid is 99.03%, and the yield is 90.05%; the purity of histidine in the raffinate was 98.16% with a yield of 99.83%.
Example 5
Preparing a double-aqueous-phase system consisting of polyethylene glycol 400, sodium carbonate and water, wherein the volume ratio of the polyethylene glycol 400 to the water is 3. A mixture of carnosine and histidine (carnosine/histidine =18.77% by mass) was dissolved in the light phase to prepare a raw material solution. And (2) performing fractional extraction at 25 ℃ by taking the heavy phase as an extracting agent and the light phase as a washing agent, wherein the flow ratio of the extracting agent to the raw material liquid to the washing liquid is 1.05. The purity of carnosine in the extraction liquid is 88.3 percent, and the yield is 90.85 percent; the purity of histidine in the raffinate was 98.27% with a yield of 97.74%.
Example 6
Preparing a double-aqueous-phase system consisting of polyethylene glycol 400, sodium sulfate and water, wherein the volume ratio of the polyethylene glycol 400 to the water is 3. A mixture of the raw material carnosine and histidine (carnosine/histidine =18.77% by mass) was dissolved in the light phase to prepare a raw material solution. And (2) carrying out fractional extraction at 25 ℃ by taking the heavy phase as an extracting agent, the light phase as a washing agent and the flow ratio of the extracting agent to the raw material liquid to the washing liquid of 1.6, wherein the extraction section has 13 stages and the washing section has 17 stages, and collecting the extraction liquid and the raffinate. The purity of carnosine in the extract is 95.55 percent, and the yield is 95.15 percent; the purity of histidine in the raffinate was 99.09% with a yield of 99.17%.
Furthermore, it should be understood that various changes and modifications can be made by one skilled in the art after reading the above description of the present invention, and equivalents also fall within the scope of the invention as defined by the appended claims.
Claims (6)
1. A method for separating carnosine and histidine is characterized in that a mixture containing the carnosine and the histidine is added into an aqueous two-phase system for extraction, so that the separation of the carnosine and the histidine is realized;
the aqueous two-phase system comprises a heavy phase serving as an extracting agent and a light phase serving as a washing agent, wherein the heavy phase consists of inorganic salt and water, and the light phase consists of a polymer and water or an organic solvent and water;
the inorganic salt is at least one of sulfate, phosphate, carbonate and chloride;
the polymer is at least one of polyethylene glycol, polypropylene glycol, polyvinylpyrrolidone and ethylene oxide-propylene oxide copolymer; the molecular weight of the polymer is 400-10000;
the organic solvent is at least one of low carbon alcohol with carbon number of 1-5, acetone and acetonitrile;
the double aqueous phase system is prepared from a polymer, inorganic salt and water, the polymer is liquid at normal temperature and normal pressure, the volume ratio of the polymer to the water in the double aqueous phase system is 0.2-2.5; or the double-aqueous-phase system is prepared from a polymer, inorganic salt and water, the polymer is solid at normal temperature and normal pressure, the mass ratio of the polymer to the water in the double-aqueous-phase system is 1-3:5, and the mass ratio of the inorganic salt to the water is 3-6; or the aqueous two-phase system is prepared from an organic solvent, inorganic salt and water, wherein the volume ratio of the organic solvent to the water in the aqueous two-phase system is 0.2-2.5, and the mass ratio of the inorganic salt to the water is 3-6;
the pH value of the aqueous two-phase system is 3-9;
the extraction temperature is 15-40 ℃.
2. The separation method according to claim 1, wherein the mass fraction of carnosine in the mixture comprising carnosine and histidine is between 1% and 88%.
3. The separation method according to claim 1 or 2, characterized in that a multi-stage fractional extraction device is adopted, the multi-stage fractional extraction device comprises an N-stage extraction section and an M-stage washing section, N, M are both positive integers, and an extractant and a washing agent in the multi-stage fractional extraction device are in countercurrent contact;
the extractant enters from the first stage of the extraction section, flows out from the Nth stage of the extraction section, enters into the Mth stage of the washing section, and finally flows out from the first stage of the washing section;
the detergent enters from the first stage of the washing section, flows out from the Mth stage of the washing section, enters into the Nth stage of the extraction section and finally flows out from the first stage of the extraction section;
the separation method specifically comprises the following steps:
(1) Dissolving a mixture containing carnosine and histidine with the light phase to obtain a raw material solution;
(2) Mixing the raw material liquid with a detergent flowing out of the Mth stage of the washing section, feeding the mixture into the Nth stage of the extraction section, flowing out of the first stage of the extraction section, collecting a raffinate rich in histidine, flowing out of the first stage of the washing section, collecting an extract rich in carnosine, and realizing the separation of the carnosine and the histidine.
4. The separation method according to claim 3, wherein N is 3 to 30.
5. The separation method according to claim 3, wherein M is 3 to 30.
6. A separation process according to claim 3, wherein the flow ratio of detergent/extractant is 0.1 to 10 and the flow ratio of feed liquid/extractant is 0 to 5.
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