JPS5946500B2 - Amino acid separation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for rapidly and accurately separating each amino acid, particularly threonine and serine, from a mixture of amino acids by liquid chromatography.

Conventionally, it is well known that each amino acid can be separated from a mixture of amino acids by a liquid chromatography method using a sulfonic acid type cation exchange resin of styrene-civinylbenzene copolymer as a filler.

However, although this method is good at separating certain mixtures of amino acids, such as glycine and alanine, the separation of threonine and serine, and isoleucine and leucine is not always satisfactory, and in particular, the mutual separation of threonine and serine is not satisfactory. It is difficult to satisfy the separation of these amino acids at the same time. In recent years, it has become known that mutual separation of each amino acid, especially threonine and serine, can be improved from a mixture of amino acids by mixing an organic solvent such as ethanol with the first buffer. However, although this method improves the mutual separation of threonine and serine to some extent, in order to improve the separation rate of threonine and serine, a large amount of organic solvent such as ethanol must be added to the first buffer solution. On the other hand, this has the disadvantage that mutual separation of other amino acid mixtures, such as glycine and alanine, becomes poor, and the viscosity of the first buffer increases, which increases the pressure in the separation column, causing damage to the column, packing material, and pump. If the pressure resistance of the pump is low, accurate separation is impossible, and there is a difference in viscosity between the first buffer containing an organic solvent and the second buffer containing no organic solvent. When the liquid is delivered using a resiprokatin tablancher pump, there is a difference in the flow rates of the first and second buffer solutions, and the column eluate and reaction test solution contain hydrin (or orthophthalaldehyde-mercaptoethanol). The mixing ratio of the column eluate and the reaction time between the column eluate and the reaction test solution change, the baseline fluctuates, and speed and quantitative performance are adversely affected. In addition, silica gel, alumina, and glass spheres into which ion exchange groups (sulfone groups) have been introduced are widely used as separating agents for thin layer chromatography and packing materials for high performance liquid chromatography because they have high pressure resistance and are inexpensive. .

However, these separating agents and fillers are sensitive to alkalis because their base materials are acidic, and if the pH is higher than 9, the separating agents and fillers will gradually dissolve and be destroyed. the impossibility of regenerating the base;
Chemical bonding methods and adhesion methods in which ion exchanger fine powder is physically attached are known methods for introducing ion exchange groups into substrates, but fillers obtained by chemical bonding methods are susceptible to acids. The bond is weak, and bonds are gradually broken at pH below 2. On the other hand, with the packing material obtained by the adhesion method, the deposits peel off in a relatively short time due to fluctuations in column temperature and liquid feeding pressure. In addition, the separation agents and packing materials obtained by these chemical bonding methods and attachment methods have a small exchange capacity and are easily saturated and poor separation when the sample concentration is high.Separating agents and packing materials for ion exchange chromatography However, it has shortcomings such as short life and impractical. Furthermore, silica gel into which an octadecyl group has been introduced has a high pressure resistance, is inexpensive, and has the amino acid PTH (Ph
enylthiOhydantOinOfamnOac
ids) It is used as a packing material in partition chromatography because it can be denatured into amino acids and then separated in partition chromatography separation mode.

However, this method has the fatal disadvantage that certain amino acids are not separated from other amino acids at all (for example, isoleucine and leucine), and that P
Apart from cases in which HT amino acids can be obtained, in the case of amino acids in protein hydrolysates or free amino acids in biological components, it is not only complicated to denature these amino acids into PHT amino acids, but the denaturation rate is not 100%. As the value fluctuates, there is a problem with quantitative performance.As the packing properties are acidic, even if contaminated with undegraded peptides and other impurities, they cannot be regenerated with alkali. In the case of ion-exchange chromatography, the solubility of the substance to be separated in the eluent is high, so even if the concentration of the substance to be separated is high, it does not cause problems. In the case of partition chromatography, the solubility of the substance to be separated in the syneresis liquid is poor, and if the concentration of the substance to be separated is high, precipitates will form in the column, clogging the column, resulting in poor separation and It has drawbacks such as problems especially when the concentration is unknown. The present inventors conducted various studies on a method for quickly and accurately separating each amino acid from a mixture of amino acids using liquid chromatography. The present invention was achieved based on the discovery that when used as a filler, amino acids are separated from each other very effectively. That is, the present invention has a sulfone group and a carboxyl group in the structural unit.

Based on a liquid chromatography method characterized in that a reticulated porous cation exchange resin made of a monovinyl aromatic compound-(meth)acrylic acid ester-polyvinyl aromatic compound copolymer is used as a column packing material. This invention relates to a method for separating each amino acid from a mixture of amino acids. The reticulated porous cation exchange resin having sulfone groups and carboxyl groups as the packing material for the liquid chromatography column used in the method of the present invention has a particle size of 3 to 5 μm.
1 Specific surface area is 50~200i/9, bore size is 50~
500 people, ion exchange capacity 0.5-10meq/9,
The molar ratio of the sulfone group to the carboxyl group is 10 to 70 mol% for the sulfone group and 90 to 3 mol% for the carboxyl group.
It contains 0 mol% and is based on a copolymer of a monovinyl aromatic compound, a (meth)acrylic acid ester, and a polyvinyl compound.

Such a network porous cation exchange resin having a sulfone group and a carboxyl group can be obtained by, for example, (1) suspending polymerizing a monovinyl aromatic compound and a (meth)acrylic acid ester in an aqueous medium in the presence of a polyvinyl aromatic compound. (2) partially polymerizing a monovinyl aromatic compound; (2) partially polymerizing a monovinyl aromatic compound;
Or, convert the produced linear polymer into a monovinyl aromatic compound,
(3) A method of dissolving a (meth)acrylic acid ester and a polyvinyl aromatic compound, carrying out suspension polymerization in an aqueous medium, and extracting the added linear polymer from the resulting polymer with an appropriate organic solvent. Iwantux is dissolved in a monovinyl aromatic compound and a (meth)acrylic acid ester, a polyvinyl aromatic compound is added thereto, and suspension polymerization is carried out in an aqueous medium.
It can be produced by sulfonating and carboxylating a polymer obtained by, for example, extracting paraffin wax added from the produced polymer with an appropriate organic solvent.

Examples of the monovinyl aromatic compound used as a starting material include styrene, methylstyrene, ethylstyrene, talolomethylstyrene, chlorostyrene, vinyltoluene, vinylnaphthalene, and the like.

Examples of (meth)acrylic esters include methyl acrylate, ethyl acrylate, butyl acrylate, methyl methacrylate, ethyl methacrylate, and butyl methacrylate. Examples of polyvinyl aromatic compounds include divinylbenzene, divinyltoluene, and divinylnaphthalene. The monovinyl aromatic compound and the (meth)acrylic ester are mixed in a ratio of 90 to 30% by weight of the (meth)acrylic ester to 10 to 70% by weight of the monovinyl aromatic compound.

The polyvinyl aromatic compound is used in a proportion of 5 to 1501) based on the total weight of the mixture of monovinyl aromatic compound and (meth)acrylic acid ester. The matrix reticulated porous resin obtained by the above method has a particle size of 3 to 50 tons.
tm1 specific surface area is 50-2007TI/f! , the bore size is 50-500 people. The method for introducing sulfone groups and carboxyl groups into the above-mentioned matrix porous resin is to mix concentrated sulfuric acid and the matrix porous resin, and then heat the mixture at 50 to 100°C for 2 hours.
This can be done by heating and holding for a period of time.

At the same time as the sulfone group is introduced by the reaction with concentrated sulfuric acid, the ester group already introduced is hydrolyzed and converted to a carboxyl group. The reticulated porous cation exchange resin made of a monovinyl aromatic compound mono(meth)acrylic acid ester-polyvinyl aromatic compound-based copolymer having a sulfone group and a carboxyl group produced in this way is
It has the following advantages.

(1) In the case of amino acid analysis, the concentration of organic medium such as ethanol in the first buffer can be reduced to zero.

(2) It has a wide usable pH range and can withstand regeneration treatment with acids and alkalis.

(3) Column life is long because there is little adsorption contamination by proteins.

(4) Since the structure has a high proportion of hydrophilic parts, in amino acid analysis, substances to be separated with many hydrophobic parts are eluted quickly.

(5) Since it has a strongly acidic sulfone group and a weakly acidic carboxyl group as cation exchange groups, its characteristics, for example, the total ion exchange capacity is larger in an alkaline state than in an acidic state.

When each amino acid is separated from a mixture of amino acids according to the method of the present invention, a commonly used chromatography method may be used.

For example, a reticulated porous cation exchange resin having a sulfone group and a carboxyl group is packed in a column under pressure, and a mixture of amino acids and a citrate buffer are supplied thereto while changing the pH ionic strength stepwise or continuously. Individual amino acids flowing out of the column can be detected using an ultraviolet absorber detector, or can be detected by measuring the degree of color development or fluorescence intensity obtained by reacting with ninhydrin or orthophthalaldehyde-mercaptoethanol. The method of the present invention can be widely applied to the separation and purification of each amino acid from a mixture of amino acids, but it can be particularly effectively used to separate threonine and serine, glycine and alanine, and isoleucine and leucine. can.

Moreover, according to the method of the present invention, compared to the conventional method using liquid chromatography using a column packed with a sulfonic acid type cation exchange resin of styrene-divinylbenzene copolymer, it is possible to Amino acids can be separated in an extremely short time and accurately. Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 (1) Production of matrix reticulated porous resin for this resin Styrene 70
9, methyl methacrylate 309, divinylbenzene (
Paraffin wax (purity 55%) 15.9, melting point 54.8°C (manufactured by Nippon Oil Co., Ltd., 125° wax) 20
9 and 1.5 g of benzoyl peroxide were mixed to obtain a homogeneous liquid.

Next, this homogeneous liquid was charged into an aqueous solution prepared by dissolving 2f1 of polyvinyl alcohol (degree of saponification 96%) in 1000 ml of water. The monomer phase was dispersed by thorough stirring, and polymerization was carried out by heating at 80° C. for 16 hours while introducing nitrogen gas. After the reaction was completed, the product was washed with hydrochloric acid, separated from the oven, washed with water, and dried.

The product was packed in a column and maintained at a temperature of 90° C. or below, and mineral spray was poured from the top to wash out the paraffin wax, followed by baking with acetone and drying to obtain a reticulated porous resin. The resulting reticulated porous resin had a particle size range of 5 to 30 μm, a specific surface area of 150/9, and a bore size of 200 μm.

(2) Sulfonation and carboxylation of the parent reticulated porous resin After charging the reticulated porous resin 1009 obtained in (1) into a separable flask, add concentrated sulfuric acid 500a,
It was kept at 70° C. for 24 hours under stirring.

The product was then filtered off and mixed with water, 3N aqueous NaOH, 3
After adding N-HCl water and 3N-NaOHl water, it was dried. The ion exchange capacity of the resulting porous network cation exchange resin was 3.8 meq/9, and the molar ratio of carboxyl groups to sulfone groups was 30 mol% for carboxyl groups and 70 mol% for sulfone groups. (3) Analysis of amino acids The network-like porous cation exchange resin having sulfone groups and carboxyl groups obtained in (2) was
The mixture was packed into a stainless steel chromatography column.

Next, a sodium citrate buffer solution (sodium concentration 0.2 Ns PH 3.25, no addition of organic medium) was poured into the chromatographic column and thoroughly equilibrated. Take 0.5ml of mixed water solution (PH2.2) for protein hydrolyzate amino acid analysis containing 0.1μMOL/d of each amino acid.
A total of 2 d of diluted solution (sodium citrate buffer) was introduced into a chromatographic column using a sandwich solution, and the above sodium citrate buffer with pH 3.25 was supplied at a flow rate of 71TI11/Hr.

The temperature of the chromatographic column was maintained at 55°C. After the flow out from the lower end of the chromatographic column, a ninhydrin test solution was introduced at a flow rate of 30 d/Hr, and the mixed solution was mixed into a 0.6 mL tube with an inner diameter of 1 L. It was introduced into a reaction coil with a length of 25 m and a temperature of 95° C. to cause a ninhydrin coloring reaction, and the colored liquid was detected with a detector and recorded on a recorder.

The results are shown in Table 1. Comparative Example 1 As a packing material for a chromatographic column, (
Example 1 except that the reticulated porous resin obtained in 1) was used.
Amino acid analysis was performed under the same conditions as in (3).

The results are shown in Table 1. Example 2 (1) Production of matrix reticulated porous resin for this resin Example 1 (
In 1), polymerization was carried out under the same conditions as in Example (d) (1) except that styrene 509 and methyl methacrylate 509 were used instead of styrene 709 and methyl methacrylate 309.

The particle size range of the obtained reticulated porous resin was 3 to 40 μm, the specific surface area was 2007rI/9, and the bore size was 120λ.

(2) Sulfonation and carboxylation of the base reticulated porous resin After charging the reticulated porous resin 1009 obtained in (1) into a separable flask, 500 d of concentrated sulfuric acid was added, and the mixture was stirred at 50°C for 20 hours. held.

Then, the product was separated and mixed with water, 3N-NaOHl water, 3
After washing with N-HCl water and 3N-NaOHl water in this order, it was dried. The obtained reticulated porous cation exchange resin had an ion exchange capacity of 4.5 me. The molar ratio of sulfone groups to carboxyl groups was 50 mol% for sulfone groups and 50 mol% for carboxyl groups.

(3) Amino acid analysis Amino acid analysis under the same conditions as in Example 1 (3) except that the porous cation exchange resin having sulfone and carboxyl groups obtained in (2) was used as the chromatographic packing material. The results are shown in Table 1.

Comparative Example 2 As a packing material for a chromatographic column, (
Example 1 except that the reticulated porous resin obtained in 1) was used.
Amino acid analysis was performed under the same conditions as in (3).

The results are shown in Table 1. Comparative Example 3 Bore size 5 as packing material for chromatography column
Amino acid analysis was carried out under the same conditions as in Example 1 (3), except that a reticulated porous resin made of a styrene-divinylbenzene copolymer having a sulfone group and an ion exchange capacity of 4.0 meq/g was used. Summer.

The results are shown in Table 1. Connect the vertices of two incompletely separated peaks that are close to each other with a straight line, and calculate the distance (b) from the intersection of this straight line and a perpendicular line passing through the valley points of the two incompletely separated peaks to the valley point. It is expressed as a percentage of the value divided by (a).

Claims (1)

[Claims] 1. A network porous cation exchange resin made of a monovinyl aromatic compound-(meth)acrylic acid ester-polyvinyl aromatic compound copolymer having a sulfone group and a carboxyl group in its structural unit is used as a column packing material. A method for separating each amino acid from a mixture of amino acids using a liquid chromatography method.