JP2010089001A - Chromatograph separation method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a chromatograph separation method of preferably separating a nonelectrolyte and zwitterion. <P>SOLUTION: The chromatograph separation method performs the separation by feeding a raw liquid and eluent to a packed layer packed with a packing material having a distribution coefficient of an electrolyte to a fixed phase smaller than the distribution coefficient of a nonelectrolyte to the fixed phase. The raw liquid contains the nonelectrolyte and an electrolyte having a basic functional group and acidic functional group, the raw liquid and eluent are adjusted to a pH value higher than pKa of the basic functional group of the electrolyte, or the raw liquid and eluent are adjusted to a pH value lower than pKa of the acidic functional group of the electrolyte, to separate into a fraction containing the nonelectrolyte and a fraction containing the electrolyte. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a chromatographic separation method.

  Conventionally, a method of separating (chromatographic separation) a plurality of components in a liquid by a chromatographic technique has been used industrially. In the chromatographic separation, a solid packing material is used, and components in the raw material liquid are separated by utilizing a difference in distribution coefficient with respect to the packing material. The principle of chromatographic separation is that a raw material liquid containing two or more components to be separated is supplied to a packed bed packed with a packing material, and this raw material liquid is caused to flow down with an eluent such as water. Due to the difference in the distribution coefficient with respect to the filler, the component with a small distribution coefficient flows down relatively quickly, and the component with a large distribution coefficient flows down relatively slowly, so that the fraction containing each component is separated.

  Examples of chromatographic separation methods include a fixed bed method and a moving bed method. The fixed bed system is a system in which a raw material liquid is passed through a packed bed filled with a filler to separate and recover a plurality of components. The fixed bed method can easily change the type and concentration of the eluent, so it is suitable for separating many types of useful substances. On the other hand, if you try to improve the separation accuracy, the concentration of the resulting substance will be reduced and concentrated. There is a problem that the operation is expensive.

  In the moving bed method, for example, the packing material is moved in the direction opposite to the moving direction of the eluent (the moving direction of the two components to be separated) at a speed intermediate between the moving speeds of the two components to be separated. Supply and separate. That is, the two components to be separated move in the packed bed in opposite directions from the supply point of the raw material liquid, and components having a small difference in distribution coefficient that are difficult to separate in the fixed layer method can be separated well. In such a moving bed system, the two components in the raw material liquid can be continuously separated with high purity. On the other hand, in a large industrial apparatus, it is extremely difficult to move the filler while maintaining the band of each component.

  As a chromatographic separation method using the principle of the moving bed method described above, there is a pseudo moving bed method. In the pseudo moving bed system, for example, the unit packed bed filled with the filler is connected so as to form a closed loop, and the supply position of the raw material liquid and the eluent and the extraction position of each component fraction are set to the unit packed bed. Separation is performed continuously while switching to the downstream side of the liquid circulation flow. By dividing the packed bed into several columns and sequentially moving the supply position of the raw material liquid in the direction in which the eluent flows, it is possible to obtain separation performance substantially equivalent to that of the moving bed system.

In chromatographic separation, a chromatographic separation method and a packing material are selected according to the type of component to be separated and the scale of the apparatus, and the target component is separated with high purity. For example, when chromatographic separation of a raw material containing a non-electrolyte such as sugar, a high-purity sugar can be obtained using a cation exchange resin having a size exclusion function, an ion exclusion function, and a ligand function as a filler. . In chromatographic separation of sugars, water or a dilute basic aqueous solution can be used as an eluent, so that the process can be adapted as a food additive. For this reason, chromatographic separation can be used favorably for food processing. In the simulated moving bed method, the sugar purification method uses a cation exchange resin having a specific particle size as a filler, and controls the supply amount of the raw material liquid and the eluent, and the fraction extraction amount of each component, A method for desalting a sugar solution to purify sucrose is disclosed (for example, Patent Document 1).
Japanese Patent Laid-Open No. 11-313699

However, chromatographic separation using a cation exchange resin as a filler can satisfactorily separate non-electrolytes such as sugars and salts with a high degree of ionization (such as sodium chloride). However, non-electrolytes and amino acids such as amino acids can be separated. It was difficult to separate from an electrolyte having a basic functional group and an acidic functional group (hereinafter sometimes referred to as an amphoteric ion).
Therefore, an object of the present invention is to provide a chromatographic separation method that satisfactorily separates non-electrolytes and zwitterions.

  According to the chromatographic separation method of the present invention, a packed bed packed with a packing material (hereinafter, sometimes referred to as an ion exclusion packing material) whose distribution coefficient to the stationary phase of the electrolyte is smaller than that of the non-electrolytic stationary phase. A chromatographic separation method performed by supplying a raw material solution and an eluent, wherein the raw material solution includes a non-electrolyte and an electrolyte having a basic functional group and an acidic functional group, and the raw material solution and the eluent solution Is adjusted to a pH higher than the pKa of the basic functional group of the electrolyte, or the raw material solution and the eluent are adjusted to a pH lower than the pKa of the acidic functional group of the electrolyte, and the fraction containing the non-electrolyte is adjusted. And a fraction containing the electrolyte.

The fraction containing the non-electrolyte and the fraction containing the electrolyte are preferably separated by a pseudo moving bed method.
The electrolyte may contain an amino acid, and the non-electrolyte may be a saccharide or a saccharide derivative. The filler is preferably an ion exchange resin, and more preferably a cation exchange resin.
The filler is preferably a salt-form cation exchange resin, and the pH of the raw material liquid and the eluent is preferably higher than the pKa of the basic functional group of the electrolyte and to 12 or less.

  According to the chromatographic separation method of the present invention, the non-electrolyte and the zwitterion can be favorably separated.

Hereinafter, the chromatographic separation method of the present invention will be described with reference to examples, but the present invention is not limited thereto.
In the chromatographic separation method of the present invention, a raw material liquid containing a non-electrolyte and an amphoteric ion and an eluent are supplied to a packed bed filled with an ion-exclusion-type filler, and the raw material liquid and the eluent are supplied to the electrolyte. In this method, the pH is adjusted to a pH higher than the pKa of the basic functional group, or the raw material solution and the eluent are adjusted to a pH lower than the pKa of the acidic functional group of the electrolyte for separation.

(Chromatography equipment)
An example of a chromatographic separation apparatus used in the chromatographic separation method of the present invention will be described with reference to FIG. FIG. 1 is a schematic diagram of a fixed bed type chromatographic separation apparatus 8. As shown in FIG. 1, the chromatographic separation device 8 includes a unit packed column 10, a raw material liquid storage tank 20, an eluent storage tank 30, a first fraction storage tank 40, and a second fraction storage tank 50.

The raw material liquid storage tank 20 is connected to the upper part of the unit packed tower 10 by a pipe 24 provided with a valve 22. The eluent storage tank 30 is connected to the upper part of the unit packed tower 10 by a pipe 34 provided with a valve 32. The unit packed tower 10 is filled with a filler to form a packed bed 12.
A pipe 60 is connected to the lower part of the unit packed tower 10. The pipe 60 is connected to the pipe 44, the pipe 54, and the pipe 64 at a branch 62. The pipe 44 is provided with a valve 42, and the pipe 44 is connected to the first fraction storage tank 40. The pipe 54 is provided with a valve 52, and the pipe 54 is connected to the second fraction storage tank 50. The pipe 64 is provided with a valve 66, and the pipe 64 is connected to a discharge port (not shown).

<Filler>
The filler filled in the packed bed 12 is a filler (ion exclusion type filler) in which the distribution coefficient of the electrolyte to the stationary phase is smaller than that of the non-electrolyte stationary phase. Examples of the ion exclusion filler include various ion exchangers such as ion exchange resins, ion exchange fibers, and monolithic porous ion exchangers. Of these, ion exchange resins, which are the most versatile, are preferred. Examples of the ion exchange resin include an anion exchange resin and a cation exchange resin. These may be used alone or in combination of two or more. It is preferable to determine according to the component to be separated.

  Examples of the anion exchange resin include strong basic anion exchange resins and weak basic anion exchange resins. Further, the anion exchange resin may be in the OH form or in the salt form such as Cl form. The type of anion exchange resin to be used is preferably selected according to the component to be separated and the type of eluent. For example, when sodium chloride is contained in the raw material liquid, sodium chloride and a non-electrolyte can be satisfactorily separated by selecting a Cl-based anion exchange resin. Examples of commercially available anion exchange resins include Amberlite (trade name) manufactured by Rohm and Haas.

  Examples of the cation exchange resin include strong acid cation exchange resins and weak acid cation exchange resins. Further, the cation exchange resin may be in the H form, or may be in a salt form such as Ca form or Na form. For example, when sodium chloride is contained in the raw material liquid, sodium chloride and a non-electrolyte can be well separated by selecting a Na-type cation exchange resin. The kind of cation exchange resin to be used can be selected according to the component to be separated and the kind of eluent. Examples of commercially available cation exchange resins include Amberlite (trade name) manufactured by Rohm and Haas.

When using an ion exchange resin as a filler, it is preferable to use a cation exchange resin. In general, the cation exchange resin has a higher exchange capacity than the anion exchange resin, that is, the number of exchange groups per unit volume is large. For this reason, it is possible to efficiently separate the electrolyte and the non-electrolyte.
Among the cation exchange resins, it is more preferable to use a salt cation exchange resin. When an H-type cation exchange resin is used as the filler, H ⁺ is released by exchanging the cation component in the raw material liquid or the eluent with the counter ion of the cation exchange resin. This is because when the raw material liquid contains a component that is easily hydrolyzed, it may be decomposed by the released H ⁺ .

(Chromatograph separation method)
Next, the chromatographic separation method of the present invention will be described with reference to FIG.
First, the pH of the raw material liquid in the raw material liquid storage tank 20 is adjusted. At this stage, when the pH is adjusted to be lower than the pKa of the acidic functional group of the zwitterion contained in the raw material liquid, the zwitterion is in the form of a cation. When the pH is adjusted to be higher than the pH of the basic functional group of the zwitterion contained in the raw material liquid, the zwitterion is in the form of an anion. The valves 22 and 66 are opened and the valves 32, 42 and 52 are closed, and an arbitrary amount of raw material liquid is supplied from the raw material liquid storage tank 20 to the unit packed tower 10. Next, the valves 32 and 66 are opened and the valves 22, 42 and 52 are closed, and the eluent is supplied from the eluent storage tank 30 to the unit packed column 10. After supplying an arbitrary amount of the eluent, the valves 32 and 42 are opened and the valves 22, 52 and 66 are closed, and the eluent is supplied to the unit packed column 10. Then, the raw material liquid and the eluent flow through the packed bed 12. Here, the filling material filled in the filling layer 12 has a smaller distribution coefficient of the electrolyte in the raw material liquid to the filling material than the distribution coefficient of the non-electrolyte filling material. For this reason, the electrolyte in the raw material liquid, that is, the zwitterion in the form of an anion or cation by pH adjustment, mainly flows down in the mobile phase (eluent, solvent of the raw material liquid, etc.). On the other hand, the non-electrolyte flows down while moving between the filler which is a stationary phase and the mobile phase. The amphoteric ions in the raw material liquid flow out from the unit packed column 10 as the first fraction at an early stage, and are stored in the first fraction storage tank 40 via the pipes 60 and 44.

  Next, the valves 32 and 52 are opened, the valves 22, 42 and 66 are closed, and the eluent is supplied to the unit packed column 10. As described above, since the non-electrolyte in the raw material liquid has a larger distribution coefficient to the filler than the electrolyte, the non-electrolyte flows down in the filling layer 12 more slowly than the electrolyte. For this reason, the non-electrolyte flows out from the unit packed tower 10 as the second fraction and is stored in the second fraction storage tank via the pipes 60 and 54.

  After collecting the second fraction, the valves 32 and 66 are opened, the valves 22, 42 and 52 are closed, and the eluent is circulated through the packed bed 12 to regenerate and wash the filler. The eluent flowing through the packed bed 12 is discharged from a discharge port (not shown) via the pipes 60 and 64.

<Raw material liquid>
The raw material liquid in the present invention contains a non-electrolyte and an electrolyte (a zwitterion) having a basic functional group and an acidic functional group. For example, a sugar solution, a biological extract in the middle of sucrose production, a culture solution obtained by microbial fermentation, and the like can be mentioned.

  The non-electrolyte contained in the raw material liquid is not particularly limited, and examples thereof include sugars and sugar derivatives, alcohols and the like. Among these, the non-electrolyte in the raw material liquid is preferably a saccharide and a saccharide derivative. This is because a remarkable effect is exhibited in the separation of such components. Here, saccharides include not only monosaccharides but also disaccharides, oligosaccharides, and polysaccharides, and examples of saccharide derivatives include amino sugars. Examples of amino sugars include N-acetylglucosamine.

  The electrolyte (amphoteric ion) having a basic functional group and an acidic functional group contained in the raw material liquid is not particularly limited, and examples thereof include amino acids. An amino acid is not limited to an α-amino acid, but means an organic substance having an amino group and a carboxyl group, and includes those polymerized by peptide bonds or other bonds. The raw material solution containing amino acids often has a pH of around 7 due to the buffering effect of amino acids, and amino acids often have no charge in this environment. For this reason, in the raw material liquid containing an amino acid, the effect of this invention is exhibited notably.

  The pH of the raw material liquid is adjusted to a pH higher than the pKa of the basic functional group of the zwitterion contained in the raw material liquid or a pH lower than the pKa of the acidic functional group of the zwitterion. This is because the zwitterion can be in the form of a cation or an anion by adjusting to such pH. Especially, it is preferable to adjust to pH higher than pKa of the basic functional group of zwitterion. This is because, when the non-electrolyte is a saccharide, if the pH is lower than the pKa of the acidic functional group, it tends to be subject to hydrolysis, and there is a concern that the resulting saccharide may deteriorate in quality and decrease in purity.

  Moreover, when using a salt cation exchange resin for a filler, it is preferable to adjust the pH of the raw material liquid to be higher than the pKa of the basic functional group in the amphoteric ions and to pH 12 or less. By making the pH higher than the pKa of the basic functional group, the zwitterion can be made into an anion, and the change in the ion form of the cation exchange resin can be suppressed. This is because when the pH is adjusted to exceed pH 12, the amount of the basic component added increases, and the purity of the resulting component may be lowered.

The method for adjusting the pH of the raw material liquid is not particularly limited. For example, the pH may be adjusted by adding a basic aqueous solution such as a sodium hydroxide aqueous solution or an acidic aqueous solution such as a hydrochloric acid aqueous solution to the raw material liquid in the raw material liquid storage tank 20. A basic substance or an acidic substance may be added. Further, the pH may be adjusted by bringing the raw material liquid into contact with the ion exchanger.
As a method for adjusting the pH by bringing it into contact with the ion exchanger, for example, the raw material liquid is previously brought into contact with the OH-type anion exchange resin and the pH of the raw material liquid is adjusted to the alkaline side, and then stored in the raw material liquid storage tank 20. Alternatively, the raw material liquid in which the pH of the raw material liquid is adjusted to the acidic side by contacting with the H-type cation exchange resin may be stored in the raw material liquid storage tank 20. Further, an ion exchange tower filled with an ion exchange resin for pH adjustment may be provided in the front stage of the packed bed 12.

  The raw material liquid may contain components such as inorganic salts and organic acids in addition to the components described above. Examples of inorganic salts include sodium chloride, potassium chloride, calcium chloride, sodium sulfate and the like. Examples of the organic acid include formic acid, acetic acid, oxalic acid, and citric acid. Even if such a component is contained, it can be separated as the first fraction by selecting an appropriate filler and pH.

<Eluent>
The eluent is not particularly limited as long as it allows each component in the raw material liquid to flow down into the packed bed 12 and the components to be separated can be appropriately separated, and can be determined according to the purpose. . Examples include water, basic aqueous solutions such as sodium hydroxide aqueous solution, acidic aqueous solutions such as hydrochloric acid aqueous solution, and alcohols such as methanol, ethanol, n-propanol, isopropanol, butanol, and isobutanol. As the eluent, one kind of these may be used alone, or a mixed solution obtained by mixing two or more kinds may be used as the eluent. The eluent may contain an acid, a base, a buffer, and a salt.

  The pH of the eluent is adjusted to a pH higher than the pKa of the basic functional group of the zwitterion contained in the raw material liquid or a pH lower than the pKa of the acidic functional group of the zwitterion. This is because the amphoteric ions in the packed bed 12 are in the form of cations or anions by setting such pH. Among the components of the raw material liquid, it is preferable to adjust to a pH higher than the pKa of the zwitterionic basic functional group together with the raw material liquid. This is because, when the non-electrolyte is a saccharide, if the pH is lower than the pKa of the acidic functional group, it tends to be subject to hydrolysis, and there is a concern that the resulting saccharide may deteriorate in quality and decrease in purity.

  When a salt cation exchange resin is used for the filler, it is preferable to adjust the pH of the eluent to be higher than the pKa of the basic functional group of the zwitterion in the raw material solution and to 12 or less. By making the pH higher than the pKa of the basic functional group, the zwitterion can be made into an anion, and the change in the ion form of the cation exchange resin can be suppressed. If the adjustment exceeds pH 12, the content of the basic component in the eluent increases, and the basic component is mixed into each fraction after chromatographic separation. is there.

When a basic aqueous solution is used as the eluent, the concentration of the basic substance is not particularly limited, but is preferably 0.001 N or less, and more preferably 0.0001 N or less. This is because when the concentration of the basic substance in the eluent is high, the concentration of the basic substance in the separated fraction is high, and the purity of the component to be separated is lowered.
When an acidic aqueous solution is used as the eluent, the concentration of the acidic substance is not particularly limited, but is preferably 0.001 N or less, and more preferably 0.0001 N or less. This is because when the concentration of the acidic substance in the eluent is high, the concentration of the acidic substance in the separated fraction is high, and the purity of the component to be separated is lowered.

  In the chromatographic separation, the temperature of the raw material liquid and / or eluent to be circulated in the packed bed is not particularly limited, and can be determined in consideration of components in the raw material liquid. For example, when sugar is purified, the temperature is preferably 50 to 80 ° C. If it is less than 50 ° C., the viscosity of the liquid in the packed bed 12 becomes high, and it is necessary to install a liquid feed pump having a high discharge pressure, which is not economically preferable. In addition, depending on the type of sugar in the raw material liquid, it tends to rot. On the other hand, if it exceeds 80 ° C., the amount of heat required for the chromatographic separation device 8 increases, which is not economically preferable. Moreover, it is because it will be easy to decompose | disassemble depending on the kind of sugar in a raw material liquid.

  The flow rate of the eluent for collecting the first fraction is not particularly limited and can be determined according to the component obtained as the first fraction. For example, the effluent obtained at the outlet of the unit packed column 10 is analyzed, and the collection of the second fraction may be started when the concentration of the component obtained as the second fraction becomes a certain level or more. You may switch to collection | recovery of a 2nd fraction with the flow volume of the eluent calculated | required empirically. The flow rate of the eluent for collecting the second fraction is not particularly limited, and can be determined according to the component obtained as the second fraction. For example, the effluent obtained at the outlet of the unit packed column 10 is analyzed, and when the concentration of the component obtained as the second fraction falls below a certain value, it may be switched to washing, which has been determined empirically. You may switch to washing | cleaning with the flow volume of eluent.

In a conventional chromatographic separation method, for example, when a sugar solution containing an electrolyte is desalted by chromatographic separation to purify the sugar, it is performed under neutral pH conditions. However, when the electrolyte is a zwitterion such as an amino acid, the zwitterion exists as a zwitterion in the neutral range. For this reason, when chromatographic separation is performed in the neutral region, the zwitterion flows into the same fraction as the sugar that is a non-electrolyte.
In the present invention, the amphoteric ion is in the form of an anion under a pH condition higher than the pKa of the basic functional group, or the amphoteric ion is in the form of a cation under a pH condition lower than the pKa of the acidic functional group. By doing so, the distribution coefficient to the ion exclusion type filler can be reduced. As a result, the difference of the distribution coefficient with respect to the filler of nonelectrolytes, such as saccharides, and a zwitterion can be enlarged, and both components can be isolate | separated favorably.

  In the above-described chromatographic separation method, a fixed-bed type chromatographic separation apparatus is used. However, the chromatographic separation method of the present invention is not limited to a fixed-bed type chromatographic separation apparatus, and a moving-bed type or pseudo-moving-bed type chromatographic separation is used. An apparatus may be used. In addition, a chromatographic separation method as disclosed in Japanese Patent No. 1998860, Japanese Patent No. 20000830, and Japanese Patent No. 1954744 can also be used. Furthermore, the present invention is not limited to this, and any other chromatographic separation method, that is, using a solid packing material and separating the components in the raw material liquid by utilizing the difference in the distribution coefficient to the packing material. It can be used in separation methods. Among them, the pseudo moving bed type chromatographic apparatus or the chromatographic apparatus improved with the pseudo moving bed type has a small amount of the eluent and high utilization efficiency of the packing material, and therefore contains the separated components in higher concentration and higher purity. A fraction can be obtained.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, it is not limited to an Example.
(Chromatograph separation device)
The chromatographic separation apparatus 100 used for the chromatographic separation of Example 1 and Comparative Example 1 will be described with reference to FIG. As shown in FIG. 2, the chromatographic separation apparatus 100 includes a jacketed column 110, a syringe 120, and an eluent storage tank 130. A pipe 127 is connected to the upper part of the jacketed column 110, and the pipe 127 is connected to the eluent storage tank 130 via the pump 132. The pipe 127 has an inlet 122. The syringe 120 is connected to the inlet 122 by a pipe 121. A pipe 118 is connected to the lower part of the jacketed column 110, and the pipe 118 is connected to the sorting container 140.

  The jacketed column 110 includes a jacket 114 on the outer periphery of the column main body 112. The column main body 112 is a glass column having an inner diameter of 20 mm and a height of 1000 mm. The jacket 114 is connected to a hot water supply device (not shown).

  As the pump 132, a double plunger plunger pump with little pulsation was used.

Example 1
Using the chromatographic separation apparatus 100 described above, N-acetylglucosamine (NAG) is obtained from a raw material solution containing amino acids (1 × 10 ⁻³ mol / L), N-acetylglucosamine (0.37% by mass) and sodium chloride. Chromatographic separation was performed as a second fraction. The column body was filled with CR1310Na (Rohm and Haas), which is a cation exchange resin.
First, warm water of 60 ° C. was circulated through the jacket 114. A 10N sodium hydroxide aqueous solution was added to the raw material liquid to adjust the pH to 10 to obtain a raw material liquid A. Next, the pump 132 was started and the eluent (1 × 10 ⁻⁴ N sodium hydroxide aqueous solution) in the eluent storage tank 130 was circulated from the pipe 127 to the packed bed 116 at a flow rate of 26 mL / min. Thereafter, 15 mL of the raw material liquid A was caused to flow from the inlet 122 to the pipe 127 with the syringe 120. And the effluent from the piping 118 was fractionated with the fractionation container 140 every 0.4 minutes. For each fractionation container 140, the fractionated effluent was measured by HPLC for the concentration of impurities including amino acids and sodium chloride, and the concentration of NAG, and the results are shown in FIG.

(Comparative Example 1)
Chromatographic separation was performed in the same manner as in Example 1 except that the raw material liquid B without adjusting the pH of the raw material liquid was used. The pH of the raw material liquid B was 7. The separated effluent was measured for the impurity concentration including amino acid and sodium chloride and the NAG concentration by HPLC, and the results are shown in FIG.

(Measuring method)
<Measurement of impurity concentration and NAG concentration>
The impurity concentration and NAG concentration in the raw material liquid, the first fraction, and the second fraction are measured as a solid content with a Brix meter (RA-500N, manufactured by Kyoto Electronics Industry Co., Ltd.) as the total concentration of impurities and NAG. The ratio of impurities to NAG in the solid content was measured by HPLC, and the impurity concentration and NAG concentration were determined.

<Measurement of the content ratio of impurities and NAG>
The ratio of impurities and NAG was measured by HPLC (LC-10Avp, Shimadzu Corporation) under the following conditions.
[Measurement condition]
Column: TSK-GEL SCX-Na type (φ6.0 × L150mm × 5)
Eluent: 1 × 10 ⁻⁴ N-NaOH aqueous solution Flow rate: 0.8 mL / min
Sample volume: 10 μL
Temperature: 70 ° C
Detector: Differential refractometer (RI)

<Measurement of amino acid concentration>
The amino acid concentration in the raw material solution was determined based on the “food analysis handbook” (Tetsujiro Ohara, Hiroyuki Iwao, Takao Suzuki, 2nd edition, Kenshisha Co., Ltd., issued April 1, 1973). It measured according to the formol titration method described in 58-60. For amino acid concentration measurement by the formol titration method, the sample is neutralized with 0.1N sodium hydroxide or 0.1N hydrochloric acid, and formalin is added to make an oxymethyl derivative, and phenolphthalein is used as an indicator or potentiometer. And titrated with 0.1N sodium hydroxide standard solution.

FIG. 3 is a graph showing the analysis results of NAG concentration and impurity concentration in the outlet liquid in Example 1, where the horizontal axis represents the integrated flow rate of the eluent (L / LR), and the vertical axis represents the normalized concentration. (C / C0) is taken. The legend (X1) indicates the impurity concentration, and the legend (Y1) indicates the NAG concentration. The normalized concentration (C / C0) is a concentration obtained by dividing the component concentration (C) in the effluent by the component concentration (C0) in the stock solution. The integrated flow rate of the eluent is the integrated flow rate of the eluent with respect to the unit resin volume (LR), and is a value represented by the unit L / LR.
In FIG. 3, the first fraction is an integrated flow rate from 0.30 L / LR to 0.40 L / LR, and the second fraction is an integrated flow rate from 0.43 L / LR to 0.60 L / L-. Up to R. In the first fraction, the impurity concentration (X1) was contained at a relatively higher concentration than the NAG concentration (Y1). On the other hand, in the second fraction, the NAG concentration (Y1) was contained at a concentration about 3 times or more the impurity concentration (X1). From this, it was found that the impurities containing sodium chloride and amino acids in the raw material liquid were separated into the first fraction, and the second fraction containing NAG at a high concentration could be separated.

FIG. 4 is a graph showing the analysis results of the NAG concentration and the impurity concentration of the outlet liquid in Comparative Example 1. The horizontal axis represents the eluent integrated flow rate (L / LR), and the vertical axis represents the raw material liquid. The normalized concentration (C / Co) expressed by the ratio of the concentration of the outlet liquid to the concentration is taken. The legend (X2) indicates the impurity concentration, and the legend (Y2) indicates the NAG concentration.
In FIG. 4, the first fraction is an integrated flow rate from 0.27 L / LR to 0.43 L / LR, and the second fraction is an integrated flow rate from 0.47 L / LR to 0.93 L / L-. Up to R. In the first fraction, the impurity (X2) was contained at a relatively high concentration with respect to the NAG concentration (Y2). On the other hand, in the second fraction, although the NAG concentration (Y2) is relatively higher than the impurity concentration (X2), the impurity concentration (X2) is moving in parallel with the decrease of the NAG concentration (Y2). , It was found that the fraction containing NAG at a high concentration could not be separated.

It is a schematic diagram which shows an example of the chromatographic separation apparatus concerning embodiment of this invention. 1 is a schematic diagram of a chromatographic separation apparatus used in Example 1 and Comparative Example 1. FIG. 3 is a graph showing a state of separation of NAG and impurities in Example 1. 6 is a graph showing a separation state of NAG and impurities in Comparative Example 1.

Explanation of symbols

8 Chromatographic separator 12 Packed bed

Claims (7)

A chromatographic separation method in which a raw material solution and an eluent are supplied to a packed bed filled with a packing material whose partition coefficient to the electrolyte stationary phase is smaller than the partition coefficient to the non-electrolyte stationary phase,
The raw material liquid includes a non-electrolyte and an electrolyte having a basic functional group and an acidic functional group,
The raw material liquid and the eluent are adjusted to a pH higher than the pKa of the basic functional group of the electrolyte, or the raw material liquid and the eluent are adjusted to a pH lower than the pKa of the acidic functional group of the electrolyte. ,
The chromatographic separation method which isolate | separates into the fraction containing the said nonelectrolyte, and the fraction containing the said electrolyte.   The chromatographic separation method according to claim 1, wherein the fraction containing the non-electrolyte and the fraction containing the electrolyte are separated by a pseudo moving bed method.   The chromatographic separation method according to claim 1, wherein the electrolyte contains an amino acid.   The chromatographic separation method according to claim 1, wherein the non-electrolyte is a saccharide or a saccharide derivative.   The chromatographic separation method according to claim 1, wherein the filler is an ion exchange resin.   The chromatographic separation method according to claim 1, wherein the filler is a cation exchange resin.   The filler is a salt-type cation exchange resin, and the pH of the raw material liquid and the eluent is adjusted to be higher than the pKa of the basic functional group of the electrolyte and to 12 or less. Item 7. The chromatographic separation method according to any one of Items 1 to 6.

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