JPS592523B2 - Concentration method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for concentrating an aqueous solution, and more specifically, the present invention relates to a method for concentrating an aqueous solution. It also relates to a method for concentrating a target substance by transferring certain contaminants into the organic solvent. The present invention provides an efficient and simple method for concentrating substances while preventing the decomposition or denaturation of heat-labile substances and removing certain types of contaminants.

Conventionally, methods for concentrating heat-labile substances include - Freeze concentration method, in which water in an aqueous solution of heat-labile substances is frozen and removed; - Dehydration by mechanical pressure or osmotic pressure using a membrane such as a molecular sieve membrane. (Membrane concentration method such as reverse osmosis method) ■ Method to evaporate water using cooled dry air and concentrate.

Among these methods, the membrane concentration method using a molecular sieve membrane is relatively convenient for concentrating high molecular compounds, but requires high pressure when concentrating low molecular compounds, especially solutions containing salts. Therefore, the strength of the membrane must be taken into consideration, and not only that, but also the loss of the target substance may be unavoidable at the same time as concentration, making it impossible to achieve efficient concentration.

Also, freeze concentration and concentration methods using dry air are not efficient.

In view of the above-mentioned technical background, the present inventor investigated various methods for concentrating an aqueous solution, particularly an aqueous solution of a thermally unstable substance, and as a result, the inventors determined that the aqueous solution is brought into contact with a specific hydrophilic organic solvent through a semipermeable membrane. An unexpected phenomenon is that water and certain impurities in the solution migrate into the hydrophilic organic solvent, but other components in the solution do not migrate into the hydrophilic organic solvent regardless of their molecular weights. They discovered this and completed the present invention.

In the present invention, an aqueous solution is passed through a semipermeable membrane having a carbon number of 1 to
A method for concentrating an aqueous solution, which comprises contacting the solution with the alcohol according to item 3 above, and concentrating the solution by transferring some kind of contaminant to the alcohol, either only or together with the water in the solution. It is.

The present invention will be explained in detail below.

In addition, in the following description, "hydrophilic organic solvent" may mean "alcohol having 1 to 3 carbon atoms."

Aqueous solution to be concentrated The aqueous solution to be concentrated in the present invention is an aqueous solution containing the target substance or the target substance and a certain type of contaminant.

Any substance may be used as long as it does not migrate into the hydrophilic organic solvent when the target substance 6 and an aqueous solution containing the substance are brought into contact with the hydrophilic organic solvent through a semipermeable membrane.

The effects of the method of the present invention are particularly significant when the target substance is a thermally unstable substance.

Note that the target substance that can be concentrated by the method of the present invention varies depending on the type of membrane, the type of organic solvent, and concentration conditions.
It is difficult to define it with unambiguous properties.

For example, the relationship between the molecular weight of the target substance and the membrane pore size regarding membrane permeability cannot be directly applied to the relationship between the two in conventional membrane processing.

Furthermore, the membrane permeability of these substances may differ depending on whether the aqueous solution contains the target substance alone or together with other impurities.

However, the target substance that can be concentrated is generally a substance that is relatively sparingly soluble in the hydrophilic organic solvent used in the present invention.

According to the present inventor's empirical findings, the specific target substance is nucleic acid dinucleoside triphosphate.

Nucleoside diphosphate, nucleoside monophosphate,
Cyclic nucleotides and their derivatives (e.g. dibutyryl cyclic nucleonate, etc.) and other nucleic acid-related substances (e.g. S-adenosyl-L-methionine, etc.), vitamins such as folic acid, various enzymes C hydrophilic organic Most enzymes that are stable to solvents are suitable.

)1. Various amino acids (mixtures may be used.

), various hormones, and various sugars.

Furthermore, the term "contaminant" in the present invention refers to a substance that passes through the semipermeable membrane together with water and migrates into the hydrophilic organic solvent when an aqueous solution is brought into contact with a hydrophilic organic solvent through the semipermeable membrane. Generally, it is a substance that is relatively easily soluble in the hydrophilic organic solvent used in the present invention.

Specifically, nucleosides, ammonia, and free organic acids (
Examples include lactic acid, acetic acid, etc.) and mineral acids (eg, hydrochloric acid, sulfuric acid, etc.).

Types of semipermeable membranes Semipermeable membranes in the present invention! : is stable against hydrophilic organic solvents, and when brought into contact with a specific hydrophilic organic solvent through this semipermeable membrane, water and certain impurities in the aqueous solution are selectively removed from the hydrophilic organic solvent. However, in normal membrane treatment methods such as reverse osmosis or water dialysis, the target substance may pass through the membrane, and selective permeability to water and contaminants is not necessarily required. pressure resistance, molecular weight cut-off, salt rejection rate, etc. (not limited to 4).

Such membranes include ordinary dialysis membranes, ion exchange membranes,
Among the membranes for reverse osmosis and the membranes for ultrafiltration, membranes that are stable in organic solvents can be mentioned, and membranes for dialysis are particularly suitable.

Membrane materials include (1) polyvinyl alcohol, cellulose such as cellophane, cellulose acetate, polyester, polyacrylonitrile, polyamide,
animal stomach.

Examples include tissue membranes such as intestines and bladder, but among these, polyvinyl alcohol and cellulose, which are stable against organic solvents, are particularly preferred.

Examples of commercial products corresponding to these membranes include A of polyvinyl alcohol hollow fiber membranes KL-1 and KL-2.
, B and C types (manufactured by Kuraray Co., Ltd.), and VISKING tube, which is a cellophane membrane (manufactured by VISKING Co., Ltd., USA).

Note that any form of membrane can be used, such as a flat membrane, a tube, or a hollow fiber, but a hollow fiber is preferable for continuous concentration on an industrial scale, and for small-scale batch concentration. When concentrating in a formula, a tube-shaped one is convenient.

Types of hydrophilic organic solvents The specific hydrophilic organic solvent used in the present invention does not have high solubility of the target product, has strong hydrophilic properties, and has the property of forming a homogeneous phase by mixing with water in any proportion. It is a solvent.

Examples of such hydrophilic organic solvents include monohydric alcohols such as ethanol and methanol, and alcohols having 1 to 3 carbon atoms such as polyhydric alcohols such as glycerin, ethylene glycol, and propylene glycol.

Concentration and Usage Amount of Hydrophilic Organic Solvent In the present invention, the concentration of the hydrophilic organic solvent is one of the important factors, and one with a low water content and high concentration must be used.

If the concentration of the hydrophilic organic solvent decreases, the loss of the target substance will increase, which is undesirable.

The preferred concentration range varies somewhat depending on the type of hydrophilic organic solvent, but the object of the present invention can be achieved if it is about 80% or more, preferably 90% or more.

The amount of hydrophilic organic solvent used is 7 to 1 of the aqueous solution (before concentration).
Approximately 0 times the amount is appropriate.

Contact Method The method of bringing an aqueous solution into contact with a hydrophilic organic solvent through a semipermeable membrane is not particularly limited and does not require any special equipment, but known contact methods commonly employed in dialysis methods are suitable. It is.

However, in the process of concentration, the hydrophilic organic solvent is diluted with water, so a contact method that can constantly supply a high concentration of hydrophilic organic solvent, that is, a method of dehydrating the hydrophilic organic solvent by flowing it over the membrane surface, is recommended. preferable.

The diluted hydrophilic organic solvent ζ4 may be discharged as is and used for other purposes, but it is advantageous for large-scale concentration to concentrate (dehydrate) it using a distillation method and/or a dehydrating agent and recycle it. be.

Further, a contact method may be adopted in which the aqueous solution is allowed to flow through the membrane surface when concentrating the aqueous solution continuously, and is allowed to remain on the membrane surface when the aqueous solution is concentrated batchwise.

When flowing both the aqueous solution and the hydrophilic organic solvent, they may be caused to flow in either countercurrent or cocurrent flow.

Contact Temperature When an aqueous solution and a hydrophilic organic solvent are brought into contact with each other through a semipermeable membrane, the temperatures of both may be maintained at approximately the same level.

If the contact temperature is high, the concentration rate will be faster, but the target substance will be more susceptible to decomposition or denaturation.

Conversely, if the contact temperature is low, the target substance is stable;
Concentration rate is slow.

Therefore, the contact temperature cannot be uniformly specified, but the upper limit may be a temperature lower than the decomposition temperature or denaturation temperature of the target substance and the boiling point of the organic solvent, and the lower limit may be a temperature higher than the freezing temperature of the aqueous solution.

Features of the present invention (1) The method of the present invention utilizes the dehydration power of a highly concentrated hydrophilic organic solvent and does not require any other thermal or mechanical energy.

(2) Since concentration can be performed under mild conditions, there is almost no decomposition or denaturation of the target substance.

(3) Hydrophilic organic solvents can be easily dehydrated or distilled even when diluted.

(4) It is possible to concentrate the target substance and separate it from contaminants (purification) in a single operation.

(5) The present invention is completely different in concept from conventional membrane treatment methods such as dialysis, reverse osmosis, or ultrafiltration, and is an invention based on a novel technical idea.

Each component analysis method (1) If the nucleic acid good luck substance component is single, 0. Ultraviolet light in IN hydrochloric acid (
It was calculated from the absorbance at 260 nm).

In the case of two or more components, each component is separated by filter paper electrophoresis and/or paper chromatography, and each spot is divided into 0. After extraction with IN hydrochloric acid, it was calculated from the absorbance of ultraviolet rays in the same manner as above.

In addition, a spectrophotometer model 124 manufactured by Hitachi, Ltd. was used to measure the absorbance.

(2) Enzyme (activity) (a) Nucleolytic enzyme (nuclease P1) Using ribonucleic acid as a substrate, perform an enzymatic reaction at 70°C and pH 4, 8 for 30 minutes, and calculate the titer from the absorbance at 260 nm of the uranium reagent-soluble portion. did.

(b) Using sodium phosphatase 5'-cytidylate as a substrate, at 45°C, p
After enzymatic reaction with H4,8 for 30 minutes and stopping the reaction with 60% perchloric acid, color was developed with amitor reagent and molybdate reagent, and the amount of inorganic phosphoric acid released was measured from the absorbance at 750 nm, and the titer was calculated. did.

(3) Folic acid Calculated from absorbance at 265 nm.

Hereinafter, the present invention will be specifically explained with reference to Examples.

Example 1 50 ml of an aqueous solution containing 348 mg of 5'-inosinic acid (free form) was placed in a Wiesking tube (manufactured by Wiesking, USA, contact area 91 crIL = 7czX 13
cm) and collected ethanol (concentration 95%) 600m
As a result of soaking in # and leaving it at room temperature for 2 hours while stirring,
The membrane fluid was concentrated to 6.8 m#.

As a result of analyzing the 57-inosinic acid in the membrane fluid and the membrane fluid (in ethanol), it was found that 343.82 mg (recovery rate 98.8%) of 57-inosinic acid remained in the membrane fluid, and 57-inosinic acid remained in the membrane fluid. The amount of leached 5'-inosinic acid was 4.17 mg (
1.2%), and no decomposition occurred.

Example 2 A hollow fiber membrane, KL-1, A type (manufactured by Kurashi Co., Ltd., contact area 240i) was mixed with the reagent grade 1 ethanol (concentration 99.5
%,ice? '+) 500 mA', and an aqueous solution of S-adenosyl-L-methionine with a concentration of 6.5 mVml (
As a result of passing the liquid (water-cooled) inside the membrane at a flow rate of 40 m11/hour, the result was 25 m! It was concentrated continuously at a rate of l/h.

When the concentrated solution was analyzed for 1 hour after the start, the recovery rate of S-adenosyl-L-methionine was 98.8%.

Example 3 50 m# of an aqueous solution containing 50 mg of folic acid was packed in a Wiesking tube (contact area 96.2 CrIt), immersed in 500 m7 of reagent primary ethanol (concentration 99.5%), and left at room temperature for 2 hours with stirring. As a result, the membrane fluid was 7
．． It was concentrated to 6ml.

As a result of analyzing folic acid in the intramembrane fluid and extramembrane fluid, it was found that 4
7.9 mg (recovery rate 95.8%) of folic acid remained, and the folic acid leached into ethanol was 2.2 mg (4.4%).
)Met.

Example 4 Nuclease P1 (nucleolytic enzyme derived from Penicillium citrinum, manufactured by Yamasa Soy Sauce Co., Ltd.) crude enzyme solution (5
08000Unit/mA) 50mlj VIS KING
Packed into a tube (contact area 91 cIIt), immersed in 500 m# of reagent grade 1 ethanol (concentration 99.5%),
As a result of being left at 3℃ for 4 hours with stirring, the liquid inside the membrane? j
It was concentrated to 17.8 m#.

The membrane solution and washing solution were returned to 50 m#, and the roller used to measure the enzyme activity was heated to 495,000 Units/mA (the membrane solution was 17.8
When converted to ml, it was 1390449 Units7mi, and the recovery rate of activity was 97.44%.

Example 5 Crude enzyme solution (13929Un) of phosphatase (derived from black koji mold, Aspergillus niger, manufactured by Yamasa Soy Sauce Co., Ltd.)
It/m#) 50m# was packed in a VISKING tube (contact area 91CrIt), immersed in 50077L# of reagent primary ethanol (concentration 99.5%), and left at 1-3℃ for 4 hours with stirring. , membrane liquid is 13.6mA
was concentrated in.

When the membrane solution and washing solution were returned to 50ml and the enzyme activity was measured, it was found to be 13869 Unit/mA' (13.6m
When converted to A, it was 50989 Unit/m#), and the recovery rate of activity was 99.67%.

Example 6 50 ml of an aqueous solution containing 347 mg (1 mmol) of 5'-adenylic acid and 267 mg (1 mmol) of adenosine
Pack 1J into a Wiesking tube (contact area 91d) and add the reagent primary methanol (concentration 99%) to 500mi! As a result of immersing the membrane in water and leaving it for 2 hours at room temperature while stirring, the liquid in the membrane was concentrated to 9.4 ml.

When we measured the ultraviolet absorbance of the extra-membrane liquid, we found that 46.2% of the ultraviolet-absorbing substances had migrated to the extra-membrane liquid, but analysis of the extra-membrane liquid revealed that 95% of the ultraviolet-absorbing substances were adenosine; '-Adenylic acid was only slightly detected.

Example 7 50 mA of aqueous solution containing 500 mJ of adenosine diphosphate
'Visking tube (contact area 91 cr!
) and reagent 1st grade ethanol (concentration 99.5%) 50
As a result of immersing the membrane in a water temperature of 0.0 m/m and leaving it for 2 hours and 30 minutes at 1 to 3° C. with stirring, the liquid in the membrane was concentrated to 22 ml.

When the ultraviolet absorbance of the membrane liquid was measured, 97.0% of the ultraviolet absorbing substance was recovered.

Furthermore, no decomposition of adenosine diphosphate was observed.

Claims (1)

[Scope of Claims] 1. An aqueous solution containing a target substance is brought into contact with an alcohol having 1 to 3 carbon atoms through a semipermeable membrane, and water in the solution is transferred into the alcohol to transform the solution. A method for concentrating an aqueous solution characterized by concentrating it. 2. An aqueous solution containing the target substance and impurities is brought into contact with an alcohol having 1 to 3 carbon atoms through a semipermeable membrane, and water in the solution passes through the semipermeable membrane and is transferred to the alcohol. 1. A method for concentrating an aqueous solution, which comprises transferring contaminants having the properties and being easily soluble in the alcohol into the alcohol, concentrating the target substance, and separating the contaminants at the same time.