Aqueous two-phase separation liquid and organic matter separation method using said aqueous two-phase separation liquid

Abstract

To provide an aqueous two-phase separation liquid allowing organic material to be separated efficiently, and an organic material separation method employing the aqueous two-phase separation liquid.SOLUTION: An aqueous solution contains a carboxybetaine represented by general formula (1) and / or an amine N-oxide represented by general formula (2), and a water-soluble salt. The solution has excellent phase separation capability for separation into two phases, and a high partition coefficient of organic material in the carboxybetaine phase after the two-phase separation, and thus is useful in separating and recovering the organic material.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an aqueous two-phase separation liquid using carboxybetaine and / or amine N-oxide, and also to a method for separating organic substances using the aqueous two-phase separation liquid. [Background technology]

[0002] Separation is a well-known method for separating and recovering organic substances. Separation utilizes the difference in the partition coefficients of various organic substances between phases to separate and recover organic substances. Separation generally uses an oil-water two-phase system, with organic solvents used in the oil phase. However, this method poses safety concerns due to its flammability and toxicity, as well as environmental concerns due to its volatility.

[0003] On the other hand, it is known that aqueous two-phase systems can be formed without the use of organic solvents by using additives such as polyethylene glycol (PEG), sulfobetaine, and tetraalkylammonium salts and water-soluble salts (e.g., Non-Patent Documents 1-3). Because aqueous two-phase systems do not use organic solvents, they do not pose environmental problems due to solvent evaporation, and there is no risk of ignition, making them highly useful from a safety perspective. However, the partition behavior of aqueous two-phase systems has not been fully elucidated, and aqueous two-phase systems using the additives have the disadvantage of having low partition coefficients for organic substances. Therefore, aqueous two-phase systems have not yet been put to practical use in the industrial field.

[0004] Against this background of conventional technology, there is a strong demand for the development of an aqueous two-phase system that has a high partition coefficient for organic substances and can efficiently separate organic substances. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Sara C. Silverio, Oscar Rodriguez, Jose A. Teixeira, and Eugenia A. Macedo, J.Chem.Eng.Data.,58,3528-3535(2013) [Non-patent document 2] Ana M.Ferreira,Helena Passos,Akiyoshi Okafuji,Mara G.Freire,Joao APCoutinho and Hiroyuki Ohno,Green Chem.,19,4012-40(2017) [Non-patent document 3] Yoshifumi Akama, Ahat Sali, Talanta, 57, 681-686 (2002) Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide an aqueous two-phase separation liquid that can efficiently separate organic substances, and a method for separating organic substances using the aqueous two-phase separation liquid. [Means for solving the problem]

[0007] The present inventors have conducted extensive research to solve the above problems and have found that an aqueous solution containing a carboxybetaine and / or amine N-oxide of a specific structure and a water-soluble salt has excellent phase separation ability, and has a high partition coefficient of organic matter in the carboxybetaine phase after separation, making it useful for separating and recovering organic matter. The present invention was completed based on this finding and through further research.

[0008] That is, the present invention provides the following aspects. Item 1. An aqueous two-phase separation liquid comprising a carboxybetaine represented by the following general formula (1) and / or an amine N-oxide represented by the following general formula (2), and a water-soluble salt: [ka] [In general formula (1), R1, R2, and R3 each represent the same or different alkyl groups, and the alkyl groups of R1, R2, and R3 have a total of 9 to 15 carbon atoms, and R4 is an alkylene group having 1 to 5 carbon atoms.] [ka] [In general formula (2), R5, R6, and R7 each represent the same or different alkyl groups, and the total number of carbon atoms in the alkyl groups of R5, R6, and R7 is 9 to 15.] Item 2. The aqueous two-phase separation liquid according to Item 1, wherein in general formula (1), R1, R2, and R3 are the same or different and each represent an alkyl group having 3 to 5 carbon atoms, and R4 is a methylene group. Item 3. The aqueous two-phase separation liquid according to Item 1 or 2, wherein in general formula (1), R1, R2, and R3 are alkyl groups having 5 carbon atoms, and R4 is a methylene group. Item 4. The aqueous two-phase separation liquid according to Item 1, wherein in general formula (2), R5, R6, and R7 are the same or different and each represent an alkyl group having 3 to 5 carbon atoms. Item 5. The aqueous two-phase separation liquid according to Item 1 or 4, wherein in general formula (2), R5, R6, and R7 are alkyl groups having 5 carbon atoms. Item 6. The aqueous two-phase separation liquid according to any one of Items 1 to 5, wherein the anion constituting the water-soluble salt is a carbonate ion, a hydrogen carbonate ion, a sulfate ion, a hydrogen sulfate ion, a phosphate ion, a hydrogen phosphate ion, a dihydrogen phosphate ion, or an acetate ion, and the cation constituting the water-soluble salt is a guanidinium ion, a magnesium ion, a calcium ion, a lithium ion, or a sodium ion. Item 7. The aqueous two-phase separation liquid according to any one of Items 1 to 5, wherein the water-soluble salt is at least one selected from the group consisting of sodium carbonate, sodium hydrogencarbonate, calcium carbonate, magnesium carbonate, sodium sulfate, magnesium sulfate, calcium sulfate, trisodium phosphate, tripotassium phosphate, disodium hydrogenphosphate, dipotassium hydrogenphosphate, sodium dihydrogenphosphate, potassium dihydrogenphosphate, sodium acetate, magnesium acetate, and calcium acetate. Item 8. The aqueous two-phase separation liquid according to any one of Items 1 to 7, which is used for separating organic substances. Item 9. A method for separating organic matter, comprising the following first and second steps: a first step of preparing and mixing an organic substance-containing material to be treated with an aqueous two-phase separation liquid containing a carboxybetaine represented by the following general formula (1) and / or an amine N-oxide represented by the following general formula (2), and a water-soluble salt; [ka] [In general formula (1), R1, R2, and R3 each represent the same or different alkyl groups, and the alkyl groups of R1, R2, and R3 have a total of 9 to 15 carbon atoms, and R4 is an alkylene group having 1 to 5 carbon atoms.] [ka] [In general formula (2), R5, R6, and R7 each represent the same or different alkyl groups, and the total number of carbon atoms in the alkyl groups of R5, R6, and R7 is 9 to 15.] And The second step is to separate the mixture obtained in the first step into two phases: an aqueous phase containing the carboxybetaine represented by general formula (1) and / or the amine N-oxide represented by general formula (2) and an aqueous phase containing a water-soluble salt, and recover the aqueous phase containing the carboxybetaine represented by general formula (1) and / or the amine N-oxide represented by general formula (2). Item 10. The method for separating an organic substance according to Item 9, wherein the organic substance has an octanol / water partition coefficient of −7 to 14. [Effects of the Invention]

[0009] According to the present invention, by using a carboxybetaine and / or amine N-oxide with a specific structure and a water-soluble salt, an aqueous two-phase system capable of efficiently separating organic substances can be formed, and therefore the method is highly useful as a separation method that does not have the environmental and safety concerns that arise in the case of an oil-water two-phase system. [Brief explanation of the drawings]

[0010] [Figure 1] 1 is a photographic image showing the state of phase separation immediately after sodium sulfate becomes insoluble in an aqueous solution of PEG600 and carboxybetaines 1 to 5, which was added until sodium sulfate became insoluble. [Figure 2] 2 is a diagram showing binodal curves of sodium sulfate with carboxybetaine 4, 5, or PEG 600. In FIG. 2, the unit of sodium sulfate concentration (%) is % by mass. [Figure 3] This figure shows a comparison of the binodal curves of carboxybetaine 5 and various water-soluble salts when the cation is fixed to sodium and the anion is changed. In Figure 3, the unit of salt concentration (%) is mass %. [Figure 4] This figure shows a comparison of the binodal curves of carboxybetaine 5 and various water-soluble salts when the anion is fixed to sulfate or nitrate and the cation is changed. In Figure 3, the unit of salt concentration (%) is mass %. [Figure 5] FIG. 1 shows a comparison of the binodal curves of carboxybetaine 5 with trisodium phosphate, tripotassium phosphate, or dipotassium hydrogen phosphate. [Figure 6] FIG. 1 shows the partition coefficients of various organic substances in aqueous two-phase systems of carboxybetaine 5 or PEG600 and sodium sulfate. [Figure 7] This is a photographic image showing the appearance of an aqueous two-phase system of carboxybetaine 5 or PEG600 and sodium sulfate when azobenzene is added. [Figure 8] FIG. 1 shows the results of comparing the effects of carboxybetaine 5 and sodium sulfate concentrations on the partition coefficients of caffeine, coumaric acid, or flavone. [Figure 9] FIG. 1 shows the effect of the type of water-soluble salt and the concentrations of carboxybetaine 5 and the water-soluble salt on the partition coefficient of caffeine in aqueous two-phase systems formed by carboxybetaine 5 and various salts. [Figure 10] FIG. 1 shows the partition coefficient of bovine serum albumin in aqueous two-phase systems of carboxybetaine 5, PEG600, or sulfobetaine 5 with sodium sulfate. [Figure 11] 11 is a photograph showing the distribution of BSA in the experiment of FIG. 10. [Figure 12] FIG. 1 shows the partition coefficient of salmon sperm DNA in aqueous two-phase systems of carboxybetaine 5, PEG600, or sulfobetaine 5 with sodium sulfate. DETAILED DESCRIPTION OF THE INVENTION

[0011] 1. Aqueous two-phase separated liquid The aqueous two-phase separation liquid of the present invention is characterized by containing a carboxybetaine represented by general formula (1) and / or an amine N-oxide represented by general formula (2), and a water-soluble salt. The aqueous two-phase separation liquid of the present invention will be described in detail below.

[0012] [Carboxybetaine and / or amine N-oxide] The carboxybetaine used in the present invention is a compound having a structure represented by the following general formula (1). [ka]

[0013] In general formula (1), R1, R2, and R3 each represent the same or different alkyl groups, and the total number of carbon atoms in the alkyl groups of R1, R2, and R3 is 9 to 15. In the present invention, the "total number of carbon atoms in the alkyl groups of R1, R2, and R3" refers to the sum of the number of carbon atoms in the alkyl group of R1, the number of carbon atoms in the alkyl group of R2, and the number of carbon atoms in the alkyl group of R3. By using a carboxybetaine in which the total number of carbon atoms in the alkyl groups of R1, R2, and R3 falls within the above range, it becomes possible to form an aqueous two-phase system in the presence of a water-soluble inorganic salt and / or organic acid salt. From the viewpoint of providing even better phase separation ability, the total number of carbon atoms in the alkyl groups of R1, R2, and R3 is preferably 11 to 15, more preferably 12 to 15, even more preferably 13 to 15, even more preferably 14 or 15, and particularly preferably 15.

[0014] The number of carbon atoms in each of the alkyl groups R1, R2, and R3 is not particularly limited as long as the total number of carbon atoms falls within the above range, but examples include 1 to 8, preferably 2 to 7, more preferably 3 to 6, even more preferably 3 to 5, still more preferably 4 or 5, and particularly preferably 5.

[0015] When the alkyl group of R1, R2, and R3 has 3 or more carbon atoms, the alkyl group may be either linear or branched, but is preferably linear.

[0016] In general formula (1), R4 is an alkylene group having 1 to 5 carbon atoms. R4 is preferably an alkylene group having 1 to 3 carbon atoms, more preferably a methylene group (-CH2-) or an ethylene group (-CH2-CH2-), and even more preferably a methylene group.

[0017] Suitable examples of carboxybetaines represented by general formula (1) include compounds in which, in general formula (1), R1, R2, and R3 are the same or different and each is an alkyl group having 3 to 5 carbon atoms, and R4 is a methylene group; more preferably, compounds in general formula (1), in which R1, R2, and R3 are the same or different and each is an alkyl group having 4 or 5 carbon atoms, and R4 is a methylene group; and even more preferably, compounds in general formula (1), in which R1, R2, and R3 are an alkyl group having 5 carbon atoms (preferably a linear pentyl group), and R4 is a methylene group.

[0018] A method for producing carboxybetaine represented by general formula (1) is known, for example, in JP-A-2009-96766, and can be derived from a known organic synthesis method.

[0019] The amine N-oxide used in the present invention is a compound having a structure represented by the following general formula (2). [ka]

[0020] In general formula (2), R5, R6, and R7 each represent the same or different alkyl groups, and the total number of carbon atoms in the alkyl groups of R5, R6, and R7 is 9 to 15. In the present invention, the "total number of carbon atoms in the alkyl groups of R5, R6, and R7" refers to the sum of the number of carbon atoms in the alkyl group of R5, the number of carbon atoms in the alkyl group of R6, and the number of carbon atoms in the alkyl group of R7. By using a carboxybetaine in which the total number of carbon atoms in the alkyl groups of R5, R6, and R7 falls within the above range, it becomes possible to form an aqueous two-phase system in the presence of a water-soluble inorganic salt and / or organic acid salt. From the viewpoint of providing even better phase separation ability, the total number of carbon atoms in the alkyl groups of R5, R6, and R7 is preferably 11 to 15, more preferably 12 to 15, even more preferably 13 to 15, even more preferably 14 or 15, and particularly preferably 15.

[0021] The number of carbon atoms in each of the alkyl groups R5, R6, and R7 is not particularly limited as long as the total number of carbon atoms falls within the above range, but examples include 1 to 8, preferably 2 to 7, more preferably 3 to 6, even more preferably 3 to 5, still more preferably 4 or 5, and particularly preferably 5.

[0022] When the alkyl group of R5, R6, and R7 has 3 or more carbon atoms, the alkyl group may be either linear or branched, but is preferably linear.

[0023] Suitable examples of amine N-oxides represented by general formula (2) include compounds in which R5, R6, and R7 in general formula (2) are the same or different and each represent an alkyl group having 3 to 5 carbon atoms; more preferably, compounds in general formula (2) are the same or different and each represent an alkyl group having 4 or 5 carbon atoms; and even more preferably, compounds in general formula (2) are the same or different and each represent an alkyl group having 5 carbon atoms (preferably a linear pentyl group).

[0024] The method for producing the amine N-oxide represented by the general formula (2) can be derived from known organic synthesis methods.

[0025] In the aqueous two-phase separation liquid of the present invention, one type of structure of the carboxybetaine represented by general formula (1) and the amine N-oxide represented by general formula (2) may be used alone, or two or more types of structures may be used in combination.

[0026] [Water-soluble salts] The water-soluble salt used in the present invention may be either a water-soluble inorganic salt or a water-soluble organic acid salt.

[0027] The type of anion constituting the water-soluble salt used in the present invention is not particularly limited, and examples thereof include sulfate ion, hydrogen sulfate ion, phosphate ion, hydrogen phosphate ion, dihydrogen phosphate ion, nitrate ion, carbonate ion, hydrogen carbonate ion, fluoride ion, chloride ion, bromide ion, iodide ion, hydroxide ion, hypochlorite ion, chlorite ion, chlorate ion, perchlorate ion, hypobromite ion, bromite ion, bromate ion, perbromate ion, hypoiodite ion, iodite ion, iodate ion, periodate ion, sulfite ion, hydrogen sulfite ion, thiosulfate ion, hydrogen thiosulfate ion, sulfide ion, hydrogen sulfide ion, thiocyanate ion, tetrahydroxide aluminate ion, tetrachloridogold(III) ion, tetrahydroxyborate ion, boric acid dihydrate ion, and the like. Examples of the cation ion include inorganic ions such as sodium ion, hydrogen borate ion, borate ion, metaborate ion, tetraborate ion, and tetrahydroborate ion; and organic acid ions such as formate ion, acetate ion, propionate ion, butyrate ion, lactate ion, tartrate ion, malonate ion, succinate ion, fumarate ion, malate ion, pyruvate ion, oxalate ion, citrate ion, adipate ion, itaconate ion, gluconate ion, glucuronate ion, galacturonate ion, saccharinate ion, glycerinate ion, xylonate ion, ascorbate ion, neuraminic acid ion, aspartic acid ion, glutamic acid ion, quinic acid ion, glycolic acid ion, coumaric acid ion, caffeic acid ion, chlorogenic acid ion, orotate ion, salicylic acid ion, and benzoic acid ion. Among these anions, the stronger the kosmotropic property in the Hofmeister series, the better the phase separation ability that can be provided, and therefore, anions with strong kosmotropic property are suitable as anions constituting water-soluble salts.Specific examples of strong kosmotropic anions include carbonate ion, hydrogen carbonate ion, sulfate ion, hydrogen sulfate ion, phosphate ion, hydrogen phosphate ion, dihydrogen phosphate ion, acetate ion, etc.The order of chaotropic and kosmotropic properties of anions is well known and is described, for example, in the literature (Satoshi Nihonyanagi, Shoichi Yamaguchi, and Tahei Tahara, J. Am. Chem. Soc., 136, 6155-6158 (2014)).

[0028] The type of cation constituting the water-soluble salt used in the present invention is not particularly limited, and examples thereof include alkali metal ions such as lithium ion, sodium ion, and potassium ion; alkaline earth metal ions such as magnesium ion and calcium ion; transition metal ions such as aluminum ion, copper ion, iron ion, nickel ion, manganese ion, chromium ion, and zinc ion; and organic ions such as ammonium ion, guanidium ion, pyridinium ion, imidazolium ion, tetrazolium ion, sulfonium ion, and phosphonium ion. Among these cations, the stronger the chaotropic property in the Hofmeister series, the better the phase separation ability can be provided, so that cations with strong chaotropic property are preferred as cations constituting the water-soluble salt. Specific examples of strong chaotropic cations include guanidium ion, magnesium ion, calcium ion, lithium ion, and sodium ion. The hierarchy of chaotropic and kosmotropic properties of cations is well known and is described, for example, in the literature (Satoshi Nihonyanagi, Shoichi Yamaguchi, and Tahei Tahara, J. Am. Chem. Soc., 136, 6155-6158 (2014)).

[0029] Specific examples of water-soluble salts used in the present invention include sodium carbonate, potassium carbonate, sodium hydrogencarbonate, ammonium carbonate, ammonium hydrogencarbonate, calcium carbonate, magnesium carbonate, sodium sulfate, sodium hydrogensulfate, ammonium sulfate, trisodium phosphate, tripotassium phosphate, disodium hydrogenphosphate, dipotassium hydrogenphosphate, sodium dihydrogenphosphate, potassium dihydrogenphosphate, sodium acetate, potassium acetate, ammonium acetate, sodium chloride, potassium chloride, sodium nitrate, potassium nitrate, ammonium nitrate, sodium bromide, potassium bromide, magnesium sulfate, ammonium sulfate, magnesium nitrate, and the like.

[0030] As described above, the stronger the kosmotropic property of the anion constituting the water-soluble salt, the more improved the phase separation ability, and the stronger the chaotropic property of the cation constituting the water-soluble salt, the more improved the phase separation ability. Therefore, suitable examples of the water-soluble salt used in the present invention include water-soluble salts composed of a strongly kosmotropic anion and a strongly chaotropic cation. Specific examples of such water-soluble salts include sodium carbonate, sodium hydrogencarbonate, calcium carbonate, magnesium carbonate, sodium sulfate, magnesium sulfate, calcium sulfate, trisodium phosphate, disodium hydrogenphosphate, sodium dihydrogenphosphate, sodium acetate, magnesium acetate, calcium acetate, etc.

[0031] The water-soluble salts used in the present invention may be in the form of an anhydrous salt or a solvate such as a hydrate.

[0032] In the aqueous two-phase separation liquid of the present invention, the water-soluble salt may be used alone or in combination of two or more kinds.

[0033] [Concentration of carboxybetaine represented by general formula (1) and / or amine N-oxide represented by general formula (2) and water-soluble salt] The aqueous two-phase separation liquid of the present invention is prepared by dissolving a carboxybetaine represented by general formula (1) and / or an amine N-oxide represented by general formula (2) and a water-soluble salt in water.

[0034] In the aqueous two-phase separation liquid of the present invention, the concentrations of the carboxybetaine represented by general formula (1) and / or the amine N-oxide represented by general formula (2) and the water-soluble salt may be appropriately set within a range in which two phases can be formed, depending on the type of carboxybetaine and / or amine N-oxide and the type of water-soluble salt. Specifically, the concentration range in which two phases can be formed can be set using a binodal curve created for each type of carboxybetaine and / or amine N-oxide and water-soluble salt used. The binodal curve is a curve showing the relationship between the concentration of the additive and the water-soluble salt at which a homogeneous phase changes to two phases (causing phase separation), and can be created by varying the concentrations of the additive and the water-soluble salt to determine the boundary between the conditions under which two phases can be formed and the conditions under which a homogeneous phase results. When the concentration of the additive (carboxybetaine represented by general formula (1) and / or amine N-oxide represented by general formula (2)) is plotted on the vertical axis and the concentration of the water-soluble salt on the horizontal axis, the upper right region of the binodal curve is the range in which two phases can be formed, and the lower left region is the range in which a homogeneous phase is formed.

[0035] In the aqueous two-phase separation liquid of the present invention, the concentrations of the carboxybetaine represented by general formula (1) and / or the amine N-oxide represented by general formula (2) and the water-soluble salt can be set by creating a binodal curve depending on the type of carboxybetaine and water-soluble compound used, as described above. For example, the concentration of the carboxybetaine represented by general formula (1) and / or the amine N-oxide represented by general formula (2) can be set within the range of 0.1 to 86% by mass, preferably 10 to 50% by mass, and more preferably 15 to 40% by mass, and the concentration of the water-soluble salt can be set within the range of 0.1 to 50% by mass, preferably 1 to 40% by mass, and more preferably 5 to 35% by mass.

[0036] [Characteristics / Applications] When the aqueous two-phase separation liquid of the present invention is stirred and allowed to stand, it becomes a two-phase separation liquid consisting of an upper aqueous phase containing a carboxybetaine represented by general formula (1) and / or an amine N-oxide represented by general formula (2) and a lower aqueous phase containing a water-soluble salt.

[0037] The aqueous two-phase separation liquid of the present invention has a high partition coefficient for organic substances and is therefore suitable for use in a separation method for separating organic substances. The method for separating organic substances using the aqueous two-phase separation liquid of the present invention will be described later.

[0038] 2. Organic matter separation method The separation method of the present invention is a method for separating organic matter using an aqueous two-phase separation liquid. Specifically, the separation method of the present invention is characterized by comprising: a first step of preparing and mixing an organic-containing material to be treated with an aqueous two-phase separation liquid containing a carboxybetaine represented by general formula (1) and / or an amine N-oxide represented by general formula (2), and a water-soluble salt; and a second step of separating the resulting mixture into two phases: an aqueous phase containing the carboxybetaine represented by general formula (1) and / or the amine N-oxide represented by general formula (2) and an aqueous phase containing the water-soluble salt; and recovering the aqueous phase containing the carboxybetaine represented by general formula (1) and / or the amine N-oxide represented by general formula (2). The separation method of the present invention is described in detail below.

[0039] [Organic matter] In the separation method of the present invention, organic matter is separated from a material to be treated that contains organic matter. In the present invention, the organic matter to be separated is not particularly limited, as long as it is soluble in an aqueous phase containing a carboxybetaine represented by general formula (1) and / or an amine N-oxide represented by general formula (2). Examples of organic matter to be separated include organic matter having an octanol / water partition coefficient (logP) in the range of -7 to 14. Organic matter to be separated preferably has an octanol / water partition coefficient in the range of -2 to 14, more preferably -1 to 14. The octanol / water partition coefficient is the ratio of the concentration of a compound dissolved in the 1-octanol phase to the concentration of a compound dissolved in water in a two-phase system of 1-octanol and water, and is a value used as an index of the degree of hydrophilicity or hydrophobicity of a compound. The octanol / water partition coefficient can be measured by the method described in Japanese Industrial Standard JIS 7260-107:200, "Measurement of partition coefficient (1-octanol / water) - Shake flask method."

[0040] The type of organic matter to be separated is not particularly limited, but examples include aliphatic compounds, aromatic compounds, alicyclic compounds, monosaccharides, oligosaccharides, polysaccharides, nucleic acids (RNA, DNA), peptides, proteins, synthetic polymers (synthetic polymers, synthetic fibers), functional organic dyes (organic semiconductors) and their complexes, microorganisms, etc.

[0041] More specifically, examples of organic substances to be separated include polysaccharides such as starch, cellulose, chitin, chitosan, pectin, inulin, agarose, carrageenan, xanthan gum, guar gum, and alginic acid; fatty acids such as butyric acid, valeric acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, lauric acid, myristic acid, pentadecanoic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, arachidic acid, and arachidonic acid; oils and fats in which fatty acids and glycerin are bound to form triglycerides, diglycerides, or monoglycerides; glycanases, nucleases, and lipases. , proteases, phosphotases, oxidases, reductases, kinases, cellulases, polymerases, lyases, isomerases, synthetases, transcription factors, receptors (adrenergic receptors, glucose receptors, dopamine receptors, angiotensin receptors, serotonin receptors, opioid receptors, acetylcholine receptors, etc.), Rubisco, growth factors (EGF, IGF, TGF, BDNF, VEGF, EPO, TPO, etc.), antibodies, cytokines (interferons, interleukins, etc.), biopharmaceuticals (insulin, somatropin, erythropoietin, etc.) ) and other proteins; microorganisms such as bacteria (Escherichia coli, Staphylococcus aureus, Salmonella, Pseudomonas aeruginosa, Streptococcus, Mycobacterium tuberculosis, Vibrio cholerae, Clostridium botulinum, etc.), fungi (Tinea fungus, Candida, etc.), phages, viruses (influenza virus, coronavirus, hepatitis virus, AIDS virus, rubella virus, measles virus, etc.); phenols, catechins, anthocyanins, flavanones, flavonols, chlorogenic acid, coumarin, lignin, coumaric acid, ferulic acid, tannins, hydroquinone, curcumin, flavones, isoflavones, tocopherol, rutin, limonene, lignans, Low molecular weight compounds such as sesamin, lutein, xanthophyll, fucoxanthin, astaxanthin, lycopene, beta-carotene, retinol, retinoic acid, caffeine, theophylline, and melanin; synthetic dyes such as porphyrin dyes, phthalocyanine dyes, indigo dyes, cyanine dyes, stilbene dyes, azo dyes, leuco dyes, and aniline dyes; synthetic polymers such as polythiophene, polyester, polyamide, polyethylene, polypropylene, polyacrylic acid and its esters, polymethacrylic acid and its esters, polysilicone, and polyurethane;Examples include functional dyes such as PEDOT·PSS, anthraquinone, scurialium, [tris(2-phenylpyridine)iridium(III)], and tris(8-quinolinolato)aluminum, and their complexes;

[0042] In the separation method of the present invention, the "material to be treated containing organic matter" that is the treatment target is something that contains organic matter and is desired to be separated from. The material to be treated containing organic matter may be in any form, such as a liquid, a paste, or a solid. The type of material to be treated containing organic matter is not particularly limited, and examples thereof include extracts of plants, biological tissues, cells, etc.; cultures of microorganisms, cells, etc.; reaction products of enzyme reactions, chemical reactions, etc.; and water treatment of wastewater, etc.

[0043] [1st step] In the first step, an aqueous two-phase separation liquid containing an organic substance-containing material to be treated, a carboxybetaine represented by general formula (1) and / or an amine N-oxide represented by general formula (2), and a water-soluble salt is prepared and mixed.

[0044] The types and concentrations of the carboxybetaine represented by general formula (1) and / or the amine N-oxide represented by general formula (2) and the water-soluble salt to be added to the aqueous two-phase separation liquid are as described in the section "1. Aqueous two-phase separation liquid" above.

[0045] The amount of the material to be treated containing organic matter to be added may be set appropriately depending on the type of organic matter to be separated, etc., but may be set, for example, so that the concentration of the organic matter to be separated in the aqueous two-phase separation liquid is about 0.001 to 30 mass %, preferably about 0.01 to 20 mass %, and more preferably about 0.1 to 15 mass %.

[0046] Thus, an aqueous two-phase separated liquid containing an organic substance is prepared and mixed, and then subjected to the second step described below.

[0047] [Second process] In the second step, the mixture obtained in the first step is separated into two phases: an aqueous phase containing the carboxybetaine represented by general formula (1) and / or the amine N-oxide represented by general formula (2), and an aqueous phase containing a water-soluble salt, and the aqueous phase containing the carboxybetaine represented by general formula (1) and / or the amine N-oxide represented by general formula (2) is recovered.

[0048] To separate the mixture obtained in the first step into two phases, an aqueous phase containing the carboxybetaine represented by general formula (1) and / or the amine N-oxide represented by general formula (2) and an aqueous phase containing the water-soluble salt, the mixture may be allowed to stand. Alternatively, to accelerate the phase separation, centrifugation may be performed.

[0049] The two-phase separation results in a two-phase separated liquid consisting of an upper aqueous phase containing carboxybetaine represented by general formula (1) and / or amine N-oxide represented by general formula (2), and a lower aqueous phase containing a water-soluble salt. The organic matter to be separated is distributed in the aqueous phase containing carboxybetaine represented by general formula (1) and / or amine N-oxide represented by general formula (2) (i.e., the upper layer), and the organic matter is separated and recovered by recovering the aqueous phase.

[0050] After the second step, the organic matter in the recovered aqueous phase may be subjected to treatment such as concentration and purification, if necessary. [Example]

[0051] The present invention will be specifically explained below by way of examples, but the present invention is not limited to these examples in any way.

[0052] Manufacturing example: Manufacturing of various carboxybetaines Carboxybetaines 1 to 5 shown below were synthesized with reference to the method described in JP 2009-96766 A. Carboxybetaine 1 is a compound in which R1 to R3 are methyl groups and R4 is a methylene group in general formula (1), Carboxybetaine 2 is a compound in which R1 to R3 are ethyl groups and R4 is a methylene group in general formula (1), Carboxybetaine 3 is a compound in which R1 to R3 are propyl groups and R4 is a methylene group in general formula (1), Carboxybetaine 4 is a compound in which R1 to R3 are butyl groups and R4 is a methylene group in general formula (1), and Carboxybetaine 5 is a compound in which R1 to R3 are pentyl groups and R4 is a methylene group in general formula (1). [ka]

[0053] Example 1: Confirmation of the formation of an aqueous two-phase system of carboxybetaine and a water-soluble salt The ability to form an aqueous two-phase system (the ability to cause phase separation) was confirmed by adding solid sodium sulfate (NaSO) (Fujifilm Wako Pure Chemical Industries, Ltd., model number 197-03345) to an aqueous solution of carboxybetaines 1 to 5 prepared at a concentration of 1.0 M until it no longer dissolved. PEG600 (Tokyo Chemical Industry, model number P0903) (mass percent concentration: 28.5%) was used as a comparative additive, as it has been shown in Non-Patent Document 1 that the addition of sodium sulfate causes phase separation.

[0054] The results are shown in Figure 1. Figure 1 is a photograph showing the phase separation immediately after sodium sulfate ceased to dissolve. PEG600 underwent phase separation, as previously reported. On the other hand, the results for carboxybetaines 1 to 5 were as follows: no phase separation was observed for carboxybetaines 1 and 2, but carboxybetaines 3 to 5 were confirmed to undergo phase separation, similar to PEG600.

[0055] Example 2: Comparison of binodal curves of carboxybetaine and sodium sulfate The phase separation ability of carboxybetaines 3 to 5, for which phase separation was confirmed in Example 1, was compared using binodal curves. PEG600 was used as a comparative additive. Sodium sulfate was used as a water-soluble salt. The binodal curve is a curve that shows the relationship between the additive that changes from a homogeneous phase to two phases (causing phase separation) and the salt concentration, with the region in the upper right corner of the curve representing two phases and the region in the lower left corner representing the homogeneous phase. The high phase separation ability of additives such as carboxybetaine and PEG can be considered to be the fact that phase separation occurs with a smaller amount of additive or salt concentration, and the closer the binodal curve is to the origin (lower left), the higher the phase separation ability.

[0056] The results are shown in Figure 2. Figure 2 shows the binodal curves for carboxybetaine 4, 5, or PEG600 with sodium sulfate. (Carboxybetaine 3 precipitated during the measurement, making it impossible to plot a binodal curve. This suggests that its phase separation ability is lower than that of carboxybetaine 4 and 5.) Comparing the binodal curves, the curves approached the origin in the order of carboxybetaine 4, PEG600, and carboxybetaine 5. This indicates that carboxybetaine 5 has a higher phase separation ability than the other two additives. Comparing the phase separation abilities, for example, at a sodium sulfate concentration of 10% by mass, PEG600 and carboxybetaine 4 underwent phase separation at additive concentrations of 15% and 20% by mass, respectively, whereas carboxybetaine 5 underwent phase separation at only 3.6% by mass (one-fifth to one-quarter of the additive concentrations of the other additives). These results demonstrate that carboxybetaine 5 has significantly superior phase separation ability.

[0057] Example 3: Comparison of phase separation ability by combination of carboxybetaine 5 and water-soluble salt Example 2 demonstrated that carboxybetaine 5 has excellent phase separation ability. Therefore, binodal curves were compared to determine whether the phase separation ability differs when combined with water-soluble salts other than sodium sulfate. The water-soluble salts used were sodium carbonate (Na2CO3, Fujifilm Wako Pure Chemical Industries, Ltd., Model No. 199-01585), sodium sulfate (Na2SO4), trisodium phosphate dodecahydrate (Na3PO4, Fujifilm Wako Pure Chemical Industries, Ltd., Model No. 191-02885), tripotassium phosphate (K3PO4, Fujifilm Wako Pure Chemical Industries, Ltd., Model No. 161-04325), dipotassium hydrogen phosphate (K2HPO4, Fujifilm Wako Pure Chemical Industries, Ltd., Model No. 164-04295), trisodium citrate dihydrate (C(OH)(CH2COONa)2COONa, Hayashi Pure Chemical Industries, Ltd., Model No. 19001975), sodium acetate (CH3COONa, Fujifilm Wako Pure Chemical Industries, Ltd., Model No. 19001975), and sodium hydroxide (NaOH). The following reagents were used: sodium chloride (NaCl, Fujifilm Wako Pure Chemical Industries, Ltd., model number 192-01075), sodium nitrate (NaNO, Fujifilm Wako Pure Chemical Industries, Ltd., model number 198-01675), sodium bromide (NaBr, Fujifilm Wako Pure Chemical Industries, Ltd., model number 195-02545), sodium bromide (NaBr, Fujifilm Wako Pure Chemical Industries, Ltd., model number 193-01505), anhydrous magnesium sulfate (MgSO, Fujifilm Wako Pure Chemical Industries, Ltd., model number 132-00435), ammonium sulfate ((NH)SO, Fujifilm Wako Pure Chemical Industries, Ltd., model number 019-03435), and magnesium nitrate hexahydrate (Mg(NO), Kanto Chemical Industries, Ltd., model number 25015-30).

[0058] Sodium sulfate (Na2SO4), trisodium phosphate (Na3PO4), trisodium citrate (C(OH)(CH2COONa)2COONa), sodium acetate (CH3COONa), sodium chloride (NaCl), sodium nitrate (NaNO3), and sodium bromide (NaBr) were used to fix the cation to the sodium ion and evaluate the difference in phase separation ability due to the difference in anion.

[0059] In addition, sodium sulfate (Na2SO4), magnesium sulfate (MgSO4), ammonium sulfate ((NH4)2SO4), sodium nitrate (NaNO3), and magnesium nitrate (Mg(NO3)2) were used to fix the anions to sulfate ions or nitrate ions and evaluate the difference in phase separation ability due to differences in cations.

[0060] Furthermore, to verify whether the order of the phase separation effects of cations and anions correlates with the order of the kosmotropicity of anions and the chaotropicity of cations, binodal curves were created for the cases where tripotassium phosphate (K3PO4), which has weaker cation chaotropicity than sodium phosphate (Na3PO4), and dipotassium hydrogen phosphate (K2HPO4), which has slightly weaker anion kosmotropicity than tripotassium phosphate (Na3PO4), with carboxybetaine 5, and the phase separation ability was compared. The results are shown in Figures 3 to 5.

[0061] Figure 3 compares the binodal curves when the cation is fixed to sodium ion and the anion is changed to carbonate ion, phosphate ion, sulfate ion, citrate ion, acetate ion, chloride ion, bromide ion, or nitrate ion. The binodal curves changed significantly when the anion was changed. The order of phase separation ability by anion was carbonate ion > phosphate ion > sulfate ion > citrate ion > acetate ion > chloride ion > nitrate ion > bromide ion. This order was consistent with the Hofmeister series (kosmotrope > chaotrope) (Satoshi Nihonyanagi, Shoichi Yamaguchi, and Tahei Tahara, J. Am. Chem. Soc., 136, 6155-6158 (2014)).

[0062] Figure 4 compares the binodal curves obtained when the anion is fixed at sulfate or nitrate and the cation is changed to sodium, ammonium, or magnesium. For sulfate, which has a strong kosmotropic property, the binodal curve did not change even when the cation was changed. On the other hand, when nitrate, which has a less strong kosmotropic property, was used as the anion, a ranking of magnesium ion > sodium ion was observed. This shows the opposite relationship (chaotrope > kosmotrope) to the Hofmeister series ranking observed for anions, suggesting that cations and anions act differently on the additive, carboxybetaine 5. It was also shown that when the anion exhibits a strong kosmotropic property, the difference in cation does not appear in the binodal curve.

[0063] Figure 5 compares the binodal curves of trisodium phosphate, tripotassium phosphate, and dipotassium hydrogen phosphate. When tripotassium phosphate, in which the cation chaotropic property of trisodium phosphate is replaced by potassium, was used, the binodal curve shifted to the upper right, indicating a weakened phase separation ability. Furthermore, when the phosphate ion in tripotassium phosphate was replaced by hydrogen phosphate ion, which has a weaker kosmotropic property, the binodal curve shifted further to the upper right.

[0064] From the above, it became clear that the phase separation ability of carboxybetaine 5 and water-soluble salts varies depending on the type and combination of cations and anions of the water-soluble salts. A comparison of cations and anions revealed that anions have a stronger effect on phase separation ability. Furthermore, preferred anions were those with strong kosmotropic properties in the Hofmeister series. On the other hand, preferred cations were those with strong chaotropic properties.

[0065] Example 4: Comparison of phase separation ability between carboxybetaine, amine N-oxide, and sulfobetaine, tetraalkylammonium salt

[0066] Additives with confirmed phase separation ability include sulfobetaine and tetraalkylammonium salts (see Non-Patent Documents 2 and 3). We compared these additives to determine whether the carboxybetaine used in the present invention has superior phase separation ability. We also used amine N-oxides, which are known to activate enzymes through hydration, similar to carboxybetaine (Takuma Aoki, Yuichi Nakagawa, Ryutaro Genjima, Kazuya Koumoto, Bioprocess Biosys. Eng., 43, 541-548 (2020)). The structures of sulfobetaine 4 and 5, tetraalkylammonium salts 4 and 5, and amine N-oxide 5, which were used for comparison with carboxybetaine, are shown below. [ka] [ka] [ka]

[0067] Specifically, a carboxybetaine 5 solution containing 57% by mass of carboxybetaine 5 and an additive solution containing 60% by mass of each additive other than carboxybetaine 5 (amine N-oxide 5, PEG600, sulfobetaines 4 and 5, and tetraalkylammonium salts 4 and 5) were prepared. Separately, saturated aqueous solutions of each water-soluble salt were prepared. 100 μL of the additive solution was mixed with 100 μL of the saturated aqueous solution of the water-soluble salt to determine whether phase separation occurred. Five types of water-soluble salts were used in the experiment: sodium sulfate, ammonium sulfate, sodium acetate, sodium chloride, and sodium bromide.

[0068] The results are shown in Table 1. The results are marked with "○" if phase separation was confirmed, and "×" if it was not confirmed. When PEG600, a known additive, was used as an additive, phase separation occurred with sodium sulfate and ammonium sulfate, but not with the other three types of water-soluble salts. On the other hand, carboxybetaine 5 according to the present invention caused phase separation with all five types of water-soluble salts used. Furthermore, amine N-oxide 5 also caused phase separation with all five types of water-soluble salts used, just like carboxybetaine 5, confirming its high phase separation ability.

[0069] For sulfobetaines, sulfobetaine 4 showed similar results to PEG600 and carboxybetaine 4, while sulfobetaine 5 caused phase separation with three types of water-soluble salts, including sodium acetate. It was revealed that the ability to cause phase separation weakened when the anionic functional group was changed from a carboxy group to a sulfonic acid group.

[0070] Furthermore, with tetraalkylammonium salts, phase separation occurred with three types of tetraalkylammonium salts: sodium sulfate, ammonium sulfate, and sodium bromide with tetraalkylammonium salt 4, and with four types of tetraalkylammonium salts: sodium sulfate, ammonium sulfate, sodium acetate, and sodium chloride with tetraalkylammonium salt 5. It was confirmed that all of them had lower phase separation ability than carboxybetaine 5.

[0071] [Table 1]

[0072] Example 5: Partitioning of organic compounds using an aqueous two-phase system formed by carboxybetaine 5 and sodium sulfate We investigated the partitioning behavior of organic substances in aqueous two-phase systems formed by carboxybetaine 5 and sodium sulfate, which had the highest phase separation ability among the additives. The organic substances whose partitioning behavior was evaluated were theophylline (Fujifilm Wako Pure Chemical Industries, Ltd., product number 209-09932) (logP = -1.12), caffeine (Fujifilm Wako Pure Chemical Industries, Ltd., product number 161-0125) (logP = 0.07), hydroquinone (Fujifilm Wako Pure Chemical Industries, Ltd., product number 085-01212) (logP = 1.25), ferulic acid (Tokyo Chemical Industry Co., Ltd., product number H0267) (logP = 1.42), phenol (Fujifilm Wako Pure Chemical Industries, Ltd., product number 031-06792) (logP = 1.46), and coumaric acid. Eleven compounds were used: benzophenone (Tokyo Chemical Industry Co., Ltd., product number C0393) (log P = 1.54), curcumin (Tokyo Chemical Industry Co., Ltd., product number C2302) (log P = 2.74), 9-aminoacridine (Tokyo Chemical Industry Co., Ltd., product number A2905) (log P = 2.56), flavone (Tokyo Chemical Industry Co., Ltd., product number F0015) (log P = 3.07), azobenzene (Tokyo Chemical Industry Co., Ltd., product number A0565) (log P = 3.22), and probucol (Sigma-Aldrich, product number P9672-1) (log P = 11.62). Each compound was hydrophilic or hydrophobic, with a log P (octanol / water partition coefficient), used as an index of hydrophilicity and hydrophobicity, ranging from -1 to 12 (the higher the log P, the more hydrophobic it is considered). [ka]

[0073] To form the aqueous two-phase system used in the extraction experiments, the ratio of additive to sodium sulfate was 24.2:8.6 (mass percent concentration, the two-phase region of the binodal curve in Figure 2), and PEG600 was used as a comparative additive.

[0074] In a 1.0 ml shell vial, 0.42 g of 2.0 M carboxybetaine 5 aqueous solution or 0.40 g of 60% (by mass) PEG 600 aqueous solution was mixed with 0.39 g of 22% (by mass) sodium sulfate aqueous solution, 1.0 mg of organic matter, and distilled water (0.18 g for carboxybetaine 5, 0.20 g for PEG 600) and stirred until dissolved using a vortex mixer. The solution was centrifuged (5000 rpm, 5 minutes, C1612, Benchmark Scientific) to separate into two phases and allowed to stand at room temperature for 1 hour. The absorbance of the organic matter contained in each phase (upper: betaine (or PEG 600) phase, lower: sodium sulfate phase) was measured using a UV-visible spectrophotometer, and the partition coefficient (absorbance of the upper layer / absorbance of the lower layer) was calculated.

[0075] The results are shown in Figure 6. Figure 6 compares the partition coefficients of each organic substance between carboxybetaine 5 and PEG600. For phenol and azobenzene, the absorbance of the organic substance in the lower layer (aqueous salt phase) was zero, and the partition coefficient was infinite, so no bar graphs are shown. Furthermore, when PEG600 was used, the organic substances curcumin, 9-aminoacridine, flavone, and azobenzene, indicated with an asterisk, did not dissolve in either the upper or lower phase and remained undissolved (Figure 7 shows the distribution and precipitation of azobenzene as an example). No such insoluble matter was observed in the aqueous two-phase system using carboxybetaine 5. Furthermore, the partition coefficients of the organic substances (partition to the upper layer) were higher for carboxybetaine 5 than for PEG600 for all organic substances. This demonstrates that carboxybetaine 5 can create an excellent aqueous two-phase system that can extract a variety of hydrophilic and hydrophobic substances into the upper layer (carboxybetaine phase) without precipitating them.

[0076] Example 6: Effect of Carboxybetaine 5 and Sodium Sulfate Concentration on the Distribution Equilibrium of Organic Matter Using Aqueous Two-Phase Systems The additive and salt concentrations required to form an aqueous two-phase system can be varied, potentially affecting the distribution of organic compounds. Therefore, in addition to the carboxybetaine 5:sodium sulfate ratio of 24.2:8.6 (the two-phase region of the binodal curve in Figure 2), similar extraction experiments were conducted using two different concentrations of carboxybetaine 5:sodium sulfate: 21.0:6.0 and 26.9:9.8. The experimental procedure was the same as in Example 5, and three organic compounds with different logP values ​​(caffeine, coumaric acid, and flavone) were used. The notation "carboxybetaine 5:sodium sulfate = X:Y" indicates that the carboxybetaine 5 is X% by mass and the sodium sulfate is Y% by mass.

[0077] The results are shown in Figure 8, which shows the relationship between composition and partition coefficient for three types of organic matter. As can be seen from Figure 8, for all organic matter, the partition coefficient increased as the concentrations of carboxybetaine 5 and sodium sulfate increased. In other words, the higher the concentrations of carboxybetaine 5 and water-soluble salts, the more the organic matter partitions to the upper layer. Furthermore, because a similar trend was observed for all organic matter, it is thought that the relationship between carboxybetaine 5 and salt concentration on the partition coefficient applies similarly to a variety of organic matter.

[0078] Example 7: Confirmation of the partition behavior of caffeine using aqueous two-phase systems formed by carboxybetaine 5 and various water-soluble salts As shown in Figure 3, the binodal curve of carboxybetaine 5 changed depending on the combination with a water-soluble salt. Therefore, an aqueous two-phase system was formed using sodium sulfate (NaSO), sodium acetate (CHCOONa), sodium chloride (NaCl), sodium nitrate (NaNO), and sodium bromide (NaBr), and an extraction experiment using caffeine as an organic substance was carried out in the same manner as in Example 5.

[0079] The aqueous two-phase systems were formed under the following conditions: carboxybetaine 5:sodium sulfate = 24:2 and 26.9:9.8, carboxybetaine 5:CH3COONa = 10:16 and 11:17, carboxybetaine 5:NaCl = 11:17 and 12:18, carboxybetaine 5:NaNO3 = 10:28 and 11:29, and carboxybetaine 5:NaBr = 10:33 and 11:34. Note that the notation "carboxybetaine 5:water-soluble salt = X:Y" indicates that the carboxybetaine 5 is X% by mass and the water-soluble salt is Y% by mass.

[0080] The results are shown in Figure 9. Figure 9 shows the partition coefficient of caffeine in aqueous two-phase systems formed with each water-soluble salt and its composition. It can be seen that the partition coefficient decreases from sodium sulfate, which has high phase separation ability, to sodium bromide, which has low phase separation ability. This means that conditions with high phase separation ability result in a higher extraction efficiency of caffeine into the upper layer. Furthermore, similar to the results in Example 6, the partition coefficient increases with increasing concentrations of carboxybetaine 5 and water-soluble salt. In other words, when organic matter is partitioned in an aqueous two-phase system formed by carboxybetaine 5 and a water-soluble salt, a water-soluble salt with high phase separation ability (chaotrope for the cation and kosmotrope for the anion) is used, and a greater amount of organic matter is extracted into the upper layer (betaine phase) under conditions where the concentrations of carboxybetaine 5 and the water-soluble salt are high.

[0081] Example 8: Confirmation of organic solubility in aqueous phase containing carboxybetaine 5 As shown in Examples 5 to 7, it was revealed that carboxybetaine 5 exhibits excellent partitioning properties and solubility for low-molecular-weight organic substances. Therefore, the solubility of flavone was investigated to determine the extent to which organic substances can be partitioned into the betaine phase.

[0082] Flavone was added in small amounts to a 57% by weight aqueous solution of carboxybetaine 5 at room temperature and stirred with a vortex mixer until dissolved. This procedure was repeated until no more flavone was dissolved, resulting in a saturated flavone solution. A flavone concentration calibration curve was created using a UV-visible spectrophotometer, and the concentration of the saturated flavone solution was determined by diluting the saturated solution based on this calibration curve, resulting in a concentration of 12.99% by weight.

[0083] In Example 5, the experiment was carried out at a low organic matter concentration (0.1% by mass) in order to measure the absorption spectrum, but it was confirmed that similar extraction could be performed even when the organic matter concentration was increased to about 13% by mass.

[0084] Example 9: Confirmation of the partitioning behavior of proteins and nucleic acids using an aqueous two-phase system formed by carboxybetaine 5 and sodium sulfate As shown in Examples 5 to 7, carboxybetaine 5 demonstrated excellent partitioning and solubility properties for low-molecular-weight organic compounds. To investigate whether similar extraction capabilities were also observed for polymeric compounds, the effects of carboxybetaine 5 on the extraction of biopolymers, proteins and nucleic acids, were investigated using bovine serum albumin (BSA) (Sigma, model number A7030-109) and salmon sperm DNA (Fujifilm Wako Pure Chemical Industries, model number 049-17321) in the same manner as in Example 5. Furthermore, phase separation was performed under conditions of 24.2% by mass of carboxybetaine 5 and 8.6% by mass of sodium sulfate. Furthermore, PEG600 and sulfobetaine 5, which has been reported to be optimal for protein extraction (see Non-Patent Document 2), as well as PEG600, were used as comparative additives.

[0085] The experimental results for BSA extraction are shown in Figures 10 and 11. As shown in Figure 10, when carboxybetaine 5 was used, the partition coefficient of BSA was 12 times higher than that of PEG600 and 1.7 times higher than that of sulfobetaine 5. Furthermore, as shown in Figure 11, precipitation was observed at the interface with PEG600 and sulfobetaine 5. On the other hand, no precipitation was observed with carboxybetaine 5. Thus, compared to sulfobetaine 5, which is known to have a high protein extraction ability, carboxybetaine 5 demonstrated superiority in both the solubility of organic matter in the betaine phase and the partition coefficient between the aqueous salt phase and the PEG600. Furthermore, because the PEG phase volume is larger with PEG600, it is inferior to carboxybetaine 5 in terms of the concentration of organic matter in the PEG phase. Furthermore, even with such a large PEG phase, BSA could not be completely dissolved.

[0086] The experimental results for DNA extraction are shown in Figure 12. As shown in Figure 12, when carboxybetaine 5 was used, the partition coefficient of salmon sperm DNA was 4.8 times higher than that of PEG600 and 3.6 times higher than that of sulfobetaine 5. As with its extraction properties for proteins, carboxybetaine 5 also showed excellent extraction properties for organic substances (biopolymers) when it came to DNA.

Claims

1. The present invention relates to a composition comprising a carboxybetaine represented by the following general formula (1) and / or an amine N-oxide represented by the following general formula (2), and a water-soluble salt thereof: The aqueous two-phase separation liquid, wherein the anion constituting the water-soluble salt is a carbonate ion, a hydrogen carbonate ion, a sulfate ion, a hydrogen sulfate ion, a phosphate ion, a hydrogen phosphate ion, a dihydrogen phosphate ion, or an acetate ion, and the cation constituting the water-soluble salt is a guanidium ion, a magnesium ion, a calcium ion, a lithium ion, or a sodium ion. 【Chemical 1】 [In general formula (1), R 1 , R 2 , and R 3 are the same or different and each represent a linear alkyl group having 4 or 5 carbon atoms, and R 4 is a methylene group. 【Chemistry 2】 [In the general formula (2), R 5 , R 6 , and R 7 are each a linear alkyl group having 5 carbon atoms.

2. In the general formula (1), R 1 , R 2 , and R 3 The aqueous two-phase separation liquid according to claim 1 , wherein is a linear alkyl group having 5 carbon atoms.

3. 3. The aqueous two-phase separation liquid according to claim 1, wherein the water-soluble salt is at least one selected from the group consisting of sodium carbonate, sodium bicarbonate, calcium carbonate, magnesium carbonate, sodium sulfate, magnesium sulfate, calcium sulfate, trisodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium acetate, magnesium acetate, and calcium acetate.

4. The aqueous two-phase separation liquid according to any one of claims 1 to 3, which is used for separating organic substances.

5. A method for separating organic matter, comprising the following first and second steps: a first step of preparing and mixing an aqueous two-phase separation liquid containing an organic substance-containing material to be treated, a carboxybetaine represented by the following general formula (1) and / or an amine N-oxide represented by the following general formula (2), and a water-soluble salt, wherein the anion constituting the water-soluble salt is a carbonate ion, a hydrogen carbonate ion, a sulfate ion, a hydrogen sulfate ion, a phosphate ion, a hydrogen phosphate ion, a dihydrogen phosphate ion, or an acetate ion, and the cation constituting the water-soluble salt is a guanidium ion, a magnesium ion, a calcium ion, a lithium ion, or a sodium ion; 【Chemistry 3】 [In general formula (1), R 1 , R 2 , and R 3 are the same or different and each represent a linear alkyl group having 4 or 5 carbon atoms, and R 4 is a methylene group. 【Chemistry 4】 [In the general formula (2), R 5 , R 6 , and R 7 are each a linear alkyl group having 5 carbon atoms; and The second step is to separate the mixture obtained in the first step into two phases: an aqueous phase containing the carboxybetaine represented by general formula (1) and / or the amine N-oxide represented by general formula (2) and an aqueous phase containing a water-soluble salt, and recover the aqueous phase containing the carboxybetaine represented by general formula (1) and / or the amine N-oxide represented by general formula (2).

6. 6. The method for separating organic matter according to claim 5, wherein the organic matter has an octanol / water partition coefficient of -7 to 14.

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