JP7742997B2 - Aqueous two-phase separation liquid and organic matter separation method using said aqueous two-phase separation liquid - Google Patents

Description

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

Lapse separation is a well-known method for separating and recovering organic substances. This method utilizes the differences in the distribution 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 raises safety concerns due to flammability and toxicity, as well as environmental concerns due to volatility.

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, along with 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 above-mentioned additives also have the disadvantage of having a low partition coefficient for organic substances. Therefore, aqueous two-phase systems have not yet been put to practical use in the industrial field.

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.

Sara C. Silverio, Oscar Rodriguez, Jose A. Teixeira, and Eugenia A. Macedo, J. Chem. Eng. Data. , 58, 3528-3535 (2013) Ana M. Ferreira, Helena Passos, Akiyoshi Okafuji, Mara G. Freire, Joao A. P. Coutinho and Hiroyuki Ohno, Green Chem. , 19, 4012-40 (2017) Yoshifumi Akama, Ahat Sali, Talanta, 57, 681-686 (2002)

The object of the present invention is to provide an aqueous two-phase separation liquid that can efficiently separate organic matter, and a method for separating organic matter using the aqueous two-phase separation liquid.

The inventors conducted extensive research to solve the above-mentioned problems and discovered 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 distribution coefficient for 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.

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:
[In general formula (1), R ₁ , R ₂ , and R ₃ each represent the same or different alkyl groups, and the total number of carbon atoms in the alkyl groups of R ₁ , R ₂ , and R ₃ is 9 to 15, and R ₄ is an alkylene group having 1 to 5 carbon atoms.]
[In general formula (2), R ₅ , R ₆ , and R ₇ each represent the same or different alkyl groups, and the total number of carbon atoms in the alkyl groups of R ₅ , R ₆ , and R ₇ is 9 to 15.]
Item 2. The aqueous two-phase separation liquid according to Item 1, wherein in general formula (1), R ₁ , R ₂ , and R ₃ are the same or different and each represent an alkyl group having 3 to 5 carbon atoms, and R ₄ is a methylene group.
Item 3. The aqueous two-phase separation liquid according to Item 1 or 2, wherein in general formula (1), R ₁ , R ₂ , and R ₃ are alkyl groups having 5 carbon atoms, and R ₄ is a methylene group.
Item 4. The aqueous two-phase separation liquid according to Item 1, wherein in general formula (2), R ₅ , R ₆ , and R ₇ 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 the general formula (2), R ₅ , R ₆ , and R ₇ 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;
[In general formula (1), R ₁ , R ₂ , and R ₃ each represent the same or different alkyl groups, and the total number of carbon atoms in the alkyl groups of R ₁ , R ₂ , and R ₃ is 9 to 15, and R ₄ is an alkylene group having 1 to 5 carbon atoms.]
[In general formula (2), R ₅ , R ₆ , and R ₇ each represent the same or different alkyl groups, and the total number of carbon atoms in the alkyl groups of R ₅ , R ₆ , and R ₇ 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.

According to the present invention, by using a carboxybetaine and/or amine N-oxide with a specific structure and a water-soluble salt, it is possible to form an aqueous two-phase system that can efficiently separate organic substances, making it a highly useful separation method that does not have the environmental or safety concerns that arise with oil-water two-phase systems.

This is a photographic image showing the state of phase separation immediately after sodium sulfate becomes insoluble in an aqueous solution of PEG 600 and carboxybetaines 1 to 5, which was added until sodium sulfate became insoluble. 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. 3 is a diagram showing 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 FIG. 3, the unit (%) of salt concentration is % by mass. 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 % by mass. FIG. 1 shows a comparison of the binodal curves of carboxybetaine 5 with trisodium phosphate, tripotassium phosphate, or dipotassium hydrogen phosphate. FIG. 1 shows the partition coefficients of various organic substances in aqueous two-phase systems of carboxybetaine 5 or PEG 600 and sodium sulfate. 1 is a photographic image showing the appearance of an aqueous two-phase system of carboxybetaine 5 or PEG 600 and sodium sulfate when azobenzene is added. 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. 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. FIG. 1 shows the partition coefficient of bovine serum albumin in aqueous two-phase systems of carboxybetaine 5, PEG 600, or sulfobetaine 5 with sodium sulfate. 11 is a photograph showing the distribution of BSA in the experiment of FIG. 10. FIG. 1 shows the partition coefficient of salmon sperm DNA in aqueous two-phase systems of carboxybetaine 5, PEG 600, or sulfobetaine 5 with sodium sulfate.

1. Aqueous Two-Phase Separation 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.

[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):

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.

The number of carbon atoms in each of the alkyl groups R ₁ , R ₂ , and R ₃ 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.

When the alkyl group of R ₁ , R ₂ and R ₃ has 3 or more carbon atoms, the alkyl group may be either linear or branched, but is preferably linear.

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.

Preferred examples of carboxybetaines represented by general formula (1) include compounds in which, in general formula (1), R ₁ , R ₂ , and R ₃ are the same or different and each is an alkyl group having 3 to 5 carbon atoms, and R ₄ is a methylene group; more preferred are compounds in general formula (1), in which R ₁ , R ₂ , and R ₃ are the same or different and each is an alkyl group having 4 or 5 carbon atoms, and R ₄ is a methylene group; and even more preferred are compounds in general formula (1), in which R ₁ , R ₂ , and R ₃ are an alkyl group having 5 carbon atoms (preferably a linear pentyl group), and R ₄ is a methylene group.

Methods for producing carboxybetaine represented by general formula (1) are publicly known, for example, in JP 2009-96766 A, and can be derived from known organic synthesis methods.

The amine N-oxide used in the present invention is a compound having a structure represented by the following general formula (2):

In general formula (2), R ₅ , R ₆ , and R ₇ each represent the same or different alkyl groups, and the total number of carbon atoms in the alkyl groups of R ₅ , R ₆ , and R ₇ is 9 to 15. In the present invention, the "total number of carbon atoms in the alkyl groups of R ₅ , R ₆ , and R ₇ " refers to the sum of the number of carbon atoms in the alkyl group of R ₅ , the number of carbon atoms in the alkyl group of R ₆ , and the number of carbon atoms in the alkyl group of R _7. By using a carboxybetaine in which the total number of carbon atoms in the alkyl groups of R ₅ , R ₆ , and R ₇ 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 R ₅ , R ₆ , and R ₇ 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.

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.

When the alkyl group of R ₅ , R ₆ , and R ₇ has 3 or more carbon atoms, the alkyl group may be either linear or branched, but is preferably linear.

Suitable examples of the amine N-oxide represented by general formula (2) include compounds in which, in general formula (2), R ₅ , R ₆ , and R ₇ are each the same or different and are an alkyl group having 3 to 5 carbon atoms; more preferably, compounds in general formula (2), in which R ₅ , R ₆ , and R ₇ are each the same or different and are an alkyl group having 4 or 5 carbon atoms; and even more preferably, compounds in general formula (2), in which R ₅ , R ₆ , and R ₇ are each an alkyl group having 5 carbon atoms (preferably a linear pentyl group).

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

In the aqueous two-phase separation liquid of the present invention, one type of structure from 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.

[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.

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, borate 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 achieved, so anions with strong kosmotropic properties are preferred as anions that constitute water-soluble salts. Specific examples of strong kosmotropic anions include carbonate ion, bicarbonate ion, sulfate ion, hydrogensulfate ion, phosphate ion, hydrogenphosphate ion, dihydrogenphosphate ion, and acetate ion. The hierarchy of anions in terms of chaotropic and kosmotropic properties is well known and is described, for example, in literature (Satoshi Nihonyanagi, Shoichi Yamaguchi, and Tahei Tahara, J. Am. Chem. Soc., 136, 6155-6158 (2014)).

The type of cation constituting the water-soluble salt used in the present invention is not particularly limited, but examples 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 group 13 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, cations with strong chaotropic properties in the Hofmeister series are preferred as cations constituting the water-soluble salt, since the stronger the chaotropic properties of these cations in the Hofmeister series, the better the phase separation ability they can provide. 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)).

Specific examples of water-soluble salts for use in the present invention include sodium carbonate, potassium carbonate, sodium bicarbonate, ammonium carbonate, ammonium bicarbonate, calcium carbonate, magnesium carbonate, sodium sulfate, sodium hydrogen sulfate, ammonium sulfate, trisodium phosphate, tripotassium phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, 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, etc.

As mentioned above, the stronger the kosmotropic properties of the anions that make up the water-soluble salt, the better the phase separation ability; and the stronger the chaotropic properties of the cations that make up the water-soluble salt, the better the phase separation ability. Therefore, suitable examples of water-soluble salts for use 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 bicarbonate, calcium carbonate, magnesium carbonate, sodium sulfate, magnesium sulfate, calcium sulfate, trisodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium acetate, magnesium acetate, calcium acetate, etc.

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

Furthermore, in the aqueous two-phase separation liquid of the present invention, one type of water-soluble salt may be used alone, or two or more types may be used in combination.

[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.

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 that allows the formation of two phases, depending on the type of carboxybetaine and/or amine N-oxide and the type of water-soluble salt. Specifically, the concentration range that allows the formation of two phases can be set using binodal curves created for each type of carboxybetaine and/or amine N-oxide and water-soluble salt used. A binodal curve is a curve that shows 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). It can be created by varying the concentrations of the additive and the water-soluble salt to determine the boundary between the conditions that allow the formation of two phases and the conditions that result in a homogeneous phase. 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 region in the upper right corner of the binodal curve is the range in which two phases can be formed, and the region in the lower left corner is the range in which a homogeneous phase is formed.

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 determined by creating a binodal curve based 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.

[Characteristics/Applications]
When the aqueous two-phase separation liquid of the present invention is stirred and allowed to stand, it separates into two phases: 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.

The aqueous two-phase separation liquid of the present invention has a high distribution coefficient for organic substances and is suitable for use in liquid separation methods for separating organic substances. A method for separating organic substances using the aqueous two-phase separation liquid of the present invention is described below.

2. Organic Substance Separation Method The separation method of the present invention is a method for separating organic substances 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 substance-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.

[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 the organic matter to be separated include organic matter having an octanol/water partition coefficient (log P) 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."

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.

Specific 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; fats and oils in which fatty acids and glycerin are bonded together as triglycerides, diglycerides, or monoglycerides; glycanases, nucleases, lipases, proteases, phosphotases, oxidases, and enzymes. Proteins such as ductases, kinases, cellulases, polymerases, lyases, isomerases, synthetases, transcriptional regulators, 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.); bacteria (Escherichia coli, Staphylococcus aureus, Salmonella, Pseudomonas aeruginosa, Streptococcus, Mycobacterium tuberculosis, Vibrio cholerae), etc. Microorganisms such as fungi (tinea fungus, Candida fungus, etc.), phages, and viruses (influenza virus, coronavirus, hepatitis virus, AIDS virus, rubella virus, measles virus, etc.); phenol, catechin, anthocyanin, flavanone, flavonol, chlorogenic acid, coumarin, lignin, coumaric acid, ferulic acid, tannin, hydroquinone, curcumin, flavones, isoflavones, tocopherol, rutin, limonene, lignan, sesamin, lutein, xanthophyll, fucoxanthin, astaxanthin, lycopene, β-carotene, retinol, retinoic acid, caffeine, theophilin low molecular weight compounds such as porphyrin dyes, phthalocyanine dyes, indigo dyes, cyanine dyes, stilbene dyes, azo dyes, leuco dyes, and aniline dyes; synthetic dyes such as polythiophene, polyester, polyamide, polyethylene, polypropylene, polyacrylic acid and its esters, polymethacrylic acid and its esters, polysilicone, and polyurethane; and functional dyes and complexes thereof such as PEDOT/PSS, anthraquinone, sculirium, [tris(2-phenylpyridine)iridium(III)], and tris(8-quinolinolato)aluminum.

In the separation method of the present invention, the "material to be treated containing organic matter" refers to a material that contains organic matter and from which separation of the organic matter is desired. The material to be treated containing organic matter may be in any form, such as a liquid, paste, or solid. There are no particular limitations on the type of material to be treated that contains organic matter, but examples 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.

[First 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.

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

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

After preparing and mixing an aqueous two-phase separation liquid containing organic matter, it is subjected to the second step described below.

[Second step]
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.

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 can be allowed to stand. Alternatively, centrifugation can be performed to accelerate the phase separation.

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 this aqueous phase.

Furthermore, after the second step, if necessary, the organic matter in the recovered aqueous phase may be subjected to treatment such as concentration and purification.

The present invention will be explained in detail below using examples, but the present invention is not limited to these in any way.

Production Example: Production 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 of general formula (1) in which R ₁ to R ₃ are methyl groups and R ₄ is a methylene group, Carboxybetaine 2 is a compound of general formula (1) in which R ₁ to R ₃ are ethyl groups and R ₄ is a methylene group, Carboxybetaine 3 is a compound of general formula (1) in which R ₁ to R ₃ are propyl groups and R ₄ is a methylene group, Carboxybetaine 4 is a compound of general formula (1) in which R ₁ to R ₃ are butyl groups and R ₄ is a methylene group, and Carboxybetaine 5 is a compound of general formula (1) in which R ₁ to R ₃ are pentyl groups and R ₄ is a methylene group.

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 property of causing phase separation) was confirmed by adding solid sodium sulfate ( _Na2SO4 ) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., model number 197-03345) to an aqueous solution of carboxybetaine 1 to 5 prepared at a concentration of 1.0 _M until it no longer dissolved. Furthermore, as a comparative additive, PEG600 (manufactured by Tokyo Chemical Industry, model number P0903) (mass percent concentration: 28.5%) was used, which has been shown in Non-Patent Document 1 to cause phase separation when sodium sulfate is added.

The results are shown in Figure 1. Figure 1 is a photograph showing the phase separation that occurred immediately after sodium sulfate ceased to dissolve. PEG 600 underwent phase separation, as has already been 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, just like PEG 600.

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. PEG 600 was used as the comparative additive. Sodium sulfate was used as the 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 ability to cause phase separation with a lower additive or salt concentration, and the closer the binodal curve is to the origin (lower left), the higher the phase separation ability.

The results are shown in Figure 2. Figure 2 shows the binodal curves for carboxybetaine 4, 5, or PEG 600 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 or 5.) Comparing the binodal curves, the curves approached the origin in the order of carboxybetaine 4, PEG 600, 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, PEG 600 and carboxybetaine 4 underwent phase separation at additive concentrations of 15% and 20% by mass, respectively, whereas carboxybetaine 5 underwent phase separation at an additive concentration of only 3.6% by mass (one-fifth to one-quarter of the other additive concentrations). These results demonstrate that carboxybetaine 5 has significantly superior phase separation ability.

Example 3: Comparison of phase separation ability by combination of carboxybetaine 5 and water-soluble salt In Example 2, it was shown that carboxybetaine 5 has excellent phase separation ability. Therefore, a comparison of binodal curves was carried out to see whether the phase separation ability differs depending on the combination with a water-soluble salt other than sodium sulfate. Examples of water-soluble salts include sodium carbonate (Na ₂ CO ₃ , manufactured by Fujifilm Wako Pure Chemical Industries, model number 199-01585), sodium sulfate (Na ₂ SO ₄ ), trisodium phosphate dodecahydrate (Na ₃ PO ₄ , manufactured by Fujifilm Wako Pure Chemical Industries, model number 191-02885), tripotassium phosphate (K ₃ PO ₄ , manufactured by Fujifilm Wako Pure Chemical Industries, model number 161-04325), dipotassium hydrogen phosphate (K ₂ HPO ₄ , manufactured by Fujifilm Wako Pure Chemical Industries, model number 164-04295), trisodium citrate dihydrate (C(OH)(CH ₂ COONa) ₂ COONa, manufactured by Hayashi Pure Chemical Industries, model number 19001975), sodium acetate (CH ₃ The following substances were used: COONa (Fujifilm Wako Pure Chemical Industries, model number 192-01075), sodium chloride (NaCl (Fujifilm Wako Pure Chemical Industries, model number 198-01675), sodium nitrate (NaNO ₃ (Fujifilm Wako Pure Chemical Industries, model number 195-02545), sodium bromide (NaBr (Fujifilm Wako Pure Chemical Industries, model number 193-01505)), anhydrous magnesium sulfate (MgSO ₄ (Fujifilm Wako Pure Chemical Industries, model number 132-00435), ammonium sulfate ((NH ₄ ) ₂ SO ₄ (Fujifilm Wako Pure Chemical Industries, model number 019-03435)), and magnesium nitrate hexahydrate (Mg(NO ₃ ) ₂ (Kanto Chemical Industries, model number 25015-30)).

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 sodium ion and evaluate _the difference in phase separation ability due to the difference in anion.

_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 different cations.

Furthermore, to verify whether the order of the phase separation effects of cations and anions correlates with _the order of the kosmotropic properties of anions and the chaotropic properties of cations, binodal curves were prepared for the cases where tripotassium phosphate ( _K3PO4 ), which has weaker cation chaotropic properties than sodium, was used in comparison with trisodium phosphate ( _Na3PO4 ), and where dipotassium hydrogen phosphate ( _K2HPO4 ) _, which has slightly weaker anion kosmotropic properties than tripotassium phosphate, was used, and the phase separation ability was compared. The results are shown in Figures 3 to ₅ .

Figure 3 compares the binodal curves when the cation is fixed to sodium ion and the anion is carbonate ion, phosphate ion, sulfate ion, citrate ion, acetate ion, chloride ion, bromide ion, or nitrate ion. Changing the anion significantly altered the binodal curve. 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)).

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

Figure 5 compares the binodal curves of trisodium phosphate, tripotassium phosphate, and dipotassium hydrogen phosphate. When tripotassium phosphate, in which the cation has been substituted with potassium, which has weaker chaotropic properties than trisodium phosphate, was used, the binodal curve shifted to the upper right, indicating a weakened phase separation ability. Furthermore, when dipotassium hydrogen phosphate, in which the phosphate ion in tripotassium phosphate has been substituted with hydrogen phosphate ion, which has weaker kosmotropic properties, was used, the binodal curve shifted even further to the upper right.

From the above, it became clear that the phase separation ability of carboxybetaine 5 and water-soluble salts varies depending on the type of cation and anion in the water-soluble salt and their combination. When comparing cations and anions, it became clear that anions have a stronger effect on phase separation ability. Furthermore, ions with strong kosmotropic properties in the Hofmeister series were preferred as anions. On the other hand, ions with strong chaotropic properties were preferred as cations.

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

Additives that have been confirmed to have phase separation ability include sulfobetaine and tetraalkylammonium salts (see Non-Patent Documents 2 and 3). The carboxybetaine used in the present invention was compared to these additives to determine whether it has superior phase separation ability. Amine N-oxides, which are known to cause enzyme activation through hydration similar to carboxybetaine (Takuma Aoki, Yuichi Nakagawa, Ryutaro Genjima, Kazuya Koumoto, Bioprocess Biosys. Eng., 43, 541-548 (2020)), were also used. The structures of sulfobetaine 4 and 5, tetraalkylammonium salts 4 and 5, and amine N-oxide 5, which were used as comparisons to carboxybetaine, are shown below.

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, PEG 600, 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 and 100 μL of the saturated aqueous solution of the water-soluble salt were mixed to check 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.

The results are shown in Table 1. "○" indicates that phase separation was confirmed, and "×" indicates that phase separation was not confirmed. When PEG 600, a known additive, was used as the 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, like carboxybetaine 5, amine N-oxide 5 also caused phase separation with all five types of water-soluble salts used, confirming its high phase separation ability.

For sulfobetaines, sulfobetaine 4 produced results similar to those of PEG 600 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 changed from a carboxy group to a sulfonic acid group.

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

Example 5: Partition of Organic Compounds Using an Aqueous Two-Phase System Formed by Carboxybetaine 5 and Sodium Sulfate The partition behavior of organic compounds in an aqueous two-phase system formed by Carboxybetaine 5 and sodium sulfate, which exhibited the highest phase separation ability among additives, was investigated. The organic compounds whose partition behavior was evaluated were theophylline (Fujifilm Wako Pure Chemical Industries, Ltd., Model No. 209-09932) (log P = -1.12), caffeine (Fujifilm Wako Pure Chemical Industries, Ltd., Model No. 161-0125) (log P = 0.07), hydroquinone (Fujifilm Wako Pure Chemical Industries, Ltd., Model No. 085-01212) (log P = 1.25), ferulic acid (Tokyo Chemical Industry Co., Ltd., Model No. H0267) (log P = 1.42), phenol (Fujifilm Wako Pure Chemical Industries, Ltd., Model No. 031-06792) (log P = 1.46), and coumaric acid. Eleven compounds were used: benzophenone (Tokyo Chemical Industry Co., Ltd., model number C0393) (log P = 1.54), curcumin (Tokyo Chemical Industry Co., Ltd., model number C2302) (log P = 2.74), 9-aminoacridine (Tokyo Chemical Industry Co., Ltd., model number A2905) (log P = 2.56), flavone (Tokyo Chemical Industry Co., Ltd., model number F0015) (log P = 3.07), azobenzene (Tokyo Chemical Industry Co., Ltd., model number A0565) (log P = 3.22), and probucol (Sigma-Aldrich Co., Ltd., model 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 larger the log P, the more hydrophobic it is considered to be).

To form the aqueous two-phase system used in the extraction experiments, the additive:sodium sulfate ratio was 24.2:8.6 (mass percent concentration ratio, the two-phase region of the binodal curve in Figure 2). PEG 600 was also used as a comparative additive.

0.42 g of 2.0 M carboxybetaine 5 aqueous solution or 0.40 g of 60% PEG 600 aqueous solution was mixed with 0.39 g of 22% 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) in a 1.0 ml shell vial 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 layer: betaine (or PEG 600) phase, lower layer: sodium sulfate phase) was measured using a UV-visible spectrophotometer, and the partition coefficient (absorbance of upper layer/absorbance of lower layer) was calculated.

The results are shown in Figure 6. Figure 6 compares the partition coefficients of each organic substance between carboxybetaine 5 and PEG 600. 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 PEG 600 was used, the organic substances curcumin, 9-aminoacridine, flavone, and azobenzene, marked 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 organic substances (partition to the upper layer) were higher for carboxybetaine 5 than for PEG 600 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.

Example 6: Effect of Carboxybetaine 5 and Sodium Sulfate Concentration on the Distribution Equilibrium of Organic Compounds Using an Aqueous Two-Phase System. The additive and salt concentrations that can form an aqueous two-phase system can be varied, and this influence may also affect 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 log P 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.

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, the partition coefficient increased for all organic matters 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, since a similar trend was observed for all organic matters, it is thought that the relationship between carboxybetaine 5 and salt concentration on the partition coefficient applies similarly to a variety of organic matters.

Example 7: Confirmation of the distribution 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 ( _Na2SO4 ), sodium acetate ( _CH3COONa ), sodium chloride (NaCl), sodium nitrate ( _NaNO3 ), 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.

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.

The results are shown in Figure 9. Figure 9 shows the distribution coefficient of caffeine in aqueous two-phase systems formed with each water-soluble salt and its composition. It can be seen that the distribution 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, as in Example 6, the distribution coefficient increases with increasing concentrations of carboxybetaine 5 and water-soluble salt. In other words, when partitioning organic matter in an aqueous two-phase system formed by carboxybetaine 5 and water-soluble salt, using water-soluble salts with high phase separation ability (chaotrope for the cation and kosmotrope for the anion) and conditions with high concentrations of carboxybetaine 5 and water-soluble salt results in more organic matter being extracted into the upper layer (betaine phase).

Example 8: Confirmation of organic substance solubility in an 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.

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, preparing 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, which was found to be 12.99% by weight.

In Example 5, an experiment was conducted at a low organic concentration (0.1% by mass) to measure absorption spectra, but it was confirmed that similar extractions could be performed even when the concentration was increased to approximately 13% by mass.

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 was found to exhibit excellent partitioning and solubility properties for low-molecular-weight organic compounds. Therefore, to investigate whether similar extraction capabilities are also observed for high-molecular-weight compounds, the effects of carboxybetaine 5 on the extraction of biopolymers, proteins and nucleic acids, were investigated using bovine serum albumin (BSA) (manufactured by SIGMA, model number A7030-109) and salmon sperm DNA (manufactured by Fujifilm Wako Pure Chemical Industries, model number 049-17321) in a manner similar to that of Example 5. Furthermore, the phase separation conditions were 24.2% by mass of carboxybetaine 5 and 8.6% by mass of sodium sulfate. Furthermore, PEG 600, sulfobetaine 5, which has been reported to be optimal for protein extraction (see Non-Patent Document 2), and PEG 600 were used as comparative additives.

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 PEG 600 and 1.7 times higher than that of sulfobetaine 5. Furthermore, as shown in Figure 11, precipitation was observed at the interface with PEG 600 and sulfobetaine 5. On the other hand, no precipitation was observed with carboxybetaine 5. In other words, compared to sulfobetaine 5, which is known to have high protein extraction ability, carboxybetaine 5 demonstrated superiority in both the solubility of organic matter in the betaine phase and the partition coefficient with the aqueous salt phase. Furthermore, because PEG 600 has a larger PEG phase volume, it is inferior to carboxybetaine 5 in terms of the concentration of organic matter in the partition. Furthermore, even with such a large PEG phase, BSA could not be completely dissolved.

The experimental results for DNA extraction are shown in Figure 12. As shown in Figure 12, when carboxybetaine 5 was used, the partition coefficient for salmon sperm DNA was 4.8 times higher than that of PEG 600 and 3.6 times higher than that of sulfobetaine 5. As with its extraction properties for proteins, carboxybetaine 5 also exhibited excellent extraction properties for organic substances (biopolymers) when it came to DNA.

Claims (6)

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 .
[In general formula (1), R ₁ , R ₂ , and R ₃ are the same or different and each represents a linear alkyl group having 4 or 5 carbon atoms, and R ₄ is a methylene group .]
[In general formula (2), R ₅ , R ₆ , and R ₇ each represent a linear alkyl group having 5 carbon atoms .] 2. The aqueous two-phase separation liquid according to claim 1 , wherein in the general formula (1), R ₁ , R ₂ , and R ₃ are linear alkyl groups having 5 carbon atoms. 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. The aqueous two-phase separation liquid according to any one of claims 1 to 3 , which is used for separating organic substances. 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 ;
[In general formula (1), R ₁ , R ₂ , and R ₃ are the same or different and each represents a linear alkyl group having 4 or 5 carbon atoms, and R ₄ is a methylene group .]
[In general formula (2), R ₅ , R ₆ , and R ₇ each represent 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. The method for separating organic matter according to claim 5 , wherein the organic matter has an octanol/water partition coefficient of -7 to 14.