JP7163691B2 - A method for measuring the pore distribution of an ion exchanger, a determination method using the measuring method, and a selection method using the measuring method. - Google Patents

Description

The present invention relates to a method for measuring the pore size distribution of an ion exchanger, a determination method using the measuring method, and a selection method using the measuring method.

Conventionally, the nitrogen adsorption method and the mercury intrusion method have been widely used as methods for measuring the pore structure of porous bodies. A method of performing size exclusion chromatography (gel filtration chromatography) using a so-called inverse size exclusion chromatography (ISEC) method has come to be used.

As a method for measuring the pore structure of a porous material by reverse size exclusion chromatography, for example, the measuring method described in Non-Patent Document 1 is disclosed.

Journal of Chromatography A, 883 (2000), p39.

The nitrogen adsorption method and the mercury intrusion method, which have been conventionally used as methods for measuring the pore structure of porous bodies, require the porous bodies to be measured to be in a dry state. Therefore, for resins that are used in a wet state, such as ion exchange resin, the pore structure disappears due to shrinkage of the resin due to drying. There is a problem that the pore structure is not reflected.

In addition, the method for measuring the pore structure of a porous material by reverse size exclusion chromatography described in Non-Patent Document 1 is an ion-exchange separation agent for separation of high-molecular-weight proteins, and the pore size is also measured. It is limited to those that are large and have a highly hydrophilic skeleton. Therefore, it is not suitable for those with small pore diameters or those with low hydrophilicity of the skeleton, and it is not necessarily suitable for measuring the pore structure of ion exchange resins in general, including polystyrene resin-based industrial ion exchange resins, in a wet state. .

The present invention has been made in view of such problems, and an object of the present invention is to provide a method for measuring the wet state pore distribution of ion exchangers in general, including industrial ion exchange resins, and a method for measuring the pore size distribution. It is an object of the present invention to provide a method for determining the degree of deterioration of an ion exchanger and a method for selecting an ion exchanger suitable for the application using the measuring method.

As described above, a method suitable for measuring the wet state pore size distribution of ion exchangers in general, including industrial ion exchange resins, has not been found. By selecting a specific substance as a sample with a known molecular size for use in size exclusion chromatography, we discovered a method suitable for measuring the wet pore size distribution of all ion exchangers, including industrial ion exchange resins. rice field.

That is, the gist of the present invention is as follows: [1] A method for measuring the wet pore size distribution of an ion exchanger by reverse size exclusion chromatography using a sample with a known molecular size, comprising : The exchanger is a polystyrene-based
An on - exchange resin with a known molecular size
A measurement method consisting only of monosaccharides, glycerol, ethylene glycol, methanol and heavy water.
[2] Ion exchange by reverse size exclusion chromatography using a sample of known molecular size
A method for measuring the wet state pore size distribution of an ion exchanger, wherein the ion exchanger is a polystyrene-based ion
An on-exchange resin with a known molecular size
A measurement method consisting of only monosaccharides and heavy water.
[3] Nonionically dissociable polysaccharides include dextran, pullulan, maltooligosaccharide, maltose
is at least one selected from the group consisting of sugar, isomaltose and sucrose, [1
] or the measurement method according to [2].
[4] Nonionically dissociable monosaccharides are glucose, galactose, mannose, fructose
erythritol, xylitol, xylose, D-mannoheptulose and sorbitol
Any one of [1] to [3], which is at least one selected from the group consisting of
measurement method.
[5] The pore distribution of a new ion exchanger and the pore distribution of the used ion exchanger are compared with [1]
Measured by the measurement method according to any one of [4] to determine the degree of deterioration of the ion exchanger
determination method.
[6] The pore distribution of the ion exchanger is measured by the measurement method according to any one of [1] to [4].
A selection method that measures more and selects an ion exchanger suitable for the application.

The measuring method of the present invention is suitable for measuring the wet pore size distribution of general ion exchangers including industrial ion exchange resins. Further, by using the measuring method of the present invention, it becomes possible to determine the degree of deterioration of the ion exchanger and to select an ion exchanger suitable for its application.

2 is a diagram showing the relationship between the molecular weight and elution volume of each sample in Example 1. FIG. 1 is a diagram showing the relationship between the pore radius and pore volume of an ion exchanger in Example 1. FIG. 2 is a diagram showing the relationship between the molecular weight and elution volume of each standard in Comparative Example 1. FIG. FIG. 10 is a diagram showing the relationship between the molecular weight of each sample and the elution volume in Example 2. FIG. FIG. 4 is a diagram showing the relationship between the pore radius and pore volume of an ion exchanger in Example 2; FIG. 10 is a diagram showing the relationship between the molecular weight of each sample and the elution volume in Comparative Example 2; FIG. 10 is a diagram showing the relationship between the molecular weight of each sample and the elution volume in Example 3; FIG. 10 is a diagram showing the relationship between the pore radius and pore volume of an ion exchanger in Example 3; FIG. 10 is a diagram showing the relationship between the molecular weight of each sample and the elution volume in Example 4; FIG. 10 is a diagram showing the relationship between the pore radius and pore volume of an ion exchanger in Example 4; FIG. 10 is a diagram showing the relationship between the molecular weight of each sample and the elution volume in Example 5. FIG. FIG. 10 is a diagram showing the relationship between the pore radius and pore volume of an ion exchanger in Example 5; FIG. 10 is a diagram showing the relationship between the molecular weight of each sample and the elution volume in Example 6. FIG. FIG. 10 is a diagram showing the relationship between the pore radius and pore volume of an ion exchanger in Example 6; FIG. 10 is a diagram showing the relationship between the molecular weight of each sample and the elution volume in Example 7; FIG. 10 is a diagram showing the relationship between the pore radius and pore volume of an ion exchanger in Example 7; FIG. 10 is a diagram showing the relationship between the molecular weight of each standard and the elution volume in Example 8. FIG. FIG. 10 is a diagram showing the relationship between the pore radius and pore volume of an ion exchanger in Example 8; FIG. 10 is a diagram showing the relationship between the pore radius and the pore volume of new, used 1, and used 2 ion exchangers in Example 9. FIG. FIG. 10 is a chromatogram of a pigment component of steviol glycosides in Example 10. FIG. FIG. 10 is a diagram showing the relationship between the molecular size of each sample and the elution time in Example 10. FIG. FIG. 10 is a graph showing changes in absorbance in Example 10;

Although the present invention will be described in detail below, the present invention is not limited to the following embodiments, and can be implemented with various modifications within the scope of the gist thereof. In addition, when the expression "~" is used in this specification, it is used as an expression including numerical values or physical property values before and after it.

(Ion exchanger)
The ion exchanger is not particularly limited as long as it has an ion exchange capacity, and examples thereof include inorganic ion exchangers and organic ion exchangers.

Examples of inorganic ion exchangers include silica-based ion exchangers having a silica skeleton; alumina-based ion exchangers having an alumina skeleton, and the like.

Examples of organic ion exchangers include polysaccharide ion exchangers having skeletons such as cellulose, dextran, and agarose; poly(styrene-divinylbenzene), poly(styrene-ethylstyrene-divinylbenzene), poly((meth) acrylic acid-divinylbenzene), polystyrene-based ion exchange resins having a skeleton of polydivinylbenzene, etc.; poly(2,3-dihydroxypropyl methacrylate-ethylene dimethacrylate), poly(hydroxyethyl methacrylate-trimethylolpropane trimethacrylate) ), acrylic ion exchange resin having a skeleton of poly (meth) acrylic acid ester, etc.; polyvinyl alcohol ion exchange resin having a skeleton of poly (vinyl alcohol-triallyl isocyanurate), etc.; poly (2-hydroxyethyl vinyl ether- diethylene glycol vinyl ether), poly(chloroethyl vinyl ether-triethylene glycol vinyl ether) and the like as a backbone of polyvinyl ether-based ion exchange resins.

Among these ion exchangers, organic ion exchangers are preferred because they are often used industrially, polystyrene ion exchange resins and acrylic ion exchange resins are more preferred, and polystyrene ion exchange resins are particularly preferred.

Since the pore radius distribution of the ion exchanger is within a range that can be sufficiently measured by the measurement method of the present invention, it is preferably 0.1 nm to 1000 nm, more preferably 0.1 nm to 600 nm, and further preferably 0.1 nm to 200 nm. preferable.

(reverse size exclusion chromatography)
Inverse size exclusion chromatography (hereinafter sometimes abbreviated as "ISEC") can measure the pore structure of a porous material using a sample with a known molecular size. A specific method is exemplified in Non-Patent Document 1.

Examples of the eluent used in ISEC for the purpose of measuring the pore size distribution of the ion exchanger include water and an aqueous solution containing a water-soluble solvent. Also, the eluent used in ISEC may contain inorganic salts, organic compounds, etc. as buffer components. In order to suppress contraction of the ion exchanger, the concentration of the water-soluble solvent and the concentration of the buffer component are preferably low.

The temperature at which ISEC is performed is preferably a constant temperature in a column oven or the like in order to stabilize the elution volume, and is preferably 0° C. to 100° C. from the viewpoint of practical use of the ion exchanger.

(Preparation with known molecular size)
In the measuring method of the present invention, it is important to select a compound to be used as a standard with a known molecular size.
It is preferable to use a non-ion dissociating compound because the sample with a known molecular size needs to have substantially no interaction with the ion exchange groups of the ion exchanger. In addition, since a sample with a known molecular size needs to have substantially no interaction with the skeleton that constitutes the ion exchanger, It is preferred to use a compound that does not have a In this way, by selecting a sample that does not substantially interact with the ion exchange group of the ion exchanger or the skeleton that constitutes the ion exchanger, when ISEC is performed, Delayed elution is suppressed, and more accurate pore distribution can be measured.
In view of the above factors, the sample with a known molecular size for measuring the pore size distribution of the ion exchanger is preferably a nonionically dissociative compound, which has substantially no hydrophobic interaction. Sugars are more preferred, and nonionically dissociable polysaccharides and nonionically dissociable monosaccharides are even more preferred.

The non-ion dissociating compound refers to a substantially non-ion dissociating compound, specifically a compound having a lower ion dissociating property than water and heavy water. A compound having a dissociation constant with a negative common logarithm (pKa) of 14 or more, and a basic compound with a negative common logarithm (pKb) of a base dissociation constant of 14 or more. Examples of nonionically dissociating compounds include nonionically dissociating polysaccharides, nonionically dissociating monosaccharides, hydrolysates of poly(ethylene carbonate), polyhydric alcohols, and methanol.
Nonionically dissociating saccharides refer to substantially nonionically dissociating saccharides, and examples thereof include nonionically dissociating polysaccharides, nonionically dissociating monosaccharides, and the like.

The molecular weight of the non-ion dissociating compound is preferably 32 to 50,000,000, more preferably 32 to 30,000,000, even more preferably 32 to 10,000,000.

Examples of nonionically dissociable polysaccharides include dextran, agarose, amylose, pullulan, isomaltooligosaccharide, maltooligosaccharide, fructooligosaccharide, galactooligosaccharide, raffinose, isomaltose, maltose, sucrose, and lactose. These nonionic dissociative polysaccharides may be used alone or in combination of two or more. Among these nonionically dissociable polysaccharides, dextran, pullulan, malto-oligosaccharides, maltose, isomaltose, and sucrose are preferable because molecular size information is known in literature, etc. The combined use with malto-oligosaccharide, the combined use of dextran, malto-oligosaccharide and maltose, and the combined use of pullulan, malto-oligosaccharide and maltose are more preferred.

Examples of nonionically dissociable monosaccharides include glucose, galactose, mannose, fructose, erythritol, xylitol, xylose, D-mannoheptulose, sorbitol and the like. These nonionically dissociable monosaccharides may be used singly or in combination of two or more. Among these nonionically dissociable monosaccharides, glucose is preferred from the viewpoint of structural similarity to dextran, pullulan and maltooligosaccharides.

In addition, in order to measure an ion exchanger with a wide range of pore distributions, which is used, for example, for simultaneously desalting and decolorizing a natural product extract, a standard with a known molecular size is required. It is preferable to use a compound whose molecular size is smaller than that of the ionically dissociable monosaccharide.
In view of the above factors, a polyhydric alcohol and methanol are preferable as a sample having a known molecular size for measuring the pore size distribution of the ion exchanger.

Examples of polyhydric alcohols include glycerol, ethylene glycol, pentaerythritol and the like. These polyhydric alcohols may be used alone or in combination of two or more. Among these polyhydric alcohols, glycerol and ethylene glycol are preferred because molecular size information is known in literature and the like.

Since the polyhydric alcohol has substantially no hydrophobic interaction with the organic ion exchanger, it is preferable not to include polyethylene glycol having a molecular weight higher than that of diethylene glycol, its monoalkyl ethers, and its dialkyl ethers. .

It is preferable not to use a monohydric alcohol having a molecular weight equal to or higher than that of ethanol as a standard having a known molecular size because it substantially has a hydrophobic interaction with an organic ion exchanger.

Furthermore, it is preferable to use heavy water as a sample with a known molecular size, for example, in order to determine the desalting performance of inorganic ions.

For the above reasons, non-ion dissociating compounds and heavy water are preferable as samples with known molecular sizes. At least two samples selected from the group consisting of nonionic dissociative saccharides, polyhydric alcohols, methanol and heavy water are preferable as the preparations with known molecular sizes.

The combination of samples with known molecular sizes can be used to measure ion exchangers with a wide range of pore distributions. Combined use of dissociative monosaccharide, polyhydric alcohol and heavy water, combined use of nonionic dissociative polysaccharide, polyhydric alcohol and heavy water, combined use of nonionic dissociable polysaccharide, nonionic dissociable monosaccharide and heavy water, non-ionic Combined use of ionic dissociative polysaccharides and polyhydric alcohols, combined use of nonionic dissociative polysaccharides and methanol, combined use of nonionic dissociative polysaccharides, polyhydric alcohols and methanol, nonionic dissociable polysaccharides and nonionics Combined use of dissociative monosaccharides and polyhydric alcohols, combined use of nonionically dissociative polysaccharides, nonionically dissociated monosaccharides and methanol, combined use of nonionically dissociated polysaccharides, nonionically dissociated monosaccharides, polyhydric alcohols and methanol is preferably used in combination with the organic ion exchanger, since it has substantially no hydrophobic interaction with the organic ion exchanger. A combination of nonionically dissociating polysaccharide, polyhydric alcohol and heavy water, a combination of nonionically dissociating polysaccharide, nonionically dissociating monosaccharide and heavy water is more preferable. combined use of saccharides, glycerol, ethylene glycol and heavy water, combined use of nonionically dissociative polysaccharides, glycerol, ethylene glycol and heavy water, combined use of nonionically dissociative polysaccharides, nonionically dissociable monosaccharides, glycerol and heavy water, Combined use of non-ionically dissociative polysaccharides, glycerol and heavy water, combined use of non-ionically dissociated polysaccharides, non-ionically dissociated monosaccharides, ethylene glycol and heavy water, combined use of non-ionically dissociated polysaccharides, ethylene glycol and heavy water More preferably, a nonionically dissociable polysaccharide, a nonionically dissociable monosaccharide, and heavy water are used in combination.

The ion-exchange groups constituting the ion exchanger are preferably in the dissociated state because the ion-exchange groups themselves become hydrophobic when they are in the non-dissociated state.

(molecular size of sample)
The molecular size of the sample can be measured by, for example, a viscosity method, a vapor pressure method, an osmotic pressure method, a light scattering method, or the like.
In addition, the molecular size of the standard is also described in known literature. For example, the molecular size of dextran is described in Journal of Chromatography A, 743 (1996), p33, and the molecular size of maltooligosaccharide is described in Biomacromolecules, 6 (2005), p143, (poly)ethylene glycol, polyethylene oxide, ethanol and methanol. The molecular size of glycerol is described in Journal of Chromatography, 206 (1981), p. 449, the molecular size of glycerol is described in Journal of Chemical Physics, 90 (1989), p. -Component technology and application system-, Haruhiko Oya, 1985, published by Saiwai Shobo, page 3 describes the molecular size and its calculation method.
Furthermore, regarding the molecular size of the sample, when it is used as a standard for size exclusion chromatography, information on the molecular weight is described for each production lot.
The molecular size of heavy water is treated as the same as that of water.

The molecular size (radius) of the sample is preferably 0.1 nm to 1000 nm, more preferably 0.1 nm to 600 nm, even more preferably 0.1 nm to 200 nm.

(Method for measuring pore distribution)
The method of measuring the pore size distribution is to fill a column with the ion exchanger to be measured, inject a sample with a known molecular size, perform size exclusion chromatography, and calculate the elution volume obtained from the elution time and flow rate. They are obtained by arranging them in order of molecular size and plotting the difference in the elution volume per unit volume of the ion exchanger and the volume average value of the molecular sizes constituting the difference.

(Application of the measuring method of the present invention)
By using the measuring method of the present invention, it becomes possible to determine the degree of deterioration of the ion exchanger and to select an ion exchanger suitable for its application.

The degree of deterioration of the ion exchanger is determined by measuring the pore distribution of a new ion exchanger and the pore distribution of the used ion exchanger by the measurement method of the present invention and comparing the obtained pore distributions. This makes it possible to check the degree of deterioration from a new product. Specifically, the rate of decrease in the cumulative pore volume of the ion exchanger as a whole, the decrease in the pore volume in the pore diameter region corresponding to the molecular size of the compound or compound group to be separated or removed from the ion exchanger By obtaining the ratio, it is possible to determine whether to replace the ion exchanger with a new one or to continue using the ion exchanger in use.

The ion exchanger suitable for the application can be selected by measuring the pore distribution of the ion exchanger by the measurement method of the present invention and comparing the obtained pore distribution with the purpose of the application. . Specifically, when using an ion exchanger for a certain application, the molecular size of the compound or group of compounds to be separated or removed is compared with the pore size distribution of each brand of ion exchanger, and the corresponding molecular size range is determined. , preferably an ion exchanger with a pore volume larger than the molecular size of interest, or an ion exchanger with a pore size distribution greater than the molecular size of interest. By doing so, it is possible to narrow down the screening work for the optimum brand.

The present invention will be described in more detail below using examples, but the present invention is not limited to the description of the following examples unless it departs from the gist thereof.

(molecular size)
For the molecular size of each sample shown in Table 1, numerical values or calculation formulas described in the following documents were used.
Molecular size of dextran: Journal of Chromatography A, 743 (1996), p33
Molecular size of malto-oligosaccharides: Biomacromolecules, 6 (2005), p143
Molecular size of (poly)ethylene glycol, polyethylene oxide, ethanol and methanol: Journal of Chromatography, 206 (1981), p449
Molecular size of glycerol: Journal of Chemical Physics, 90 (1989), p1200
Molecular size of water: Pure water/ultra-pure water production method - element technology and application system -, supervised by Haruhiko Oya, 1985, published by Saiwai Shobo, p3

[Example 1]
In a polycarbonate column with an inner diameter of 18 mm and a length of 200 mm, "Diaion SAF11AL" (trade name, manufactured by Mitsubishi Chemical Corporation, gel-type anion exchange resin, skeleton: poly(styrene-ethylstyrene-divinylbenzene), as an ion exchanger, ion-exchange group: trimethylammonium group) was packed in the chloric form.
The configuration apparatus and evaluation conditions for size exclusion chromatography were as follows, and an autosampler was used to inject a sample having a known molecular size (the compound shown in FIG. 1), and the elution time was measured.
HPLC pump: "LC-20AT" (model name, manufactured by Shimadzu Corporation)
Auto sampler: "SIL-20A" (model name, manufactured by Shimadzu Corporation)
Column oven: "CTO-10ASvp" (model name, manufactured by Shimadzu Corporation)
Differential refractive index detector: "RID-10A" (model name, manufactured by Shimadzu Corporation)
Eluent: deionized water Flow rate: 1.0 mL/min Temperature: 30°C

Molecular weight of each sample, common logarithm of molecular weight, elution time, elution volume, difference in elution volume (0 for negative values), molecular size (radius), pore radius (elution volume per unit volume of ion exchanger) Table 1 shows the volume average value of the molecular sizes that constitute the difference) and the pore volume (the difference in the elution volume per unit volume of the ion exchanger).

The relationship between the common logarithm of the molecular weight of each standard and the elution volume is shown in FIG. Figure 2 shows the relationship between the volume average molecular size (pore radius) that constitutes the difference in elution volume per unit volume of ion exchanger and the difference in elution volume per unit volume of ion exchanger (pore volume). show.
As can be seen from FIG. 1, the pore size distribution of the ion exchanger was able to be measured as shown in FIG. 2, because the sample with the known molecular size specified in the present invention was selected.

[Comparative Example 1]
The same operation as in Example 1 was performed except that ethanol and isopropanol were added as standards.
The relationship between the common logarithm of the molecular weight of each standard and the elution volume is shown in FIG.
As can be seen from FIG. 3, since ethanol and isopropanol were used, the elution volume increased due to hydrophobic interaction with the ion exchanger, and the pore size distribution could not be calculated.

[Example 2]
The ion exchanger was changed to "Diaion HPA25L" (trade name, manufactured by Mitsubishi Chemical Corporation, high porous type anion exchange resin, skeleton: poly (styrene-ethylstyrene-divinylbenzene), ion exchange group: trimethylammonium group). , The same operation as in Example 1 was performed except that glycerol, ethylene glycol and methanol were omitted as samples.
The relationship between the common logarithm of the molecular weight of each standard and the elution volume is shown in FIG. FIG. 5 shows the relationship between the pore radius and pore volume of the ion exchanger.
As can be seen from FIG. 4, the pore size distribution of the ion exchanger was able to be measured as shown in FIG. 5, because the sample with the known molecular size specified in the present invention was selected.

[Comparative Example 2]
The procedure was carried out in the same manner as in Example 2, except that the standard was changed to the compound shown in FIG. Polyethylene oxide (molecular weight 2,000,000 to 21,000) and polyethylene glycol (molecular weight 10,000 to 400) were used as polyethylene glycol having a molecular weight higher than that of diethylene glycol.
The relationship between the common logarithm of the molecular weight of each standard and the elution volume is shown in FIG.
As can be seen from FIG. 6, since polyethylene glycol having a molecular weight higher than that of ethanol, isopropanol, and diethylene glycol was used, the elution volume increased or fluctuated due to hydrophobic interaction with the ion exchanger, and the pore distribution could not be calculated. .

[Example 3]
The ion exchanger was changed to "Diaion HPA512L" (trade name, manufactured by Mitsubishi Chemical Corporation, high porous type anion exchange resin, skeleton: poly (styrene-ethylstyrene-divinylbenzene), ion exchange group: trimethylammonium group). , The same operation as in Example 1 was performed except that glycerol, ethylene glycol and methanol were omitted as samples.
The relationship between the common logarithm of the molecular weight of each standard and the elution volume is shown in FIG. FIG. 8 shows the relationship between the pore radius and pore volume of the ion exchanger.
As can be seen from FIG. 7, the pore size distribution of the ion exchanger was able to be measured as shown in FIG. 8 because the sample with the known molecular size defined in the present invention was selected.

[Example 4]
Change the ion exchanger to "Diaion PA308" (trade name, manufactured by Mitsubishi Chemical Corporation, porous anion exchange resin, skeleton: poly (styrene-ethylstyrene-divinylbenzene), ion exchange group: trimethylammonium group), The same operation as in Example 1 was performed except that glycerol, ethylene glycol and methanol were omitted as samples.
The relationship between the common logarithm of the molecular weight of each standard and the elution volume is shown in FIG. FIG. 10 shows the relationship between the pore radius and pore volume of the ion exchanger.
As can be seen from FIG. 9, the pore size distribution of the ion exchanger was able to be measured as shown in FIG. 10 because the sample with the known molecular size specified in the present invention was selected.

[Example 5]
Change the ion exchanger to "Diaion WA30" (trade name, manufactured by Mitsubishi Chemical Corporation, porous anion exchange resin, skeleton: poly (styrene-ethylstyrene-divinylbenzene), ion exchange group: dimethylamino group), The procedure was carried out in the same manner as in Example 1 except that glycerol, ethylene glycol and methanol were omitted as standards and the hydrochloride form was filled.
The relationship between the common logarithm of the molecular weight of each standard and the elution volume is shown in FIG. FIG. 12 shows the relationship between the pore radius and pore volume of the ion exchanger.
As can be seen from FIG. 11, the pore size distribution of the ion exchanger was able to be measured as shown in FIG. 12, because the sample with the known molecular size specified in the present invention was selected.

[Example 6]
Change the ion exchanger to "Diaion UBK510L" (trade name, manufactured by Mitsubishi Chemical Corporation, gel type cation exchange resin, skeleton: poly (styrene-ethylstyrene-divinylbenzene), ion exchange group: sulfonic acid group), The same operation as in Example 1 was performed except that glycerol, ethylene glycol and methanol were excluded as standards and filled as sodium form.
The relationship between the common logarithm of the molecular weight of each standard and the elution volume is shown in FIG. FIG. 14 shows the relationship between the pore radius and pore volume of the ion exchanger.
As can be seen from FIG. 13, the pore size distribution of the ion exchanger was able to be measured as shown in FIG. 14 because the sample with the known molecular size defined in the present invention was selected.

[Example 7]
The ion exchanger was changed to "Diaion RCP160M" (trade name, manufactured by Mitsubishi Chemical Corporation, high porous type cation exchange resin, skeleton: poly (styrene-ethylstyrene-divinylbenzene), ion exchange group: sulfonic acid group). , The same operation as in Example 1 was carried out, except that glycerol, ethylene glycol and methanol were used as standards and the sodium form was filled.
The relationship between the common logarithm of the molecular weight of each standard and the elution volume is shown in FIG. FIG. 16 shows the relationship between the pore radius and pore volume of the ion exchanger.
As can be seen from FIG. 15, the pore size distribution of the ion exchanger was able to be measured as shown in FIG. 16, because the sample with the known molecular size defined in the present invention was selected.

[Example 8]
Change the ion exchanger to "Diaion WK10S" (trade name, manufactured by Mitsubishi Chemical Corporation, porous cation exchange resin, skeleton: poly (methacrylic acid-ethylstyrene-divinylbenzene), ion exchange group: carboxyl group), The same operation as in Example 1 was performed except that glycerol, ethylene glycol and methanol were excluded as standards and filled as sodium form.
The relationship between the common logarithm of the molecular weight of each standard and the elution volume is shown in FIG. FIG. 18 shows the relationship between the pore radius and pore volume of the ion exchanger.
As can be seen from FIG. 17, the pore size distribution of the ion exchanger was able to be measured as shown in FIG. 18, because the sample with the known molecular size defined in the present invention was selected.

[Example 9] Determination of degree of deterioration of ion exchanger In a polycarbonate column with an inner diameter of 18 mm and a length of 200 mm, "FPA90CL" (trade name, Dow Chemical Company, anion exchange resin, skeleton: polystyrene, Ion exchange groups: strong basic type) and two used products (referred to as used product 1 and used product 2, respectively) used for decolorizing the sucrose solution were packed as chlorine type.
After that, size exclusion chromatography was performed under the same conditions as in Example 1, except that glycerol, ethylene glycol and methanol were removed as samples, and the elution time was measured.
FIG. 19 shows the relationship between the pore radius and the pore volume of the new, used 1, and used 2 ion exchangers.
As can be seen from FIG. 19, used product 1 (total ion exchange capacity: 0.95 eq/L) exhibits almost the same pore distribution as new product (total ion exchange capacity: 1.13 eq/L). , Product 2 used (total ion exchange capacity: 0.78 eq/L) had a small pore volume in the pore radius region of 1 nm to 2 nm, and clogging of pores could be confirmed.

[Example 10] Selection of ion exchanger suitable for decolorization step This example relates to the selection of an anion exchange resin used in the anion exchange desalting and decolorization steps in the production of a stevia sweetener derived from stevia leaves.
The method for obtaining a steviol glycoside-containing liquid from stevia leaves followed "Diaion Manual 2" (p.329, published by Mitsubishi Chemical Corporation). Specifically, stevia leaves were extracted at 60° C., calcium chloride and magnesium oxide were added, and filtration was performed to obtain an extract. This extract was passed through a synthetic adsorbent "Sepabeads SP700" (trade name, manufactured by Mitsubishi Chemical Corporation) to adsorb steviol glycosides, followed by washing and extrusion. The fractions eluted with 85% methanol in water and the additional water wash extruded fractions were then combined to give approximately 40% methanol in water of steviol glycosides. This aqueous solution is passed through a cation exchange resin "Diaion SK1BH" (trade name, manufactured by Mitsubishi Chemical Corporation) to desalinate the cation component, and about 40% of the steviol glycoside is subjected to anion exchange desalting and decolorization. An aqueous methanol solution was obtained.

The physical properties of about 40% methanol aqueous solution of steviol glycoside obtained are as follows.
Stevioside concentration: 17.5mg/mL
Rebaudioside A concentration: 1.3 mg/mL
Electrical conductivity: 61 μS/cm
pH: 3.5
420 nm absorbance: 4.55 AU (cell optical path length: 1 cm)

Size exclusion chromatography was performed on the dye component in about 40% methanol aqueous solution of the obtained steviol glycoside. The configuration apparatus and evaluation conditions for size exclusion chromatography are as follows.
HPLC pump: "LC-10AS" (model name, manufactured by Shimadzu Corporation)
Degasser: "DGU-12A" (model name, manufactured by Shimadzu Corporation)
Auto sampler: "SIL-12AF" (model name, manufactured by Shimadzu Corporation)
Ultraviolet/visible absorbance detector: "SPD-6AV" (model name, manufactured by Shimadzu Corporation)
Differential refractive index detector: "SE-71" (model name, manufactured by Showa Denko K.K.)
Column: "Superdex 75" (trade name, manufactured by GE Healthcare)
Column size: inner diameter 10 mm, length 300 mm
Eluent: 15 mM sodium phosphate buffer (pH 7.0)
Flow rate: 0.8 mL/min Temperature: 25°C

A chromatogram measured at a wavelength of 420 nm is shown in FIG. Comparison between molecular size and elution time of each sample (various dextran standard products for GPC analysis with known molecular weights, various maltooligosaccharide standard products, maltose, glucose and heavy water) obtained from the chromatogram measured with a differential refractive index detector. The relationship is shown in FIG.
From a comparison of FIGS. 20 and 21, excluding the abnormal peak before about 10 minutes, which is the exclusion limit elution time of the column, the molecular size of the dye component in about 40% methanol of the steviol glycoside is It was found to exist up to about 5 nm.

"Diaion PA308" (trade name, manufactured by Mitsubishi Chemical Corporation), "Diaion HPA512L" (trade name, manufactured by Mitsubishi Chemical Corporation) and "Diaion HPA25L" (trade name, manufactured by Mitsubishi Chemical Corporation) as anion exchange resins ), an about 40% methanol aqueous solution of the obtained steviol glycoside was passed under the following conditions, fractions were collected, and various physical properties were measured.
Column diameter: 15mm
Resin volume: 40 mL
Flow rate: 280 mL/hour Temperature: 25°C

FIG. 22 shows changes in absorbance measured using an ultraviolet spectrometer at a wavelength of 420 nm and an optical path length of 1 cm. As the amount of liquid passing increases, the decolorization ability decreases and the absorbance increases. "Diaion HPA25L" had the largest flow rate until the absorbance increased.
Comparing this result with the pore distributions in FIG. 5 (“Diaion HPA25L”), FIG. 8 (“Diaion HPA512L”), and FIG. "Diaion PA308", which has almost no , has the smallest amount of liquid permeation until the absorbance increases, and although there is a pore distribution in the pore radius region of 1 to 10 nm, most of it is present in the range of 1 to 5 nm. The absorbance of the ion HPA512L' then increases. On the other hand, "Diaion HPA25L" has a uniform pore distribution in the pore radius range of 1 to 20 nm, so pigment components with various molecular weights can diffuse and penetrate into the resin pores, increasing the absorbance. The amount of liquid flowed up to became the largest.
In addition, as described above, from a comparison between FIG. 20 and FIG. 21, the molecular size of the dye component in about 40% methanol of the steviol glycoside exists up to a radius of about 5 nm. It was found that for decolorization, it is necessary to select "Diaion HPA25L", which is an anion exchange resin having a sufficient pore volume even with a pore radius of 5 nm.

The measuring method of the present invention is suitable for measuring the wet pore size distribution of general ion exchangers including industrial ion exchange resins. Further, by using the measuring method of the present invention, it becomes possible to determine the degree of deterioration of the ion exchanger and to select an ion exchanger suitable for its application.

Claims (6)

Ion Exchanger by Reverse Size Exclusion Chromatography Using Standards of Known Molecular Size
moistA method for measuring pore size distribution,
The ion exchanger is a polystyrene-based ion exchange resin,
A sample with a known molecular size isNonionically dissociable polysaccharides, nonionically dissociable monosaccharides and glycerol
and ethylene glycol and methanolheavy waterconsisting only of,Measuring method. Ion Exchanger by Reverse Size Exclusion Chromatography Using Standards of Known Molecular Size
A method for measuring wet state pore size distribution, comprising:
The ion exchanger is a polystyrene-based ion exchange resin,
Is the sample with known molecular size only nonionically dissociable polysaccharides, nonionically dissociable monosaccharides, and heavy water?
measurement method. Nonionically dissociable polysaccharides are dextran, pullulan, maltooligosaccharide, maltose,
It is at least one selected from the group consisting of somaltose and sucrose, claim 1 or
2. The measurement method described in 2. Nonionically dissociable monosaccharides are glucose, galactose, mannose, fructose,
litritol, xylitol, xylose, D-mannoheptulose and sorbitol
The measurement according to any one of claims 1 to 3, which is at least one selected from the group consisting of
Method. Claims 1 to 4, wherein the pore distribution of the new ion exchanger and the pore distribution of the used ion exchanger are
Measured by the measurement method according to any one of the above to determine the degree of deterioration of the ion exchanger,
judgment method. The pore size distribution of the ion exchanger is measured by the measuring method according to any one of claims 1 to 4.
and selecting an ion exchanger suitable for that application.

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