JPS6171354A - Separating method of saccharide - Google Patents

Abstract

PURPOSE:To make it possible to separate isomaltose, maltose and glucose by liquid chromatography suitably, by using two kinds of ions as counter ions of SO3<->, and using aqueous solution as an elute. CONSTITUTION:The copolymer particles of sulfonated porous styrene and divinylbenzene are swelled by water and mounted on a glass filter. An aqueous solution of silver nitrate is made to flow in the particles. The counter ions of SO3<-> are completely made to be Ag<+>. After washing with water, the product is swelled with water and filled in a column A. The copolymer particles are swelled by water and mounted on the glass filter, and an aqueous solution of sodium chloride is made to flow in the particles. The counter ions of the SO3<-> are completely made to be Na<+>. After washing with water, the product is swelled with water and filled in a column B. The columns A and B are connected in series one by one. By using high performance liquid chromatography and a differential refractometer, the sample liquid is injected. Then water is used as an elute, and measurement is performed.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for separating sugars, particularly inmaltose, maltose, and glucose contained in small amounts in sugar, molasses, etc., by liquid chromatography.

(Prior art) As a method for separating saccharides by liquid chromatography, 1) a packing material having chemically bonded amino-7 groups (for example, silica gel, etc. having chemically bonded aminopropylsilyl groups) f; Separation [Journal Chromatography (J, Chromatogr, ) Volume 144 1
69 pages (1977)], 2) Separation by molecular sieve using cross-linked dextran gel etc. [written by S.C.Churms].

Atopansies◆Advances in Carbohydrate Chemistry
rateChemistry) Volume 25, page 13, Academic Press
) Published in 1970, USA].

3) Separation as boric acid complex ions using anion exchange resin (Analytical Chemistry
em, ) vol. 52, p. 1079 (19SO)].

4) Separation by a compromise mode of molecular sieving and partitioning using a salt-type cation exchange resin [Journal Chromatography (J, Chromatogr, ) IQ Volume 3 2
29 pages (1975)] are known. Among these methods, methods 2) and 4) are easy to operate because they use water as an eluent. But isomaltose.

Maltose and glucose cannot be separated by the method 2) above, and even by the method 4) above, satisfactory separation performance cannot be obtained.

(Problems to be Solved by the Invention) Generally, when saccharides are separated using salt-type cation exchange resin particles, the separation performance depends on the type of counter ion (salt t - type of cations forming the salt) It is known that it is influenced by

In the separation of disaccharides, cations other than Ag + ions, such as Ll, Na, K, Rb, Cs
Alkali metal ions such as + Mg' h Ca'' I
Alkaline earth metal ions such as S r and Ba +
Fe”+Fe+Co.

Transition metal ions such as Ni and Cu have no effect as counter ions, and Ag+ ions are suitable counter ions for separating isomaltose and maltose, which are disaccharides. but,
The counter ion is -Ag+ ion.

The elution times of isomaltose and glucose are similar9
．． Separation of isomaltose and glucose becomes impossible.

In addition, the counter ion suitable for separating disaccharides and monosaccharides is L
Alkali metal ions such as i'', Na'', K'', Rb'', Cs+ + Mg + Cal Sr
l Ba and other alkaline earth metal ions,
By using one or more of these as a counter ion, it becomes possible to separate disaccharides and monosaccharides, that is, inmaltose and glucose;

Separation of maltose and isomaltose is not possible.

Therefore, the present invention solves the problems of the prior art described above,
The object of the present invention is to provide a method for suitably separating isomaltose, maltose, and glucose by liquid chromatography.

(Means for Solving the Problems) The present invention provides SO3-group-containing cation exchange resin particles filled with Ag + alkali metal ions and/or alkaline earth metal ions as counter ions to the 5Oa-groups. The present invention relates to a method for separating saccharides by liquid chromatography, which is characterized by using one or more columns filled with an agent and using water or an aqueous solution as an eluent.

The cation exchange resin particles in the present invention are SO3'''
It is preferable to have 2 milliequivalents (2 me (1/g) or more of the group per 1 g of resin, particularly preferably 3 to 5 meq/g. When the 5Os group is less than 2 me (1/g),
Hydrophilicity tends to be insufficient. In addition, cation exchange resins have substantially 5O3H groups or SOs in terms of separation performance.
It is preferable that the H group is neutralized by an inorganic cation other than those mentioned above and does not have nine groups. here.

"Substantially" means that by neutralization titration, etc.! l) 5O3H
This means that the amount of the group or its neutralized group is undetectable.

In addition, the particle diameter of the cation exchange resin particles in the present invention is 1 to 20 μm for liquid chromatography analysis.
and 20 for preparative separation by liquid chromatography.
A thickness of ~300 μm is preferred.

In addition, there is no particular limit to the degree of pores of the cation exchange resin particles in the present invention, but the exclusion limit is 400 to 1 × 10
It is preferable to set it within the range of 7.

The SO3-group-containing cation exchange resin particles are preferably sulfonated porous styrene-divinylbenzene copolymer particles. In such cation exchange resin particles, the SOs'' group is bonded to a styrene/divinylbenzene copolymer, but the copolymer is a copolymer of divinylbenzene and a styrene monomer. It is preferable to contain 2 to 60% by weight of divinylbenzene. The styrenic monomers include (c) styrene, ethyl heptanovinylbenzene, vinyltoluene, α-Me-styrene, etc., and (l) other monomers such as vinyl acetate, methacrylic acid, acrylic acid, etc. within a range of 5% by weight or less. It may also be contained.

1 ft-, , X, f range dipinylbenzene copolymer c.

It is a particulate material with pores.

The sulfonated porous styrene-divinylbenzene copolymer particles can be produced by a conventionally known method.

Examples, t, divinylbenzene and styrenic monomer C4f
Suspension polymerization is carried out in the presence of a water-insoluble organic solvent such as amyl alcohol or toluene, the resulting particles are isolated and swollen with a swelling agent such as dichloroethane or trichloroethane, concentrated sulfuric acid or chlorosulfuric acid is added, and the resulting particles are incubated at room temperature. K can also be obtained by carrying out a sulfonation reaction at ~120°C. The ion exchange capacity described above can be adjusted by appropriately adjusting the reaction conditions during the reaction with concentrated sulfuric acid or chlorosulfuric acid.1 The degree of pores of the cation exchange resin in the present invention is determined by the suspension polymerization described above. Sometimes, it can be adjusted by adjusting the conditions, mainly the type and amount of the water-insoluble organic solvent.9 The particle size can be adjusted by appropriately selecting the suspension polymerization conditions.

Incidentally, it is preferable to use divinylbenzene in an amount of 5 to 60% by weight based on it of the above reaction components.

Furthermore, as the styrene monomer, other monomers such as those mentioned above, vinyl acetate, methacrylic acid, acrylic acid, etc. may be used in an amount of 5% by weight or less based on the total amount of the reaction components. In addition, peroxides such as benzoyl peroxide are used during suspension polymerization.

Commonly used polymerization initiators can be used.

The sulfonated porous styrene-divinylbenzene copolymer particles preferably have an ion exchange capacity of 2 meq/g or more, particularly preferably 3 to 5 meq/H.

The filler in the present invention is SO3- group-containing cation exchange resin particles, which simultaneously contain Ag+ and an alkali metal ion and/or alkaline earth metal ion as a counter ion to the 5Os- group. , those having only Ag+ as a counter ion, and those having only alkali metal ions and/or alkaline earth metal ions as counter ions can be used. However, for those having only Ag+ as a counter ion and those having only alkali metal ions and/or alkaline earth metal ions as counter ions, these may be mixed and packed into a column, or each may be packed into a column separately. These are used in conjunction.

That is, it is sufficient that the filler as a whole contains Ag+, which is a counter ion of the 5Os- group, and an alkali metal ion and/or an alkaline earth metal ion.

Ag+ is a counter ion of the SO3- group present in the filler.
and the alkali metal ion and/or the alkaline earth metal ion, the former/latter is preferably present in an equivalent ratio of 3/7 to 7/3. If this ratio is too large, the separation of maltose and isomaltose will be good, but the separation of inmalt-stuclucose will be poor, and if this ratio is too small, the separation of isomaltose and glucose will be good.

Separation of maltose and isomaltose deteriorates.

The above equivalent ratio is preferably around 515.

Preferably, the filler is prepared and used as follows to adjust the ratio of the counterions.

That is, SO3' having only Ag+ as a counter ion
`` group-containing cation exchange resin particles and SO3- group-containing cation exchange resin particles having only alkali metal ions and/or alkaline earth metal ions as counter ions were prepared separately, and K was attached to each.

Measure the equivalent amount of counter ions per unit weight or per unit volume, use this as a guide, decide the weight ratio or volume ratio to be used, and either mix them and fill the column, or separate columns. The method of filling and then connecting is simple and preferable in terms of use. For this purpose, SO3 with almost the same properties such as particle size, ion exchange capacity, porosity, etc.
It is particularly preferable to prepare two cation exchange resin particles containing a ``group'' and to have the same equivalent amount of the niger loop ion present in each as a counter ion.

Cation exchange resin particles in which the counter ion of the SO3 group is dissolved in Ag are cation exchange resin particles containing SOs groups in which the counter ion of the SO3 group is Na'', H+, etc. In addition, 5Os- group-containing cation exchange resin particles in which the counter ion of the SO3 group is an alkali metal ion and/or alkaline earth metal ion are obtained by treatment with an aqueous solution of the SO3 group. “Na”
+ By treating SOs- group-containing cation exchange resin particles such as H+ with an aqueous solution of a water-soluble alkali metal salt, alkaline earth metal salt, alkali metal hydroxide, alkaline earth metal hydroxide, etc. can get.

Furthermore, the 5Os-group-containing cation exchange resin particles are such that the counter ions of the 5Os-groups are simultaneously dissolved in Ag and in alkali metal ions and/or alkaline earth metal ions.

A suitable amount of alkali metal ions and/or
or treated with an aqueous solution containing alkaline earth metal ions, or treated with an appropriate amount of Ag'' ( or the counter ion of the SO3- group is Na”
, H+, etc. SO3- group-containing cation exchange resin particles are mixed with an appropriate amount of Ag+ ions and an appropriate amount of alkali metal ions and/or
Alternatively, it can be obtained by treatment with an aqueous solution containing alkaline earth metal ions.

The alkali metal ions and/or alkaline earth metal ions used in the present invention include +Li"+Na".

So, alkali metal ions such as b+ and aS+, Mg".

Examples include alkaline earth metal ions such as 2+ Ca .Sr and B3. One or more of these can be used, but in consideration of the separability of inmaltose and glucose, versatility, etc., it is most preferable to use Na+ ions.

The eluent used in the present invention is maltose.

Considering the solubility of samples such as inmaltose and glucose, water is preferable. However, if necessary, organic solvents such as methanol, ethanol, gropanol, acetonitrile, tetrahydrofuran, etc.

An aqueous solution in which a salt, acid, or alkali is dissolved in water and is dilute to the extent that the counter ion of the SO3'' group of the SOs-group-containing cation exchange resin is not replaced may be used.

In the method of the present invention, a sample is injected through a sample injection port, the eluent is passed through a column filled with the column packing material, and the passed-through is detected by a detector.
It can be applied to high-performance liquid chromatography analysis in which a chromatogram is drawn, preparative separation in which a sample is passed through a column filled with Ui4J and each fraction is collected. In such a separation method, the column packing material used in the present invention does not function as an ion exchange resin.

Also, to fill the column with column packing material.

Conventionally known methods can be used, and it is particularly preferable to press-fit in the form of a slurry.

(Function) In the present invention, in the column packing material, the counter ion of the SO3- group dissolves in Ag), which helps in the separation of maltose and inmaltose. Ikegata: When the counter ion of the 5Os group is an alkali metal ion and/or an alkaline earth metal ion, it is useful for separating inmaltose and glucose. Separation of these sugars is made possible synergistically by using the above two types of counterions.

Therefore, if there is too much Ag as a counter ion, the separation of maltose and isomaltose will be improved.

Separation of isomaltose and glucose becomes worse.

Moreover, when there are too many alkali metal ions and/or alkaline earth metal ions as counter ions.

Although the separation of isomaltose and glucose is improved.

Separation of maltose and isomaltose deteriorates.

(Example) Next, the present invention will be described in detail based on Examples and Comparative Examples.

Example 1 Sulfonated porous styrene-divinylbenzene copolymer particles 259 having an average particle diameter of 10 μm, an ion exchange capacity of 4.3 meq/g, and a degree of crosslinking (weight ratio of divinylbenzene component in the copolymer) of 10% were prepared. It was swollen with water and placed on a glass filter. Add to this 5% silver nitrate aqueous solution II!
was applied to completely change the counter ion of the SOs- group to Ag+,
After washing with water, swell with water and make into a slurry, diameter 10.7
A stainless steel column with a height of 300 mm and a height of 300 mm was packed. This is the column prisoner.

Separately, average particle size 10 μm, ion exchange capacity 4.3 meq
Sulfonated porous styrene-divinylbenzene copolymer particles 259 having a crosslinking degree of 10%/g and a crosslinking degree (weight ratio of the divinylbenzene component in the copolymer) were swollen with water and placed on a glass filter. Add to this 5% sodium chloride aqueous solution SO0m! ! The counter ion of the 5Os- group was completely changed to Na+, and after washing with water, it was swollen with water to form a slurry, and the slurry was packed into a stainless steel column with a diameter of 10.7 mm and a height of 300 onn. This is referred to as column fB).

Column country and (Bl) are connected one by one in series, high performance liquid chromatograph (Hitachi 635A model) and 5hodex
Using a SE 11 type differential refractometer (Showa Denko M), the following sample solution was injected, water was used as the eluent, and the flow rate was 1. Measurements were performed at oml/min and column temperature of 60°C.

0.1 each of maltose, isomaltose and hyglucose
FIG. 1 shows a chromatogram obtained by injecting 100 μl of a sample solution containing %. As is clear from FIG. 1, good separation was achieved.

Example 2 Chromatogram obtained by injecting 100 μl of the sample solution in the same manner as in Example 1 except for using a sample solution containing 0.1% each of sucrose, maltose, isomaltose, glucose, and fructose. is shown in Figure 2.

It can be seen from FIG. 2 that good separation was achieved.

Example 3 Average particle size 10 μm, ion exchange capacity 4.3 meq/g
and 25 g of sulfonated porous styrene-divinylbenzene copolymer particles having a degree of crosslinking (weight ratio of divinylbenzene component in the copolymer) of 10% were swollen in water and placed on a glass filter.

Add to this 5% silver nitrate aqueous solution 11! The counter ion of the 5Os- group is completely changed to Ag, and the slurry is swollen with soaked water and made into a slurry. This is called sura IJ-(A').

Average particle size 10 μm, ion exchange capacity i 4.3 meq/
Sulfonated porous styrene-divinylbenzene copolymer particles with a crosslinking degree of 10% and a crosslinking degree of 10% were swollen in water and placed on a glass filter. Pour so ml of a 5% aqueous sodium chloride solution into this.

The counter ion of the SO3- group is completely swollen with NaKL and washed with soaking water to form a slurry. This is referred to as slurry (B').

Mix slurry (A') and slurry (B') well.

A 10.7 mmφ×300 mm stainless steel column was packed to obtain a column (C). Using this column (C1, 2
Saccharides were separated under the following conditions.

o Reagents Wako Pure Yakusei reagent was used for sucrose, maltose, glucotose, and fructose, and a reagent manufactured by Tokyo Kasei was used for isomaltose. The eluent has a specific resistance of 5. O
Ion-exchanged water with a resistance of 10'Ω or more was used.

0 Equipment: High performance liquid chromatograph is Hitachi model 635A, and differential refractometer is 5hodexSE 1 as a detector.
1 (manufactured by Showa Denko), and two columns (C1') were connected in series.

0 condition: Use water as the eluent, flow rate 1.0 ml/”
Measurements were performed at an In+ column temperature of 60°C.

Figure 3 shows maltose, inmaltose, and glucose in 1
．． This figure shows a chromatogram obtained by injecting a sample solution containing 0% at 20μ, and it can be seen that good separation can be obtained.

Figure 4 shows a chromatogram obtained by injecting a sample solution containing 1.0% each of sucrose, maltose, isomaltose, glucose, and fructose at 20μ, and good separation can be obtained. I understand that.

Comparative Example 1 The same operation as in Example 1 was performed except that only one column (A) was used and the column (Bl) was not used.

0.1 each of maltose, isomaltose and hyglucose
FIG. 5 shows a chromatogram obtained by injecting t-SOμl of a sample solution containing %.

As can be seen from FIG. 5, it was not possible to separate inmaltose and glucose.

Comparative Example 2 The operation was carried out in the same manner as in Example 1, except that only one column (B) was used and no column cap was used.

0.1 each of maltose, isomaltose and hyglucose
FIG. 6 shows a chromatogram obtained by injecting SOμl of a sample solution containing %.

As can be seen from Figure 6, maltose and isomaltose could not be separated.

(Effects of the Invention) According to the present invention, saccharides, particularly maltose, isomaltose, and glucose contained in small amounts in sugar, molasses, etc., can be separated very well. That is, according to the present invention, not only trisaccharides and monosaccharides can be separated, but also multiple strains of trisaccharides can be individually separated.

[Brief explanation of the drawing]

Figure 1 is a taromadogram showing the analysis results of Example 1, Figure 2 is a chromatogram showing the analysis results of Example 2, and Figure 3 is a chromatogram showing the analysis results of Example 2.
The figure shows the analysis results of Example 3. Chromatogram showing the results of a sample solution containing maltose, isomaltose and glucose, FIG. 4 is Example 3
Among the analysis results, sucrose. Chromatogram showing the results of a sample solution containing maltose, inmaltose, glucose, and hifructose; FIG. 5 is a chromatogram showing the analysis results of Comparative Example 1;
The figure is a chromatogram showing the analysis results of Comparative Example 2. Explanation of symbols 1. 5, 9, 13. 17... Maltose peak 2.
6,10.14...Inmaltose peak 3.7,
11,15.20...Glucose peak 4.12.
...Sucrose peak 8.16...Fructose peak 18...Joint peak of isomaltose and glucose 1
9...Joint peak of maltose and isomaltose￥
1c2] 1-M lutose trk-”-7 elution 2 halls (starvation z)-4・pass 5-70-s#) 1m'-75 each output volume (-
1) - l8... in 11 Leto people Hiku° Lukosuri m and -
7 elutions each 1 (ml) 6th (2) io 20 3 soil volume (-1) -

Claims (1)

[Claims] 1. SO_3^- group-containing ion exchange resin particles filled with a filler having Ag^+ and an alkali metal ion and/or alkaline earth metal ion as a counter ion to the SO_3^- group. 1. A method for separating saccharides by liquid chromatography, which comprises using one or more columns and using water or an aqueous solution as an eluent. 2.SO_ where the counter ion of SO_3^- group is Ag^+
Filling the same column with 3^- group-containing cation exchange resin particles and SO_3^- group-containing cation exchange resin particles in which the counter ion of the SO_3^- group is an alkali metal ion and/or an alkaline earth metal ion. 2. The method for separating saccharides according to claim 1, wherein the saccharide separation method is used by packing the saccharides into separate columns. 3.SO_3^-based cation exchange resin particles are SO_3^
The method for separating saccharides according to claim 1 or 2, which comprises porous styrene-divinylbenzene copolymer particles containing - groups. 4. SO_ where the counter ion of SO_3^- group is Ag^+
The volume ratio of 3^- group-containing cation exchange resin particles and SO_3^- group-containing cation exchange resin particles in which the counter ion of the SO_3^- group is an alkali metal ion and/or an alkaline earth metal ion is 7/3 to 7/3. The method for separating saccharides according to any one of claims 1 to 3, using a column packed at 3/7. 5. The method for separating saccharides according to any one of claims 1 to 4, wherein the alkali metal ion is Na^+.