JPH0339592B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to liquid chromatography. Conventionally, methods for analyzing sugars by liquid chromatography include (1) partition-type separation using a packing material with chemically bonded amino groups (e.g., diethylaminoethyl cellulose), (2) cross-linked dextran gel, etc. (3) Separation as a borate complex ion using an anion exchange resin, (4) A compromise mode of molecular sieve and distribution using a salt-type cation exchange resin as a column packing material. Separation is known. Among these methods, methods (2) and (4) are easy to operate because they use water as an eluent. However, with method (2) above, it is impossible to separate sugars with different molecular weights. In contrast, (4) above
This method is possible. However, as a result of further study by the present inventors on the method (4) above, it was found that when water is used as an eluent, the separation ability decreases with prolonged use. That is, in the method (4) above, when water is used as an eluent, there is a drawback that the life of the column packing material is short. The present invention solves these problems. That is, the present invention relates to a liquid chromatography method for analyzing sugars using porous styrene-divinylbenzene copolymer particles having SO ^- ₃ groups and divalent metal cations as counter ions as a column packing material. , relates to a liquid chromatography method for analyzing sugar, characterized in that an aqueous solution having an alkaline substance concentration of 5 x 10 ^-5 to ^{10 -2} M/ is used as an eluent. The column packing material of the present invention has a divalent metal cation such as Ca ⁺⁺ , Sr ⁺⁺ , Pb ⁺⁺ , Ba ⁺⁺ , Mg ⁺⁺ or the like as a counter ion to the SO ⁻ ₃ group. In particular, as counterions (a) at least one ion selected from the group consisting of Ca ⁺⁺ and Sr ⁺⁺ and (b) at least one ion selected from the group consisting of Pb ⁺⁺ and Ba ⁺⁺ ( It is preferable that the equivalent ratio of the ions of a) to the ions of (b) is (a)/(b) in the range of 1/2 to 2/1.
In this case, glucose, galactose, mannose and fructose, which have the same molecular weight, can be separated. Also, when Ca ⁺⁺ or Sr ⁺⁺ is used as a counterion,
Glucose, mannose and fructose can be separated, and when Pd ⁺⁺ or Ba ⁺⁺ is used as a counterion, glucose, galactose and mannose can be separated. The column packing material in the present invention preferably has SO ^- ₃ groups in an amount of 2 milliequivalents (meq/g) or more per gram of the column packing material, particularly preferably 3 to 5 meq/g. When it is less than 2meq/g, hydrophilicity tends to be insufficient. In addition, the particle size of the column packing material of the present invention is 1 to 20 μm for liquid chromatography analysis.
For fractional separation by liquid chromatography, those having a diameter of 20 to 300 μm are preferable. Further, although there is no particular restriction on the degree of pores in the column packing material of the present invention, it is preferable that the exclusion limit is in the range of 400 to 1×10 ⁷ . The SO ^- ₃ group described above is bonded to a styrene-divinylbenzene copolymer, but the copolymer is
It is a copolymer of divinylbenzene and a styrene monomer, and preferably contains 5 to 60% by weight of divinylbenzene. The styrenic monomers include styrene, ethyl monovinylbenzene, vinyltoluene, α-Me-styrene, vinyl acetate,
Other monomers such as methacrylic acid and acrylic acid may be used in amounts up to 5% by weight. The column packing material of the present invention comprises sulfonated porous styrene-divinylbenzene copolymer particles and the general formula () M(OH) ₂ () (where M is a metal that can be a divalent cation). It can be obtained by reacting a hydroxide represented by The above M is Ca, St, Pd,
There are Ba, Mg, etc. More specifically, it can be obtained by mixing an aqueous solution in which the above hydroxide is dissolved with sulfonated porous styrene-divinylbenzene copolymer particles or a salt thereof, filtering the mixture, and repeating the same operation. can. Alternatively, it can be obtained by filling a column with sulfonated porous styrene-divinylbenzene copolymer particles or a salt thereof or by passing an aqueous solution through the funnel in the same manner as above. In this case, sulfonated porous styrene
It is preferable to use the hydroxide in such a manner that the amount of metal cations in the hydroxide is equivalent or more to the SO ^- ₃ groups in the divinylbenzene copolymer particles. Sulfonated porous styrene-divinylbenzene copolymer particles are produced by a conventionally known method. For example, divinylbenzene and styrenic monomers, and optionally other monomers, are polymerized in suspension in the presence of a water-insoluble basic solvent such as amyl alcohol or toluene, and the resulting particles are isolated to produce dichloroethane, trichloroethane, etc. It can be obtained by swelling with a swelling agent such as, adding concentrated sulfuric acid or chlorosulfuric acid, and carrying out a sulfonation reaction at room temperature to 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, and the degree of pores of the column packing material in the present invention can be adjusted during the suspension polymerization. The conditions can be adjusted mainly by adjusting the type and amount of the wastewater-soluble organic solvent, and the particle size can be adjusted by appropriately selecting the suspension polymerization conditions. In addition, the sulfonated porous styrene-divinylbenzene copolymer has an ion exchange capacity.
2 meq/g or more is preferable, especially 3 to
5meq/g is preferable. Further, in the column packing material of the present invention, each of the porous styrene-divinylbenzene copolymer particles having SO ^- ₃ groups has the above-mentioned (a) as a counter ion for the SO ^- ₃ groups.
In terms of separation performance, it is preferable to have the ion of (a) and the ion of (b), but the following may also be used. i.e. SO ^- ₃ group and its counterion as Ca ⁺⁺ or
Porous styrene-divinylbenzene copolymer particles (A) containing Sr ⁺⁺ (ion (a)), SO ^- ₃ group and Pb ⁺⁺ or as its counter ion
Porous styrene-divinylbenzene copolymer particles (B) having Ba ⁺⁺ (ions of (b)) are formed by ions of (a) and ions of (b) (a)/(b) ( The column packing material of the present invention can be used as a column packing agent in the present invention if it is blended at an equivalent ratio of 1/2 to 2/1. It is preferable that the copolymer particles (A) and (B) are mixed as uniformly as possible, and each copolymer particle has the same particle size, degree of pores, degree of swelling, and content of SO ^- ₃ groups. It is preferable to use For this purpose, copolymer particles (A) and (B) are neutralized using the same lot of sulfonated porous styrene-divinylbenzene copolymer particles in the same manner as described above. preferable. Such copolymer particles
When using (A) and (B), they are in volume ratio
They may be blended so that the ratio of (A)/(B) is 1/2 to 2/1, which facilitates the mixing operation. When the column packing material of the present invention has ions (a) and ions (b) as counterions, SO ^- ₃
It is most preferable that the ions (a) and (b) are present in each particle of the porous styrene-divinylbenzene copolymer particles having a group, in a uniform distribution. For this purpose, the column packing is composed of sulfonated porous styrene-divinylbenzene copolymer particles, (c) at least one compound selected from the group consisting of Ca(OH) ₂ and Sr(OH) ₂ , and ( d) At least one compound selected from the group consisting of Pb(OH) ₂ and Sr(OH) ₂ is dissolved in a molar ratio of (c)/(d) from 1/2 to 2/1. Neutralized with aqueous solution. The eluent used in the present invention is an aqueous solution of an alkaline substance. Alkaline substances are substances that exhibit alkalinity when made into aqueous solutions such as hydroxides, hydrogen carbonates, and carbonates of alkali metals and alkaline earth metals, and specifically include LiOH, NaOH,
KOH, Sr(OH) ₂ , Ba(OH) ₂ , _NaHCO3 ,
Examples include KHCO ₃ , NA ₂ CO ₃ and K ₂ CO ₃ . These alkaline substances are used at a concentration of 5×10 ⁻⁵ to ^{10 −2} M/. If the concentration is less than 5 x 10 ^-5 , the life of the column packing material will be shortened, and if it exceeds 10 ^-2 M/, ion exchange with the counter ion of the column packing material cannot be ignored, and the separation performance will change. When a column packing material obtained by reacting sulfonated porous styrene-divinylbenzene copolymer particles or a salt thereof with a hydroxide represented by the above general formula () is used as a column packing material of the present invention, This problem is particularly noticeable. The column packing material of the present invention comprises sulfonated porous styrene-divinylbenzene copolymer particles.
It can be obtained by reacting metal halides such as MCl ₂ , MBr ₂ (where M is the same as in the general formula ()), but in this case, when the above hydroxide is used, However, it is preferable to use the eluent of the present invention, although the problem of longevity is less pronounced. In the liquid chromatography method according to the present invention, a sample is injected from a sample injection port, the eluent is passed through a column filled with the column packing material, and the passed material is detected by a detector. It is applied to high-performance liquid chromatography analysis that draws a chromatogram, and preparative separation that collects each fraction by passing a sample through a column filled with column packing material. Next, examples of the present invention will be shown. Example 1 Sulfonated porous styrene-ethyl monovinylbenzene-divinyl having a particle size of 8 to 15 μm, an ion exchange capacity of 4.3 meq/g, and a degree of crosslinking (polymerization ratio of the divinylbenzene component in the copolymer, the same shall apply hereinafter) of 10%. Swell 24.0 g of benzene copolymer particles in about 150 g of water to form a slurry, and add Sr to this slurry.
An aqueous solution of 13.7 g _{of (OH) 2.8H 2 O} dissolved in water to make 500 ml was mixed and stirred for 1 hour. Note that the sulfone group and Sr ⁺⁺ were blended at an equivalent ratio of 1/1. After that, it was filtered and washed with water to obtain a column packing material with an exclusion limit of 1000. The obtained column packing material was swollen with water to form a slurry and packed into a 10.7 mmφ x 30 cm stainless steel column, which was then set in a high-performance liquid chromatography analyzer to collect raffinose, maltose, glucose, galactose, and mannose. and a sample containing fructose was subjected to analysis. The analysis conditions are flow rate
A chromatogram was obtained using a differential refractometer as a detector at a rate of 1.0 ml/min, an eluent of 10 ^-4 M/NaOH aqueous solution, and a column temperature of 60°C. Note that the eluent was passed continuously. FIG. 1 shows the chromatogram obtained by sampling immediately after the start of the eluent flow, FIG. 2 shows the chromatogram obtained by sampling 3 hours later, and Table 1 shows the change in the number of theoretical plates over time. In Figures 1 and 2, peak 1 is roughinose,
Peak 2 is maltose, peak 3 is glucose,
Piece 4 is galactose, peak 5 is mannose and peak 6 is fructose. No change was observed in peak shape or separation, and in Table 1,
It can be seen that there is no decrease in the number of theoretical plates. Example 2 The same procedure was used as in Example 1 except that 16.2 g of Ba(OH) _{2.8H 2 O} was used instead of 13.7 g _of Sr(OH) 2.8H ₂ O in Example 1, but the exclusion limit of 1000 was achieved. A column packing material was obtained. Note that here, the sulfone group and
Ba ⁺⁺ was blended at an equivalent ratio of 1/1.
Thereafter, in the same manner as in Example 1, a column was filled and analysis by high performance liquid chromatography was performed. FIG. 3 shows the chromatogram obtained by sampling immediately after the start of the eluent flow, FIG. 4 shows the chromatogram obtained by sampling 3 hours later, and Table 1 shows the change in the number of theoretical plates over time. In FIGS. 3 and 4, each peak name is the same as in Example 1. No change in peak shape or separation was observed, and Table 1 shows that no decrease in the number of theoretical plates was observed. Example 3 In Example 1, instead of 13.7 g of Sr(OH) _{2.8H 2} O, 6.9 g of Sr(OH) 2.8H _{2 O} and Ba
A column packing material was produced in the same manner as in Example 1 except that 8.1 g of (OH) ₂ was used. Here, the equivalent ratio of the total amount of Sr ⁺⁺ and Ba ⁺⁺ to the sulfone group was 1/1, and the equivalent ratio of Sr ⁺⁺ and Ba ⁺⁺ was also 1/1. Thereafter, in the same manner as in Example 1, a column was filled and analysis by high performance liquid chromatography was performed. Samples for analysis were samples containing mannose, arabinose, and fructose. Figure 5 shows the chromatogram obtained by sampling the sample immediately after the start of eluent flow, Figure 6 shows the chromatogram obtained by sampling 3 hours later, and Figure 7 shows the chromatogram obtained by sampling the sample 3 hours later. Chromatograms are shown in the figure and FIG.
Table 1 also shows the change in the number of theoretical plates over time. In FIGS. 5 and 6, each peak is the same as in Example 1. In FIGS. 7 and 8, peak 7 is mannose, peak 8 is arabinose, and peak 9 is fructose. No change in peak shape or separation was observed with elapsed time, and Table 1 shows that no decrease in the number of theoretical plates was observed. Comparative Example 1 A column packing material was produced in the same manner as in Example 1. Thereafter, a column was filled in the same manner as in Example 1, and analysis by high performance liquid chromatography was performed in the same manner as in Example 1. The analysis conditions are
Flow rate 1.0 ml/min, eluent water and column temperature 60
℃, and a chromatogram was obtained using a differential refractometer as a detector. A chromatogram obtained by sampling 3 hours after the start of passing the eluent is shown in FIG. 9, and a change in the number of theoretical plates over time is shown in Table 1. In FIG. 9, each peak is the same as in Example 1. It can be seen in Table 1 that the separation between galactose in peak 4 and mannose in peak 5 has deteriorated, and that the number of theoretical plates has decreased by about 25% after 3 hours. Comparative Example 2 A column packing material was produced in the same manner as in Example 2. Thereafter, a column was filled in the same manner as in Example 1, and liquid chromatography analysis was performed under the same analysis conditions as in Comparative Example 1. FIG. 10 shows a chromatogram obtained by sampling 3 hours after the start of eluent flow, and Table 1 shows the change in the number of theoretical plates over time. In FIG. 10, each peak is the same as in Example 1. It can be seen in Table 1 that the peak shape became broader and the separation of mannose in peak 5 and fructose in peak 6 became significantly worse, and that the number of theoretical plates decreased by about 45% after 3 hours. Comparative Example 3 A column packing material was produced in the same manner as in Example 3. Thereafter, a column was filled in the same manner as in Example 1, and liquid chromatography analysis was performed under the same analysis conditions as in Comparative Example 1. Samples for analysis were used for analysis.
The chromatogram obtained by sampling the sample 3 hours after the start of the eluent flow is shown in the 11th column.
Similarly, the sample is shown in FIG. 12. Table 1 also shows the change in the number of theoretical plates over time. In FIG. 11, each peak is the same as in Example 1, and in FIG. 12, each peak is the same as in Example 3. It can be seen that as time elapses, the peak shape becomes broader and the separation becomes worse, especially for the sample, the separation becomes significantly worse. Table 1
It can be seen that the number of theoretical plates decreased by about 50% after 3 hours.

[Table] * Counter ion raw material Note) The theoretical plate number was calculated by sampling fructose after a certain period of time after starting the flow and using the peak of fructose in the obtained chromatogram using the following formula. N=5.54×(t _R /W) ² (where, t _R is the length from the sample injection point to the peak apex (mm), and W is the half-width of the peak (mm)). Schematic diagram first
Shown in Figure 3. The eluent is stored in a container 10 and is drained by a pump 11 through a pipe 12, through a column 13 and a detector 14 into a container 15. A sample is introduced through an injection port 16 (for example, a three-way inlet), and a chromatogram recorder 17 is connected to the detector 14 . As is clear from the above, when porous styrene-divinylbenzene copolymer particles having SO ^- ₃ groups and a divalent metal cation as a counter ion are used as a column packing material, the liquid chromatograph according to the present invention According to the graph method, the lifetime of the column packing material can be extended.

[Brief explanation of the drawing]

Figures 1 and 2 are chromatograms obtained by sampling immediately after and 3 hours after the start of eluent passage in Example 1, respectively, and Figures 3 and 4 are chromatograms obtained by sampling in Example 2. Chromatograms, FIGS. 5 and 6, which show the same results as in Example 1, are chromatograms, FIGS. 7 and 6, respectively, which show the same results as in Example 1 when sample ( Figure 8 is a chromatogram showing the same results as in Example 1 when sample () was used in Example 3, Figure 9 is a chromatogram showing the results of Comparative Example 1, and Figure 10 is a comparison. Chromatogram showing the results of Example 2, No. 11
The figures and FIG. 12 are for Comparative Example 3 and Example 1.
Chromatogram showing similar results as well as the 13th
The figure shows a schematic diagram of an analyzer used in Examples and Comparative Examples. Explanation of symbols, 1...Roughinose peak, 2
... Maltose peak, 3 ... Glucose peak, 4 ... Galactose peak, 5 ... Mannose peak, 6 ... Fructose peak,
7... Mannose peak, 8... Arabinose peak, 9... Fructose peak.

Claims (1)

[Claims] 1. A liquid chromatography method for analyzing sugars using porous styrene-divinylbenzene copolymer particles having SO ^- ₃ groups and a divalent metal cation as a counter ion as a column packing material. In,
As an eluent, the alkaline substance concentration is 5 × 10 ^-5 ~
A liquid chromatography method for analyzing sugar, characterized by using a 10 ^-2 M/aqueous solution. 2 The column packing material is composed of sulfonated porous styrene-divinylbenzene copolymer particles or a salt thereof and the general formula () M(OH) ₂ () (However, in the formula, M can be a divalent cation. A liquid chromatography method for analyzing sugar according to claim 1, which is obtained by reacting a hydroxide represented by a metal.