JPS59178359A - Liquid chromatograph - Google Patents

Abstract

PURPOSE:To extend the life of a column filler by employing an aqueous solution with a specified concentration of alkaline substances as eluent. CONSTITUTION:A column filler can be obtained by reaction of a hydroxide as given by the general formula M(OH)2 (where, M is metal which can be a bivalent cation) with a sulfonated porous stylene-divinyl benzene based copolymer particle. M herein used include Ca, St, pd, Ba and Mg. An eluent herein used is an aqueous solution of alkaline substances. These alkaline substances are used at the concentration of 5X10<-5>-10<-2>M/l. When the concentration is under 5X 10<-5>, the life of the column filler will be shortened while when it exceeds 10<-2>M/ l, ion exchange of the column filler with an ion becomes more than ignorable value and thus, the separability will change.

Description

[Detailed description of the invention] The invention relates to liquid chromatography.

Conventionally, methods for analyzing sugars by liquid chromatography include: 1) photoreagents with chemically bonded amine groups, etc.
22) Separation using molecular sieves using cross-linked distrangel, 3) Separation as borate complex ions using anion exchange resin, 4) Salt-type cation exchange Separation using a compromise mode of molecular sieving and distribution using a resin-gold column packing material is known. 2 of these
Method 4) uses water as an eluent and is therefore easy to operate.

However, the above method 2) is incapable of separating sugars having different molecular weights. On the other hand, the method 4) above results in a bald dragon.

However, as a result of further study by the inventors on the above method 4), it was found that when water is used as an eluent, the 9-separation ability decreases with long-term 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.

Unexploded bone” 1. There is a solution to such problems.

That is, the invention is based on a liquid chromatography method using porous styrene-divinylbenzene copolymer particles having an SOi group and a divalent metal cation as its counter ion as a column packing. The present invention relates to a liquid chromatography method characterized in that an aqueous solution having a substance concentration of 5XIO' to 10-''M/l is used.

of metal cations.

In particular, as counter ions (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 - k(a) ion and ( The equivalent ratio of ions in b) is (a)
/(b) in a ratio of 172 to 2/1.

In this case, glucose, galactose, mannose and fructose, which have the same molecular weight, can be separated.

The column packing material according to the present invention is capable of separating counter ions such as Pd or Ba, and preferably has a sO-based column packing material 1y-2S equivalent (meq/P) or more, particularly 3 to 5 It is preferable to have meq/9-. If it is less than 2 meq/7, the hydrophilicity tends to be insufficient.

The particle size of the column packing material of the present invention is 1 to 2 o/im for liquid chromatography analysis and 20 to 300 o/im for preparative separation by liquid chromatography.
Preferably, the diameter is μm.

Although there is no particular limit to the degree of pores in the column packing material of the present invention, it is advisable to set the exclusion limit to a range of 400-IXIO7.

The fcstch group mentioned above is bonded to a styrene-dihinylbenzene copolymer.

It is preferable that the copolymer of divinylbenzene and a styrene monomer contains 25 to 60% by weight of divinylpuffene. Examples of the styrene monomer include styrene, ethyl heptanovinylbenzene, vinyltoluene, α-Me-stynon, and other monomers such as vinyl acetate, methacrylic acid, and acrylic acid in a range of 5 weight percent or less. May be used in

The column packing material of the present invention comprises sulfonated porous styrene-divinylbenzene copolymer particles and general formula (I) M(OH)2 (I) (However, 1M in the two formulas can be a divalent cation. It can be obtained by reacting hydroxides represented by metals. The above M is Ca, St, and pa.

There are Ba, Mg, etc.

More specifically, water in which the above hydroxide was dissolved was mixed with sulfonated porous styrene-divinylbenzene copolymer particles or a salt thereof, and the mixture was passed through nine mouths.

Further, it can be obtained by repeating the same operation. Alternatively, it can be obtained by filling a column with sulfonated porous styrene-divinylbenzene copolymer particles or a salt thereof or placing them on a funnel, and passing the entire aqueous solution through the column in the same manner as described above. In this case, 803 in the sulfonated porous styrene-divinylbenzene copolymer particles
against the base.

It is preferable to use the above-mentioned hydroxide so that the amount of metal cations is equal to or more than the same amount.

Sulfonated porous styrene-divinylbenzene copolymer particles are produced by a conventionally known method.

fltjL d + divinylbenzene, styrene monomer, and optionally other monomers are suspended and polymerized in the presence of a water-insoluble organic solvent such as amyl alcohol or toluene, and the resulting particles are entirely isolated and treated with dichloroethane or trichloromethane. It can be obtained by swelling with a swelling agent such as ethane, adding concentrated panic acid or chlorosulfuric acid, and carrying out a sulfonation reaction at room temperature to 20°C. The above-mentioned ion exchange capacity can be adjusted by adjusting the reaction conditions as appropriate during the reaction with concentrated acid or chlorosulfuric acid.

What is the degree of pores in the column porosity in non-invention?

During the above suspension polymerization, the particle size can be adjusted by adjusting the conditions, mainly the type and amount of water-insoluble organic bath agent, and the particle size can be adjusted by appropriately selecting the suspension polymerization conditions. .

The sulfonated porous styne-dihinylbenzene copolymer preferably has an ion exchange capacity of 2 meq/P or more, particularly 3 to 5 meq/P. Preferably.

In addition, in the column packing material of the present invention, each of the porous styrene-divinylbenzene copolymer particles having an SO3' group has the above-mentioned ions (a) and (b) as counter ions to the SOs group. In terms of separation performance, it is preferable to have the following properties, but it is also possible to use a second-order one.

Namely.

Ca- or S as the SOs group and its counterion
Porous styrene-divinylbenzene copolymer particles (A) formed by disintegrating r+1 (ion of (a)) and 803 group and Pb++ or Ba as its counter ion
+ “Porous styrene having ((b) no ion) −
Divinylbenzene copolymer particles (B) are combined with (a) ions and (bl ions (a)/(b)
A mixture having a ratio of 1/2 to 2/1 (total ratio) can be used as a non-inventive column packing agent.

It is preferable that the copolymer particles (A) and (B) are mixed as uniformly as possible, and the particle size of each copolymer particle.

It is preferable to use those having the same degree of pores, moisture content, and content of 803 groups. For this purpose, the copolymer particles (A) and CB) are preferably neutralized in the same manner as described above using sulfonated porous styrene-divinylbenzene co-ff1 integral particles from the same lot. .

When such copolymer particles (A) and (B) are used, they have a volume ratio of (A)/FB) of 1/2 to 2/2.
It is only necessary to blend the components so that the total amount becomes 1, which facilitates the mixing operation.

In the column packing material of the present invention, the ions of (a) and (
b) If you have ions of ions as opposite ions, then SOs
It is most preferable that the ions (a) and (b) are present in a uniform distribution in each particle of the porous styrene-divinylbenzene copolymer particles having the group Ij. For this purpose, the column packing material is a sulfonated porous styrene-divinylbenzene-based compound, -fX coalesced particles k(C) Ca
(01-1) At least one compound selected from the group consisting of 2 and Sr(OH)2, and (d) at least one compound selected from the group consisting of Pb(OH)2 and 5r(OH)z. (C)/Id) is dissolved in a molar ratio of 1/2 to 2/1 and neutralized with a water bath solution consisting of 4-.

The T6 bath solution used in the present invention is an aqueous solution of an alkaline substance. Alkaline substances include hydroxides of alkali metals, alkaline earth metals, etc., hydrogen carbonates, carbonates, etc., which exhibit alkalinity when made into a water bath solution, and specifically include OH, NaOH, and the like.

KOH, Sr(OH)2, Ba(OH)2,
NaHCO3, KHCO3.

There are Na2CO3, 2CO3, etc. These Arca 1
Lunar material c, 5 X 10-5~10 Mo t'
7) Used at 9 degrees. If the concentration is less than 5 x 10 Mon, the life of the column packing material will be shortened, and if it exceeds 10 Mon, the ion exchange with the counter ion of the column packing material cannot be ignored and the resolution will change.

As the column packing material of the present invention, the sulfonated porous styrene-divinylbenzene copolymer particles obtained by reacting the sulfonated porous styrene-divinylbenzene copolymer particles with its salt and the hydroxylated P/Ik represented by the above general formula (I) were used. sometimes.

The issue of longevity is particularly noticeable.

The column packing material of the present invention comprises sulfonated porous styrene-divinylbenzene copolymer particles, MC12, MBr
2 (here, 9M is the same as in the case of general formula (1)), etc. can be obtained by reacting a metal halide such as Although the problem is not significant, it is preferable to use the eluent of the present invention.

In the liquid chromatography method according to the present invention, a sample is injected from a sample injection port, passed through a column filled with the above-mentioned column packing material while passing the above-mentioned eluent, and detected by a 2-passage detector, This method is applicable to high-performance liquid chromatography analysis in which a complete chromatogram is used, and dispersion in which a sample is passed through a column filled with a column packing material and collected every 7 fractions.

Next, an example of the present invention will be shown.

Example 1 Particle size 8-15 μm, ion exchange capacity 4.3 meQ/i%
and sulfone (IJlL
Styrene-ethyl monovinylbenzene-divinylbenzene copolymer particles 24
H2O13,7Pk 5o dissolved in water.

The aqueous solution prepared in a was mixed and stirred for 1 hour. In addition.

The sulfone group and Sr were blended at an equivalent ratio of 1/1. After this, rinse with two mouths of water, and the exclusion limit is 1. An OOO column packing material was obtained. The obtained column packing material was swollen with water to form a slurry and filled into a stainless steel column of 10.7 mm diameter x 30 cm, and then sent to an analyzer for high performance liquid chromatography to contain centiole, raffinose, maltose, crucose, Sample 814 (sample ■) containing caractose, mannose, and fructose was subjected to analysis. Analysis conditions ij, k quantity i, Onll/
%, eluent u10-'M,/L NaOH aqueous solution and column temperature 60°C, using a differential refractometer as a detector, clotgellum was obtained. In addition.

The eluent was passed through continuously.

Figure 1 shows the taromadogram obtained by sampling immediately after the start of eluent flow, and Figure 2 shows the complete chromatogram obtained by sampling 3 hours later.

Table 1 shows the change in the number of theoretical plates over time. In FIGS. 1 and 2, peak 1 is raffinose, peak 2 is maltose, peak 3 is glucose, heath 4 is galactose, peak 5 is mannose, and peak 6 is fructose. peak shape.

No change was observed in the separability, and Table 1 shows that no change was observed when the number of theoretical plates was low.

Example 2 In Example 1, 5r(OH)+ ・8H2013
,7y- instead of Ba (OH)2 ・8H2016
, 2f A column packing material with an exclusion limit of 1.000 was obtained in the same manner as in Example 1 except that f was used. Here, the sulfone group and Ba were mixed at a ratio of 1/1. Thereafter, a column was filled in the same manner as in Example 1, and analysis by high performance liquid chromatography was performed.

Figure 3 shows the chromatogram obtained by sampling immediately after the start of eluent flow, and Figure 4 shows the chromatogram obtained by sampling 3 hours later.

Table 1 shows the change in the number of theoretical plates over time. In FIGS. 3 and 4, each peak salt is the same as in Example 1. It can be seen from Table 1 that no change in peak shape or separation was observed, and no decrease in the number of theoretical plates was observed.

Example 3 In Example 1, Sr (OH)2 ・8H201
Instead of 3,7ft, Sr (OH)2 '8H2
06,9'! A column packing material was produced in the same manner as in Example 1 except that - and Ba(OH) 28°1z were used. In addition, here, the total amount of Sr+1 and Ba"+ and the sulfone group were adjusted so that the equivalent ratio was 1/1, and S
The equivalent ratio of r and Ba was also set to 1/1. Thereafter, a column was filled in the same manner as in Example 1, and analysis by high performance liquid chromatography was performed.

Samples for analysis were Sample I and a sample containing mannose, arabinose, and fructose (Sample ■).

Figure 5 shows the taromadogram obtained by sampling sample ① immediately after the eluent started flowing.

The entire chromatogram obtained by sampling after 3 hours is shown in FIG. 6, and the chromatogram of sample U is also shown in FIGS. 7 and 8. Also.

Table 1 shows the change in the number of theoretical plates over time.

In FIGS. 5 and 6, each peak awn is the same as in Example 1. In Figures 7 and 8, peak f is mannose and peak sweat is arabino? -gose and peak are fructose. No change was observed in the peak shape or separability 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. What are the analysis conditions?

Process-1. Qm/min, eluent was water and column temperature #60
℃, and a talomadogram was obtained using a differential refractometer as a detector. Figure 9 shows a chromatogram obtained by sampling 3 hours after the start of eluent flow.

Table 1 shows the change in the number of theoretical plates over time.

In FIG. 9, each peak reference is the same as in Example 1.

The separation of galactose in peak 4 and mannose in peak 5 is poor, and in Ibara 1, the number of theoretical plates is 3.
It can be seen that the value has decreased by about 25 coefficients after an hour.

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.

Each peak value is the same as in Example 1. The peak shape became broad, and the separation of mannose in peak 5 and fructose in peak 6 became significantly worse.9 In Table 1, it can be seen 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 talomadography analysis was performed under the same analysis conditions as in Comparative Example 1.

Samples for analysis were Sample I and Sample ■.

Figure 11 shows the chromatogram obtained by sampling 3 hours after the start of eluent flow for sample I, and the chromatogram for sample H.
The same is shown in FIG. 12. Table 1 also shows the change in the number of theoretical plates over time. In FIG. 11, each peak field is the same as in Example 1.

In Fig. 12, each peak density is the same as in Example 3.Depending on the elapsed time, the peak shape becomes broad and 2 min μ
m-sexuality is,,i,! +. It can be seen that, especially for sample 1, the 4-separability becomes significantly worse. It can be seen that in the coating 1, the number of theoretical plates decreased by about 50 factors after 3 hours.

Note) The number of theoretical plates was calculated by sampling fructose a certain time after the start of liquid flow and using the fructose peak in the obtained chromatogram using the following formula.

A schematic diagram of the apparatus used for the analyzes above is shown in FIG. 13. The eluent is stored in a container 10, and the eluent is pumped to a pump 11.
through pipe 12 to column 13 and detector 1
4, and the liquid is drained into the container 15. The sample is inlet 16
(for example, a three-way cock), and a chromatogram recorder 17 is connected to the detector 14.

As is clear from the above, the porous styrene group containing the SO3 group and the divalent metal cation as its counter ion.
According to the liquid chromatography method according to the present invention when divinylbenzene copolymer particles are used as a column packing material, the life of the column packing material can be extended.

[Brief explanation of drawings]

FIG. 1 and FIG. 2 are for Example 1, respectively. The chromatograms, Figures 3 and 4, obtained by sampling immediately after the start of passing the eluent and after 3 hours are the chromatograms, Figures 5 and 4, respectively, showing the same results as in Example 1 in Example 2. FIG. 6 is a chromatogram showing the same results as in Example 1 when sample (1) was used in Example 3, and FIGS. Example 1 when used
A chromatogram showing the same results as in Figure 9 is Comparative Example 1.
10 is a chromatogram showing the results of Comparative Example 2, FIGS. 11 and 12 are chromatograms showing the same results as Example 1 in Comparative Example 3, and FIG. 13 is a chromatogram showing the results of Comparative Example 2. , shows a schematic diagram of the analyzer used in Examples and Comparative Examples. Explanation of symbols 1... Raffinose 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 Dissolved powder)
figure

Claims (1)

[Claims] 1. In a liquid chromatography method using porous styrene-divinylbenzene copolymer particles having an SO3 group and a divalent metal cation as its counter ion as a whole column packing material, an eluent A liquid talomagography method characterized in that an aqueous solution having an alkaline substance concentration of 5 x 10-5 to 10-2 M/l is used. 2. The column packing material is sulfonated porous styrene.
The divinylbenzene copolymer particles are crushed and their salts are reacted with a hydroxide represented by the general formula (1) MOO12II (in which 9M is a metal base that can become a divalent cation). The liquid chromatography method according to claim 1, which is obtained separately.