JPS6128302B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a packing material for chromatography. More specifically, the present invention relates to a method for producing a packing material for high performance liquid chromatography (hereinafter abbreviated as HLC) that has excellent separation performance for organic oligomer substances.

In recent years, with the progress of research and practical application of so-called oligomers with molecular weights ranging from several hundred to several thousand, their chemical structural characteristics have been characterized by infrared absorption analysis, plasma absorption analysis, nuclear magnetic resonance absorption, mass spectrometry, etc. However, research on oligomers as a single species (molecule) has not progressed sufficiently because no satisfactory method has been established to isolate oligomers as a single species (molecule).

Methods for separating oligomers include conventional distillation methods, solvent extraction methods, crystallization methods, fractional precipitation methods,
Centrifugal separation method, liquid chromatography (LC)
LC (abbreviated as LC) method is known, but LC has the advantage of having fewer restrictions on the applicable molecular weight range and chemical stability, and of being able to separate each single chemical species.
The law is excellent. Among HLC, gel permeation chromatography (hereinafter abbreviated as GPC) is particularly suitable because separation occurs based on differences in molecular weight. GPC is a type of liquid chromatography in which the development speed is delayed because the solute molecules permeate the solvent held in the pores of the column packing material (hereinafter referred to as gel). Separation of each molecule is performed based on the difference in molecular weight. Therefore, oligomer molecules are separated and eluted from the column in order from those with a high degree of polymerization that are difficult to penetrate into the gel, and in the chromatogram obtained thereby, the peak interval is narrower as the elution volume is smaller.

Furthermore, if the logarithm of the degree of polymerization of each molecule separated by GPC and the elution volume are plotted on the vertical and horizontal axes, a straight line with a negative slope will be drawn. Therefore, as the degree of polymerization increases, it becomes difficult to separate each component of the oligomer into single chemical species.

On the other hand, in recent years there has been a strong demand for faster analysis and separation, and hard gels with high mechanical strength are involved.
With the spread of HLC equipment, high-speed GPC, which can perform analysis in several tens of minutes, has become widely used. In order to enable high-speed GPS separation of each oligomer component with a high degree of polymerization, improvements have been made to the operating conditions, such as a recycling method in which the solute to be separated is repeatedly passed through the column, but it takes a long time for the desired analysis or separation. However, there were limitations such as requiring expensive equipment and requiring a high level of experience in setting operating conditions. This is because the separation performance of columns packed with styrene-divinylbenzene gel used in high-speed GPC in the oligomer region is insufficient. With known techniques, it has not been possible to obtain a gel that satisfies both strength and separation performance.

The inventors of the present invention have intensively studied methods for realizing the separation of oligomers with a high degree of polymerization without the above-mentioned restrictions, and as a result, have arrived at a method for producing a gel with a wide peak spacing without compromising other performance, and have completed the present invention. did.

Conventionally, methods for producing gels having a styrene-divinylbenzene skeleton suitable for separating oligomers can be roughly divided into the following two methods.

(1) Significantly less divinylbenzine (e.g. 5
A method of obtaining particles by suspension polymerizing a mixture of styrene, divinylbenzene, and a polymerization initiator in a composition (weight% or less).

(2) A method in which the amount of divinylbenzene is larger than in the case of (1), suspension polymerization of styrene and divinylbenzene is carried out in the coexistence of a gel swelling solvent, and the solvent is removed from the particles after polymerization.

Among these, the gel produced by method (1) has a low weight percentage of divinylbenzene units (hereinafter expressed as
It is inappropriate for Furthermore, when synthesized using method (2), a type of porous gel called expanded network gel can be obtained, which is a type of porous gel that has been used in recent years for high-speed oligomer separation.
Many GPC gels are made using this method. However, the gel produced by this method has a high degree of crosslinking, so although it has excellent mechanical strength, its separation performance is insufficient.

Suspension polymerization for producing a gel is generally carried out until the polymerization reaction is completely completed. For example, in USP 3,326,875, benzoyl peroxide was used as a polymerization initiator to synthesize a gel.
Suspension polymerization is carried out by heating for a long time, and in the latter half of the polymerization, heating is performed at a high temperature for a long time to complete the polymerization.

However, in method (2), the present inventors achieved a method for high-speed GPC with excellent separation performance without substantially reducing mechanical strength by stopping polymerization while leaving some unpolymerized monomers. The gel was successfully produced.

That is, the present invention provides a total of 100 parts by weight of at least 65 to 90 parts by weight of one or more monovinyl aromatic monomers, 35 to 10 parts by weight of one or more polyvinyl aromatic monomers, and one or more solvents for dissolving polystyrene. When carrying out radical polymerization by suspending a liquid mixture consisting of 30 to 200 parts by weight in water,
The present invention relates to a method for producing a filler for chromatography, characterized in that polymerization is stopped when the polymerization rate reaches a range of 85 to 98%.

Here, monovinyl aromatic monomers include styrene, vinyltoluene, α-methylstyrene,
One or more types are selected from compounds such as ethylvinylbenzene, and one or more types from divinylbenzene and trivinylbenzene are used as the polyvinyl aromatic monomer.

The monovinyl aromatic monomer is used in an amount of 65 to 90 parts by weight, preferably 65 to 80 parts by weight, and the polyvinyl aromatic monomer is used in an amount of 35 to 10 parts by weight, preferably 35 to 20 parts by weight.

The amounts of the monovinyl aromatic monomer and the polyvinyl aromatic monomer are selected so that the total amount of both is 100 parts by weight. Generally, commercially available divinylbenzene contains 40% by weight or more of ethylvinylbenzene, but when using commercially available divinylbenzene, pure divinylbenzene and the remaining monomer components, mainly ethylvinylbenzene, are mixed into polyvinyl aromatic monomers. , shall be calculated as monovinyl aromatic monomer.

If the amount of the monovinyl aromatic monomer exceeds the above range, the mechanical strength of the gel will decrease, making the gel unsuitable for high-speed GPC, while if it is less, the separation performance will deteriorate.

Furthermore, other monomers copolymerizable with these monomers are produced, such as acrylic acid, acrylic ester, methacrylic acid, methacrylic ester, acrylonitrile, methacrylonitrile, vinylpyridine vinylimidazole, and vinylpyrrolidol. It may be added to the extent that the physical properties of the particulate crosslinked copolymer are not substantially changed.

The average molecular weight of the solvent that dissolves polystyrene
10 ⁴ to ^{10 6} linear polystyrene at room temperature
A solvent that dissolves at least % by weight and is selected from aromatic hydrocarbons, ketones, esters, nitriles, and halogenated hydrocarbons, specifically benzene, toluene, xylene, cyclohexanone, and methyl. Benzoate, benzonitrile, n-
Butyl acetate and the like are preferred, and toluene is particularly preferred from the viewpoint of ease of handling and price.

The amount of solvent is based on 100 parts by weight of the total amount of monovinyl aromatic monomer and polyvinyl aromatic monomer.
It is used in a range of 30 to 200 parts by weight. If the amount of solvent is less than this range, the number of micropores with a pore size suitable for separating oligomers will decrease, resulting in a decrease in separation performance.
If the amount is too large, the mechanical strength of the gel decreases, making the gel unsuitable for high-speed GPC. From a practical point of view, the amount of solvent is
A range of 80 to 150 parts by weight is preferred.

The polymerization initiator is 2,2′-azobis-(1-methoxy2,4-dimethylvaleronitrile), 2,
2'-azobis-(2,4-dimethylvaleronitrile), 2,2'-azobisisobutyronitrile,
Azo initiators such as 1,1'-azobiscyclohexane-1-carbonitrile, or isobutyryl peroxide, diisopropyl peroxydicarbonate, di-2-ethylhexyl dicarbonate, 2,4-dichlorobenzoyl peroxide, t -Butyl peroxypivalate, 3,5,
5-trimethylhexanoyl peroxide, octanoyl peroxide, decanoyl peroxide, lauroyl peroxide, stearoyl peroxide, propionyl peroxide, acetyl peroxide, t-butylperoxy-2-ethylhexanoate, benzoyl peroxide One or more types of organic peroxide-based initiators such as oxides are used. The initiator is used in an amount of 0.1 to 5.0 parts by weight based on 100 parts by weight of all monomers.

The suspension stabilizer is selected from commonly known organic polymer stabilizers such as polyvinyl alcohol and methylcellulose.

The volume ratio or weight ratio of the organic phase to the aqueous phase during polymerization is not particularly limited, and may be within a range normally selected when carrying out suspension polymerization.

Polymerization is carried out as follows. Weigh out a predetermined amount of the monovinyl aromatic monomer, polyvinyl aromatic monomer, polymerization initiator, and solvent for dissolving polystyrene, mix to make a homogeneous solution, add it to the water in which the suspension stabilizer has been dissolved, and add it to the water using a disperser or homogenizer. Stir and mix using a stirrer with high shearing force called .

Stirring raises the temperature of the liquid, and in some cases the container is cooled from the outside to prevent polymerization from starting before the monomer-containing oil droplets reach the target particle size. The temperature when adjusting the diameter of the oil droplet varies depending on the amount and type of polymerization initiator or inhibitor contained in the oil droplet, but it is usually best to keep it below 20°C. After the oil has reached a predetermined size, it is heated while stirring to a predetermined temperature to carry out a polymerization reaction. At this time, stirring may be performed to the extent that the composition and temperature within the system are kept uniform. The polymerization temperature is not particularly limited and may be within the temperature range normally set when carrying out suspension polymerization. Polymerization has a polymerization rate of 85-98%
It will stop when it reaches this range. If the polymerization rate is lower than this range, the mechanical strength of the gel will be low, and if it is higher than this range, the separation performance of the gel will be poor. From a practical standpoint, the polymerization rate is preferably in the range of 90 to 96%.

Termination of polymerization is carried out by commonly known methods such as adding a polymerization inhibitor or cooling the polymerization solution, and is not particularly limited. It is best to filter out particles, wash with water, and cool.

The obtained particles are separated and thoroughly washed with water, hot water, acetone, etc. to remove the suspension stabilizer that accompanies the particles.
Remove solvent, residual monomer, etc. Furthermore, by classifying the particles if necessary, it becomes possible to use them as a gel for high-speed GPC.

In the present invention, the polymerization rate is expressed as a value obtained by subtracting from 100 the percentage of the amount of monomer remaining at a certain time after the start of polymerization relative to the total amount of monomers before polymerization, and is a value obtained by subtracting from 100 the percentage of the amount of monomer remaining at a certain time after the start of polymerization with respect to the amount of total monomer before polymerization. It is determined by extracting the polymerization liquid with an organic solvent different from the components contained in the polymerization liquid and analyzing the monomer components in the extract using gas chromatography.
The timing of polymerization termination can be easily determined by determining the relationship between polymerization time and polymerization rate in advance.

In order for the gel obtained according to the present invention to exhibit sufficient separation performance in GPC, the volume average particle size of the gel is preferably in the range of 2 to 50 μm, preferably 5 to 30 μm.

The particle size of a gel is determined as a volume average particle size, and in the present invention, the volume average particle size refers to the cumulative volume of an integral curve in which the horizontal axis is the particle size and the vertical axis is the cumulative volume of particles up to a certain particle size. This value represents the particle size at 50% of the particle size, and is determined using a device such as a Coulter Counter (Coulter Electronics, Inc., USA).

In order to measure the physical properties of the gel obtained by the method of the present invention, GPC analysis was performed and a calibration curve was obtained.
The measurement conditions at that time were as follows.

Column made of stainless steel, inner diameter 7-8 mm, length
50cm Solvent Chloroform Sample Polystyrene 0.5%-Chloroform solution 20μl Flow rate 0.1-2.0ml/min Temperature Room temperature Detection method UV254nm The gel was charged into the column by upward flow in the chloroform solvent.

A calibration curve in GPC generally represents the relationship between the molecular weight of a substance to be separated and the elution volume in a chromatogram, but in the present invention, as shown in Figure 1, the vertical axis represents the molecular weight (M) of each component of a polymer or oligomer. The logarithm refers to the line obtained by plotting the value (Ve/Vt) obtained by dividing the elution volume (Ve) by the column volume (Vt) on the horizontal axis.

In FIG. 1, the value on the vertical axis at the point where the extension of the inclined straight line intersects the extension of the line parallel to the vertical axis is called the exclusion limit molecular weight (hereinafter referred to as Mlim). Preferably 1000-15000,
More preferably, it is suitable for obtaining a gel in the range of 1500 to 12000.

Examples of chromatograms obtained by using the gel obtained by the method of the present invention for GPC separation of styrene oligomers (Figures 2, 3, and 4) and corresponding examples of the conventional method (Figures 5, 6, and 7) Comparing the two methods shown in Figure), the former shows better separation performance as oligomers with a higher degree of polymerization are separated. In this way, by stopping the polymerization a little before the polymerization is completed, a gel with good separation performance can be obtained with almost no change in other physical properties. 2nd, 3rd, 4th
The number of separated oligomers in each figure is larger than in Figures 5, 6, and 7, but when looked at in more detail, the intervals between the peaks of the separated components are wider. The wide peak interval indicates that the slope of the straight line of the calibration curve is gentle, and the reason why the gel obtained by the present invention has excellent separation performance is that the calibration curve defined by the following equation This is considered to be based on the fact that the absolute value of the gradient (hereinafter referred to as α) is small.

α=｜△logM/△(Ve/Vt)｜ Although it is not necessarily clear why a gel with a low α and good separation performance can be obtained by the method of the present invention, some unpolymerized monomers remain It is thought that this process caused changes in the skeleton and micropore structure to stop polymerization, resulting in a gel with a unique particle structure.

According to the research of the present inventors, when comparing the physical properties of gels polymerized to a polymerization rate of 100% by changing only the amount of solvent coexisting during polymerization, it was found that the amount of solvent equivalent to the amount of unpolymerized monomer of the present invention was compared. Even if it increases,
Although Mlim increases somewhat, α remains almost unchanged. Therefore, the reason why a gel with a low α can be obtained in the present invention is not because the unpolymerized monomer simply functions as a solvent.

The packing material for chromatography obtained by the method of the present invention can be used not only as a packing material for GPC but also as a packing material for HLC based on adsorption and distribution effects.

The invention will be explained in further detail by the following examples.

Example 1 1 Put 700 ml of water containing 4.2 g of polyvinyl alcohol into a flask, add 36.4 g of styrene, 36.4 g of divinylbenzene (purity 56%), and 69.8 g of toluene.
and 2 g of 2,2'-azobisisobutyronitrile
A homogeneous mixture consisting of the following was added. The flask was placed in an ice-water bath, and the polymerization liquid in the flask was stirred at high speed using a laboratory disperser while sufficiently cooling the flask and keeping the internal temperature below 20°C. Next, the flask was placed in a water bath kept at 60°C, and polymerization was carried out by heating at 60°C for 6 hours while stirring with a boat-shaped bladed stirring bar to maintain a uniform temperature inside the flask. The polymerization rate at the end of the polymerization was determined to be 91% by gas chromatography analysis of the remaining monomer. The obtained polymer particles were separated, and after sufficient water and acetone were added in that order, they were dispersed in acetone and simple classification was performed using the difference in sedimentation rate. The volume average particle size of the particles thus obtained was adjusted to 1% using a Coulter Calanter ZB type (Coulter Electronics, Inc., USA).
When measured in a saline solution, it was 9.2 μm. Using this gel, values of α=5.5 and Mlim=5200 were obtained from a calibration curve obtained under the conditions described in the specification. Using this packed column and using benzene as the solute, the number of theoretical plates (hereinafter referred to as N _B ) was measured and found to be 11,200 plates. Furthermore, using this packed column, a chloroform solution of styrene oligomers mainly consisting of tetramers was analyzed by GPC at a solvent flow rate of 1 ml/min, and a chart (Fig. 2) separated into hexamers was obtained.

Example 2 In Example 1, styrene 33.8 was used as the polymerization liquid.
g, divinylbenzene (purity 56%) 39.0 g, toluene 94.5 g and 2,2′-azobis-(2,4-
A gel was prepared in the same manner as in Example 1, except that a mixture containing 1 g of dimethylvaleronitrile was used and polymerized at 60° C. for 7 hours. The polymerization rate at the end of the polymerization was determined to be 95% by gas chromatography analysis of the remaining monomer. The volume average particle diameter of the obtained gel was 9.5 μm, and values of α=4.7 and Mlim=10000N _B =12500 plates were obtained by measurement using a column packed with this gel. Using this packed column, styrene oligomers mainly consisting of tetramers were analyzed in the same manner as in Example 1. Charts (tertiary
Figure) was obtained.

Example 3 In Example 1, styrene 41.6 was used as the polymerization liquid.
g, divinylbenzene (purity 56%) 31.2 g, toluene 69.8 g and benzoyl peroxide 1 g
A gel was prepared in the same manner as in Example 1, except that the mixture was polymerized at 75°C for 7 hours. The polymerization rate at the end of the polymerization was determined to be 93% by gas chromatography analysis of the remaining monomer. The volume average particle diameter of the obtained gel was 9.7 μm, α = 4.3, Mlim = from measurements using a column packed with this gel.
Values of 3800 and N _B =7200 stages were obtained. When styrene oligomers, mainly tetramers, were analyzed using this packed column in the same manner as in Example 1, a chart (FIG. 4) in which the oligomers were separated was obtained.

Comparative Example 1 The same procedure as in Example 1 was carried out except that the polymerization was carried out at 60°C for 6 hours and then at 80°C for 4 hours.

The polymerization rate at the polymerization end point was determined to be 100% by gas chromatography analysis of the remaining monomer.

The volume average particle diameter of the obtained gel was 9.1 μm, and when GPC analysis was performed using this gel in the same manner as in Example 1, α = 6.5, Mlim = 7000, N _B = 93000.
The analysis chart of styrene oligomers mainly consisting of tetramers was as shown in Figure 5. The chart obtained in Example 1 (Fig. 2) and the gel of Comparative Example 1 were separated only up to the pentamer, whereas the gel of Example 1 was separated up to the hexamer, and the present invention You can see how good the separation performance of this gel is.

Comparative Example 2 A gel was prepared in the same manner as in Example 2, except that the polymerization was carried out at 60°C for 10 hours and then at 80°C for 10 hours. The polymerization rate at the end of the polymerization was determined to be 99% by gas chromatography analysis of the remaining monomer. The volume average particle diameter of the obtained gel was 9.3 μm, and from measurements using a column packed with this gel, α = 5.8, Mlim = 11000, N _B = 13000.
I got the value of the stage. Furthermore, as a result of analysis of styrene oligomers mainly consisting of tetramers, which was carried out in the same manner as in Example 2, only hexamers were separated. (Figure 6) Comparative Example 3 A gel was prepared in the same manner as in Example 3, except that the polymerization was carried out at 7.5°C for 10 hours and then at 85°C for 10 hours. The polymerization rate at the end of polymerization was 100%. The volume average particle diameter of the obtained gel was 9.8 μm, and values of α=5.3 and Mlim=4200N _B =9000 plates were obtained by measurement using a column packed with this gel. In addition, it was carried out in the same manner as in Example 3.
As a result of analysis of styrene oligomers mainly composed of hexamers, only hexamers were separated. (7th
(Figure) Comparative Example 4 Polymer particles were obtained in the same manner as in Example 1 except that the amounts of styrene and divinylbenzene with a purity of 56% were changed to 20.8 g and 52 g, respectively. The polymerization rate at the end of polymerization was 93%. The particles are classified to determine the average particle size.
A gel of 8.9 μm was obtained. From the calibration curve obtained using this gel under the conditions stated in the specification, α = 8.5,
Mlim got a value of about 30000. From these values, it was thought that this gel was not necessarily suitable for separating oligomers, but when styrene oligomers were actually analyzed, only tetramers could be separated.

Comparative Example 5 Polymer particles were obtained in the same manner as in Example 1 except that the amounts of styrene and divinylbenzene (purity 56%) were changed to 66.3 g and 6.5 g, respectively. The polymerization rate at the end of polymerization was 92%. The particles were classified to obtain a gel with an average particle size of 8.8 μm. An attempt was made to measure the performance by filling a column with this gel, but due to the low strength of the gel, it was not possible to increase the solvent flow rate to 1 ml/mm.

Comparative example 6 Example 1 except that the amount of toluene used was 10 g
Polymer particles were obtained in the same manner as above. The polymerization rate was 94%. The particles were classified to obtain a gel of 8.5 μm. A value of α=7.0 Mlim500 was obtained from a calibration curve obtained using this gel under the conditions described in the specification. When styrene oligomers were analyzed using a column packed with this gel, only double bodies were separated.

Comparative Example 7 Polymer particles were obtained in the same manner as in Example 1 except that the amount of toluene used was 182 g. Polymerization rate is 89%
It was hot. The average particle size obtained by classifying these particles is 9.0
An attempt was made to measure the performance by filling a column with a μm gel, but the strength of the gel was so low that it was not possible to increase the solvent flow rate to 1 ml/mm.

[Brief explanation of the drawing]

Figure 1 shows a calibration curve representing the relationship between the molecular weight (M) of the substance to be separated and the elution volume/column volume ratio (Ve/Vt) in the chromatogram, and Figures 2 to 7 are examples of examples, respectively. 1 to 3, using a column filled with the gel obtained in Comparative Examples 1 to 3.
A chart obtained by GPC analysis is shown.

Claims (1)

[Claims] 1 A total of 100 parts by weight of at least 65 to 90 parts by weight of one or more monovinyl aromatic monomers, 35 to 10 parts by weight of one or more polyvinyl aromatic monomers, and one or more solvents for dissolving polystyrene.
When radical polymerization is carried out by suspending a liquid mixture consisting of 30 to 200 parts by weight in water, the polymerization rate is 85 to 98.
A method for producing a packing material for chromatography, characterized in that polymerization is stopped when the polymerization reaches a range of %.