JPS5817526B2 - Method for producing packing material for chromatography - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a packing material for chromatography, particularly a packing material for high performance liquid chromatography (hereinafter abbreviated as HLC).

More specifically, HL is made of an aromatic vinyl compound-based crosslinked copolymer that has excellent separation performance for organic oligomer substances.
The present invention relates to a method for producing a filler for C.

In recent years, with the progress in research and practical application of so-called oligomers with molecular weights ranging from several hundred to several thousand, their chemical structural characteristics have been clarified by infrared absorption analysis, ultraviolet absorption analysis, nuclear magnetic resonance absorption analysis, mass spectrometry, etc. However, research on oligomers as a single species (molecule) has not progressed sufficiently because a satisfactory method for isolating oligomers as a single species (molecule) has not been established.

Conventionally known methods for separating oligomers include distillation, solvent extraction, crystallization, fractional precipitation, centrifugation, and liquid chromatography (hereinafter abbreviated as LC).

Among these separation methods, the LC method is superior because it has a relatively large applicable molecular weight range, fewer restrictions on chemical stability, and the ability to separate single chemical species. There is.

Among LC methods, gel permeation chromatography (
(hereinafter abbreviated as GPC) is suitable.

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 intervals between peaks are narrow on the side where the elution volume is small.

Furthermore, if the logarithm of the degree of polymerization and the elution volume of each molecule separated by GPC 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 or separation, and the development of hard gels with high mechanical strength and HCL
With the spread of equipment, high-speed GPC, which can perform analysis in several minutes, is now widely used.

High-speed processing of each oligomer component with a high degree of polymerization. In order to make separation by GPC possible, improvements have been made to the operating conditions, such as recycling methods in which the solutes to be separated are repeatedly passed through columns, but the desired analysis or separation still takes a long time and requires expensive equipment. In addition, there were restrictions such as 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, gels that satisfy both strength and separation performance have not been obtained.

did not exist.

The inventors of the present invention have conducted intensive studies on technology to realize the separation of oligomers with a high degree of polymerization without the above-mentioned limitations, and as a result, they have arrived at a method for producing a gel that exhibits wide peak spacing without compromising other performance. completed.

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

(1) A method of obtaining particles by suspension polymerization of a mixed solution containing styrene, divinylbenzene, and a polymerization initiator in a composition with a significantly low content of divinylbenzene (for example, 5% by weight or less).

(2) The amount of divinylbenzene is larger than in the case of (1), and styrene and divinylbenzene are subjected to suspension polymerization in the coexistence of a gel swelling solvent, and after polymerization, the particles are separated.

How to remove solvent.

Among these methods, method (1) produces a gel that has a low weight percentage of divinylbenzene units (hereinafter expressed as X) in all the monomer units constituting the gel, so it It is unsuitable for GPC.

Furthermore, when synthesized by method (2), a type of porous gel called an expanded network gel can be obtained.

Many of the high-speed GPC gels used for oligomer separation in recent years are prepared using method (2).

However, the gel made by this method has a high degree of crosslinking, so
Although the mechanical strength was excellent, the separation performance was insufficient.

Incidentally, the polymerization temperature in suspension polymerization is generally set at a temperature at which the half-life of the radical polymerization initiator used is approximately 10 hours.

In gel synthesis, US Pat. No. 3,326,875 uses benzoyl peroxide as a polymerization initiator to synthesize 75
The half-life of benzoyl peroxide in toluene at 75 °C is 10 hours (C,E,H,Bawnetaj, Trans.
araday Soc, ±111216 (1951)
).

However, in the method (2) above, the present inventors used a polymerization initiator that separates at a relatively low temperature, and at least in the initial stage of polymerization, the decomposition rate of the polymerization initiator was much higher than the conditions normally used. By polymerizing the gel, we succeeded in producing a gel for high-speed GPC with excellent separation performance without substantially reducing mechanical strength.

That is, the method for producing a chromatography filler according to the present invention comprises: 100 parts by weight in total of 65 to 90 parts by weight of at least one type of monovinyl aromatic monomer and 35 to 10 parts by weight of at least one type of polyvinyl aromatic monomer; When carrying out radical polymerization by suspending in water a mixture of 30 to 200 parts by weight of a solvent capable of dissolving polystyrene and more water, at least one initiator having a half-life of 2 to 60 minutes at 70°C is used, and at least Polymerization rate is 50
%, the half-life of the polymerization initiator used is 5 to 12%.
It is characterized in that the polymerization is carried out at a temperature in the range of 0 minutes.

Here, examples of the monovinyl aromatic monomer include compounds such as styrene, vinyltoluene, α-methylstyrene, and ethervinylbenzene, which may be used alone or in combination of two or more.

Examples of polyvinyl aromatic monomers include divinylbenzene and trivinylbenzene, and these may be used alone or in combination.

The monovinyl aromatic monomer is used in an amount of 65 to 9 Q 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 (total of both monomers; (100 parts by weight).

Generally, commercially available divinylbenzene contains about 40% by weight or more of ethylvinylbenzene, but when using this commercially available divinylbenzene, pure divinylbenzene and the remaining monomer components, mainly ethylvinylbenzene, are mixed separately. It shall be calculated as polyvinyl aromatic monomer and monovinyl aromatic monomer.

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

Furthermore, other monomers copolymerizable with these monomers, such as acrylic acid, acrylic ester, methacrylic acid, methacrylic ester, acrylonitrile, methacrylonitrile, vinylpyridine, vinylimidazole,
It may be added to the extent that it does not substantially adversely affect the separation performance of the particulate crosslinked copolymer filler that produces monomers such as vinylpyrrolidone.

"A solvent that can dissolve polystyrene" means an average molecular weight of 10
Refers to a solvent that dissolves 1% by weight or more of linear polystyrene having a molecular weight of 4 to 106 at room temperature, and such solvents are selected from among aromatic hydrocarbons, ketones, esters, nitriles, and logenated hydrocarbons. selected, specifically benzene,
Toluene, xylene, cyclohexanone, methyl benzoate, pentonitrile, n-butyl acetate, etc. are preferable, among which toluene is preferred from the viewpoint of ease of handling and price.

These solvents may be used alone or in combination of two or more.

The amount of solvent is 30 to 200 parts by weight based on 100 parts by weight of the total amount of monovinyl aromatic monomer and polyvinyl aromatic monomer.
Used in parts by weight.

If the amount of solvent is less than this range, there will be fewer micropores with a pore size suitable for oligomer separation (resulting in a decrease in separation performance, and if it is too much, the mechanical strength of the gel will decrease, making the gel unsuitable for high-speed GPC). become.

Practically speaking, the amount of the solvent is preferably in the range of 80 to 150 parts by weight.

At least one type of polymerization initiator used has a half-life of 2 to 60 minutes at 70°C.

Specific examples of initiators that can be used include 2,2'-azobis-(4-methoxy-2,4-dimethylvaleronitrile)
, 2,2'-azobis-(2,4-dimethylvaleronitrile), inbutyryl peroxide, diisopropyl peroxydicarbonate, di-2-ethylhexyl peroxydicarbonate, among others, 2,2'-
Azobis-(2,4-dimethylvaleronitrile) and diisopropyl peroxydicarbonate are preferred.

Even if an initiator with a half-life at 70°C outside the above range is used alone, the effects of the present invention cannot be obtained, but when used in combination with an initiator within the above range, the same effects as the present invention can be obtained. .

70℃ when using a mixture of two or more types of polymerization initiators
It is preferable that an initiator having a half-life in the range of 2 to 60 minutes is included in an amount of 60% by weight or more of the total initiator.

The initiator is used in a total 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 a solvent capable of dissolving the monovinyl aromatic monomer, polyvinyl aromatic monomer, polymerization initiator, and polystyrene, mix to make a homogeneous solution, add it to water in which the suspension stabilizer has been dissolved, and use a disperser or homogenizer. Stir and mix with a stirrer with a high shear force, which is called by the name of .

The temperature of the liquid increases due to stirring, and the container is optionally 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 droplets varies depending on the amount and type of polymerization initiator or inhibitor contained in the oil droplets.
It is usually best to keep the temperature below 20°C.

After the oil droplets have reached a predetermined size, the mixture is heated to a predetermined temperature while stirring 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 is carried out at a temperature where the half-life of the initiator in toluene is in the range of 5 to 120 minutes until the polymerization rate reaches 50%. 2,4-dimethylvaleronitrile) at a temperature where the half-life is in the range of 8 to 90 minutes, and when diisopropyl peroxydicarbonate is used, the half-life is in the range of 9 to 100 minutes. Preference is given to carrying out the polymerization at temperature.

If the polymerization temperature until the polymerization rate reaches 50% is lower than this range, the gel separation performance will be poor, and if it is higher, a gel with low mechanical strength will be obtained, both of which are not preferred.

The polymerization temperature after the polymerization rate reaches 50% or more is not particularly limited, and the polymerization may be continued at the same temperature or within the temperature range normally used for polymerization.

Polymerization is carried out until substantially no polymerization progresses.

The particles obtained after polymerization are separated from each other and thoroughly washed with water, hot water, acetone, etc. to remove suspension stabilizers, solvents, residual monomers, etc. that accompany the particles.

Furthermore, by classifying the gel if necessary, it becomes possible to use it as a gel for high-speed GPC.

The relationship between the half-life of a polymerization initiator and temperature is given in J.

Brandrup, E., H., Immergut, E.
ditors “POLYMER HANDBOOK 2n
d EditionJA Wiley-Inter
It is determined from the data described in Science Publication, and among them, the data in toluene, which has a similar chemical structure to vinyl aromatic monomer, will be used.

The type and amount of the initiator used in the polymerization can be determined by analyzing the gel using pyrolysis gas chromatography or infrared absorption spectrum.

In the present invention, "polymerization rate" is expressed as a value obtained by subtracting from 100 the percentage of residual monomer at a certain time after the start of polymerization with respect to the total monomer amount before polymerization, and the amount of residual monomer is the amount of granular crosslinked copolymer, It is determined by extracting a polymerization solution such as a suspension with an organic solvent different from the components contained in the polymerization solution, and analyzing the limonomer components in the extract using gas chromatography.

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

Here, the "volume average particle size" refers to the particle size corresponding to 50% of the cumulative volume in an integral curve in which the horizontal axis is the particle size and the vertical axis is the cumulative total of particle volumes up to a certain particle size.

This value is determined using a device such as a Coulter Counter (Coulter Electronics, Inc., USA).

G for measuring the physical properties of the gel obtained by the method of the present invention
A calibration curve was determined by PC analysis, and the measurement conditions at that time were as follows.

Column: Stainless steel, inner diameter 7-8 mm, length 50α cylindrical Solvent: Chloroform Sample: Holimethylene 70.5% = -Chloroform solution,
20 μl Flow rate: 0.1 to 2.01 nl/min Temperature: Room temperature Detection method: '[JV 254 μm Note that the gel was filled into the column by upward flow in a 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 (CM) of each component of the polymer or oligomer. It refers to a curve obtained by plotting the logarithm of , on a graph in which the value (V, /Vj) obtained by dividing the elution volume (Vo) by the column volume (Vt) is scaled 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 with the extension of the line parallel to the vertical axis is called the exclusion limit molecular weight (hereinafter referred to as Mlim).

In the method of the present invention, Mlim is 500 to 20,000, preferably 1,000 to 15,000, more preferably 1,500.
Suitable for obtaining gels in the range of ~12,000.

An example of a chromatogram obtained by using the gel obtained by the method of the present invention for GPC separation of styrene oligomers (see Figure 3 in Example 1 below) and a corresponding example of the conventional method (see Figure 8 in Comparative Example 1 below) Comparing the two, it can be seen that the latter can only separate up to pentamers, while the former can separate up to heptamers, showing superior separation performance.

In FIG. 3, the number of separated oligomers is greater than in FIG. 8, 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 due to the slope of the calibration curve defined by the following equation. This is considered to be based on the fact that the absolute value (hereinafter referred to as α) is small.

According to the research conducted by the present inventors, when the type of monomer used for gel production is constant, the weight percent of polyvinyl aromatic monomer units (hereinafter referred to as There is a correlation with , and as X increases, α also increases (Figure 2).

As shown in Figure 2, the gel (line A) obtained by the method of the present invention is obtained by polymerization under the relatively long half-life of the polymerization initiator of about 10 hours, which is the conventional method. Compared to the gel (line B) shown in FIG.

That is, the gel obtained by the present invention has improved separation performance compared to the corresponding gel obtained by the conventional method, with almost no change in other properties.

The granular crosslinked copolymer obtained by the method of the present invention is GP
It can be used not only as a filler for C, but also as a filler for HCL based on its adsorption and distribution effects.

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

Example 1 Into 11 flasks, add 4.2 parts of polyvinyl alcohol and 7 parts of water.
Add 00ml of solution, 36.4P of styrene, and 36.4t of divinylbenzene (purity 56%). , toluene 69.
1' and diisopropyl peroxydicarbonate 2
A homogeneous mixture of ingredients 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 lab body sparser while sufficiently cooling the flask and keeping the internal temperature below 20°C.

Next, the flask was placed in a water bath maintained at 60°C, and while stirring with a boat-shaped stirring bar to maintain a uniform temperature inside the flask, the polymerization was carried out by heating at 60°C for 6 hours and then at 80°C for 4 hours. I did it.

diisopropyl peroxydicarbonate at 60°C
- Half-life in luene is 100 minutes.

The obtained polymer particles were taken from house to house, thoroughly washed with water and acetone in that order, and then simply classified using the difference in sedimentation rate.

The volume average particle size of the particles thus obtained was measured in a 1% saline solution using a Coulter Counter ZB model (Coulter Electronics, Inc., USA).
It was 0 μm.

From a calibration curve obtained using this gel under the conditions described in the specification, values of α=4.5 and Ml im =4000 were obtained.

Using this packed column and using benzene as a solute, the number of theoretical plates (hereinafter referred to as NB) was measured and found to be 14,800 plates.

In addition, using this packed column, a chloroform solution of styrene oligomers containing mainly heavy weights was prepared at a solvent flow rate of 1 ml/
Chart of GPC analysis with mrn and separation of heavy bodies (
Figure 3) was obtained.

Example 2 In Example 1, 33.8 g of styrene and 39.8 g of divinylbenzene (purity 56%) were used as the polymerization liquid. Using a mixed solution consisting of OS', 94.5f toluene and 2,2'-azobis-(2,4-dimethylvaleronitrile) 2tjI,
A gel was prepared in the same manner as in Example 1, except that the polymerization was carried out at 80° C. for 10 hours.

The half-life of 2,2'-azobis-(2,4-dimethylvaleronitrile) in toluene at 80°C is 15 minutes.

The volume average particle size of the obtained gel was 9.0 μm, and from the calibration curve obtained using a column packed with this gel (1-4
,6, the value of Mlim=11000 is obtained, and NB is given to Sarah.
= 12,000 stages.

Using this packed column, styrene oligomers mainly consisting of heavy bodies were analyzed in the same manner as in Example 1, and a chart (Fig. 4) in which octamers were separated was obtained.

Example 3 In Example 1, the polymerization liquid used was 36.4 g of styrene, 36.4 S' of divinylbenzene (purity 56%), and 69.8 g of toluene. , and 2 g of 2,2'-azobis-(2,4-dimethylvaleronitrile).
A gel was prepared in the same manner as in Example 1 except that the polymerization was carried out at 0°C for 10 hours.

The volume average particle diameter of the obtained gel was 8.2 μm, and α=4.0 from the calibration curve obtained using a column packed with this gel.
3. A value of Ml im = 3000 is obtained, and further NB
-15600.

Using this packed column, styrene oligomers mainly consisting of heavy bodies were analyzed in the same manner as in Example 1, and a chart (FIG. 5) in which the heavy bodies were separated was obtained.

Example 4 A gel was prepared in the same manner as in Example 3 except that the polymerization was carried out at 70° C. for 10 hours.

The half-life of 2,2'-azobis-(2,4-dimethylvaleronitrile) in toluene at 70°C is 47 minutes.

The volume average particle diameter of the obtained gel was 8.6 μm, and from the calibration curve obtained using a column packed with this gel, a-=4.
7, Ml im = 3800 (7) values were obtained, and NB = 14500 stages.

When styrene oligomers, mainly tetramers, were analyzed using this packed column in the same manner as in Example 1, a chart (FIG. 6) in which heptamers were separated was obtained.

Example 5 In Example 1, the polymerization liquid used was styrene 41.68 divinylbenzene (purity 56%) 31.2P, toluene 69
．． Using a mixture of 8 g, 2,2'-azobis-(2,4-dimethylvaleronitrile) and 0.1' of 2,2'-azobisisobutyronitrile, the mixture was heated at 80°C for 10
A gel was prepared in the same manner as in Example 1, except that the polymerization was carried out for a certain period of time.

The volume average particle diameter of the obtained gel was 83 μm, and α = 3.9 from the calibration curve obtained using a column packed with this gel.
, Mlim=2300 is obtained, and furthermore, NB=
It had 15,100 steps.

Furthermore, using this packed column, styrene oligomers mainly consisting of tetramers were analyzed in the same manner as in Example 1.
The chart (Fig. 7) in which the octamer was separated was forced to slide.

Comparative Example 1 The same procedure as in Example 2 was carried out except that 2,2'-azobisisobutyronitrile was used as the polymerization initiator in Example 1.

The half-life of 2,2'-azobisisobutyronitrile in toluene at 60°C is 17 hours.

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, it was found that a = 6.5, Mlim = 7000, NB = 9300 stages, 4 The analysis chart of the styrene oligomer mainly composed of oligomers was as shown in Fig. 8.

When compared with Char 1 obtained in Example 1 (Fig. 3), the gel of Comparative Example 1 was separated only up to the pentamer, whereas the gel of Example 1 was separated up to the heptamer. This shows how good the separation performance of the gel of the present invention is.

[Brief explanation of the drawing]

Figure 1 shows the molecular weight (
M) and elution volume/column volume ratio (Ve/Vj)
FIG. 2 shows a comparison between the present invention (line A) and the conventional method (line B) regarding the relationship between α and X. In addition, FIGS. 3 to 8 show GPC using columns filled with the gels obtained in Examples 1 to 5 and Comparative Example 1, respectively.
A chart obtained from the analysis is shown.

Claims (1)

[Claims] 1 A small amount (also a kind of monovinyl aromatic monomer 65~
A mixed solution consisting of a total of 100 parts by weight of 90 parts by weight and 35 to 10 parts by weight of at least one type of polyvinyl aromatic monomer and 30 to 200 parts by weight of a solvent capable of dissolving polystyrene is suspended in water to carry out radical polymerization. In doing so,
At least one initiator having a half-life of 2 to 60 minutes at 70°C is used, and the polymerization is carried out at a temperature where the half-life of the polymerization initiator used is in the range of 5 to 120 minutes until the polymerization rate reaches at least 50%. A method for producing a packing material for chromatography, characterized in that: