JPS5845658B2 - Packing material for liquid chromatography - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a packing material for use in liquid chromatographs, particularly gel permeation chromatographs (hereinafter abbreviated as GPC).

Conventionally, GPC, a type of liquid chromatography, has been widely used as a method for rapidly measuring the molecular weight and molecular weight distribution of natural and synthetic polymers. The types of solvents that can be used are limited, and therefore the types of polymers to be measured are also restricted.

Currently, polystyrene gel is most widely used as a packing material for GPC.

Polystyrene gel is a copolymer of styrene and divinylbenzene, and has advantages such as excellent resolution, excellent resolving power over a wide molecular weight distribution, and high mechanical strength. Since it is a molecule and is hydrophobic, it can be applied to a sample solution dissolved in almost any organic solvent, but it cannot be applied to an aqueous solution to measure the molecular weight.

Therefore, various hydrophilic fillers, such as porous silica, porous glass, or crosslinked dextran gel, have been used instead of the styrene gel as fillers to which aqueous samples can be applied.

However, the above-mentioned inorganic fillers such as porous silica and porous glass have high mechanical strength but have low molecular weight separation ability and have a narrow molecular weight separation range. It has the disadvantage that it has low mechanical strength and cannot be used as a packing material for high performance liquid chromatography used under relatively high pressure conditions.

The present invention was made for the purpose of solving the above-mentioned drawbacks of conventional packing materials for GPC, and providing a packing material that can be used with aqueous sample solutions and has high resolution and a high number of theoretical plates. It is.

That is, the gist of the present invention is that the general formula CH2=C-C-0(-CH2-CH2-0)nC-C
100 parts by weight of a compound represented by =CH2111 100R2 (in the formula, R,, R2 is hydrogen or a methyl group, and n is an integer of 3 to 18) and X methylolalkyl y (meth)acrylate (in the formula, X and y are integers) and X≧y≧3) is subjected to aqueous suspension polymerization in the presence of an organic solvent in which the mixture is dissolved but the polymer is not dissolved. It consists in a filler for liquid chromatography made of polymer.

The compound represented by the general formula % used in the present invention is a glycol (meth) in which R1 and R4 are hydrogen or a methyl group, and n is an integer from 3 to 18.
Acrylic acid esters, such as triethylene glycol diacrylate, triethylene glycol dimethacrylate, tetraethylene glycol diacrylate, tetraethylene glycol dimethacrylate, nonaethylene glycol diacrylate, nonaethylene glycol dimethacrylate, tetradecaethylene glycol diacrylate , tetradecaethylene glycol dimethacrylate, and the like.

The compound represented by the above general formula is highly hydrophobic when n is 1 or 2 and cannot be applied to hydrophilic samples, and when n is 19
If the particle size is larger, the particles become softer and lack mechanical strength as a filler for GPC, so n is limited to 3 to 18, preferably 4 to 14.

The X methylolalkyl y (meth)acrylate used in the present invention has the formula, where X and y are positive integers, and X≧y≧
3 and having at least three methylol groups and three or more acrylate or methacrylate groups in the molecule, such as tetramethylolmethanetetraacrylate, tetramethylolmethanetetramethacrylate, tetramethylolmethanetriacrylate,
Tetramethylolmethane trimethacrylate, trimethylolmethane triacrylate, trimethylolmethane trimethacrylate, trimethylolpropane triacrylate, trimethylolpropane trimethacrylate, etc. Tetramethylolmethane tetraacrylate, tetramethylolmethane triacrylate and tetramethylolmethane L; Rimeku acrylate is preferably used.

The compound represented by the general formula and the methylolalkyl y(meth)acrylate are made into a mixture, but if the amount of X methylolalkyl y(meth)acrylate added is small, the resulting porous polymer becomes soft. If the amount is too large, phenomena such as adsorption and distribution will occur and the resolution will be reduced. Therefore, it is added in an amount of 5 to 60 parts by weight per 100 parts by weight of the compound represented by the above general formula, and preferably 1
It is 0 to 40 parts by weight.

In the present invention, aqueous suspension polymerization is carried out in the presence of an organic solvent that dissolves the mixture but does not dissolve the polymer. Organic solvents can be used, such as aromatic hydrocarbons such as toluene, xylene, diethylbenzene, and dodecylbenzene, hexane,
Examples include saturated hydrocarbons such as heptane, octane, and decane, and alcohols such as isoamyl alcohol, hexyl alcohol, and octyl alcohol.The amount added is not limited in any way, but 15 to 15% of the mixture is added.
Preferably, 200% by weight is used, more preferably 20-150 parts by weight.

The organic solvent is uniformly dissolved in the mixture, and since the mixture is subjected to aqueous suspension polymerization, it exists dispersed in the resulting polymer particles, and after the polymerization is completed, the organic solvent is removed from the particles. By removing it, a porous polymer is obtained.

Therefore, the size of the pores of the porous polymer can be arbitrarily changed by using various organic solvents having different compatibility with the above mixture.

Any known method can be used for aqueous suspension polymerization, for example, the mixture and radical generating catalyst are dissolved in the organic solvent, and the resulting solution is mixed with suspension polymerization stabilizer such as polyvinyl alcohol or calcium phosphate. This is carried out by adding the agent to the aqueous phase in which it is dispersed, stirring and then heating to 50-100°C.

The above radical generating catalyst is a catalyst that generates radicals as a reaction initiator, and examples of the catalyst include organic peroxides such as benzoyl peroxide and cumene peroxide, and inorganic peroxides such as hydrogen peroxide, potassium persulfate, and ammonium persulfate. Any known radical generating catalyst can be used, such as peroxide, azo compounds such as abisisobutyronitrile, and azobisisobutyramide.

The polymer particles polymerized by the above-mentioned aqueous suspension polymerization are dried by heating or the like to release the organic solvent in the particles, thereby forming a porous polymer, which is used as a filler for liquid chromatography.

In order to be used as a packing material for liquid chromatography, the particle size of the porous polymer is preferably uniform and in the range of 3 to 40 μm, and although the pores vary depending on the particle size, generally 50 μm toward the inside of the particle. ˜2000 angstroms is preferred.

The structure of the present invention is as described above, and the obtained filler contains a compound represented by the above general formula and
Since it is a polymer of (meth)acrylate, it necessarily has many hydrophilic groups and many hydrophobic groups in its molecule, so when it is used as a packing material for liquid chromatography, it has a strong resistance to hydrophilic polymers. It has good molecular weight resolution even for hydrophobic polymers.

In addition, since the compound represented by the above general formula and X methylolalkyl y (meth)acrylate both have a plurality of acrylate groups, they form a three-dimensional network crosslink when polymerized, so the mechanical strength of the particles is large, making them particularly suitable as fillers for GPC. It is.

Furthermore, since the packing material for liquid chromatography of the present invention is manufactured by aqueous suspension polymerization in the presence of an organic solvent, it has many pores in the fine particles that are the packing material, and has a high molecular weight resolution. By changing the organic solvent, pores of any size can be formed, and by changing the polymerization conditions, particles of any size can be obtained, making it possible to produce a packing material that can be applied to any liquid chromatograph. It is effective for

Next, the present invention will be explained in detail.

Example 1 2 equipped with a cooler, stirrer, thermometer and dropping funnel
1. In a separable flask, 400 ml of 4% by weight polyvinyl alcohol aqueous solution, 80 g of tetraethylene glycol dimethacrylate, and 21.0 g of tetramethylolmethane triacrylate. ) A mixed solution consisting of 100 g of toluene and 1.5 g of benzoyl peroxide was fed.

Next, the mixture was heated to 80°C while stirring at a stirring speed of 40Or, p, m.
The temperature was raised to 1, and a polymerization reaction was carried out for 10 hours, followed by cooling.

After cooling, the polymerization product was separated from the mother liquor and washed with hot water and acetone to obtain a porous polymer having a particle size of 5 to 13 μm.

35 TLl of a liquid chromatography filler with a particle size of 8 to 10 μm obtained by removing fine particles and coarse particles from the porous polymer obtained was dispersed in 80 ml of distilled water, and GP
C stainless steel column diameter 7.9mm, length 50cfr
Pour distilled water into L) using a high-pressure constant flow pump at 1.6 + 711
Filling was performed by forward feeding at a speed of /min.

The obtained packed column was packed in a high-performance liquid chromatograph (manufactured by Shimadzu Corporation, trade name: Shimadzu DuPont High-Performance Liquid Chromatograph 8).
30 type) and measured the molecular weight using standard dextran and polyethylene glycol with different molecular weights as samples and using distilled water as the eluent, it had good molecular weight resolution at molecular weights of 300,000 or less, and each peak was normal. It had a symmetrical shape.

The number of theoretical plates per column measured using ethylene glycol was 6000 plates and 10.5 doors.

Further, 351 rLl of a liquid chromatography filler with a particle size of 9 to 111 Lm obtained by removing fine particles and coarse particles from the porous polymer was dispersed in 80 ml of distilled water, and G
Sude stainless steel column for PC diameter 7.9mm, length 50cr
rL) was filled with tetrahydrofuran by force-feeding it at a rate of 2.0 ml/min using a high-pressure constant flow pump.

Using the obtained packed column and using monodispersed polystyrene samples with different molecular weights as samples, the molecular weights were measured in the same manner using tetrahydrofuran as the eluent, and it was found that it had good molecular weight resolution at molecular weights of 250,000 or less.

The number of theoretical plates per column measured using acetone was 4000 plates and 10.5 doors.

Example 2 Aqueous suspension polymerization was carried out in the same manner as in Example 1, except that a mixed solution consisting of 80 g of triethylene glycol dimethacrylate, 20 g of tetramethylolmethane tetraacrylate, 100 g of toluene, and 1.5 g of benzoyl peroxide was used, A porous polymer having a particle size of 4 to 13 μm was obtained.

35 rfLl of a liquid chromatography filler with a particle size of 5 to 7 μm obtained by removing fine particles and coarse particles from the porous polymer obtained was dispersed in 80 TrLl of distilled water.
Sude stainless steel column for PC diameter 7.9mm, length 50cr
Add 2.0 ml of distilled water to rL) using a high-pressure constant flow pump.
Filling was performed by forward feeding at a speed of /min.

The obtained packed column was connected to the high performance liquid chromatograph used in Example 1, and the molecular weights were measured using standard dextran and polyethylene glycol with different molecular weights as samples and distilled water as the eluent. It had molecular weight resolution ability.

The number of theoretical plates per column measured using glucose was 5000 plates, 10.5 plates.

Example 3 Aqueous suspension polymerization was carried out in the same manner as in Example 1, except that a mixture consisting of 65 g of nonaethylene glycol dimethacrylate, 31.1-toluene, 100 g of tetramethylolmethane tetraacrylate, and 1.5 g of pensoyl peroxide was used. A porous polymer having a particle size of 5 to 20 μm was obtained.

A filler for limb chromatography 35TLl with a particle diameter of to-15 μm obtained by removing fine particles and coarse particles from the porous polymer obtained was dispersed in distilled water 807711,
Sude stainless steel column for GPC diameter 7.9mm, length 50cm
IIL), add distilled water to L5rrt using a high-pressure constant flow pump.
It was filled by forward feeding at a rate of 1/min.

Using the obtained packed column, the molecular weight was measured in the same manner as in Example 1 using standard dextran and polyethylene glycol with different molecular weights as samples and distilled water as the eluent. It had molecular weight resolution ability.

The number of theoretical plates per column measured using ethylene glycol was 4000 plates/0.5 m.

Example 4 An aqueous suspension was prepared in the same manner as in Example 1, except that a mixed solution consisting of tetraethylene glycol dimethacrylate soy, 20 g of tetramethylolmethane trimethacrylate, isoamyl alcohol soy, 20 g of diethylbenzene, and 1.5 g of benzoyl peroxide was used. Turbid polymerization was performed to obtain a porous polymer with a particle size of 5 to 15 μm.

Filler for liquid chromatography with a particle size of 8 to 10 μm obtained by removing fine particles and coarse particles from the porous polymer obtained! 35 m7! Dispersed in 80TLl of distilled water, GP
Sude stainless steel column diameter 7.9mm for C.

Distilled water was pumped into the tube (length 50 cIft) at a rate of 2.0 ml/min using a high-pressure constant flow pump to fill the tube.

Using the obtained packed column and using standard dextrans with different molecular weights as samples and distilled water as the eluent, the molecular weights were measured in the same manner as in Example 1. As a result, good molecular weight resolution was found for molecular weights of 700,000 or less. Was.

The number of theoretical plates per column measured using glucose was 5,500 plates and 10.5 m.

Comparative example l 80 g of diethylene glycol dimethacrylate, 20 g of tetramethylolmethane triacrylate, 1 toluene
Aqueous suspension polymerization was carried out in the same manner as in Example 1 except that a mixed solution consisting of 00 g and 1.59 g of benzoyl peroxide was used to obtain a porous polymer having a particle size of 4 to 20 μm.

A liquid chromatography filler 35TLl having a particle size of 6 to 9 μm obtained by removing fine particles and coarse particles from the porous polymer obtained was subjected to GP treatment in the same manner as in Example 2.
A stainless steel column for C was loaded, polyethylene glycol was used as a sample, and the amount of separated particles was measured in the same manner as in Example 2, but polyethylene glycol did not elute and could not be measured.

Comparative Example 2 Tetraethylene glycol dimethacrylate tooy,
Aqueous suspension polymerization was carried out in the same manner as in Example 1, except that a mixed solution consisting of toluene toog and 1.5 g of benzoyl peroxide was used, and the particle size was 5 to 25 μm.
A porous polymer was obtained.

Fine particles and coarse particles were removed from the obtained porous polymer to obtain liquid chromatography filler A having a particle size of 8 zlO μm and liquid chromatography filler B having a particle size of 15 to 20 μm.

Filler A357rLl was dispersed in 80ml of distilled water, and GP
When distilled water was fed forward at a rate of 0.5 ml/min using a stainless steel column ball constant flow pump for C, it became clogged and could not be filled.

Filler B was filled in the same manner except that it was pumped at a rate of 1.6 ml/min, and standard dextran was used as a sample in Example 2.
The molecular weight was measured in the same manner as in , but the peak was asymmetric and could not be measured.

Also, using nonaethylene glycol dimethacrylate instead of tetraethylene glycol dimethacrylate, aqueous suspension polymerization was carried out in the same manner to obtain a porous polymer with a particle size of 8 to 23 μm, and an attempt was made to fill the column in the same manner, but the polymer It was too soft to fill.

Comparative Example 3 Same as in Example 1 except that a mixed solution consisting of 50 g of tetraethylene glycol dimethacrylate, 1-50 g of tetramethylolmethane trimethacrylate, 80 g of isoamyl alcohol, 21 g of badiethylbenzene, and 1.5 g of benzoyl peroxide was used. particle size 5-1
A porous polymer of 5 μm was obtained.

A packing material for limb chromatography with a particle size of 7 to 10 μm obtained by removing fine particles and coarse particles from the porous polymer obtained was packed in a stainless steel column for GPC in the same manner as in Example 4, Polyethylene glycol was used as a sample and the molecular weight was measured in the same manner as in Example 2, but polyethylene glycol did not elute and could not be measured.

Claims (1)

[Scope of Claims] 1 100 parts by weight of a compound represented by the general formula; ) acrylate (wherein x, y are integers and A packing material for liquid chromatography made of a porous polymer obtained by suspension polymerization. 2. The filler according to claim 1, wherein n of the compound represented by the general formula is 4 to 14. 3 x methylol alkyl y (meth)acrylate is 1
The filler according to claim 1, which is 0 to 30 parts by weight.