JPH0425762A - Production of copolymer particle for reversed phase chromatography and column for reversed phase chromatography - Google Patents

Abstract

PURPOSE:To allow aq. suspension polymn. by respectively specifying the specific molar ratios of specific compds. CONSTITUTION:The compd. having at least the two groups expressed by formula I, the compd. expressed by formula II (where l is 1 to 30) and the compd. expressed by formula III (where k is 6 to 24) are used. The compds. I, II, III are used at the use ratios specified in such a manner that the molar ratio of II/I+II+III attains a 0.02 to 0.3 range in order to maintain hydrophilicity and the molar ratio of III/I+II+III attains 0.07 to 0.5 in order to maintain separabili ty. The aq. suspension polymn. is progressed for 6 to 10 hours usually at 70 to 90 deg.C in the presence of a polymn. initiator and dispersant. The aq. suspension polymn. is effected in this way.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for producing copolymer particles for reversed phase chromatography and a column for reversed phase chromatography.

(Prior art) Conventionally, as a separation method of liquid chromatography,
Reversed phase partition chromatography is known, which separates samples based on the strength of retention due to differences in hydrophobicity of the sample. The following are known as column packing materials commonly used in reversed phase liquid chromatography.

(1) Chemically bonded silica gel in which the surface of silica gel is chemically modified with a silane coupling agent having an octadecyl group, etc. [GA Kirkland.

Journal of Chromatographic Satisfaction, by J.J. Destefano
of Chromatography 5science
) Volume 8, page 309, 1970].

(2) Styrene-divinylbenzene copolymer particles [M.D. Greaser, D.G. Beetzak, Analytical Chemistry
al Chemistry, Vol. 45, p. 1383, 1973].

f3) Methacrylic acid ester copolymer particles having a long fi alkyl chain (JP-A-62-90533).

(Problems to be Solved by the Invention) Silica gel has the disadvantage that it is sensitive to alkalis and cannot be used in all pH ranges because the pH of the eluent is limited to a range of 2 to 8. It is difficult to chemically bond a camping agent to a silanol group, and since unreacted silanol remains, there is a problem that basic samples are adsorbed.

However, although the above-mentioned styrene-divinylbenzene and methacrylic ester copolymers solve the above-mentioned problems with silica gel, they swell and contract violently between the various solvents, making it difficult to use the eluent in the column. There is a problem in that it is difficult to change the measurements every time.

The present invention solves these problems and provides acid resistance.

The object of the present invention is to provide an efficient method for producing copolymer particles useful as a column packing material for reversed-phase partition chromatography, which have alkali resistance and have small differences in swelling and shrinkage between various solvents.

(The invention seeks to solve! l11 [) The present invention solves (a
) A compound having at least two groups represented by the general formula (1) %formula% (in the formula, R1 represents H or CH) in one molecule.

(b) A compound represented by general formula (II) (wherein R12 and R3 each independently represent H or CH3'j&:, and I! represents an integer of 1 to 30) and (C1 general formula @ ) +x to H2C=C-CO-CkH*
@) (In the formula, R4 represents H or CH3, and is 6 to 2
represents an integer of 4) as (bl/ [: (a)-Nb)
+(c)) is the molar ratio o, 02~0.3, (c
)/C(a)+(bl+(e))] is 0.0 in molar ratio
Water-based so that it is in the range of 7 to 0.5! I! ! The present invention relates to a method for producing copolymer particles for reversed phase chromatography, which are characterized by turbid polymerization. Another aspect of the present invention relates to a column for reversed phase chromatography filled with copolymer particles obtained by the above production method.

(a) A compound having at least two groups represented by the general formula (J) in one molecule used in the present invention.

Already known ones can be used. Among these, polyvinyl esters of polyhydric alcohols and polyvinyl ethers of hypopolyhydric alcohols are preferred.

Examples of polyvinyl esters of polyhydric alcohols and polyvinyl ethers of polyhydric alcohols include 9.

Polyalkylenes such as ethylene glycol dimethacrylate, ethylene glycol diacrylate, propylene glycol dimethacrylate, alkylene glycol divinyl esters of propylene glycol diacrylate, polyethylene glycol dimethacrylate, polypropylene glycol dimethacrylate, polyethylene glycol diacrylate, polypropylene glycol diacrylate, etc. Divinyl esters of glycols, di- or triacrylates of glycerin, di- or trimethacrylates of glycerin, di- or trimethacrylates of trimethylolpropane, di- or triacrylates of trimethylolpropane, di- or tetramethacrylates of tetramethylolmethane, di- or trimethacrylates of tetramethylolmethane. Di-, tri- or tetra-acrylates, ethylene glycol diallyl ether, propylene glycol diallyl ether, polyethylene glycol diallyl ether, polypropylene glycol diallyl ether, di- or triallyl ether of glycerin.

di- or triallyl ether of trimethylolpropane,
Examples include ditri- or tetraallyl ether of tetramethylolmethane, but polymethacrylic acid ester of polyhydric alcohol is preferred in view of monoreactivity and pressure resistance of the produced copolymer particles for reversed phase chromatography.

Among these, ethylene glycol dimethacrylate, tetramethylolmethane ditrimethacrylate or tetramethacrylate are more preferred. These (a) component compounds are:
You can use one or more types of food.

(b) Ethylene glycol chain of the compound represented by general formula (II) used in the present invention (1000Hz CHx O→
r) is necessary to impart hydrophilicity to the copolymer particles for reversed phase chromatography, and for this reason, the general formula (If
) If l is 0, hydrophilicity cannot be imparted.

If B has poor resolution and t is 31 or more, it will be difficult to perform one synthesis, making it unsuitable for practical use.

(b) Examples of the compound represented by the general formula (I[) include monoacrylates and monomethacrylates of ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, nonaethylene glycol, and tricosane ethylene glycol. These compounds as component (b) can be used alone or in combinations of 2 or more.

The aliphatic chain (-CkHzk +
In the following cases, the separation ability becomes poor, and when the number of atoms per gram of carbon is 25 or more, one synthesis becomes difficult and is not suitable for practical use.

fe) Examples of the compound represented by the general formula (III) include 1.

Examples include methacrylic esters, acrylic esters, and aliphatic ethers of aliphatic alcohols having 6 to 24 carbon atoms. Considering reactivity, alkali resistance, and easy industrial availability, stearyl methacrylate and lauryl methacrylate are preferred. These (C) component compounds can be used alone or in combination of two or more.

In the present invention, when the proportion of components (&), (b) and (C) is too small, hydrophilicity will be impaired and the separation performance will be poor. (b)/C(al+(b)+
(c) The molar ratio of ) is 0.02 to 0.3. It is preferably used in a range of 0.05 to 0.2.

In addition, (if the C1 component is too small, the separation function of reversed phase partitioning will deteriorate and the separation performance will deteriorate.fe)/[f&)+(b)
The molar ratio of C1) is 0.07 to 0.5. Good luck 0.
It is used in a range of 1 to 0.3.

fan, (b) and fe) Components Fi are subjected to aqueous suspension polymerization, if necessary, in the presence of an organic solvent incompatible with water to form crosslinked polymer particles.

In aqueous suspension polymerization, suspension polymerization is carried out in an aqueous medium, and this aqueous medium requires water, and
Water in which a water-soluble organic solvent is dissolved may be used within a range that does not impede the stability of the suspension system.

Aqueous suspension polymerization is carried out in the presence of a polymerization initiator.

As a polymerization initiator, benzoyl peroxide, dichlorobenzoyl peroxide, dicumyl peroxide, di-tert-butyl peroxide, 2.5-di(peroxybenzoate) hexyne-3
, 1,3-bis(M 3 phthylberoxyisopropyl)benzene, lauroyl peroxide, tert-butyl peracetate,
λ5-dimethyl-45-di(tert-butylperoxy)hexane-3,2,5-dimethyl-2,5-di(tert-butylperoxy)hexane and tert-butyl perbenzoate, methyl ethyl ketone peroxide, methyl cyclohexanone peroxide, etc. Examples include organic peroxides, azo compounds such as azobisisophthyronitrile, and dimethylazodiin butyrate. One or more types of these can be used. The amount to be used is appropriately determined depending on the type of monomer, etc., but is preferably 0.1 to 4.
0 weight s1!2 used.

Aqueous suspension polymerization is carried out in the presence of a dispersant. Examples of dispersants include polyvinyl alcohol, water-soluble cellulose derivatives such as alkyl cellulose, hydroxyalkyl cellulose, and carboxyalkyl cellulose, and polymeric protective colloids such as sodium polyacrylate. The dispersant is preferably used in an amount of 1 to 0.001% by weight based on water.

An anionic surfactant may be added to the polymerization system as a dispersion aid if necessary to control particle size, etc.

A water-soluble inorganic salt can be added to the polymerization system to reduce the solubility of monomers or organic solutions that are incompatible with water.

(a), fb) and fc) Components: Compound 1 Dispersant 1 Polymerization initiator and dispersion aid used as necessary, organic solvent incompatible with water used as necessary, etc.

These are mixed in advance or added individually to an aqueous medium and dispersed. In this case, in order to disperse well, it is preferable to perform high-speed stirring (3000 to 8000 rPm) using a homomixer or the like, and this high-speed stirring can be continued until the initial stage of double polymerization. The subsequent polymerization is carried out under normal stirring (10 to 1000 rpm) using a propeller stirrer or the like. The aqueous medium is preferably used in an amount of 1 to 50 times the weight of the components (a), (b), and (e), and the total amount of the water-incompatible organic solvent used as necessary. In this case, water is used as the aqueous medium, but!
! ! Water containing a water-soluble organic solvent dissolved therein within a range that does not impede the stability of the turbid system may be used.

By adding an organic solvent that is incompatible with water during polymerization, the produced copolymer particles for reversed phase chromatography can be made porous.

The organic solvent used in the present invention is water-incompatible.

The amount of lubrication dissolved in 100 g of water at 25° C. is 15 g or less, such as toluene, diethylbenzene, dodecane, isoamyl alcohol, chlorobenzene, ethyl acetate, butyl acetate, butyl acetate, and cellosolve acetate. These solvents may be used alone or
Two or more types may be mixed and used. These usage amounts are
0 to total monomer amount (t8)+(b)-He))
Preferably, 300% by weight is used. If the amount of organic solvent that is incompatible with water is small, it becomes difficult to make the resulting particles porous.

If the amount is too large, the porosity of the resulting particles will increase and the pressure resistance will tend to become poor.

The polymerization reaction is usually carried out at a temperature range of 70 to 90°C.

Allow to proceed for 6-10 hours.

The nine particles obtained by K as described above have a diameter of 1 to 200 μm.

The copolymer particles for reversed phase chromatography, preferably spherical particles of 1 to 15 μm, can be classified as necessary and used as a column packing material for reversed phase chromatography.

(Example) Next, the present invention will be explained by examples.

Example 1 In the present invention (1409° of tetramethylolmethane trimethacrylate as a compound of the Kl component (19.5 g of polyethylene glycol methacrylate as a compound of the BL component, 40.5 g of stearyl methacrylate as a compound of the (e) component) ((b)/C(a
) + (bl + (c) ) = 0.07 (% le ratio)
, (C)/ [(aj+(b) ten(c))=0.2
1 (molar ratio)), acetic acid n as an organic solvent insoluble in water
-Butyl 1009. 100g of cellosolve acetate and 1.0g of 7zobisisobutyronitrile as a polymerization initiator
g mixture into 0.1% by weight aqueous methylcellulose solution Z
Suspend in l/l.

Under high speed stirring (4000 rpm) using a homomixer
.

Mix for about 10 minutes at room temperature until the particle size of the oil droplets is 1-100 μm.
I adjusted it so that After that, it was transferred to an ordinary propeller stirring device and heated to 80℃ while stirring (200 rpm).
By reacting for a period of time, copolymer particles for reverse phase chromatography of the present invention which are insoluble in water and organic solvents were obtained. The particles were filtered, washed with water at 21° C. and methanol at 21° C., and then air-dried (yield: 95%). The obtained particles were classified to have an average particle size of 6 μm, and filled in a stainless steel column with a diameter of 6.0φ×150 mm to prepare a column. Using this column, ■ acetonium as eluent) IJ/water = 70/30
FIG. 1 shows a chromatogram in which uracil and benzoic acid esters were separated using (V/V). ■Next, using the same column, the eluent was methanol/water = 80
Figure 2 shows a chromatogram in which U5 and benzoic acid esters were separated using 20 (V/V) Kf. ■Next, use Na2CO3 at pH 11 as the eluent.
-HCI buffer was passed for 250 hours, and then the chromatogram was measured in the same manner as in (2) above, and the chromatogram is shown in FIG.

■Next, after passing a 0.1% h-lifluoroacetic acid/acetonitrile = 80/20 (V/V) solution at pH 1O as an eluent for 250 hours, the chromatogram measured in the same manner as in the above (■) is shown in Figure 4. It was shown as

Differences in eluent (■ and ■), alkali and acid (■ and ■)
When swelling and contraction 9 decomposition occurs due to.

The pressure difference between the columns becomes large, or the separation ability deteriorates, resulting in a broad chromatogram, but in any case, there is almost no deterioration in performance when the copolymer particles of the present invention are used in nine columns, and changing the eluent is not necessary. It was found that the swelling and shrinkage caused by oxidation were small, and that it had excellent alkali resistance and acid resistance.

Example 2 120 g of tetramethylolmethane trimethacrylate,
53.2 g of polyethylene glycol methacrylate and 26.89 g of stearyl methacrylate were used (
b) / [(&) + (b) + (C) ) = 0.20
C molar ratio), (c)/C(a)+(b)+(c
Example 1 except l ) = 0.1 s (molar ratio))
The process from polymerization reaction to filling was carried out in the same manner as above.

Acetonitrile/water = 70/30 (V/V) as eluent
FIG. 5 shows a chromatogram separating uracil and benzoic acid esters when using T-.

The acid resistance, alkali resistance, and solvent compatibility were excellent as in Example 1.

Example 3 Tetramethylolmethane trimethacrylate 1209,
Polyethylene glycol methacrylate 26.49 and stearyl methacrylate 53.6 gtl! 'use*
((b)/[(1k)-Hbl+(e)) = 0
．． 10 (molar ratio), (cl/ [(al+(bl
+ (cl) = 0.28 <molar ratio)) The same procedure as in Example 1 was carried out from the polymerization reaction to filling.

Acetonitrile/water = 70/30 (V/V) as eluent
A chromatogram separating uracil and benzoic acid esters is shown in Figure 6.

The acid resistance, alkali resistance, and solvent compatibility were excellent as in Example 1.

Comparative Example 1 Tetramethylol trimethacrylate 159.59 and stearyl methacrylate 40.59 were used (
b)/C(a)+(b)+((ri) = 0 (molar ratio), ((ri/((&++(b)/fc)) =
The procedure from polymerization reaction to filling was carried out in the same manner as in Example 1, except for 0.2 (molar ratio)).

fg Syneresis K) p/-L/Water = 80/20 (v/V)
The separation chromatogram of uracil and benzoic acid esters when using this method is shown in Figure 7.

The copolymer particles obtained here are outside the scope of the present invention.

It has a high degree of hydrophobicity and strongly adsorbs highly hydrophobic samples, so it has poor practicality. Unfortunately, the chromatogram was broad due to strong adsorption.

Comparative Example 2 120 g of tetramethylolmethane trimethacrylate,
80 g of polyethylene glycol methacrylate was used ((b)/((&++(b)+(el) =
0.67 (molar ratio), (C)/C(a)+(b)
+ (c)] = 0 (molar ratio))
The process from polymerization reaction to filling was carried out in the same manner as J1.

When acetonitrile/water = 70/30 (V/V) was used as the eluent, uracil and benzoic acid esters were separated, and romatodalum was shown in FIG. 8.

It had no adsorption ability and its resolution was extremely poor.

In the above, the chromatographic conditions were as follows.

Furthermore, in all cases.

Flow rate 1.0-/ "i" *Sample amount 20 pl Sample: Uracil 25 μg/me (1 in the figure) Methyl benzoate as benzoate esters 0.25 μ//me Butyl benzoate 0.50 μl/mt Hexyl benzoate 0 .75 pi/rnt detector UV (
254 nm) (2 in the figure) (3 in the figure) (4 in the figure).

(Effects of the invention) The copolymer particles for reversed phase chromatography obtained by the present invention have excellent acid resistance and alkali resistance, and have small differences in swelling and shrinkage between different solvents, allowing use of a wide range of eluents. It's an excellent one.

It is useful as a column packing material for reversed phase partition chromatography.

[Brief explanation of drawings]

Figures 1, 2, 3, and 4 show the chromatographs measured under specific conditions using the column in Example IK, and Figure 5 shows the chromatographs measured using the column in Example 2. gram, Figure 6. 7 is a chromatogram measured using the column in Example 3, a chromatogram measured using the column in Comparative Example 1, and FIG. 8 is a chromatogram measured using the column in Comparative Example 1. This is a chromatogram measured using the column in Comparative Example 2. Explanation of symbols 1...Uracil 2...Methyl benzoate 3...Butyl benzoate 4...Hexyl benzoate Elution time (min) Figure 1 Elution time (min) Figure 2 Elution time (min) Figure 3 Elution time (min) Figure 4 Elution time (min) Figure 5 Elution time (min) Figure 6 Elution time (min) Figure 7 Elution time (mxn) Figure 8

Claims (1)

[Claims] 1. (a) General formula (I) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) (In the formula, R^1 represents H or CH_3) One molecule of the group represented by A compound having at least two of them. (b) General formula (II) ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (II) (In the formula, R^2 and R^3 each independently represent H or CH_3, and l is an integer from 1 to 30. (represents an integer) and (c) general formula (III) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(III) (In the formula, R^4 represents H or CH_3, and k is 6 to 24
(display the integer of )
] is 0.02 to 0.3 in molar ratio, (c)/[(a) + (
A method for producing copolymer particles for reversed phase chromatography, characterized in that aqueous suspension polymerization is carried out such that the molar ratio of [b)+(c)] is in the range of 0.07 to 0.5. 2. The method for producing copolymer particles for reversed phase chromatography according to claim 1, wherein the compound represented by formula (III) (2(c)) is lauryl methacrylate or stearyl methacrylate. 3. A column for reversed phase chromatography packed with copolymer particles obtained by the production method according to claim 1 or 2.