JPS5927865B2 - Packing material for high performance liquid chromatography - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a filler used for high performance liquid chromatography.

Liquid chromatography can be classified from the viewpoint of substance separation mechanism into adsorption chromatography, partition chromatography,
It is broadly classified into gel immersion chromatography, reversed phase partition chromatography, ion exchange chromatography, etc.

The substances to be separated are all difficult-to-volatile substances, which can be broadly classified into inorganic substances and organic substances, and organic substances are further classified into hydrophilic substances and hydrophobic substances. Next, if we classify commercially available packing materials for high-performance liquid chromatography that separate hydrophilic substances based on their separation mechanism, they are classified into packing materials for gel immersion chromatography, packing materials for reversed-phase chromatography, packing materials for ion-exchange chromatography, etc. .

Here, high-performance liquid chromatography is a general term for liquid chromatography having functions such as pumping an eluent using a high-pressure constant flow pump or the like and detecting the separation state using a purple line detector or the like.
However, the packing material for gel immersion chromatography has pores inside it, and the diffusion of the sample into the pores causes a delay in the elution time, and the difference in this lag time causes a delay in the elution time. It has a separation mechanism in which separation is performed by

Hydrophilic packing materials for high-speed gel immersion chromatography are commercially available from several companies. Furthermore, ion exchange chromatography has a mechanism in which the elution time is delayed due to electrostatic interaction between the stationary phase surface having a dissociable substituent and a sample having an opposite charge.

It is also commercially available from several companies as a packing material for high performance ion exchange liquid chromatography. In addition, in reversed-phase chromatography, the sample is retained on the stationary phase due to the interaction between the hydrophobic surface of the stationary phase and the hydrophobic groups of the sample, so the more hydrophobic the sample, the longer the elution time. .

A spherical filler made of a styrene-divinylbenzene crosslinker is generally used as a filler for gel immersion chromatography, but it can also be used as a filler for reversed phase chromatography. All of the above-mentioned fillers have hydrophobic properties, and it is impossible to use water without an organic solvent as an eluent; therefore, they are not suitable for the purpose of separating hydrophilic substances. Often appropriate.

As a packing material for high-performance liquid chromatography to separate such hydrophilic substances, a silica gel carrier with long-chain alkyl groups chemically bonded is known. What about interactions based on hydrophobicity with non-polar substances? Two intertwined separation mechanisms, the effect of prolonging the separation time and the adsorption effect between the sample and silanol groups, i.e. the effect of prolonging the elution time of polar substances, work simultaneously, so that each component in the sample is It is difficult to predict the separation behavior, and if only water is used as the separation liquid, the separation ability will decrease and it will be difficult to separate each component, so it is not sufficient for the separation of biochemical substances such as proteins. It is difficult to say that it has.

As mentioned above, as a conventional packing material for high performance liquid chromatography, there has not yet been provided one that has high performance in applications aimed at separating hydrophilic substances, particularly biochemical substances. In view of the current state of packing materials for high-performance liquid chromatography as described above, the present invention is a high-performance liquid chromatograph that is effective in separating hydrophilic substances and that can accurately separate samples containing only water as a liquid eluent. This study was made as a result of intensive research aimed at providing a filler for fillers, and the gist of the study was to develop a polymer or copolymer whose main component is tetramethylolmethane triacrylate, and a copolymer of hydroxymethyl methacrylate and styrene. A granular filler made of a synthetic polymer selected from polymers, which is used in chromatography using polyethylene glycol as a sample and water as an eluent, increases the sample exfoliation time due to temperature rise. The present invention relates to a packing material for high performance liquid chromatography, which is characterized in that it exhibits behavior.
The packing material for high performance liquid chromatography of the present invention is a granular material made of a synthetic polymer, and when the granular synthetic polymer is used as a packing material, water is eluted from polyethylene glycol as a sample. This behavior shows that in high performance liquid chromatography, when the column temperature increases, the sample separation time increases.

For example, when the column temperature is kept at room temperature (20℃) and when the column temperature is kept at room temperature (20℃),
The exfoliation time is longer when the temperature is kept at 50°C than when it is kept at 50°C. In this way, the packing material of this invention has two characteristics: water can be used as an eluent, and the dissolution time of the sample becomes longer as the column temperature increases. A packing material for high performance liquid chromatography that has both of these two characteristics has not been found in the past, and the present invention is the first to find a packing material for hydrophilic substances, especially biochemical substances such as proteins, amino acids, and enzymes. It was discovered that it is effective for the separation of Also, what is the temperature of water when separating samples using this packing material? It is preferable to adopt a temperature range suitable for use as syneresis, usually a temperature range of 0 to 85°C. The filler of the present invention has two characteristics as described above. Of these, water can be used as the eluent, in order to do this, the synthetic polymer constituting the filler has a hydrophilic group that has an affinity for water, and the surface of the filler is water-resistant. Furthermore, regarding hydrophilic groups, dissociative substituents such as carboxyl groups and amino groups have the effect of imparting hydrophilicity, but the presence of these substituents causes problems in chromatography. During implementation, the separation behavior becomes complicated due to the ion exchange mechanism.
In addition, inorganic hydrophilic groups such as silanol groups also have a strong adsorption mechanism during chromatography, which complicates the separation behavior, so these dissociative substituents and inorganic hydrophilic groups are inappropriate. It has been found that an organic non-dissociable hydrophilic group that can provide a hydrophilic effect without ion exchange mechanism or adsorption mechanism is suitable.

Examples of the non-dissociable hydrophilic group include an alcoholic hydroxyl group, an amide group, an engineered tylene oxide group, and a propylene oxide group. Furthermore, since the elution time of the sample becomes longer with increasing temperature, it can be concluded that most of the separation effect of the packing material of the present invention is based on hydrophobic interaction.

There is no known example to date in which a filler exhibits the above behavior in reverse phase chromatography using water as a eluent. Note that hydrophobic interactions are different from interactions that are mainly stabilized by bond energy such as electrostatic bonds or hydrogen bonds, and are statistical thermodynamic interactions whose main effect is an increase in entropy. In order to cause such interaction, it is necessary that hydrophobic groups such as alkyl groups, phenyl groups, and alkylene groups exist in the synthetic polymer constituting the filler.For this reason, the present filler It is necessary for the synthetic polymer 1 to contain at least both the above-mentioned non-dissociable hydrophilic group and all the above-mentioned hydrophobic groups in the molecule.

In producing the filler of the present invention, known water suspension polymerization techniques are most suitable.

Polymerized particles can be obtained by subjecting a monomer having the non-dissociable monohydrophilic group and the aforementioned hydrophobic group in its molecule to suspension polymerization in water. A specific monomer is tetramethylolthane triacrylate, and this monomer is used as a homopolymer or as a copolymer containing 50% or more of the monomer. . Alternatively, hydroxymethyl methacrylate may be used as the monomer having a non-dissociable hydrophilic group, and styrene may be used as the monomer having a hydrophobic group, and a mixture of these monomers may be subjected to water suspension polymerization.
2) Using one or a mixture of two of the above monomers, add a polymerization catalyst and a suspension stabilizer, and conduct water suspension polymerization with stirring to obtain polymerized particles with a diameter of 5 to 200 microns. be able to. As for the polymerization method, known methods can be used. In addition, during polymerization, if an organic solvent is added that dissolves the above monomer but does not dissolve the polymer, the resulting spherical polymer becomes porous and the surface area of the filler increases. and is effective for increasing separation efficiency. It is also used as a packing material for high performance liquid chromatography. For this purpose, the filler is required to have excellent mechanical strength, low swelling, etc. Therefore, it is desirable that the filler is spherical, and furthermore, it is desirable that it be crosslinked. Therefore, it is desirable that either or both of the non-dissociable hydrophilic group and the hydrophobic group be polyfunctional groups.
The polymer particles obtained by water suspension polymerization are dried by heating or the like.

Then, by dispersing the dry particles in water, it is determined whether or not they get wet with water. Particles that cannot be wetted cannot be used as a filler in the present invention because they cannot be used in high-performance liquid chromatography in which water is used as the eluent. Next, the polymer particles obtained by removing fine particles and coarse particles were packed into a liquid chromatography column, ion-exchanged water was used as the separation liquid, and polyethylene glycol (PEG4OO, with a molecular weight of 400 and an average degree of polymerization of 8) was used as a sample. The polyethylene glycol is separated using a polyethylene glycol containing each component with a degree of polymerization of 4 to 15, and the dissolution behavior is observed. To be employed as a filler in the present invention, room temperature and
It is necessary to measure the chromatogram at 0°C and obtain a result that the separation time at 50°C is longer than at room temperature. The packing material selected by the above two test methods has both excellent hydrophilicity and hydrophobic interaction, and is used as the packing material for high performance liquid chromatography of the present invention.

Since the packing material for high-performance liquid chromatography of the present invention is as described above, it is possible to accurately separate hydrophilic substances, and also to separate samples using water as a separating liquid, and it is possible to separate hydrophilic substances with high precision. Therefore, it shows excellent performance in separating biochemical substances, especially serum proteins, enzymes, vitamins, amino acids, etc.

Next, examples of the present invention will be described.

Example 1 400ml of 4% by weight polyvinyl alcohol aqueous solution was placed in a 2t separable flask equipped with a cooler, stirrer, thermometer, and dripping load! and tetramethylolmethane triacrylate 100r and benzoyl peroxide 1
．． A mixed solution consisting of 5f was supplied.

Next, the temperature was raised to 80° C. while stirring at a stirring speed of 400 rpm, reaction was carried out for 10 hours, and the mixture was cooled.

After cooling, the polymerization product was separated from the mother liquor and washed with hot water and acetone to obtain a spherical polymer having a particle size of 5 to 20 microns. Particles of 8 to 12 microns obtained by removing fine particles and coarse particles are dispersed in 800 d of ion-exchanged water, and ionized into a stainless steel column (diameter 7.9 Wr! FLl length 50 c! n) using a high-pressure constant flow pump. Replacement water was filled by pumping at a rate of 1.6 mt/min. The obtained packed column was subjected to high performance liquid chromatography (
Product name: Shimadzu DuPont High Performance Liquid Chromatograph Model 830)
Chromatographic analysis was performed using polyethylene glycol (PEG4OO, manufactured by Wako Pure Chemical Industries, Ltd.) with a molecular weight of 400 as a sample and ion-exchanged water as an eluent while maintaining the column temperature at room temperature of 35° C., 50° C., and 70° C., respectively.

The obtained chromatograms at each temperature are shown in FIG. The lower the molecular weight of the trough components in the sample, the faster they tend to extrude, and the extrusion begins with ethylene glycol, diethylene glycol, triethylene glycol, etc.
The order was as follows. It can be seen that the dissolution time of each component increases as the temperature increases, and that each component is most clearly separated at 50°C. Example 2 40t of hydroxymethyl methacrylate as monomer
A spherical polymer having a particle size of 5 to 15 microns was polymerized under the same polymerization conditions as in Example 1, except that styrene 30f was used, and the same stainless steel column as in Example 1 was filled by pressure-feeding.

The resulting packed column had good affinity for water.
Connect to a high-performance liquid chromatograph (same as in Example 1),
Chromatographic analysis was performed at room temperature (20° C.) and 50° C. using polyethylene glycol (same as in Example 1) as a sample and using ion-exchanged water as an eluent. It was observed that the elution time became longer at 50° C. for each component having a different degree of polymerization. 1, that is, for example, the separation time of a component with a polymerization degree of 8 was 12 minutes at room temperature and 32 minutes at 50°C.

Example 3 In a polymerization apparatus similar to Example 1, 400 d of 4% by weight polyvinyl alcohol aqueous solution, 50 f of tetramethylolmethane triacrylate, 50 t of neopentyl glycol dimethacrylate, 100 t of toluene were added.
A mixed solution consisting of 1.5 f of benzoyl peroxide was supplied.

Next, the temperature was raised to 75° C. while stirring at a stirring rate of 400 rpm, and the 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 spherical porous polymer having a particle size of 5 to 25 microns. 4・Ta1 obtained by removing fine particles and coarse particles
Particles of 0 to 15 microns were dispersed in 100 d of ion-exchanged water, and a stainless steel column (diameter 7.9 wn 1 length 50 cm) was used.
) to 2.01 m of ion-exchanged water using a high-pressure constant flow pump.
It was filled by force feeding at a speed of 1 n.

The obtained packed column was connected to a high performance liquid chromatograph (same as in Example 1), and chromatographic analysis was performed at room temperature and 50°C using polyethylene glycol (same as in Example 1) as a sample and ion-exchanged water as a separation liquid. I went to For each component, elution time was observed to be longer at 50°C than at room temperature. Example 4 The packing material prepared according to Example 1 was used to separate amino acids.

The terms and conditions are as follows.

The obtained chromatogram was as shown in FIG.

In this figure, 1 is the peak of alanine, 2 is the peak of leucine, 3 is the peak of tyrosine, and 4 is the peak of phenylalanine.

Example 5 Chromatographic purification of glucose oxidase was carried out using the packing material prepared according to Example 2.

The conditions were as follows, and the obtained chromatogram consisted of three peaks, the areas of which were 1:1.3:3.8 in order of peak appearance.

When the eluates were fractionated into peaks and the enzyme activity was quantified by the paraoxidase-indamine dye method, only the fraction corresponding to the final elution peak showed enzyme activity.
Therefore, it can be seen that the present packing material can be effectively used for enzyme purification. Example 6 Using the packing material produced in Example 3, major proteins in human serum were separated.

The terms and conditions are as follows.

The obtained chromatogram consisted of two peaks, which were confirmed to be γ-globulin and albumin, respectively, in the order of excretion, by identification using standards.

Comparative Example 1 A commercially available hydrophobic packing material for GPC (trade name: HSG6O, manufactured by Shimadzu Corporation) consisting of a styrene-divinylbenzene-based porous material was connected to a high-performance liquid chromatograph (same as in Example 1), and water was pumped. I tried it.

However, even when the inlet pressure reached 100 Kf, no liquid flowed out of the column, making it impossible to use this column as a packing material for aqueous GPC. Comparative Example 2 The monomer is tetraethylene glycol dimethacrylate. Ito obtained 10 to 15 micron particles obtained by removing fine particles and coarse particles from the product polymerized in the same manner as in Example 1. It was packed into a similar stainless steel column.

The obtained column was connected to a high performance liquid chromatograph (same as in Example 1), and chromatographic analysis was performed at room temperature and 50°C using polyethylene glycol (same as in Example 1) as a sample and ion-exchanged water as an eluent.

The obtained chromatogram showed that there was almost no difference in the separation time between room temperature and 50°C, and the elution time at 50°C was not longer than that at room temperature, and the separation of each component was performed well. Nakatsuta. This packing material exhibited only extremely weak hydrophobic interactions and was therefore unsuitable for the separation of biochemical substances.

For example, even if an attempt was made to perform the same separation as in Example 6, both of the two components would elute without being retained by the packing material.
Separation of the components was not possible.

[Brief explanation of drawings]

Figure 1 shows the chromatogram obtained in Example 1.
FIG. 2 shows the chromatogram obtained in Example 4. 1...Alanine, 2...Leucine, 3
...Tyrosine, 4...Phenylalanine.

Claims (1)

[Scope of Claims] 1. A granular filler made of a synthetic polymer selected from a polymer or copolymer containing tetramethylolmethane triacrylate as a main component, and a copolymer of hydroxymethyl methacrylate and styrene, A packing material for high performance liquid chromatography, characterized in that the packing material exhibits a behavior in which the elution time of the sample becomes longer as the temperature rises in chromatography using polyethylene glycol as a sample and water as an eluent. 2. The filler according to item 1, wherein the filler has a spherical shape.