JPS5930224B2 - Packing material for high performance liquid chromatography consisting of a new granular cross-linked copolymer - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a filler for high performance liquid chromatography (hereinafter abbreviated as HLC) comprising a novel particulate crosslinked copolymer.

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

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

Among these five separation methods, the LC method is the most preferred, considering that it has a relatively large applicable molecular weight range, has fewer restrictions on chemical stability, and can separate single species. It is excellent. Among the LC methods, 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 due to the solute molecules penetrating into the solvent held in the pores of the column packing material (hereinafter referred to as gel). In other words, each molecule is separated 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 or separation, and 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 GPC separation of each component of oligomers 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 still takes a long time to achieve the desired analysis or separation. It has limitations such as being time consuming, requiring expensive equipment, and requiring a high level of experience in setting operating conditions. This is because the separation performance of a column packed with a styrene-divinylbenzene gel used in high-speed GPC in the oligomer region is insufficient. No known technology has been able to provide a gel that satisfies both strength and separation performance. The present inventors have intensively investigated a method for realizing the separation of oligomers with a high degree of polymerization without the above-mentioned limitations, and as a result, they have discovered a gel that exhibits wide peak spacing without degrading other performances, and have devised the present invention. completed.

That is, the filler for high-speed chromatography according to the present invention is made of a styrene-divinylbenzene-based particulate crosslinked copolymer with a volume average particle diameter of 2 to 50 μm, and has a toluene retention amount of 0.5 to 3.0 W1t/. f, the exclusion limit molecular weight (hereinafter referred to as Mtim) of polystyrene is 500 to 20,0
00, the weight percent x of divinylbenzene units relative to all monomer units in the filler and the slope α of the polystyrene calibration curve are characterized in that they are in a range surrounded by the following three straight lines.

α=2.5 A value representing the particle size of
Determined by Coulter Counter (Coulter Electronics, USA) in 1% saline solution.

The principle of the Coulter counter and the operating conditions of the device are as follows.
"Particle Size Measurement Technology" (edited by Powder Engineering Research Group, Nikkan Kogyo Shimbun)
is shown. Gels with a volume average particle diameter of less than 2 μm are difficult to fill in a column and have a large pressure loss, making them unsuitable as gels for high-speed GPC.
Particles with a particle size exceeding m have insufficient separation performance.

Practically, the volume average particle diameter is preferably between 5 μm and 30 μm. The skeleton of the copolymer gel constituting the filler of the present invention (hereinafter abbreviated as "the gel of the present invention") is mainly composed of units derived from styrene and divinylbenzene monomers.

However, in addition to pure divinylbenzene, divininebenzene, which is usually available commercially, contains monopynyl aromatic monomers such as ethylvinylbenzene, and units derived from these monopynyl aromatic monomers are also included in the gel skeleton. It's fine. Furthermore, other pinyl monomer units may be included as long as they do not substantially change the separation performance of the gel. The weight percent of divinylbenzene units (hereinafter referred to as X) with respect to all monomer units in the gel is defined by the following formula. Here, St represents the styrene unit in the gel, DVB represents the pure divinylbenzene unit in the gel, and EVB represents the weight of the pinyl monomer unit constituting the gel other than styrene and pure divinylbenzene.

X is determined by making the gel into a tablet with potassium bromide and measuring the infrared absorption vector.

That is, the absorption of monosubstituted benzene, 750a "J
The absorption intensity of 790- which is the absorption intensity of meta-disubstituted benzene
The relationship between the ratio of the absorption intensity of 1 and the proportion of dipinylbenzene is determined in advance using a sample with known X, and X can be determined by applying the infrared absorption vector of the unknown sample to this calibration curve. Sample particles with known X are obtained by suspension polymerizing various liquids with different mixing ratios of styrene and divinylbenzene until the polymerization rate reaches 99% or more, and X of this sample is the total monomer before polymerization. Corresponds to the weight percent of pure divinylbenzene in The calibration curve is plotted on a graph in which the vertical axis is the logarithm of the molecular weight M of each component of the styrene oligomer and the horizontal axis is the value Ve/Vt, which is the elution volume Ve divided by the column volume Vt, as shown in Figure 1. It is a line obtained by chromatogram and represents the relationship between the elution volume and the molecular weight of the substance to be separated in the chromatogram.

In this graph, the slanted line part and the line part parallel to the vertical axis are both almost straight lines, and the part where both straight lines intersect is a curved line. Mtim of the present invention is expressed as a value on the vertical axis at a point where an extension of an inclined straight line intersects an extension of a line parallel to the vertical axis in FIG. Mlim is one of the physical property values unique to gels, and represents the limit molecular weight at which gels can exert a separation effect based on differences in molecular size. Substances with molecular weights larger than this value are not separated and are essentially eluted together. In the present invention, the slope of the polystyrene calibration curve (hereinafter referred to as α) is the absolute value of the slope of the inclined straight line in FIG. 1, and is expressed by the following equation.

α is a physical property value specific to gel, and is a measure of the distance between peaks on a chromatogram of a substance with a constant difference in molecular weight.
The lower α is, the greater the distance between peaks is.

The calibration curve is created by introducing a solution containing polystyrene of known molecular weight into the sample inlet near the column inlet, which is in an equilibrium state by passing the solvent through the column, and then measuring the peak of polystyrene eluted from the column near the column outlet. It is characterized by having mechanical strength that can be used in HLC with different molecular weights for the operation of determining the amount of elution solvent required to be detected by a certain detector.

α and X of the gel of the present invention are the following Omoto straight lines (1) and (2)
and (3), and T
− ▼
~37 Preferably, it exists in a region surrounded by the following three straight lines (4), (5), and (6).

Furthermore, within the region surrounded by straight lines (4), (5) and (6), X and α have values within the region surrounded by the following three straight lines (7), (8) and (9). Gels are particularly suitable for preparative separations because of their low α and extremely wide peak spacing.

Eight - "
~J' Also, the following straight line (11, (11), (12
) and (13), gels with values of x and α in the region surrounded by
Especially excellent for C analysis. However, these do not limit the use of the gel having the structure of each region.

The gels with α of 2.5 obtained so far have insufficient mechanical strength and are unsuitable for high-speed GPC.

Further, when X>35, it is difficult to obtain a gel with low α when Mtim is 500 to 20,000. The gel of the present invention has a toluene retention amount (hereinafter referred to as Sr) of 0.5 to
3.0m! , /F, particularly preferably 0.5 to 2.5 mt/f. Sr is expressed as the amount of toluene (b) that the dried gel 1r can contain, and is a value that is a guideline for the pore size of the gel at the time of swelling. Sr is 0.5d/f
Gels with Sr of less than 3 do not have sufficient separation performance;
．． Gels exceeding 0 d/f have insufficient mechanical strength and are unsuitable for high-speed GPC. Sr is obtained by centrifuging a gel that has been immersed in toluene and in a sufficiently equilibrium state to remove the toluene attached to the surface, then measuring its weight (W1), and then drying the gel to calculate the dry weight. If we find (W2), we can calculate it using ▼▼B.

Here, d represents the specific gravity of toluene. Next, a typical example of the manufacturing method for obtaining the gel of the present invention will be described, but the gel of the present invention is not limited to only this manufacturing method.

Polymerization is carried out as follows. Styrene 37-84
Parts by weight, commercially available dipinylbenzene (purity 56%) (100
- styrene) parts by weight, 0.1 to 5 parts by weight of a radical polymerization initiator, and 30 to 200 parts by weight of a solvent for dissolving polystyrene.
Weigh out parts by weight, mix and homogenize the solution, add it to water to which a suspension stabilizer has been added, and stir and mix using a stirrer with a high shearing force called a disperser or homogenizer. After the oil droplets reach a predetermined size, they are heated with continued stirring and set at a specific temperature to carry out a polymerization reaction. The polymerization temperature varies depending on the polymerization initiator used, but it is usually set higher than the initiator usage temperature recommended in catalogs and the like. Polymerization is carried out until the polymerization rate reaches at least 90%. The particles obtained after polymerization are taken from house to house, thoroughly washed with water, hot water, acetone, etc., and then classified if necessary, so that they can be used as a gel for high-speed GPC.

Here, as the radical polymerization initiator, diisopropyl peroxydicarbonate, 2,2'-azobis-(2,4
-dimethylvaleronitrile) is particularly preferred, and toluene is particularly preferred as a solvent for dissolving polystyrene.

The gel of the present invention has a structure with a lower α at the same x than a conventional gel with a styrene-divinylbenzene skeleton and an Mtim of 500 to 20,000.

Therefore, the separation performance of oligomers with a relatively high degree of polymerization is improved by increasing the interval between peaks on the chromatogram without substantially reducing other physical properties, particularly mechanical strength. The gel of the present invention is suitable not only for high-speed GPC but also as a packing material for HLC based on effects such as adsorption and distribution. Next, the gel of the present invention will be described in more detail with reference to Examples.

Example 1 Put 700rr1t of water in which 4.2f of polyvinyl alcohol was dissolved in a 1t flask, add styrene 36.4V, divinylbenzene (purity 56%) 36.4t1 toluene 69g.
．． A homogeneous mixture of 87 and diisopropyl peroxydicarbonate 2r 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 disperser 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 heated at 60°C for 6 hours and then at 80°C for 4 hours while stirring with a boat-shaped stirring rod to keep the temperature inside the flask uniform. Polymerization was carried out. The obtained polymer particles were taken from house to house, thoroughly washed with water and acetone in that order, and then dispersed in acetone to perform simple classification 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 Model ZB (Coulter Electronics, Inc., USA) and found to be 10.0 μm.

This gel was dispersed in chloroform, and the inner diameter was 7.
Packed into a 5wL, 50cm long stainless steel column,
A calibration curve was determined using the method described above. As a result, Mtim is 4
0001α was 4,5. Dry this gel and
When it was made into a tablet and its infrared absorption spectrum was measured, X was found to be 28%. Sr was determined by the method described above and was found to be 1.36 d/f. Further, when the number of theoretical plates (hereinafter referred to as NB) was measured using a column packed with this gel using benzene as a solute, it was found to be 14,800 plates.
Using this packed column, a chloroform solution of styrene oligomers mainly consisting of tetramers was analyzed by GPC. The obtained chart was as shown in FIG. Example 2 A gel was prepared in the same manner as in Example 1, except that the rotation speed of the disperser was lowered and stirred using a slider.

The physical properties of the gel obtained as a result are that the volume average particle diameter is 15.
0μm1Mtim=4,000,. d=4.5, X=
28(:fl), Sr=1.35mt/I, . NB=1
It was 0,000 steps.

Example 3 In Example 1, styrene 33 was used as the polymerization liquid.
．． 8V, divinylbenzene (purity 56%) 39.0f1
Toluene 94.5f and 2,2'-tezobis(2,
A gel was prepared in the same manner as in Example 1, except that a mixed solution of 4-dimethylvaleronitrile (2f7) was polymerized at 80° C. for 10 hours.

The physical properties of the resulting gel were as follows: volume average particle size: 9.0μ
m, Mllm=11,0001α=4.6, X=32%
, Srsl. 95r! f! /T,. NB=12,000
It was hot on the steps. Example 4 In Example 1, the polymerization liquid was styrene 41.6t1 divinylbenzene (purity 56%) 31.2y1 toluene 6
Using a mixed solution consisting of 9.8t and 2t of 2,2'-azobis-(2,4-dimethylvaleronitrile), the mixture was heated at 80°C.
A gel was prepared in the same manner as in Example 1, except that the polymerization was carried out for 10 hours.

The physical properties of the resulting gel were as follows: volume average particle size: 7.6μ
MlMllm=3,400, αS3.4, X=25%,
Sr=1.59me/F,. It was NB 25,700 steps. Comparative Example 1 Column manufactured by Company A commercially available for GPC analysis of oligomer 1 (inner diameter 7.5 wrm 1 length 50 cm)
Separation performance was investigated under the same conditions as in the examples using m).

As a result, Mllm=3,100, α=5.0, NB=
It had 15,000 steps. Furthermore, the gel packed in the column has a volume average particle size of 11.7 μM. X=21%,
Sr=1.08d/7. Using this packed column, a chloroform solution of styrene oligomers mainly consisting of tetramers was analyzed by GPC under the same conditions as in Example 1. The chart obtained was as shown in FIG. When compared with the chart (Figure 2) obtained by analysis under the same conditions using the packed column of Example 1, which has approximately the same number of theoretical plates, the gel from Company A only separated up to the hexamer. On the other hand, in the gel of the present invention, even heptamers were separated, which shows how good the separation performance of the gel of the present invention is. Example 5 A gel was prepared in the same manner as in Example 4 except that the polymerization was carried out at 70° C. for 10 hours.

The resulting gel arostability was 8.6 μm in volume average particle size.
M. Mtim = 3,500) α = 3.8, X = 24"
, Sr=1.41-/V. ．． NB=8,000 steps.

[Brief explanation of the drawing]

Figure 1 shows the molecular weight M of the substance to be separated in the chromatogram.
FIG. 2 shows a calibration curve representing the relationship between elution volume/column volume ratio Ve/Vt, and FIG. Figure 3 shows a chart obtained by GPC analysis using a conventional packed column in the same manner as in Figure 2.

Claims (1)

[Scope of Claims] 1. Comprised of a styrene-divinylbenzene-based particulate crosslinked copolymer with a volume average particle diameter of 2 to 50 μm, with a toluene retention of 0.5 to 3.0 ml/g and a polystyrene exclusion limit molecular weight of 500. ~20,000, and the weight percent X of divinylbenzene units based on the total monomer units in the filler and the slope α of the polystyrene calibration curve exist in a range surrounded by the following three straight lines. Packing material for high performance liquid chromatography. α=(2/15)X+(4/3) α=2.5 The packing material for high performance liquid chromatography according to claim 1, which is present in the range surrounded by three straight lines. α=(2/15)X+(16/15)α=3 The packing material for high performance liquid chromatography according to claim 1, which is present in the area surrounded by straight lines. α=(2/15)X+(16/15) α=4.4 X=27 X=35