JP2012008061A - Separation column for liquid chromatography and liquid chromatography - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a separation column for liquid chromatography achieving a high-speed separation and analysis without lowering a column performance even if enhancing a linear velocity, and to provide liquid chromatography.SOLUTION: A monolith type separation column incorporates a monolith rod formed into a columnar shape in the separation column, a specimen and a moving phase flow into the monolith rod, and the monolith rod is alternately provided with portions having pores and portions without pores. The monolith rod is configured such that porous bodies having pores and portions without pores are alternately disposed in the specimen flowing direction. Alternatively, the portions having pores are porous bodies and the portions without pores are non-porous beads.

Description

  The present invention relates to a separation column for a liquid chromatograph and a liquid chromatograph.

  A liquid chromatograph is a device that analyzes the components of a sample by separating the components of the sample in the eluent with a separation column and detecting the components flowing in the separated order with a detector such as a spectrophotometer. . In order to perform high-speed analysis while maintaining separation performance using a particle packed column commonly used in liquid chromatographs, the particle surface size of the column is reduced to increase the particle surface area, and the unit It is necessary to increase the amount of liquid delivered per hour. For example, the analysis time can be shortened to about 1/10 by changing the diameter of the particles to about 2 μm with respect to a conventional column in which particles having a diameter of about 5 μm are packed in a cylindrical container having an inner diameter of about 4 mm. However, if the particle diameter is reduced, the flow resistance increases, and a necessary flow rate cannot be obtained unless the liquid is fed at a high pressure. Therefore, it is necessary to increase the pressure resistance of the entire analyzer including the column.

  As a column structure for avoiding this increase in pressure resistance, a monolithic column having a structure in which a three-dimensional network skeleton and its voids are integrated is proposed instead of a conventional particle-packed column. Monolithic columns have high porosity and do not increase flow resistance. There are pores called mesopores in the order of nm in the skeleton, and these mesopores contribute to improving the separation performance because the surface area from which the sample is desorbed increases. For example, a monolithic silica column in which a monolithic rod or a monolithic silica rod is incorporated as a porous body in a narrow tube has been proposed to achieve high performance.

  Silica-type porous body is formed by sol-gel transition based on spinodal decomposition and then pores are made by ammonia treatment, but it is difficult to mold the uniformity and uniformity with high accuracy. It has the problem of being. On the other hand, as a method of controlling the pore distribution of the porous body, a structure having a solvent-rich phase that becomes a large pore by adding metal alkoxide as a starting material, adding an appropriate coexisting substance to the material, and A manufacturing method for generating the same has been proposed (see, for example, Patent Document 1).

  FIG. 5 is a cross-sectional view showing the concept of how the sample flows in the monolith column. Inside the cylindrical monolith column 1, there is a mobile phase 2, a stationary phase 3 having a structure in which a three-dimensional network skeleton and its voids are integrated, and the sample 4 has a mobile phase 2 as shown by the arrow in the figure. And back and forth between stationary phase 3. The sample 4 is separated by repeating the collection and desorption by the distribution to the stationary phase 3 and the adsorption force. The voids of the stationary phase 3 are pores having various shapes that have different diameters and depths. Due to such pores, the surface area in contact with the sample is large, the field contributing to separation is increased, and high separation performance is obtained.

  However, since the pores of the skeleton are irregularly present, some samples move to the surface of the skeleton, and some samples move in the axial direction to penetrate deeply into the pores and diffuse. Increasing the linear velocity of the mobile phase flow rate divided by the column cross-sectional area results in differences in the time at which individual samples elute to the mobile phase. It is considered that the theoretical plate height (H) is particularly large in the high flow rate region. Therefore, a monolithic column that does not deteriorate the column performance even when the linear velocity is increased is desired.

JP 7-41374 A

  An object of the present invention is to provide a liquid chromatograph separation column and a liquid chromatograph capable of performing high-speed separation analysis without lowering the column performance even when the linear velocity is increased.

  In order to solve the above-mentioned object, an embodiment of the present invention is a monolith type separation column in which a monolith rod formed in a cylindrical shape is incorporated in a separation column, and a sample and a mobile phase flow into the monolith rod. The rod is characterized by alternately having sites having pores and sites having no pores.

  The monolith rod is characterized in that a porous body having pores and a portion having no pores are alternately arranged in the flow direction of the sample.

  Alternatively, the site having pores is a porous body, and the site having no pores is non-porous beads.

  According to the present invention, it is possible to obtain a liquid chromatograph separation column and a liquid chromatograph capable of performing high-speed separation analysis without degrading column performance even when the linear velocity is increased.

It is a block diagram which shows schematic structure of a liquid chromatograph. It is a graph which shows the relationship between the linear velocity u and theoretical plate height H when a particle diameter is changed. It is sectional drawing which shows an example of the structure of a separation column. It is sectional drawing which shows an example of the structure of a separation column. It is sectional drawing which shows the concept of the flow method of the sample in a monolith column.

  Embodiments of the present invention will be described below using the drawings as appropriate.

〔Example〕
FIG. 1 is a configuration diagram showing a schematic configuration of a liquid chromatograph. The eluent stored in the eluent container 11 is fed by the liquid feed pump 13, the sample stored in the sample container 12 is injected by the autosampler 14, and is separated by the separation column 15 in the column oven 16 that is a thermostat. The sample is separated into components and detected by the detector 17. The eluent that has been detected is discharged into the waste liquid container 18.

  Next, the structure of the separation column will be examined. In a particle-packed column using particles as a filler, if the mobile phase linear velocity is u, the theoretical plate height is H, and the particle diameter is dp, the following relationship is obtained from Van Deemter's equation.

H = Adp + B / u + Cu Equation 1
Here, A is a coefficient representing the diffusion in the radial direction, B is a coefficient representing the diffusion in the axial direction, and C is a coefficient relating to the movement of the sample between the mobile phase and the stationary phase. The smaller the theoretical plate height H, the better the column performance. As the linear velocity increases, the theoretical plate height H increases and the column performance decreases.

  FIG. 2 is a graph showing the relationship between the linear velocity u and the theoretical plate height H when the particle diameter is changed. Three types of particles having particle diameters of 2 μm, 3 μm, and 5 μm were used as packing materials for the separation column, and the relationship between the linear velocity u and the theoretical plate height H was obtained by calculation and graphed. When the particle diameter is 3 μm and 5 μm, the theoretical plate height H increases as the linear velocity u increases. Therefore, the column performance tends to decrease. When the particle diameter is 2 μm, the theoretical plate height H does not increase even if the linear velocity u increases. This is because the sample moves quickly between the mobile phase and the stationary phase in the separation column having a particle size of 2 μm as compared with the separation column having a particle size of 3 μm or 5 μm. The step height H is maintained.

  According to the document "High performance of HPLC using monolithic silica column, Kimura et al., Bunkeki 200410, pp. 576-584", column performance is measured with a separation impedance defined by the reciprocal of the product of column pressure and eluent viscosity. In comparison, the monolithic column has higher performance by one digit or more than the particle packed column, but the separation velocity tends to be inferior since the linear velocity u is about 5 mm per second. According to the document “G. Desmet et al., Anal. Chem., 77, 4058-4070 (2005)”, sub-2 μm particles such as a particle diameter of 1.7 μm, and US Patent Application Publication No. US2007 / 0189944 are disclosed. Compared with core-shell type particles that are microcapsules having an outer shell with a diameter of 3 μm or more, which is made of a dendritic porous material having mesopores and macropores, as disclosed in JP-A-2008-145190 and the like, This is due to the fact that the number of theoretical plates is approximately 10,000 or less, the analysis time of the monolith column is relatively long, and the C term of Equation 1 is large. That is, in the monolith column, the rate at which the sample moves back and forth between the mobile phase and the stationary phase is large, and the theoretical plate height increases when the linear velocity is increased. Here, the particle packed column and the monolith column are compared again. The term C in the above equation 1 is a term related to mass transfer. For example, it relates to the time for the sample solute to move from the mobile phase to the stationary phase, or vice versa. The shorter the sample solute movement time, the smaller the C term and the smaller the theoretical plate height.

  In a column using core-shell type particles and a stationary phase thickness of about 1 μm, or a column using sub-2 μm particles, the migration time of the sample solute can be reduced. The sample solute can escape to the mobile phase anywhere in the stationary phase by moving the pore region a distance of 1 μm to 2 μm. On the other hand, in the case of a monolith column, the sample solute may not escape to the mobile phase even if it moves 10 μm in the axial direction. If the pore region of the skeleton of the stationary phase of the monolith column is long in the axial direction, it takes time for the sample solute to come out to the mobile phase side, and the C term of the above formula 1 in the case of the monolith column is increased. It is estimated that this is a factor.

  Next, the mobile phase is examined. The pore region of the stationary phase is ideally about 1 μm in all directions, but the volume scale of the mobile phase has an ideal state. In other words, since the macropores of the monolith column do not cause excessive pressure loss, the macropore ratio is ideally about 2 to 3 μm, which is 1.5 to 2 times of 1 to 2 μm in the skeleton region.

  On the other hand, the core-shell type particle has a particle diameter of about 2.7 μm and a gap-scale length scale corresponding to macropores of about 1.5 μm. The sub 2 μm particles have a narrow gap, and the length scale corresponding to macropores is 1 μm or less. The typical scale of these macropores determines the column permeability. Therefore, a monolith column is the best. If there is no packing along the inner wall of the column container and the mobile phase flows without obstacles, this structure must be avoided because the sample solute is not trapped in the stationary phase at all.

  From the above, the ideal stationary phase and mobile phase of the column for liquid chromatography are derived. The stationary phase as the pore region is a minute volume space of about 1 μm in all directions, the volume space occupied by the mobile phase is a hollow space such as a macropore of 2 μm to 3 μm, and obstacles are disposed. The present invention provides a liquid chromatograph column that satisfies such conditions.

  The sub-2μm particles realize an almost ideal minute volume space of about 1μm, but when filled in an empty column as it is, the macropore equivalent scale becomes about 1μm in the volume space occupied by the mobile phase, and the movement speed is increased. I can't make it bigger. The core-shell type particle is about 1.5 μm and slightly larger.

  In a monolithic column in which the pore area of the skeleton of the stationary phase is 1 μm to 2 μm, the sample solute cannot escape to the mobile phase even if it can move 10 μm in the axial direction, thus preventing an increase in the moving speed. This is because the pore region is continuous in the axial direction of the skeleton skeleton. Therefore, if the partition membrane is partially disposed in the axial direction, the sample solute cannot move in the axial direction, but once escapes to the mobile phase, it moves again to the pore region of the adjacent skeleton.

  FIG. 3 is a cross-sectional view showing an example of the structure of the separation column. The longitudinal cross section of the monolith rod inserted in the container of a separation column is shown, and the porous body arranged the 1st member 5 which has a pore, and the 2nd member which does not have a pore alternately in the direction of flow. It is a configuration. The first member 5 has pores of about 10 nm to 100 nm. The sample enters the pores of the first member 5, but the second member 6 has no pores, so the sample cannot move deeply in the axial direction and prevent the sample from moving to the mobile phase for a long time. Can do. And even if linear velocity goes up, the separation column of a liquid chromatograph and a liquid chromatograph which can perform high-speed separation analysis without degradation of column performance are realizable.

  A method for producing a porous body constituting the separation column shown in FIG. 3 will be described. First, a solution obtained by adding alkoxysilane and polyethylene glycol to an acetic acid aqueous solution and stirring the mixture is poured into a hollow tube having an inner diameter of 20 mm to 60 mm. Next, when the hollow tube filled with the stirred solution is held at a constant temperature for a certain time, silica gel is formed inside the hollow tube. Furthermore, by treating the silica gel with ammonia, pores of about 10 nm to 100 nm are produced on the skeleton surface. Thereafter, the porous first member 5 having strength by firing is formed. The first member 5 has a three-dimensional network-like skeleton-like skeleton having a gap of about 10 μm in addition to the pores.

  On the other hand, silica gel is formed in the container, and baking is performed without ammonia treatment to form the second member 6 having no pores. The second member 6 does not have pores, but has a three-dimensional network-like skeleton-like skeleton with a gap of about 10 μm.

  The first member 5 and the second member 6 are taken out of the container and cut into thicknesses of 1 μm to 2 μm, respectively. The first member 5 and the second member 6 are alternately overlapped to form a monolith rod for the separation column. A monolithic rod is covered with PEEK resin, and a functional group such as an octadecyl group is chemically bonded, so that it can be used as a separation column for liquid chromatography.

  When the gap formed when the first member 5 and the second member 6 are overlapped is large and the performance as a separation column is lowered, the monolith rod is again manufactured after coating with PEEK resin. That is, when alkoxysilane and polyethylene glycol are added to an acetic acid aqueous solution, a stirred solution is injected, held at a constant temperature for a certain period of time, and then fired, the first member 5 and the second member 6 are placed between them. To form a monolithic rod with strength. This monolith rod has a structure in which portions having pores and portions having no pores are alternately arranged, and has a three-dimensional network skeleton-like skeleton with voids of about 10 μm as a whole.

  FIG. 4 is a cross-sectional view showing an example of the structure of the separation column. The cross section of the monolith rod inserted in the container of a separation column is shown, and is composed of a porous body 7 and beads 8. The porous body 7 has a three-dimensional network skeleton void of about 10 μm, and further has pores of about 10 nm to 100 nm on the skeleton surface. The beads 8 are a polymer made of, for example, polystyrene-cyvinylbenzene, and are a non-porous type having no pores, and the particle diameter is preferably about 10 μm.

  The beads 8 have a structure in which the separation column is packed with a fine structure, and the porous body 7 is formed in the gap therebetween. As a result, the separation column has a structure in which the porous bodies 7 having pores are alternately arranged. It has become. Therefore, since the sample does not move deeply into the pores, the elution time until the mobile phase does not increase and the sample can be prevented from diffusing, so the column performance decreases even if the linear velocity increases. Thus, a separation column for liquid chromatography capable of high-speed separation analysis can be realized.

  A method for manufacturing the separation column shown in FIG. 4 will be described. First, the beads 8 are injected and filled into a hollow tube having an inner diameter of 20 mm to 60 mm. At this time, the beads 8 are filled so as to have a close-packed structure in the hollow tube. Next, an alkoxysilane and polyethylene glycol are added to an acetic acid aqueous solution and a stirred solution is injected into the hollow tube filled with the beads 8. When the hollow tube is held at a constant temperature for a certain period of time, the stirred solution forms a silica gel skeleton with voids formed by filling the beads. Further, by treating the silica gel with ammonia, pores of about 10 nm to 100 nm are produced on the skeleton surface. Furthermore, when the hollow tube is given strength by firing, a porous body 7 is formed. The porous body 7 has an integral structure in which voids formed by filling beads 8 are filled, and has a three-dimensional network skeleton and voids of about 10 μm, and pores of about 10 nm to 100 nm on the surface of the skeleton. Have. A monolithic rod composed of the porous body 7 and the beads 8 is taken out from the hollow tube, and this monolithic rod is covered with PEEK resin and functional groups such as octadecyl groups are chemically bonded to use as a separation column for liquid chromatography. be able to.

  As described above, according to the embodiment of the present invention, the formation of the portion without the pores present in the axial direction of the porous body eliminates the deep movement of the sample inside the pores, and the mobile phase. It is possible to prevent the elution time from becoming longer and to prevent the sample from diffusing in the axial direction of the porous body. Therefore, even if the linear velocity increases, a separation column for liquid chromatography capable of high-speed separation analysis can be realized without lowering the column performance.

1 Monolith Column 2 Mobile Phase 3 Stationary Phase 4 Sample 5 First Member 6 Second Member 7 Porous Body 8 Bead

Claims (14)

  A monolith type separation column in which a monolith rod formed in a cylindrical shape is incorporated, and a sample and a mobile phase flow into the monolith rod, the monolith rod having a portion having a pore and a portion having no pore A separation column for a liquid chromatograph characterized by comprising alternately.   2. The liquid chromatograph according to claim 1, wherein the monolith rod has a configuration in which a porous body having pores and a portion having no pores are alternately arranged in a flow direction of the sample. Separation column.   3. The separation column for liquid chromatography according to claim 2, wherein the pore diameter of the porous body is 10 nm to 100 nm.   3. The separation column for a liquid chromatograph according to claim 2, wherein the monolith rod has a three-dimensional network skeleton and has a gap of 1 μm or more.   2. The separation column for liquid chromatography according to claim 1, wherein the monolith rod is made of silica.   2. The separation column for a liquid chromatograph according to claim 1, wherein the porous body is configured by alternately superposing first members having pores and second members having no pores.   2. The separation column for liquid chromatography according to claim 1, wherein the part having the pores is a porous body, and the part having no pores is a non-porous bead. A pump for feeding the eluent,
A sampler for injecting a sample into the eluent,
A separation column that separates the sample by flowing an eluent into which the sample has been injected;
In a liquid chromatograph comprising a detector for detecting components separated by the separation column and analyzing the components of the sample,
The separation column is a monolith type separation column in which a monolith rod formed in a cylindrical shape is incorporated, and the sample and the mobile phase flow into the monolith rod, and the monolith rod has a portion having pores and a pore. A liquid chromatograph characterized by alternately having a portion not having s.   9. The liquid chromatograph according to claim 8, wherein the monolith rod has a configuration in which a porous body having pores and a portion having no pores are alternately arranged in a flow direction of the sample. .   The liquid chromatograph according to claim 9, wherein the pore size of the porous body is 10 nm to 100 nm.   10. The liquid chromatograph according to claim 9, wherein the monolith rod has a three-dimensional network skeleton and has a gap of 1 μm or more.   9. The liquid chromatograph according to claim 8, wherein the monolith rod is made of silica.   9. The liquid chromatograph according to claim 8, wherein the porous body is configured by alternately superposing first members having pores and second members having no pores.   9. The liquid chromatograph according to claim 8, wherein the portion having the pores is a porous body, and the portion having no pores is a non-porous bead.

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