JP2013019892A - Method and device for processing isomer compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simply and easily separate isomer compounds in a short time period by solving problems in separation of so-called isomer compounds that utilization of differences in chemical interactions cannot be expected, since differences in absorption forces, charges, and hydrophobic properties are small, and it takes time before equilibration of the whole system in which holding and separation are stably conducted in a liquid chromatography.SOLUTION: A resistor is arranged in an outlet channel of a column used in the liquid chromatography. With the actuation of the resistor, column inlet pressure is adjusted to 10 MPa to 100 MPa so as to change solute behaviors of the isomer compounds for every compound.

Description

  The present invention relates to the treatment of isomeric compounds in liquid chromatography, in particular their separation and fractionation.

 Chromatography is a widely used technique for separating components by utilizing differences in adsorption power, charge, and hydrophobicity due to differences in the types and sizes of functional groups of compounds. However, in the separation between compounds with the same molecular weight and functional group, that is, the same molecular formula, but different molecular structures, so-called isomeric compounds, the difference in adsorption power / charge / hydrophobicity is small. I cannot expect a big difference of chemical interaction. For that purpose, capillary gas chromatography with a very high basic separation performance of the column and a theoretical plate number of 200,000 or more can be expected, but it cannot be applied to pyrolysis components or high boiling point compounds. Time-consuming techniques such as derivatization are taken.

On the other hand, in the case of liquid chromatography that requires relatively little pretreatment, it is often impossible to separate isomeric compounds by simply changing the eluent to change the ratio of the organic solvent and water. For this reason, it is necessary to devise various means such as adding a high molecular compound such as cyclodextrin having high recognition ability of isomeric compounds to the eluent or using an LC column using these compounds as a stationary phase. come.
Changing to a special eluent takes time to stabilize the baseline, and in some cases, the liquid contact part of the apparatus may be damaged.

  Although a method for lowering the column temperature to 15 ° C. or lower and performing a low-temperature analysis has been proposed, an expensive cooling oven is required, and more energy is required for cooling. Depending on the time of year, damage to the device due to condensation may be considered, and it is difficult to put into practical use in consideration of the difficulty of use and the stabilization time for keeping the column at a uniform temperature.

  In liquid chromatography, it takes considerable time for the retention and separation to stabilize, in other words, to equilibrate the state of all system systems. In particular, it takes a longer time to separate isomer compounds because a special eluent is used or the analysis temperature is lowered. Furthermore, in changing the column, it takes a lot of time and effort to change the column, to select an intermediate solvent for changing the solvent in the column to the eluent, and to equilibrate the eluent.

  In contrast, changes in flow rate and pressure are fast equilibrated and can be an effective means in liquid chromatography. The change in the flow rate is only faster or slower as a whole, and the separation between the individual components does not change greatly. However, it has been found that the change in pressure affects the separation in various ways.

  Rogers et al. Reported that the retention of methyl orange and ethyl orange in a silica stationary phase and water mobile phase system decreased until 20000 psi, and increased when the pressure was higher than 20000 psi. After that, ion-pair was reported. In RPLC, when the pressure was increased to 2410 bar, the increase in retention of methyl orange was increased by 3.2 times. This was because the ion pair system was a complex equilibrium system, and the increase in ion pair production due to high pressure might have resulted in an increase in retention. Is suggested (Non-Patent Document 1).

  Jorgenson et al. Reported that the viscosity (0.89 cP) of acetonitrile (ACN) -water (40:60 v / v) mixed solution increased by 0.16 cP / kbar at 25 ° C. and 3500 bar (Non-Patent Document). 2).

  McGuffin et al. Reported that the retention of methoxycoumarin derivatives of C10-C20 fatty acids increased from 9% to 24% by increasing the column inlet pressure 36-360 bar (Non-patent Documents 3 and 4). Dispersion force is considered as an interaction along with an entropic effect, and it is believed that all of the solute, mobile phase, and stationary phase are compressed. Furthermore, it reports a 7% increase in retention for planar aromatics (PAH) and a small or rather decreased effect for non-planar aromatics.

  Jorgenson et al. Reported a 12% increase in retention at 1000 bar using an octadecyl (ODS) group-bound silica gel column for 2,3-hydroxynaphthalene (Non-patent Document 5).

  Tanaka et al., When dissociable solutes (acids, bases) are eluted at a pH near their pKa (acid dissociation constant), the pressure changes the pKa of the solute or buffer solution material, and the high pressure promotes ionization. We are reporting. As a result, in the ionization controlled reverse phase chromatography, the pH of the mobile phase and the ionization rate of the solute change with pressure, the retention of weak acid and weak base changes, and the separation pattern changes. In this case, it is considered that the pressure effect on the species that causes a larger volume change dominates the direction (increase or decrease) of the retention change (Non-Patent Document 6).

  Evans et al., In cyclodextrin-added reversed-phase chromatography system or cyclodextrin-bonded stationary phase system, increased uptake into cyclodextrin by pressurization, retention of p-nitrophenol, and preferential incorporation of optical isomer compounds. It is reported that retention may change and the separation factor may change (increase or decrease). It has been reported that the pressure effect in the equilibrium equilibrium system with such a cyclic structure host material is larger than the pressure effect in the stationary phase, and the degree of separation decreases in any case (Non-Patent Documents 7 and 8).

  McCalley et al. Increased pressure by about 50% (applying a 30 μm capillary between the column and the detector) for hydrophobic / neutral substances, about 12%, polar and high molecular substances, ions (acids, bases). ) It has been reported that an increase in retention of more than 50% was observed for sex substances. Furthermore, the influence by the stationary phase (effect in which C18 is larger than C1) is reported. We conclude that the increase in retention due to high pressure is caused by desolvation of the solute under high pressure and promoting retention. Also, much larger pressure effects (increased retention) have been reported for macromolecules such as proteins. (Non-patent documents 9, 10, 11, 12)

  As described above, various announcements including the notable views have been made. However, many reports on changes in retention of these separation columns due to pressure relate to hydrophilic compounds. Even with hydrophobic compounds, a 5-24% increase in retention was observed with respect to a pressure increase of 50 MPa, but none of the hydrophilic compounds reported a significant increase in retention.

The previous reports on the pressure effect are classified into the following four types.
Type 1: Dispersion interaction (McGuffin, McCalley) of non-polar / hydrophobic compound (long chain alkyl compound, PAH): small increase in retention (increase in dispersion power + steric exclusion) for hydrophobic (planar) solutes.
Type 2: Ionization control of weak acid and weak base at pH around pKa (Tanaka): Retention change (increase, decrease) through change in pH and pKa for weak acid and weak base (by increase in solvation (electrocondensation) Promotion of ion dissociation at high pressure)
Type 3: Desolvation of ions and hydrophilic solutes at a pH away from pKa (McCalley): Large increase in retention of hydrophilic solutes and ions (also thought to be based on desolvation of solutes) .
Type 4: In the inclusion phenomenon (increased complex formation) by the cyclic structure host (increase in complex formation), the hydrophilic solute that can be incorporated into the space of the host increases the complex formation due to pressurization, and the separation factor between optical isomer compounds is small. Change (increase or decrease).

G.Prukop, L.B.Rogers, Sep.Sci.Technol., 13 (1978) 59. J.W.Thompson, T.J.Kaiser, J.W.Jorgenson, J. Chromatogr. A 1134 (2006) 201. V.L.McGuffin, S.-H. Chen, Anal. Chem. 69 (1997) 930. V.L.McGuffin, C.E.Evans, J. Microcol. Sep. 3 (1993) 513. J.S. Mellors, J.W.Jorgenson, Anal.Chem. 76 (2004) 5441. N. Tanaka, T. Yoshimura, M. Araki, J. Chromatogr. 406 (1987) 247. L.M.Ponon, S.M.Hoenigman, M. Cai, C.E.Evans, Anal. Chem. 72 (2000) 3581. C.E.Evans, J.A. Davis, Anal. Chim. Acta 397 (1999) 163. M. M. Fallas, U. D. Neue, M. R. Hadley, D. V. McCalley, Journal of Chromatography A, 1209 (2008) 195 S.-H. Chen, C.-T. Ho, K.-Y. Hsiao, J.-M. Chen, J. Chromatogr. A 891 (2000) 207. M.A. Stadalius, H.S. Gold, L.R.Snyder, J. Chromatogr. 296 (1984) 31. X. Liu, P. Szabelski, K. Kaczmarski, D. Zhou, G. Guiochon, J. Chromatogr. A 988 (2003) 205.

  With such a background, in the separation of isomeric compounds, it is required to achieve the separation with a simple and fast eluent. Here, the column is used as a separation column, and is an analytical column or a preparative column.

  In order to realize the separation of isomer compounds, it is important to change the solute behavior (pattern) for each isomer component. As the interaction by pressure, Type 1 works universally, and in addition, stereoselectivity works, so that it can be expected to change the separation between isomeric compounds.

  In liquid chromatography, if the flow rate is increased, it is possible to increase the column inlet pressure due to the resistance in the column, but the pressure on the outlet side becomes atmospheric pressure, and at the center of the column, the inlet pressure increases by half. Stay on. The pressure effect on the stationary phase often only works at a part of the inlet and lowers the separation performance of the column. The method of decreasing the temperature for increasing the pressure has a small effect of increasing the pressure because the increase in viscosity is not large at a practical temperature.

  On the other hand, the pressure changes as the flow rate and column length change, but the column performance also changes at the same time, so it is unclear whether the change in the separation pattern is due to the effect of pressure or due to other factors. Cannot be obtained. In order to obtain a constant pressure effect, it is necessary to bring the same effect as the pressure effect on the column inlet side at the column outlet, and it is necessary to apply the same back pressure as the inlet to the outlet side. That is, the effect is most clearly obtained if the back pressure is applied so that the average pressure of the entire column is twice or more that of the normal analysis.

  For example, in the case of a column packed with 5 μm particles that are often used, when 100% acetonylyl is used as an eluent and flowed at a linear velocity of 1 mm / sec, the analysis pressure becomes 1 MPa to 5 MPa. When the back pressure is applied so that the same pressure effect works on the outlet side, the inlet pressure is doubled to 2 MPa to 10 MPa.

Means for achieving the above object are as follows.
One of them is to provide resistance at the outlet channel of the column used in liquid chromatography, and adjust the column inlet pressure to 10 MPa to 100 MPa by operating the resistance to change the solute behavior of the isomer compound for each component. It is the processing method of the characteristic isomeric compound.

  One of them is a method for treating isomeric compounds characterized in that the change in solute behavior is solute separation.

  One of them is a method for treating isomeric compounds characterized in that the change in solute behavior is a change in the order of solute appearance.

  One of them is a method for treating isomeric compounds characterized in that the change in solute behavior is solute coalescence.

    One of them is a method for treating an isomer compound, wherein the solute behavior of the isomer compound is changed while continuously lowering the column inlet pressure between 100 MPa and 10 MPa.

  As one of them, the solute behavior of the isomer compound is changed by changing the composition of the eluent to change the column inlet pressure continuously or stepwise between 100 MPa and 10 MPa. It is a processing method.

  As one of them, the column inlet pressure is changed between 100 MPa and 10 MPa by changing the column temperature to change the solute behavior of the isomer compound.

  One of them is an analysis method of an isomer compound, wherein the column is an analysis column.

  One of them is a method for fractionating an isomer compound, wherein the column is a fractionation column.

  One of them is an apparatus for treating an isomeric compound, and in the column outlet channel, a resistor is connected between the column outlet and the detector, between the detector and the discharge or both, and the column inlet pressure Is an isomeric compound processing device characterized in that the pressure can be adjusted to a predetermined pressure.

  One of them is an isomer compound analyzer using an analytical column as the column.

  One of them is an isomeric compound fractionator using a fractionation column as the column.

  One of them is an apparatus for treating an isomeric compound, wherein the resistor is a resistance tube.

  As one of them, the resistance tube is one of a tube, a monolith column, and a packed column.

  One of them is an apparatus for treating an isomer compound, wherein the resistor is a regulator capable of changing a space capacity through which a liquid flows.

  One of them is the isomeric compound analysis apparatus according to any one of the above, characterized in that an analytical column is used as the column.

  One of them is the isomeric compound processing apparatus according to any one of the above, wherein the preparative column is used as the column.

  As one of them, an apparatus for treating an isomeric compound is characterized in that a valve is provided on the column outlet channel side, a plurality of resistors are connected to the valve, and a predetermined column inlet pressure is obtained by selecting the resistor to be connected. is there.

One of them is an isomer compound separation column using a packing material in which a stationary phase is coated or chemically bonded to a particulate carrier or monolith carrier having a pressure resistance of 10 MPa or more at a density of ² μmol / m ² or more.

One of them is a packing material for an isomeric compound separation column in which a stationary phase is coated or chemically bonded to a particulate carrier or a monolith carrier having a pressure resistance of 10 MPa or more at a density of ² μmol / m ² or more.

  By connecting a resistor between the column outlet and the detector, between the detector and the discharge or both, and by changing the analysis pressure to 10 MPa or more, a change in isomeric compound elution, for example, separation, change of separation order, It became possible to make changes such as coalescence. When the analysis pressure is changed, the equilibration time is short, and it is possible to eliminate the equilibration waiting time of 30 minutes or more when the eluent and temperature are changed as in the conventional case.

  The pressure adjustment can be easily realized by using various commercially available methods.

  In addition, isomeric compounds can be separated even at room temperature with a simple eluent, so that it has become possible to easily perform fractionation, which has been difficult to control with a large amount of eluent.

Schematic illustration of an embodiment of the present invention Invention-Examples Subject Isomeric Compound Tocopherol Diagram Schematic explanatory diagram of one embodiment of the present invention Schematic explanatory diagram of one embodiment of the present invention Rotary valve diagram of one embodiment of the present invention Valve switching diagram of one embodiment of the present invention Regulator diagram of one embodiment of the present invention Chromatogram of one embodiment of the present invention Chromatogram of one embodiment of the present invention Chromatogram of one embodiment of the present invention Chromatogram of one embodiment of the present invention Chromatogram of one embodiment of the present invention Chromatogram of one embodiment of the present invention Chromatogram of one embodiment of the present invention Chromatogram of one embodiment of the present invention Chromatogram of one embodiment of the present invention Chromatogram of one embodiment of the present invention Chromatogram of one embodiment of the present invention Chromatogram of one embodiment of the present invention Chromatogram of one embodiment of the present invention Chromatogram of one embodiment of the present invention

Generally, in the dissolution of carbon dioxide, the solubility is greatly increased up to about 10 MPa with respect to the pressure, and it is said that the change is small at 10 MPa or more and is almost constant at 15 MPa.
It is considered that the solubility of the sample components in the stationary phase in HPLC shows almost the same tendency, and considering the fast stability, 10 MPa or more in which the change in solubility in pressure is small is considered ideal. In the examples of the present invention, it has been proved that the effect can be obtained at 10 MPa or more.

If the device or column allows, higher pressure will be more stable because the effect of the pressure gradient in the column will not affect the entire system. When the back pressure is applied and the system pressure is increased to 100 MPa, the pressure effect on the column portion becomes 5% or less, and the pressure change effect due to the column type and column size is almost eliminated. Therefore, if the pressure is 10 MPa or more, the isomer compound can be treated, and the upper limit pressure depends on the apparatus pressure resistance.
The HPLC apparatus with the highest pressure resistance currently on the market has a pressure resistance of 150 MPa, and the pressure influence of the column portion is 3% or less, so that more stable data can be expected. In reality, however, resin-based sealing materials are used for the wetted parts such as the plunger of the feed pump that delivers the eluent and the injector that introduces the sample. I can't make liquid. Therefore, it is appropriate to consider the upper limit in practical use of pressure adjustment as 100 MPa.

  In order to change the separation of isomeric compounds with good reproducibility, that is, to control the separation between compounds, a resistance is connected to the column outlet side, that is, the column outlet flow path, and adjusted so that the column inlet pressure is within 10 MPa to 100 MPa. It is very useful to do.

  In that case, isomer compound separation can be achieved even with an increase in resistance between the column outlet side and the detector or detector cell, but there is a concern that the peak shape may deteriorate due to the occurrence of dead volume at that portion. Ideally, resistance is added later.

The detector cell itself can be used as a resistor, and any cell can be used as long as it can withstand pressure of 10 MPa or more, and the following cells can be used.
Quartz and glass are often used as the detector cell, but since there is no pressure resistance as it is, Teflon (registered trademark), polyethylene, polypropylene, polyetheretherketone (hereinafter abbreviated as PEEK), etc. A quartz or glass cell coated with a resin that transmits light used for detection is suitable because a withstand voltage of about 50 MPa can be obtained. When a fused silica capillary cell having an inner diameter of less than 1 mm is used, a pressure resistance of up to about 100 MPa can be obtained, and the isomeric compound separation of the present invention can be realized as it is.

  However, in the case of a capillary, since the inner diameter is less than 1 mm, there is a limit that the flow rate that can be used is limited to about 1 mL / min. Considering fractionation using a higher flow rate, a metal cell such as stainless steel is preferable to withstand a back pressure of 50 MPa or more. When a high pressure is applied to the detector cell, the solute enters the surface scratch of the order of several μm and causes adsorption. There are various methods of physically polishing, but there is a limit to the mechanical inner surface processing of fine detector cells. Therefore, it is preferable to use a cell having a glass surface formed by chemical treatment with a reactive reagent such as a silicic acid compound such as tetraethoxysilane, tetramethoxysilane, or cyclosiloxane, or polysilazane.

  As a method for controlling the pressure by connecting a resistor to the column outlet channel side, it is sufficient that a pressure of 10 MPa or more is applied to the column inlet, and it is not particularly limited, but specifically, the following method can be considered.

A method of connecting a hollow resistance tube having an inner diameter of 0.15 mm or less, preferably a fused silica capillary tube having a smooth liquid contact surface is simple and suitable.
In order to prevent damage due to physical vibration, it is effective to protect the connection portion or the entire resistance tube with a resin tube such as PEEK.
Furthermore, in order to obtain a stronger resistance with a shorter length, a monolithic resistor in which a so-called monolithic body having a through-pore that penetrates 100 μm or less, for example, 50 μm, and whose skeleton is three-dimensionally connected is formed in the tube. A tube is preferred.

  Since they have a relatively small dead volume, they can be sufficiently used even if they are provided between the column and the detector or detector cell. These can be used alone, but a resistor may be connected.

  By connecting a certain resistance between the column and the detector or the detector cell, the pressure immediately becomes stable at 10 MPa or more, and isomeric compound separation can be performed stably. Furthermore, by providing different resistances that can be changed later, the pressure can be adjusted.

For example, a monolith resistance tube that is not affected by the dead volume is inserted between the column and the detector cell so that the pressure becomes 20 MPa, which is 10 MPa or more where the effect is obtained.
If the following resistance of 40 MPa is provided after such a cell, the column inlet pressure can be adjusted from 20 MPa to 60 MPa. In this case, since it takes only 40 MPa at the maximum, if a commercially available pressure resistant 40 MPa cell is used, it is possible to use it without any need for vitrification.

  As a resistance after the detector or the detector cell, an HPLC packed column can be used instead of the resistance tube. Further, the pressure can be changed sequentially by attaching a plurality of resistance tubes to the valve and sequentially switching the flow paths. In addition, a high pressure-resistant liquid needle valve that has been commercially available can also be used. Since the flow path resistance can be freely changed by changing the throttle of the needle, the pressure can be easily changed automatically.

  Any conventional technique can be used as long as the back pressure can be adjusted, such as a resistance adjustment using a conical resistance tube with a narrowed inner diameter or a monolith filter. When the effect of dead volume is considered, it is recommended to combine a low dead volume resistance tube with a volume resistance.

  The column to be used is not particularly limited, but LC packed type columns and monolithic columns having a high-density structure stationary phase have a large change in isomeric compound separation. Examples of the change in elution behavior include expansion and promotion of separation, change, change, and coalescence of the outflow order.

  Particle-packed columns and monolithic columns that use rigid structures such as oligosaccharides, polysaccharide derivatives, and proteins as a stationary phase can be effective by isolating isomeric compounds under pressure.

  Specifically, a compound stationary phase having a long alkyl chain of C16 or more, a stationary phase having an aromatic ring having a planar structure, and the like have a high-density structure and a large change in isomeric compound separation.

Depending on the stationary phase and the porous body, when a stationary phase of 1 to ² μmol / m ² is introduced, monomolecular layers are formed in a single layer (monomeric bond). If it is 1 μmol or less, it becomes sparse and low density as a stationary phase. In the stationary phase which is said to have a high density, the bonding group is in a state of being introduced in multiple layers (polymeric) and is often a stationary phase of ² μmol / m ² or more.

  Particulate inorganic carriers such as silica gel, titania and zirconia, inorganic-organic hybrid carriers containing organic substances, organic carriers such as styrenedivirbenzene polymer, inorganic monoliths, inorganic-organic hybrid monoliths, organic monoliths as carriers Any carrier having a pressure of 10 MPa or more that can hold the stationary phase can be applied.

Specifically, the following method was implemented as a method of setting the pressure at the column inlet to 10 MPa or more.
FIG. 1 shows an HPLC system as a general LC system, which includes an HPLC pump 1, an injector 2, a column 3, and a detector 4. The eluent serving as the mobile phase is sent out by the HPLC pump 1 and flows in the order of the injector 2, the column 3, and the detector 4, and is discharged.

  In the apparatus of FIG. 1, a first resistor 5 is installed after the column 3 or a second resistor 6 is installed after the detector. The pressure of the entire HPLC system is expressed as a total value of the piping injector pressure, the column pressure, the resistance 5 pressure, the detector piping pressure, and the resistance 6 pressure. The column inlet pressure is a pressure obtained by subtracting the piping injector pressure from the pressure of the entire system. Actually, when the linear velocity is 2 mm / sec or less at which the highest performance is obtained, the pipe injector pressure is 0.5 MPa or less, and the system pressure and the column inlet pressure may be considered the same.

  Separation of tocopherol isomers was performed using Inertsil (registered trademark) C30 (USP code L62 triacontyl group-bonded silica gel 5 μm GL Science) having an inner diameter of 4.6 mm and a length of 25 cm as an HPLC packed column serving as a stationary phase. Tocopherol has the structure of FIG. 2, and the isomers of β and γ are particularly difficult to separate because they differ only in the bonding position of the methyl group (abbreviated as Me in the figure). As the eluent, 100% methanol, which is the simplest composition widely used in HPLC, was used, and the flow rate was 500 μL / min (linear velocity 0.5 mm / sec) and the column temperature was 40 ° C. as analysis conditions.

  In the analysis in which the resistors 5 and 6 are not connected, the column inlet pressure is 3.1 MPa, and the isomers are not separated (FIG. 8 (1) 1: δ-tocopherol, 2: γ-tocopherol, 3: β-tocopherol, 4: α-tocopherol, and so on).

  As a result of installing a fused silica capillary tube 40 cm in inner diameter 0.05 mm on resistor 5 and not connecting resistor 6, the column inlet pressure became 13 MPa, and the elution of isomers represented by peaks 2 and 3 changed and separation occurred. It was seen (FIG. 8 (2)).

  Furthermore, by changing the resistance 5 to an inner diameter 0.03 mm fused silica capillary tube 25 cm, the column inlet pressure was 49 MPa, and elution of β-tocopherol was slower than the isomeric γ-tocopherol, resulting in separation. (FIG. 8 (3)).

  In both systems, the capacity was 1 μL or less, the delay time due to the dead volume was 0.1 seconds or less, and the peak shape was not adversely affected. That is, it was confirmed that isomer separation with a simple eluent can be performed by connecting a resistance at which the column inlet pressure becomes 10 MPa or more between the column and the cell.

Furthermore, the effect was confirmed by changing various types of resistors and connection locations.
As a result of connecting the monolith capillary tube MonoCap (registered trademark) inner diameter 0.2 mm, length 1.5 cm (capacity 0.6 μL through pore 2 μm) to the resistor 5, the pressure became 49 MPa, and the isomer was the same as in FIG. 8 (3). separated.

  A resistor 5 is connected to an inner diameter 0.05 mm fused silica capillary tube 40 cm in length, and an Inert Sustain C18 HPLC column (USP code L1 octadecyl group-bonded silica gel 2 μm, manufactured by GL Sciences Inc.) is used as a resistor 6 after the cell. Attached. The column inlet pressure was 49 MPa, and the isomers were separated as in FIG. 8 (3).

  Since the column used for the resistor 6 is after detection and does not affect the separation, a used column whose separation has deteriorated may be used and is economical. In the case of this system, the pressure applied to the cell is 30 MPa, which is convenient because it can be used in a commercial cell with a high withstand voltage. In addition, HPLC columns are more useful because they are less likely to clog than capillary tubes and resistance fluctuations are less likely to occur.

  A fused silica capillary tube having a length of 5 cm was connected to the resistor 5, and a 6-way rotary valve was connected to the resistor 6 with five types of resistors. When the rotary valve is turned, the resistance can be switched stepwise. The outlet port 9 of the rotary valve 7 has no resistance, the outlet port 10 is provided with a 0.065 mm inner diameter PEEK tube 30 and a length of 3 m, and the outlet port 11 is inner diameter 0.05 mm fused silica capillary tube 31 with a length of 1.2 m. Attached to the outlet port 12 is a 0.03 mm inner diameter fused silica capillary tube 32 length 15 cm, the outlet port 13 is attached a 0.2 mm inner diameter monolith capillary tube 33 MonoCap, a length 2 cm (through pore: 1 μm), and the outlet port 14 A column having a particle diameter of 2 μm and an HPLC column inner diameter of 2.1 mm and a length of 15 cm was attached.

  When the rotary valve 7 was turned and set to the port 10, the column inlet pressure was 13 MPa, and the same separation as in FIG. 8 (2) was obtained. When turned and set to ports 11 to 14, the column inlet pressure was 49 MPa, and the same separation as in FIG. 8 (3) was obtained. It was confirmed that any port could be applied to the separation of isomers. Further, it was confirmed that the pressure could be freely changed by turning to the ports 10 to 14 and changing the attached resistance.

  In this study, the resistance tube was not clogged, but it is possible that dirt will come out due to the valve seal, so it is recommended that the column be resistant to clogging and that the resistance be as large as possible.

  A resistor 5 is not connected to the resistor 5 as shown in FIG. 3, but a 6-way switching valve 7 is connected to the resistor 6, and resistance tubes 18 and 19 are incorporated as shown in FIG. By switching the valve, the resistance can be changed step by step. As resistance, when the inner diameter 0.05 mm fused silica capillary tube 18 length 40 cm and inner diameter 0.03 mm fused silica capillary tube length 25 cm were incorporated and switched, the column inlet pressure became 13 MPa and 49 MPa, respectively. (2) The same separation as in FIG. 8 (3) was obtained. The capacity of the switching valve is as small as 10 μL or less, the delay time is about 1 second, and it can be used before the detector cell.

  A resistor was connected to the resistor 6 without connecting a resistor to the resistor 5. The regulator uses the balance of the force due to the load of the compression spring 23 and the outlet side pressure in the structure as shown in FIG. 7 and moves the piston 21 by the action of the piston seal 22 to change the space capacity through which the liquid flows. The resistance pressure can be adjusted. In the open state, no resistance was applied and the separation state was the same as in FIG. 8A. However, when the pressure was adjusted to 13 MPa by turning the pressure adjusting screw 24, separation was observed as shown in FIG. 8B. Further, by further narrowing down, the isomers were separated as shown in FIG. 8 (3).

  If the pressure adjusting screw 24 is automatically and mechanically adjusted, the pressure can be continuously varied. In that case, if the column inlet pressure is increased to a pressure of 10 MPa or more, the isomer separation effect is exerted. Therefore, in fact, a resistance tube having a volume of 10 MPa or more and a small dead volume is connected to the previous stage, and then, It is recommended to connect a regulator. In addition, there is little pressure fluctuation and system stability is accelerated.

  Thus, in the present invention, as long as a pressure of 10 MPa or more can be applied to the column inlet, various means can be used, and the number of resistors and the connection position are not limited.

  Using a tocopherol isomer, which is the same sample as in Example 1, using an Inertsil® C30 ID 4.6 mm, 250 mm long HPLC packed column with a simple eluent of 100% methanol, A comparison was made between the temperature effect that has been said so far and the pressure effect according to the present invention. As in Example 1, β and γ tocopherol isomers are not completely separated under normal conditions as shown in FIG. 9 (1). However, it was separated as shown in FIG. 9 (3) by lowering the column temperature. Isomeric separation has been achieved at 20 ° C.

  However, the oven temperature was set to 20 ° C., and the monitor temperature reached the set value after about 30 minutes. Therefore, the actual analysis was performed, but the peak retention time was not stable, and stable data began to be obtained from the third analysis. It was found that the holding time was finally stabilized after about 30 minutes after the oven temperature was stabilized, and that equilibration time of about 1 hour was required from the oven temperature setting. Naturally, a huge amount of energy is used for cooling.

The method of the present invention was carried out using the same column, the same eluent, and the same sample.
The column temperature was fixed at 30 ° C., and a fused silica capillary tube (capacity 0.2 μL) having an inner diameter of 0.03 mm and a length of 28 cm was connected as a resistance after the column. The column inlet pressure was 57.9 MPa and stabilized in about 20 minutes, and stable data as shown in FIG. 10 (1) was obtained from the first analysis. Further, a fused silica capillary tube having an inner diameter of 0.03 mm and a length of 10 cm was connected as a resistance. The column inlet pressure was 79 MPa. Even when the capacity of the joint portion connecting the resistance tubes is included, the total capacity is 1 μL or less, and completely separated data is obtained as shown in FIG.
The stabilization time was also within 20 minutes, and it was confirmed that the isomer separation by applying pressure was simpler and faster than the conventional method.

The same method as in Example 1 was applied to HPLC analysis of a real sample commercial supplement rather than a standard sample.
A commercially available tocotrienol supplement was divided by a cutter and 10.5 mg of the content was weighed and dissolved in 1 mL of hexane / isopropanol = 1: 1 to prepare a sample for HPLC.
Inertsil (registered trademark) C30 Using an HPLC packed column having an inner diameter of 4.6 mm and a length of 250 mm, 100% methanol was analyzed at a flow rate of 0.5 mL / min, a column temperature of 30 degrees, and a detection of 295 nm. Compared with the method, the effect was confirmed.
In the conventional method, the pressure is 3.1 MPa, and the isomers 2) gamma tocotrienol and 3) beta tocotrienol are not separated. (Figure 11 top)
In the method of the present invention, in order to prevent breakage, a fused silica capillary with an inner diameter of 0.1 mm and a length of 25 cm covered with a peak tube with an inner diameter of 0.4 mm as a protective tube is used as a resistance tube, and a SUS 1/16 inch ferrule is placed behind the column. It connected between the detectors, and the others were analyzed under the same conditions as the conventional method.
The column inlet pressure was 63.3 MPa, and as shown in the lower part of FIG. 11, all four isomers of 1) delta tocotrienol, 2) gamma tocotrienol, 3) beta tocotrienol, and 4) alpha tocotrienol were separated. It was found that it was included. It was confirmed that the isomer separation according to the present invention can also be applied to an actual sample analysis of an extract from a supplement.

  1. Fatty acid methyl ester under the same conditions as in Example 1. γ-Methyl (z, z, z) -6,9,12-octadecatrienoate and 2. Analysis of isomers of α-Methyl (z, z, z) -9,12,15-octadecatetrienoate was performed.

  Inertsil® C30 inner diameter 4.6 mm length 250 mm and Inertsil® ODS-P inner diameter 3.0 mm at a flow rate of 0.5 mL / min using a 100% methanol eluent that is easy to use and fast replacement. The method of the present invention was carried out with two types of columns having a length of 200 mm. A high pressure cell was connected to the outlet channel of the column, and then a regulator was connected as a resistor, and the experiment was conducted while varying the back pressure.

  In the case of Inertsil (registered trademark) C30, inner diameter 4.6 mm, length 250 mm, first, analysis was performed without applying back pressure, and almost no separation was possible at a pressure of 7.4 MPa (FIG. 12 (1)). . When the regulator inlet was turned to adjust the column inlet pressure to 56.3 MPa, the elution time of the two components changed and separation was observed. (FIG. 12 (2)) Although it was not complete separation, it was confirmed that pressure effectively acts on the separation (peak 1: γ-Methyl (z, z, z) -6,9,12- octadecatrienoate, peak 2: α-methyl (z, z, z) -9,12,15-octadecatrienoate).

  The same was done for Inertsil (registered trademark) ODS-P, inner diameter 3.0 mm, length 200 mm. In the case where no pressure was applied, almost no separation was performed at 7.4 MPa (FIG. 12 (1)), and when the pressure was adjusted to 57.3 MPa, the elution time of the two components was changed, resulting in complete baseline separation (FIG. 12). (2)).

  The same effect was obtained by connecting a fused silica capillary tube having an inner diameter of 0.03 mm and a length of 25 cm in front of the cell. It was confirmed that the pressure works effectively in changing the separation of the isomers, although the size varies depending on the type of column.

An example in which the method and apparatus of the present invention shown in Example 1 are applied to sorting is shown below.
Inertsil (registered trademark) ODS-P (USP code L1 octadecyl group-bonded silica gel 5 μm, manufactured by GL Sciences Inc.) Using a preparative column having an inner diameter of 20 mm and a length of 250 mm, 70% acetonitrile water as a simple eluent was supplied at a flow rate of 10 mL / The solution was fed in min, and the xylene isomers 1. o-xylene, 2. 2. m-xylene; An attempt was made to fractionate p-xylene. A PEEK tube wound in a coil shape having an inner diameter of 0.13 mm and a length of 2 m was connected as a resistance to the outlet channel of the separation column, and fractionation was performed. The separation column inlet pressure was 65 MPa. The capacity of the resistance tube is 0.03 mL, but since the flow rate is fast, the actual peak delay time is 0.2 seconds or less and has no effect.

In ODS-P, it was possible to fractionate xylene isomers with a simple 70% acetonitrile water eluent without adding β-cyclodextrin to the eluent (FIG. 13).
10 minutes to 11.5 minutes as the first fraction (4), 11.6 minutes to 12.8 minutes as the second fraction (5), 12.8 minutes to 13 minutes as the third fraction (6), When the purity of each fraction was checked using gas chromatography, 100% pure o-xylene was obtained with 99% recovery in the first fraction (4), and the third fraction (6). In the second fraction (5), 70% purity m-xylene was obtained with a recovery rate of 90%, and it was found that the target component could be fractionated. It was.
In the conventional method, since β-cyclodextrin is contained in the eluent, it was necessary to remove it further after fractionation. However, in the present invention, since it can be performed with a simple eluent having no added components, only the solvent is evaporated as it is. As a result, it was found that it was very suitable for purification because it could be concentrated or dried easily.

  It was also possible to fractionate isomers by using a regulator 25 that automatically turns the knob 24 as a resistance and performing a pressure gradient in which the pressure was varied from 50 MPa to 10 MPa. Since the flow rate was high, even if the regulator 25 was provided between the column and the detector cell, there was no influence of dead volume, and it was possible to fractionate isomers. FIG. 7 shows an example of the regulator. In general, the body 20 has a structure in which a piston 21 is shielded by a compression spring 23 through a piston seal 22.

  With the apparatus and method shown in Example 1, using an analytical column of Inertsil® C30 ID 4.6 mm, length 250 mm and Inertsil® ODS-P ID 3 mm, length 200 mm, with a simple eluent 1. 50% acetonitrile-water at a constant temperature of 20 ° C. α-estradiol and 2. An attempt was made to separate the β-estradiol isomers. Inertsil (registered trademark) C30, the inner diameter was 4.6 mm, and the length was 250 mm, the flow rate was 0.35 mL / min. In the conventional analysis in which no resistor was connected, the pressure was 4.2 MPa, and the isomer peaks 1 and 2 were separated (FIG. 14 (1)).

  As a resistance, a fused silica capillary tube having an inner diameter of 0.03 mm and a length of 25 cm was connected to the column outlet, and the column pressure was analyzed as 71 MPa. As a result, it was eluted as a single peak without separation (FIG. 14 (2)). .

  In the C30 column, the functional groups that serve as the stationary phase are present in a high density. By pressurizing the stationary phase, more planar β-estradiol can easily enter the high density stationary phase, resulting in large elution. It is thought to be late. On the other hand, α-estradiol has a three-dimensional molecular shape compared to β-integral, and its influence is not so remarkable. As a result, it does not separate at 71 MPa, but to change the separation between isomers. It was confirmed that the pressure was effective.

  Inertsil (registered trademark) ODS-P with an inner diameter of 3 mm and a length of 200 mm, the flow rate was 0.13 mL / min. In a conventional analysis without connecting a resistor, the pressure is 3.4 MPa, and 2 β-estradiol is eluted first (FIG. 15 (1)). As a resistance, a fused silica capillary tube having an inner diameter of 0.03 mm and a length of 66 cm was connected to the column outlet, and the column pressure was 71 MPa. As a result, α-estradiol was eluted first (FIG. 15 (2)).

  Even in ODS-P, more planar β-estradiol is retained stronger, elution time is delayed, and peak elution is reversed compared to 3.4 MPa. From these facts, it was found that the separation can be greatly changed by adjusting the pressure, and it has been found that the preparation of separation, which has been conventionally performed by changing the eluent, can be easily performed simply by connecting a resistor and changing the pressure. .

The apparatus and method shown in Example 1 were applied to the separation of isomeric compounds having ionic hydrophilic groups using an Inertsil (registered trademark) ODS-P column having an inner diameter of 3 mm and a length of 200 mm. Is an isomer of a fatty acid with a carbonyl group at the end,
1. Cis-vaccinic acid (11Z-Octadecenoic Acid)
2. Petroselinic acid (cis-6-Octadecenoic Acid)
3. Eladic acid (trans-9-Octadecenoic Acid)
Was used as the eluent, and a mixed mobile phase of 97.5% methanol and 2.5% 10 mM phosphoric acid (H 2 O) was used, and the effect was verified at a flow rate of 0.4 mL / min.

  In a normal analysis in which no resistance is connected, the column inlet pressure is 3.2 MPa, which is 10 MPa or less, and the fatty acid isomers are not separated (FIG. 16 (1)). When a fused silica capillary tube having an inner diameter of 0.03 mm and a length of 15 cm was connected to the column outlet as a resistance, the column inlet pressure was 10 MPa or more, 40.2 MPa, and the isomers were separated (FIG. 16 (2)).

  It was confirmed that the method of the present invention is effective also in the separation of ionic isomers. As a matter of course, it was confirmed that the effect was obtained even when a buffer was included as an eluent.

  From the above, it has been found that the separation behavior can be changed by changing the elution behavior of isomers simply by adding resistance to the conventional analysis state so that the column inlet pressure is 10 MPa or more.

  According to the apparatus and method shown in Example 1, the tocopherol isomers of Example 2 (1.σ-tocopherol, 2.γ-tocopherol, 3.β-tocopherol, 4.α-tocopherol) were used as samples, and different types of immobilization were performed. Separations were performed on packed columns with phases.

  Among so-called ODS columns in which octadecyl groups are bonded to spherical silica gel, Inertsil (registered trademark) ODS-3 (USP code L1 octadecyl group-bonded silica gel, carbon content 15%, 5 μm, manufactured by GL Sciences) Inner diameter 3 mm, length 100 mm, Inertsil (Registered trademark) ODS-EP (USP code L1 octadecyl group-bonded silica gel embed type carbon content 9% 5 μm GL Science Co., Ltd.) inner diameter 3 mm length 100 mm, Inertsil (registered trademark) ODS-P (USP code L1 octadecyl group-bonded silica gel carbon Amount 29% 5 μm GL Sciences Inc.) Three types of columns with an inner diameter of 3 mm and a length of 200 mm were used, the flow rate was 0.2 mL / min with 100% methanol eluent, the inner diameter was 0.03 mm and the length was 60 c. Fused silica capillary tube connected to the column outlet of tested the effect of pressure.

  In ODS-3 and ODS-EP, there was an increase in retention time due to an increase in pressure, but no change in separation between isomers was observed, and β-form and γ-form were not separated (FIGS. 17 and 18).

On the other hand, in ODS-P, the elution of isomeric compounds is changed and separated (FIG. 19). Each small ODS-3, ODS-EP-pressure effect, the binding group density octadecyl, 1.3μmol ^/ m 2, a 1.7μmol / ^{m 2,} the high pressure effect ODS-P, 2.3μmol / M ² . Incidentally, in C30 used in Example 2, the bonding group density of the chemical bonding group is 2.5 mol / m ² , and the pressure effect in the isomer separation is effective for a high-density stationary phase of ² mol / m ² or more. I understand that.

Inertsil (registered trademark) ODS-3 1.3 μmol / m ² and ODS-EP 1.7 μmol / m ² have the same pressure effect as Type 1 and only increase in retention occurs. There has been no significant change in the separation. Isomeric compounds have the same bonding groups and differ only in their arrangement, and separation due to the effects of types 1 to 4 cannot be expected so much.

  In C30 and ODS-P, the binding arrangement is different and the elution between isomeric compounds with different molecular conformations changes. Therefore, in addition to the type 1 effect, multiple layers can be used as fillers (polymeric bonds). ) It was found that the stereoselective effect of the high-density stationary phase worked. Furthermore, when pressure is applied, it is considered that the stereoselective effect appears greatly and the separation changes drastically.

  In addition, since these packed columns are generally commercially available, they can be easily obtained, and it is not necessary to select a special stationary phase and can be applied to isomer separation.

However, it goes without saying that a similar stationary phase can be prepared by the following synthesis without using a commercially available packed column. A specific synthesis method is as follows. In a solution of 5% octadecyltrichlorosilane dissolved in toluene in silica gel having a surface area of 450 m2 / m ² , a pore size of 10 nm, and a particle size of 5 μm, a reflux reaction is performed for 10 hours or more, and an octadecyl group similar to a commercially available Inertsil ODS-EP A stationary phase having a bonding group density of 2 μmol / m ² or more was obtained (bonding group density 2.1 μmol / m ² ).

Moreover, after applying 10% triacontyl benzoate toluene solution to the same gel and removing under reduced pressure at 120 ° C. in nitrogen, a stationary phase having a bond group density of 2.2 μmol / m ² similar to that of commercially available Inertsil C30 was obtained. It was.

Even in these stationary phases, the effect of changing the separation between isomers due to an increase in pressure is obtained, and any synthesis method or carrier may be used, but if the bond group density is 2 μmol / m ² or more, the isomers It was confirmed that the effect of changing the separation between them became high.

  From the Examples so far, it was confirmed that applying an pressure of 10 MPa or more to the HPLC column is effective for isomer separation, and specifically, it can be realized by attaching a resistance after the column and further changing it.

  On the other hand, it is known that in the gradient analysis in which the conventional eluent ratio is changed, the column pressure also changes with the change in the eluent ratio. In a gradient analysis using a typical reverse phase partition column, since the elution is aimed at early by increasing the amount of the organic solvent, the pressure decreases and changes. Even if the pressure at the start of analysis is 10 MPa or more, as the gradient analysis proceeds, the organic solvent concentration increases, the eluent viscosity decreases, and the final analysis pressure drops to 5 MPa. This is not applicable to separation between isomers.

Therefore, a resistance tube was attached after the column to bring the pressure to 10 MPa or more, and it was verified whether it could be applied to separation between isomers.
As a sample, 1) cis 11-octadecyl methyl ester and 2) cis 6-octadecyl methyl ester were used, and HPLC analysis was performed with the experimental conditions set as follows.
HPLC analytical column: Inertsil (registered trademark) ODS-P 3 μm ID 3 mm Length 100 mm
Eluent A: 10 mM phosphoric acid aqueous solution Eluent B: Methanol flow rate: 0.2 mL / min
Gradient: A / B 16/84 (initial) → 15 minutes linear gradient → 1/99 → hold until 30 minutes → then to initial state Detection wavelength: UV 205 nm
When the column temperature was 19 ° C., the pressure dropped to 8.8 MPa-5 MPa, and the chromatogram of FIG. 21A was obtained. A fused silica capillary with an inner diameter of 0.1 mm and a length of 25 cm covered with a peak tube with an inner diameter of 0.4 mm as a protective tube is connected as a resistance tube between the column and the detector with a SUS 1/16 inch ferrule. The result of analysis under conditions is the chromatogram of FIG. 21B. The pressure changed from 62.4 MPa to 39.5 MPa, but it was still 10 MPa or more, the peak became higher, and the isomers were separated. From this result, it was confirmed that the pressure change due to the solvent change at the time of the gradient has the effect of the present invention.

  Even if the resistance tube was not changed, it could be changed from 62.4 MPa to 39.5 MPa only by changing the gradient eluent, and the isomer separation state could be adjusted. In this example, since it is a water-methanol system, the change is a little less than 2 times, but if it is 30% ethanol (viscosity at 10 ° C. 4) -ethyl acetate (viscosity at 10 ° C. of 0.5), it is about 8 times. Since the pressure can be changed, if it is set to 10 MPa or more at the lowest time, it is effective for isomer separation.

In HPLC, it is known that when the temperature is raised, the movement speed of the solute component in the stationary phase is increased, so that elution is accelerated and the peak shape is improved.
Therefore, analysis was performed by changing the column temperature in the state of the present invention.

  The pressure changed up to 56.2-35.8 MPa at 20 ° C. and up to 50.4-32.0 MPa at 25 ° C., and the chromatograms of FIGS. 21C and 21D were obtained. As the temperature increases, the pressure decreases, and elution of 1 and 2 approaches and the resolution becomes poor as an isomer. However, it still retained 10 MPa or more at the end of the gradient, and it was confirmed that these isomers were separated. For example, in the case of 30% ethanol, the viscosity at the column temperature is 80 ° C. and the viscosity is 0.6 ° C., and when the temperature is changed, a pressure change close to 10 times can be caused.

From this result, it was clarified that the peak shape can be changed while maintaining the separation of the isomers as long as the analysis temperature can be maintained at a pressure of 10 MPa or more.
Therefore, the pressure change of a little less than 10 times is possible by the change in the solvent ratio accompanying the gradient and the change in the column temperature, the isomer separation can be changed, and the pressure from 10 MPa to 100 MPa can be changed by combining both. It was confirmed that changes would be possible.

  It has been reported that the phase ratio of the solute changes according to the pressure change and the retention time changes, but the retention changes between the isomers of the compound, and the separation between the isomers is effective. There has never been a report to say. In order to confirm this characteristic according to the present invention, the data of the examples were put together to confirm what kind of phenomenon occurred.

Since the retention occurs depending on how much the solute remains in the stationary phase, the solute volume change ΔV occupying the stationary phase can be obtained from the following equation from the change in the retention ratio accompanying the increase in pressure.
ΔV = −gas constant × T (K) × ln (holding ratio when carrying out the present invention / holding ratio in the case of conventional analysis) / working pressure of the present invention ΔV1 is the amount of solute volume change of solute 1, Δ When V2 is the solute volume change amount of the solute 2, the ΔΔV value that is the ratio of the change amount between the solute compounds of the solute 1 and the solute 2 is a value that is not affected by the phase ratio.

  The results of analysis on two types of HPLC analysis columns are summarized in the following table. It can be seen that by applying pressure corresponding to the difference in the structure of the solute, incorporation into the stationary phase occurs, volume change occurs in the stationary phase, and the isomers are separated.

  For example, taking the case of an isomer α- / γ-linolenic acid methyl ester at a temperature of 303 K (20 ° C.) using an Inertsil (registered trademark) ODS-P column (first row in the table) as an example. The retention ratio of α-linolenic acid methyl ester in the conventional analysis is 1.00, and the retention ratio when the pressure of the present invention is applied to 50 MPa is 1.82, and ΔV1 is −30. 2 (−8.31 × 303 × In (1.82 / 1.00) / 50).

  The retention ratio of the isomer γ-linolenic acid methyl ester is 1.12, and the retention ratio when the pressure of 50 MPa of the present invention is applied is 2.45, and ΔV2 is −39.4 according to the above formula. It becomes. The difference ΔΔV between both isomers is -9.3 after rounding.

When calculation is performed in the same manner, ΔΔV is obtained for each isomeric compound. It is considered that the larger the absolute value of this difference is, the more isomeric component the effect of the present invention can be expected.
Even when an Inertsil (registered trademark) C30 column was used, ΔΔV was obtained for each compound in the same manner.

  The △△ V value varies depending on the type of stationary phase. In this result, the same △△ V value is obtained for C30 and ODS-P, and △△ V value (ODS-P)> △△ V value ( C30). In m- / p-xylene on the second line in the table and β- / α-estradiol on the sixth line in the table, almost the same value is obtained, and in β- / γ-tocopherol, almost the same absolute value is obtained.

  Although the elution order varies depending on the stationary phase, there may be a difference between + and-, but the amount of change in retention of solute in the stationary phase due to pressure change is almost the same, and the effect of elution change between the isomers is almost the same. it is conceivable that.

  On the other hand, in the compounds in the first, third, and fourth rows in the table, the absolute value of the ΔΔV value of ODS-P is larger than the absolute value of the ΔΔV value of C30.

The compounds in the 2nd and 6th rows in the table are solutes with the hydrocarbon chain as the main skeleton and rigid sites, and the compounds in the 1st, 3rd and 4th rows in the table have ester chains and are flexible sites. It is considered as a solute.
That is, it is considered that the identification site of C30 is rigid and the recognition site of ODS-P includes a rigid site and a flexible site.

  As described above, it was suggested that the ΔΔV value was used as an index for measuring the magnitude of the pressure effect. And by using this value, it became clear that the difference in solute penetration occurred due to the pressure effect and the separation between isomers changed. Furthermore, it was estimated that the recognition site between isomers was different depending on the liquid phase species, and the structure of the compound was also recognized.

Claims (20)

  In the liquid chromatography, a resistance is provided in the outlet passage of the column used, and the column inlet pressure is adjusted to 10 MPa to 100 MPa by the operation of the resistance, and the solute behavior of the isomer compound is changed for each component. For treating isomeric compounds.   The method for treating an isomeric compound according to claim 1, wherein the change in the solute behavior is solute separation.   The method for treating an isomer compound according to claim 1, wherein the change in the solute behavior is a change in a solute appearance order.   The method for treating an isomer compound according to claim 1, wherein the change in the solute behavior is solute coalescence.   5. The isomer compound according to claim 1, wherein the solute behavior of the isomer compound is changed while continuously or stepwise decreasing the column inlet pressure between 100 MPa and 10 MPa. Processing method.   5. The solute behavior of an isomer compound is changed by changing the composition of an eluent to change the column inlet pressure continuously or stepwise between 100 MPa and 10 MPa. The processing method of the isomeric compound as described in any one.   The column inlet pressure is changed between 100 MPa to 10 MPa by changing a column temperature, and the solute behavior of the isomer compound is changed. 5. Method for processing isomeric compounds.   The method for analyzing an isomer compound according to any one of claims 1 to 5, wherein the column is an analytical column.   The method for fractionating isomeric compounds according to any one of claims 1 to 5, wherein the column is a preparative column.   An apparatus for processing an isomeric compound, wherein a resistance is connected to a column outlet channel between a column outlet and a detector, between a detector and a discharge or both, An apparatus for treating an isomeric compound, wherein the apparatus is adjustable to a predetermined pressure.   The isomer compound analyzer according to claim 8, wherein an analytical column is used as the column.   The isomer compound fractionator according to claim 8, wherein a fractionation column is used as the column.   The apparatus for treating an isomer compound according to claim 8, wherein the resistor is a resistance tube.   12. The apparatus for treating an isomer compound according to claim 11, wherein the resistance tube is one of a tube, a monolith column, and a packed column.   The apparatus for treating an isomer compound according to claim 8, wherein the resistor is a regulator capable of changing a space capacity through which the liquid flows.   The apparatus for treating an isomer compound according to any one of claims 8 to 13, wherein an analytical column is used as the column.   The apparatus for treating an isomer compound according to any one of claims 8 to 13, wherein a fractionation column is used as the column.   A valve is provided on the column outlet channel side, a plurality of resistors are connected to the valve, and a predetermined column inlet pressure is obtained by selecting a resistor to be connected. An apparatus for treating an isomer compound according to 1. An isomeric compound separation column using a filler in which a stationary phase is coated or chemically bonded to a particulate carrier or monolith carrier having a pressure resistance of 10 MPa or more at a density of ² μmol / m ² or more. A packing material for an isomeric compound separation column, wherein a stationary phase is coated or chemically bonded to a particulate carrier or monolith carrier having a pressure resistance of 10 MPa or more to a density of ² μmol / m ² or more.

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