JP6131443B2 - Separation medium, column using the separation medium, liquid chromatograph equipped with the column, and method for producing the separation medium - Google Patents

Description

  The present invention relates to the field of analytical chemistry, and more particularly to a separation medium used as a packing material for a column or pretreatment cartridge used for separating sample components. The present invention provides a separation medium such as an analytical column used in a liquid chromatograph mainly including high performance liquid chromatograph (HPLC) and a column (including a cartridge shape) used for pretreatment before analysis. It also relates to the equipment used.

  One use of the separation medium is as a column packing material for liquid chromatographs. As the column packing material, silica gel and silica gel bonded with a functional group such as ODS (octadecylsilyl) group are often used. In liquid chromatography using such a packing material, residual impurities (mainly metal components) in silica gel cause deterioration of the peak shape of the analysis target component. In particular, peak tailing occurs when analyzing a component having a high metal coordination property, and it is considered that the cause is due to a secondary interaction between the metal impurity in the silica gel and the coordination compound. For this reason, in order to synthesize silica gel or a packing material in which a functional group is bonded to silica gel, or to fill a column, there has been devised to remove this metal component as much as possible.

  However, it is difficult to completely remove metal impurities in silica gel. Therefore, in the case of ODS silica gel filler, the silanol groups and metal impurities remaining in the ODS silica gel filler are inactivated by devising the secondary silylation of the ODS silica gel filler, that is, the number of end capping and the reaction reagent. (See Patent Document 1). There, primary end capping using an amine compound, secondary end capping using another amine compound, and additional end capping using a tertiary amine are performed.

  The metal impurities in the silica gel of the ODS silica gel filler currently used are controlled so as to be below a certain concentration. For example, Na, Ca, Mg, Al, Fe, Ti, and the like as metals are controlled so that each is 10 ppm or less, and the total of all metal impurities is 30 ppm or less (for example, Shimadzu Corporation, a product of Shimadzu Corporation). -pack). Therefore, normally, the total concentration of all metal impurities of 30 ppm or less is the reference value of the impurity concentration in the silica gel of the filler.

  As another use of the separation medium, there is a packing material for a column that performs a pretreatment for removing impurities and the like before subjecting the analysis target to a liquid chromatograph. Such a packing material has the same problem as the packing material of the analytical column.

Japanese Patent Laid-Open No. 2008-232802 JP-T-2006-504854

  The method described in Patent Document 1 is an effective method for removing the adverse effects caused by inactivating the silanol groups and metal impurities remaining in the ODS silica gel filler, and demonstrates only the functions of the original ODS functional groups. I will try to let you. Therefore, as the function of the ODS silica gel filler, only the function of the ODS group is exhibited, and other functions are not exhibited.

  It is an object of the present invention to provide a new silica-based separation medium that utilizes the interaction between components to be analyzed and metal ions in the separation medium.

  Another object of the present invention is to obtain an analytical column and a pretreatment column using the separation medium, and to construct a liquid chromatograph using such a column.

  Since it is difficult to completely remove the residual metal component of the separation medium, the present invention does not completely remove the residual metal component, but instead introduces the metal component at a higher concentration than the residual metal component. As a result, it was found that a separation medium having new separation characteristics can be obtained by positively using the metal component so as to affect the separation characteristics.

The separation medium of the present invention includes a carrier which is silica gel or a carrier containing silica gel as a component, and 50 ppm or more of metal bonded to the carrier so as to impart hydrophobicity to the carrier. In the present specification, the “metal” means a metal ion constituting the metal salt and a metal derived from the metal ion unless otherwise specified as “metal salt”, “metal ion”, “metal acoion”, and the like. (For example, Fe in FeCl ₃ ). The “metal” is considered to give metal ions in a carrier immersed in water or an aqueous solution at least when the separation medium is used. In the present specification, the “metal ion” is a concept including a metal acoion. In the present specification, “binding” to the carrier is a concept including not only trapping by the three-dimensional structure of the carrier constituting molecule but also interaction by intermolecular forces such as adsorption and hydrogen bonding. Is substantially synonymous with “immobilization”.

  Usually, the concentration of metal impurities in the silica gel used for the packing material of the liquid chromatograph is controlled to be 30 ppm or less in total. In the present invention, a metal is introduced into the carrier at a higher concentration than the metal impurities. Therefore, the metal concentration of 50 ppm or more in the present invention may include not only the metal introduced into the carrier but also the metal previously contained in the carrier as an impurity. It is the sum of all metal types. The metal is preferably bound to the support at a concentration that will saturate.

  The type of each metal constituting the metal that is 50 ppm or more in total is preferably a type of metal usually contained as an impurity in silica gel, and includes, for example, Al, Fe, Zn, Mn, Ni, Mg, Ag, or the like. . The type of metal to be introduced into the support is preferably selected from the types of metals usually contained as impurities in silica gel, more preferably at least selected from the group consisting of Al, Fe, Zn, Mn, Ni, Mg and Ag. One type.

  Since the present invention solves the problem caused by residual metal impurities in silica gel, the carrier having such a problem is not limited to silica gel but also includes a carrier containing silica gel as a component. Examples of such a carrier containing silica gel as a component include inorganic / organic hybrid particles as described in Patent Document 2.

  In the first form of the separation medium, a functional group that gives an affinity different from that of silica gel to a component to be analyzed is not bound to the carrier. The functional group is a functional group that characterizes a second form of a separation medium to be described later, such as an ODS group.

  In the second form of the separation medium, in addition to the metal of 50 ppm or more, a functional group that gives an affinity different from that of silica gel to the component to be analyzed is further bound to the support. Examples of such a functional group include an ODS group, an octyl group, a butyl group, and an aminopropyl group. Specifically, for example, before and after the step of bonding a functional group such as an ODS group to silica gel, an aqueous metal salt solution is added to the carrier, and the metal is also immobilized on the silica gel. As a result, the resulting separation medium has a new separation selectivity due to the presence of an overwhelmingly large amount of metal in the silica gel compared to the amount of residual metal in the silica gel before the addition of the aqueous metal salt solution. It becomes a separation medium that cannot be seen.

  Regarding end capping, it is not excluded to perform end capping at a normal level for suppressing the influence of silanol groups.

  The column of the present invention is such that the separation medium of the first form or the second form is packed in a tube. An example of the first form of the column is a cartridge shape that is filled with the separation medium of the first form and used off-line, and is suitable for use as a pretreatment column for liquid chromatography. Is. Such a pretreatment column is used, for example, to capture an analysis target component in a sample and discharge impurities, and then to elute the captured analysis target component and use it for a liquid chromatography sample.

  Another example of the first form of the column is a pretreatment column that is packed with the separation medium of the first form and configured in a column shape that is used online. Such a pretreatment column can be used as a component of a liquid chromatograph. An example of such a liquid chromatograph is a first solvent supply that supplies the pretreatment column and a first solvent that causes the pretreatment column to hold one or more components to be analyzed in the sample. A second solvent supply flow path for supplying a flow path and a second solvent for eluting the analysis target component held in the pretreatment column to the pretreatment column, the second solvent being different from the first solvent; An analytical column for separating the analyte to be analyzed connected to the flow path through which the eluate from the pretreatment column flows, a detector for detecting the analyte separated by the analytical column connected downstream of the analytical column, A first flow path that connects the first solvent supply flow path to the pretreatment column, a second solvent supply flow path is connected to the pretreatment column, and an eluate flow path from the pretreatment column is connected to the analysis column. Switch between the second flow path It is obtained by a flow path switching mechanism.

  The second form of the column of the present invention is packed with a separation medium of the second form in which a functional group giving an affinity different from that of silica gel is further bound to a component to be analyzed in addition to a metal of 50 ppm or more on a carrier. The column shape is used on-line. The column of the second form can be used as an analysis column in a liquid chromatograph regardless of whether or not a pretreatment column is provided. Such a liquid chromatograph includes a liquid chromatograph provided with the column of the first embodiment as a pretreatment column.

The separation medium of the present invention is produced by including the following steps (A) and (B).
(A) a step of immersing a carrier which is silica gel or a carrier containing silica gel as a component in an aqueous metal salt solution, and then drying the carrier; and (B) a heat treatment at a temperature in the range of 400 to 1200 ° C. Process.

  The “dipping” in the above step (A) is not particularly limited as long as it is a method in which the support is sufficiently wetted with the metal salt aqueous solution, for example, a method in which the support is immersed in the metal salt aqueous solution, a column or a cartridge is packed. And the like by a method in which an aqueous metal salt solution is dropped onto the prepared carrier.

  The metal is preferably at least one selected from the group consisting of Al, Fe, Zn, Mn, Ni, Mg, and Ag.

  The resulting separation medium is more hydrophobic than before the heat treatment. Silylation is conceivable as a method for imparting hydrophobicity to silica gel, but the above-described method of the present invention uses a hydrophobic interaction by heat treatment of silica gel (inactivation of silanol groups) without using a silylating agent. It is characterized in that the cation exchange interaction by the metal acoion existing on the surface of the silica gel is simultaneously given to the silica gel.

  In one embodiment, step (A) bonds the metal to silica gel in a saturated state.

  In another embodiment, the method further includes a step of bonding a functional group comprising an octadecylsilyl group, an octyl group, a butyl group, or an aminopropyl group to the support before or after the step (A) or after the step (B).

  The separation medium of the first form is one in which a functional group that gives an affinity different from that of silica gel, such as an octadecylsilyl group, is not bound to a carrier. This separation medium has a hydrophobic property and can interact with acidic, neutral and basic compounds by a hydrophobic interaction mechanism. Furthermore, the metal ions on the surface of the silica gel have acidic properties and can interact with basic compounds by a selective electrostatic interaction mechanism (ie, cation exchange mode). The strength of the cation exchange interaction depends on the acidity of Fe (III) ion, Al ion, Ag ion or Ni ion, that is, the pKa value, so that the acidity of the metal ion can be selected by selecting the metal species. The electrostatic interaction can be controlled by the difference in degree. This result means that metal ion affinity chromatography can be applied to the analysis of a wide range of basic compounds.

  In the separation medium of the second form, a functional group such as an ODS group that gives an affinity different from that of silica gel to the analysis target component in addition to a metal of 50 ppm or more is further bonded to the carrier. By analyzing using an analytical column packed with this separation medium, for example, the retention of a basic compound becomes stronger depending on the type of metal to be immobilized, and the selectivity is different from that of a general ODS column. Become. This is presumed to depend on the pKa value and hydrophobicity of the basic compound as the analysis target component. Moreover, since the behavior as the separation medium varies depending on the type of metal to be immobilized, the separation can be optimized by varying the metal species to be immobilized according to the component to be analyzed. Further, by optimizing various parameters such as the amount of the organic solvent in the mobile phase, the pH of the buffer solution, and the salt concentration, it becomes possible to respond more flexibly.

It is sectional drawing which shows the cartridge which is one Embodiment of a separation medium. It is a partially cutaway sectional view showing a column which is another embodiment of the separation medium. It is a flow-path block diagram which shows the liquid chromatograph by which the pre-processing column of one Example was mounted. ~ It is a graph which shows the collection | recovery rate for every analysis target component by the various pretreatment columns on acidic conditions. ~ It is a graph which shows the recovery rate for every analysis object component by various pretreatment columns under neutral conditions. It is a graph which shows the relationship between logP and the recovery rate of the analysis object component under the acidic condition by a pretreatment column. It is a graph which shows the relationship between logP and the recovery rate of the analysis object component under neutral conditions by a pre-processing column. ~ It is a graph which shows the relationship between the acidic solvent density | concentration for every analysis target component by various pre-processing columns, and a recovery rate. ~ It is a graph which shows the relationship between the neutral solvent density | concentration for every analysis target component by various pre-processing columns, and a recovery rate. It is a flow-path block diagram which shows the liquid chromatograph by which the analysis column of one Example was mounted. It is a figure which compares the chromatograph by the metal fixed ODS column of the Example in a 1st gradient analysis with the chromatograph by the ODS column of a comparative example. It is a figure which compares the chromatograph by the metal fixed ODS column of the Example in the same gradient analysis with the chromatograph by the ODS column of a comparative example. It is a figure which compares the chromatograph by the metal fixed ODS column of the Example in a 2nd gradient analysis with the chromatograph by the ODS column of a comparative example. It is a figure which compares the chromatograph by the metal fixed ODS column of the Example in the same gradient analysis with the chromatograph by the ODS column of a comparative example. It is a figure which compares the chromatograph by the metal fixed ODS column of the Example in a 3rd gradient analysis with the chromatograph by the ODS column of a comparative example. It is a figure which compares the chromatograph by the metal fixed ODS column of the Example in the same gradient analysis with the chromatograph by the ODS column of a comparative example. It is a figure which shows the chromatograph by the metal fixed ODS column of the Example in a 4th gradient analysis compared with the chromatograph by the ODS column of a comparative example. It is a figure which compares the chromatograph by the metal fixed ODS column of the Example in the same gradient analysis with the chromatograph by the ODS column of a comparative example. It is a figure which compares the chromatograph by the (Fe (III) -ODS column of an Example) and the chromatograph by the ODS column of a comparative example about the case where KCl is added as a salt to a mobile phase. It is a figure which compares the chromatograph by the (Fe (II) -ODS column of an Example) with the chromatograph by the ODS column of a comparative example about the case where KCl is not added as a salt to a mobile phase. It is a figure which compares the case where KCl is added as a salt with a mobile phase, and the case where it does not add, comparing the chromatograph by the Al-ODS column of an Example with the chromatograph by the ODS column of a comparative example. It is a figure which compares the case where KCl is added as a salt with a mobile phase, and the case where it does not add, comparing the chromatograph by the Ag-ODS column of an Example with the chromatograph by the ODS column of a comparative example. It is a figure which compares the chromatograph by the (Fe (III) -ODS column of an Example) with the chromatograph by the ODS column of a comparative example about the case where PFPA is not added as a salt to a mobile phase. It is a figure which compares the case where PFPA is added as a salt with a mobile phase, and the case where it does not add, comparing the chromatograph by the Al-ODS column of an Example with the chromatograph by the ODS column of a comparative example. It is a figure which compares the chromatograph by the Ag-ODS column of an Example with the chromatograph by the ODS column of a comparative example about the case where PFPA is not added as a salt to a mobile phase.

(First embodiment)
Separation medium in which a functional group that gives an affinity different from that of silica gel for a component to be analyzed is not bound to a carrier:
When silica gel is used as a carrier, the form of silica gel is not particularly limited, but as a preferred form, the particle diameter is 1 to 300 μm, more preferably 1 to 200 μm, even more preferably 1 to 150 μm, and the pore diameter is 2 to 100 nm. More preferably, 3-30 nm and a specific surface area are 50-800 m < ² > / g, More preferably, 200-400 m < ² > / g can be mentioned. Further, the particle diameter is particularly preferably 10 to 100 μm for pretreatment and particularly preferably 2 to 20 μm for an analytical column. As the carrier, inorganic / organic hybrid particles may be used. In the following description, the case of using silica gel will be described, but the same applies to the case of using inorganic / organic hybrid particles.

A metal is bound to the support. As the method, the support is immersed in a metal salt aqueous solution of a metal such as Al, Fe, Zn, Mn, Ni, Mg, Ag, etc., dried, and heated. Examples of the metal salt include AlCl ₃ , FeCl ₃ , FeCl ₂ , ZnCl ₂ , MnCl ₂ , NiCl ₂ , MgCl ₂ , AgNO _{3 and the} like. Although the metal salt aqueous solution is not particularly limited, for example, a 0.01 to 1% by mass aqueous solution of a metal salt, preferably a 0.05 to 0.5% by mass aqueous solution, with respect to 100 parts by mass as a dry mass of the carrier, for example, It is added to the carrier at a ratio of 0.2 to 20 parts by mass, preferably 0.5 to 10 parts by mass, and stirred for 3 to 24 hours, preferably 5 to 12 hours. Next, the metal-containing carrier is filtered through a glass filter or the like and washed with water or the like. The obtained carrier is dried at, for example, 80 to 200 ° C., preferably 100 to 170 ° C., for example, 30 minutes to 10 hours, preferably 1 to 5 hours, and subsequently, for example, 300 to 1500 ° C., preferably 400 to 1200 ° C. More preferably, the separation medium can be obtained by heating at 400 to 1100 ° C., for example, for 30 minutes to 10 hours, preferably 2 to 4 hours.

  A specific example of obtaining a separation medium is shown. Each of a 0.2% aqueous solution of ferric chloride, a 0.2% aqueous solution of aluminum chloride hexahydrate, a 0.2% aqueous solution of silver nitrate, and a 0.2% aqueous solution of nickel nitrate was separated on a separate silica gel (particle size : 50 μm, pore size: 12 nm) and stirred for 5 to 10 hours. As a result, it was estimated that the metal was bound to the silica gel in a saturated state. Next, the silica gel containing a metal was filtered with a glass filter (No. 3) and washed with 200 mL of water. Finally, the silica gel containing the metal was dried at 150 ° C. for 2 hours, and subsequently heated at 400 to 1000 ° C. for 2 to 4 hours to obtain a separation medium.

  The separation medium thus obtained may be referred to as “metal-silica gel” in the present specification. As specific metals, symbols such as Al, Fe (II) (ferrous iron), and Fe (III) (ferric iron) are included.

  The separation medium obtained in this way can be used as a pretreatment separation medium for removing impurities before introducing the sample into the liquid chromatograph, and for separation analysis of liquid chromatographs. It can also be used as a separation medium.

  One use form as a pretreatment separation medium is the cartridge 2 shown in FIG. 1A. A cylindrical container in which a spout 6 is provided at the upper end of the cylindrical main body 4 and an outlet 8 is provided at the lower end is filled with a separation medium 14 via a filter 12.

  The cartridge 2 is used offline. By injecting a sample solution containing impurities from the spout 6, the analysis target component is captured by the separation medium 14, and the impurities are discharged from the outlet 8. Thereafter, a solvent for eluting the analysis target component captured in the separation medium 14 is injected from the spout 6, whereby the analysis target component is eluted from the outlet 8, and a sample solution without impurities is obtained.

  Another form of use as the pretreatment separation medium is an online column 16 as shown in FIG. 1B. The column 16 is a column main body 18 such as a stainless steel tube filled with a separation medium 20. At both ends of the column main body 18, connection ports 22 and 24 for connection to the flow path of the liquid chromatograph are provided.

  Such a column 16 is used not only as a pretreatment column but also as an analysis column for separating an analysis target component by a liquid chromatograph. When used as an analytical column, one longer than the pretreatment column is generally used.

  An example in which the column 16 is used as a pretreatment column in a liquid chromatograph is shown in FIG. A pretreatment column 16A packed with a separation medium in which a functional group that gives an affinity different from that of silica gel for a component to be analyzed is not bonded to a carrier is interposed between two ports of a hexagonal valve 30 constituting a flow path switching mechanism. It is connected. The pretreatment column 16A is a column tube made of stainless steel (length 10 mm, inner diameter 4.0 mm) filled with a separation medium.

  The other port of the valve 30 is connected to a flow path 35 for supplying a pretreatment solvent for capturing the component to be analyzed in the pretreatment column 16A and discharging impurities and the like. A flow path consisting of a pump 38A for supplying the solvent 36A and a flow path consisting of a pump 38B for supplying the solvent 36B are connected to the flow path 35 in parallel. The channel 35 is provided with an injector 40 for injecting a sample solution.

  An analysis column 32 of a liquid chromatograph is connected to the other port of the valve 30. The analytical column 32 is housed in a column oven (not shown) and kept at a constant temperature. A detector 34 such as an ultraviolet-visible spectrophotometer that detects an analysis target component eluted from the analysis column 32 is connected downstream of the analysis column 32. The analysis column 32 is longer than the pretreatment column 16A, and the analysis column 32 is filled with another separation medium different from the pretreatment column 16A. The separation medium of the analytical column 32 is not particularly limited, and may be, for example, a separation medium in which a functional group such as metal and ODS is bonded to silica gel, which is a second embodiment described later. The liquid chromatograph apparatus is a column for reverse phase chromatography in which ODS is bonded as a functional group to silica gel.

  In order to elute the analysis target component captured by the pretreatment column 16A and guide it to the analysis column 32, a flow path 41 for supplying an analysis solvent is connected to the other port of the valve 30. The channel 41 includes a channel composed of a pump 44A for supplying the solvent 42A and a channel composed of the pump 44B for supplying the solvent 42B, which are connected in parallel so that the gradient analysis using two types of solvents can be performed. ing.

  Still another port of the valve 30 is a drain port for discharging liquid.

Any number of solvents may be used to be supplied to the pretreatment column together with the sample solution. For example, 1 to 5 types of solvents may be used. Such a solvent is not particularly limited as long as it is suitable for the sample components, and examples thereof include buffer solutions such as an acetate buffer solution and a phosphate buffer solution, and if necessary, an organic solvent such as acetonitrile, methanol, ethanol and the like. (The volume ratio is preferably organic solvent: water = 90: 10 to 1:99, more preferably 80:20 to 20:80). The solvent is supplied, for example, at a flow rate of 0.5 to 20 mL / min, preferably 1 to 10 mL / min, for example, 0.2 to 10 minutes, preferably 1 to 5 minutes.

  In the liquid chromatograph apparatus in FIG. 2, as a pretreatment, the solvent 36A or 36B is supplied by each pump 38A or 38B, and the sample solution is injected from the injector 40. The sample solution flows to the drain through the pretreatment column 16A. Meanwhile, the analysis target component is captured by the pretreatment column 16A, and impurities are discharged. In order to stabilize the analysis flow path of the liquid chromatograph even during pretreatment, the solvent is flowed from the flow path 41 to the analysis column 32 at a constant flow rate.

  After completion of the pretreatment, the valve 30 is switched, and the analysis solvent flows from the flow path 41 to the analysis column 32 through the pretreatment column 16A. As a result, the analysis target component captured by the pretreatment column 16A is eluted by the analysis solvent and introduced into the analysis column 32. The analysis target component separated by the analysis column 32 is detected by the detector 34. The gradient analysis using the analysis solvent may not be performed. However, in the liquid chromatograph apparatus in FIG. 2, the mixture ratio is changed by temporally adjusting the pumps 44A and 44B to perform the gradient analysis. it can.

  The specific example of a solvent is shown. An aqueous solution of 5 mmol / L of ammonium acetate or an aqueous solution of 0.1% acetic acid is used as the solvent 36A, and acetonitrile containing 5 mmol / L of ammonium acetate is used as the solvent 36B (shown as M4 in FIGS. 3 to 7). A mixture of water (volume ratio 6: 4) or a mixture of acetonitrile and water containing 0.1% acetic acid (volume ratio 6: 4) is used. In the pretreatment, the solvent 36A or 36B is allowed to flow at a uniform concentration for 1 minute at a flow rate of 2 mL / min.

  A 0.1% aqueous solution of trifluoroacetic acid is used as the solvent 42A, and a mixed solution (volume ratio 6: 4) of acetonitrile and water containing 0.1% trifluoroacetic acid is used as the solvent 42B. These solvents are introduced in gradient elution mode at a flow rate of 1 mL / min. The gradient conditions are a condition for changing the concentration of the solvent 42B from 0 to 100% in 12 minutes and a condition for changing the concentration of the solvent 42B from 0 to 100% in 3 minutes.

  An ODS column (length: 150 mm, inner diameter: 4.6 mm, silica gel particle size: 5 μm) is used as the analysis column 32, and the column temperature is set to 40 ° C. The amount of sample injected into the injector 40 is 10 μL. The detector 34 measures the ultraviolet absorbance at 280 nm.

(Evaluation of silica gel of the first form)
With the liquid chromatograph apparatus shown in FIG. 2, the retention characteristics of the separation medium used as the pretreatment column 16A were evaluated. A method for obtaining the recovery rate was adopted for the evaluation. The recovery rate was determined as follows. After the model compound is injected as a sample and captured in the pretreatment column 16A, the peak area (A) of each model compound when separated by the analysis column 32 and detected by the detector 34 by switching the valve 30 and the pretreatment The model compound is introduced into the analysis column 32 without passing through the column 16A, separated, and the peak area (B) of each model compound when detected by the detector 34 is measured. The recovery rate was determined by the following formula.
Recovery rate (%) = (A / B) × 100

Five types of heat-treated Fe (III) -silica gel were used as the silica gel of the first form. There are five types of heat treatment conditions as follows.
(1) 4 hours at 1000 ° C (2) 2 hours at 1000 ° C (3) 2 hours at 800 ° C (4) 2 hours at 600 ° C (5) 2 hours at 400 ° C

  For comparison, the recovery rate was also measured using a silica gel containing no metal and not heat-treated (referred to as control) as a separation medium. The fact that no metal is contained means that no metal is intentionally contained as in the embodiment, and about 30 ppm of metal may be contained as an impurity.

  The recovery rate was determined for five model compounds (acidic compound, neutral compound and basic compound). 3-4 and 6-7, (A) to (E) represent the following model compounds, respectively.

(Acidic compounds)
(A) Ketoprofen (Ketoprofen: pKa = 3.9, logP = 2.911)
(Neutral compound)
(B) Propyl paraben (log P = 2.901) (basic compound)
(C) Trimethoprim (Trimethoprim: pKa = 7.1, logP = 0.79)
(D) donepezil (Donepezil: pKa = 9.4, logP = 3.913)
(E) Imipramine (pmi = 9.4, logP = 4.355)

  The mobile phase of the pretreatment is acidic (solvent 36A is a mixed solution of 0.1% acetic acid in water and solvent 36B is a mixed solution of acetonitrile and water containing 0.1% acetic acid, and the ratio of solvent 36B varies in the range of 0 to 40%. 3A to 3E show the results when the pH was about 4), and the mobile phase of the pretreatment was neutral conditions (solvent 36A was ammonium acetate 5 mmol / L aqueous solution, solvent 36B was ammonium acetate 5 mmol / L Fig. 4 shows the results when the ratio of the solvent 36B was changed in the range of 0 to 40% with a pH of about 7).

  Fe (III) -silica gel showed an increase in the recovery rate for any model compound compared to the control, but the increase in the recovery rate was remarkable especially for the three basic compounds (trimethoprim, donepezil and imipramine) The tendency was strong under acidic conditions.

  Moreover, the recovery rate of the model compound increased as the temperature of the heat treatment of Fe (III) -silica gel increased under both acidic and neutral conditions.

  The above results indicate that the hydrophobicity of the surface of the silica gel increases when the silica gel is heat-treated, and that the hydrophobicity increases as the temperature of the heat treatment increases.

  From the above, it can be said that the recovery rate of the model compound by the heat-treated silica gel is increased by the hydrophobic interaction with the silica gel under both acidic and neutral conditions.

  Ketoprofen increased in recovery under acidic conditions than under neutral conditions. This result indicates that ketoprofen decreases hydrophobicity under neutral conditions and exists as an anion repelled by Fe (III) acoion.

  Next, when Fe (III) -silica gel that has been heat-treated (1000 ° C. for 2 hours) is used, the mobile phase is 20% under acidic conditions (A) and neutral conditions (B) and the concentration of solvent 36B is 20%. The recovery rates of various model compounds are shown in FIG. 5 in relation to the log P of the model compound. Donepezil and imipramine, which are basic compounds with high hydrophobicity (high logP), and trimethoprim, which is a basic compound with low hydrophobicity (low logP), all showed high recoveries. The acidic compound and the neutral compound were hardly retained in the heat-treated Fe (III) -silica gel, although the log P was higher than that of trimethoprim.

  From the above, since the metal ions in Fe (III) -silica gel are acidic and the recovery does not show dependence on the logP value, the basic compound has the same electrostatic interaction as that in the cation exchange mode. It can be said that it is selectively held by this mechanism.

  As described above, the recovery rates of the three basic compounds are significantly increased by both hydrophobic interaction and cation exchange interaction. In particular, donepezil and imipramine have higher pKa values than trimethoprim and are strongly retained by cation exchange interactions.

  The three basic compounds showed higher recoveries under neutral conditions than under acidic conditions. This is due to increased hydrophobicity of the basic compound under neutral conditions, increased hydrophobicity of the surface of the heat-treated silica gel, and cation exchange interaction between the basic compound and Fe (III) ions. It is estimated that However, under neutral conditions, the effect of increasing the hydrophobicity of the basic compound is greater than the cation exchange interaction between the metal ion on the heat-treated Fe (III) -silica gel and the basic compound. .

  Next, regarding the interaction between metal ions (metal species are Fe (III), Al, Ag and Ni) having different acidities (ie, pKa values) and basic compounds, heat treatment is performed at 1000 ° C. for 2 hours. Using the applied metal-silica gel, the recovery rate of five model compounds was measured. The results under acidic conditions using an acidic mobile phase for pretreatment are shown in FIG. 6, and the results under neutral conditions using a neutral mobile phase are shown in FIG.

  The three basic compounds show various recoveries with heat-treated metal-silica gel (metal species is Fe (III), Al, Ag or Ni) in pre-treatment under acidic and neutral conditions. It was. The heat-treated Fe (III) -silica gel showed the highest recovery rate for basic compounds under acidic conditions. On the other hand, all the heat-treated metal-silica gels showed higher recovery rates of basic compounds under neutral conditions than under acidic conditions.

  These results indicate that the degree of interaction between the metal acoion and the basic compound is affected by the acidity of each acoion of Fe (III), Al, Ag, and Ni. In addition, a metal acoion shows various pKa values. The pKa value depends on the size and charge of the metal ion, and further depends on the electronegativity of the element. In general, the smaller the metal ion size, and the greater the charge for the same size, the more acidic the metal ion. The higher the element electronegativity, the more acidic the metal ions. It is known that the pKa values of Fe (III), Al, Ag, and Ni ako ions are 2.2, 5.0, 12.0, and 9.9, respectively.

  Thus, using heat-treated metal-silica gel acoions (especially Fe (III) acoions), the basic compounds are retained by electrostatic interactions (ie, cation exchange mode) and become hydrophobic depending on the pH of the mobile phase. It is also retained by sexual interaction. For example, it is retained by hydrophobic interactions under acidic conditions (0.1% acetic acid; pH is about 4).

  Each heat-treated metal-silica gel was used as an on-line solid extraction column, and a test solution was injected into each column, but no decrease in chromatographic performance or noticeable change in column pressure was observed. These results indicate that these heat-treated metal-silica gels have sufficient stability and reusability.

  From the above results it can be seen that these heat-treated metal-silica gels can be adapted to hydrophobic alkyl chains and ionic functional groups when used as mixed-mode reversed-phase-cation exchange HPLC columns. is there. Conventionally, in mixed mode HPLC, two columns, a column having an ODS stationary phase and a column having a cation exchange stationary phase, are connected, or two types of stationary phases, a reverse phase stationary phase and an ion exchange stationary phase, are used as one. It has been realized by mixing in two columns or by chemically binding an ionizable functional group in the alkyl chain to a silica-based support.

  Thus, as a representative of the heat-treated metal-silica gel, Fe (III) -silica gel that had been heat-treated (2 hours at 1000 ° C.) was used, and the ability of the mixed-mode reverse-phase / cation exchange was examined. An acidic mobile phase (aqueous solution containing 0.1% acetic acid) and a neutral mobile phase (aqueous solution containing 5 mmol / L ammonium acetate) are used as a pretreatment mobile phase in a column switching HPLC system, and a concentration of 0.5 mg is used as a sample. A / mL imipramine solution was used. The relationship between the amount of imipramine injected into the heat-treated Fe (III) -silica gel and the peak area was examined. As a result, the relationship between the amount of imipramine and the peak area was almost linear up to an injection amount of 25 μg regardless of whether an acidic mobile phase was used or a neutral mobile phase was used. As a result, it can be seen that the heat-treated metal-silica gel has sufficient capacity for mixed mode reverse phase-cation exchange for basic compounds.

(Second Embodiment)
Separation medium in which a metal and a functional group such as ODS are bonded to silica gel:
When silica gel is used as a carrier, the form of silica gel is not particularly limited, but it is preferable that the chemical modification can be efficiently performed. As such a preferable form of silica gel, the particle diameter is 1 to 200 μm, more preferably 1 to 15 μm, the pore diameter is 4 to 100 nm, more preferably 4 to 30 nm, and the specific surface area is 50 to 800 m ² / g, more preferably. May be ²⁰⁰ to 400 m ² / g. As the carrier, inorganic / organic hybrid particles may be used. In the following description, the case of using silica gel will be described, but the same applies to the case of using inorganic / organic hybrid particles.

Bond metal to silica gel. As the method, silica gel is contacted with an aqueous metal salt solution of Al, Fe, Zn, Mn, Ni, Mg, or Ag, dried, and then heated. Examples of such metal salts include AlCl ₃ , FeCl ₃ , FeCl ₂ , ZnCl ₂ , MnCl ₂ , NiCl ₂ , MgCl ₂ , AgNO _{3 and the} like.

  A functional group such as octadecylsilyl group, octyl group, butyl group, cyanopropyl group or aminopropyl group is bonded to the silica gel. The reaction of bonding a functional group such as octadecylsilyl group to silica gel is a well-known method in the field of column packing production. For example, the case where an octadecylsilyl group is bonded will be described as follows. A silylating agent such as monochlorodimethyloctadecylsilane and silica gel are mixed in a nonpolar solvent such as toluene and reacted while refluxing. The reaction yield varies depending on the solvent used, temperature, time, presence or absence of catalyst addition, and the like. Further, although the bonding form varies depending on the type of silylating agent, they are not limited here.

  When the separation medium thus obtained is used as a liquid chromatograph column packing material, an end-capping process is usually performed in many cases. The end capping method is a well-known method in the column packing material manufacturing field, but the presence or absence of the end capping process and the method thereof are not limited.

  Any of the step of bonding a metal to silica gel and the step of bonding a functional group to silica gel may be performed first. Further, these steps may be performed a plurality of times in order to further strengthen the bond.

This will be more specifically shown as Example (1). Weigh about 2 g of silica gel particles. The silica gel particles are not particularly limited, and for example, those having an average particle diameter of 5 μm, an average pore diameter of 12 nm, and an average specific surface area of 350 m ² / g.

  To this, 200 mL of an aqueous solution of a metal salt (for example, ferric chloride or aluminum chloride) having an appropriate concentration (for example, 0.2% by weight) is added. The solution is stirred for 6-12 hours, then washed and filtered to collect silica gel particles. The silica gel particles are dried at about 150 ° C. for about 2 hours. Furthermore, it heats at 500-1200 degreeC for about 2 to 5 hours. The concentration and addition amount of the aqueous metal salt solution need not be exact. By changing these, it is possible to adjust the amount of metal relative to the silica gel, but in order to reduce the influence of trace metals remaining on the silica gel, it is preferable to bond to a saturated state. Therefore, a metal-immobilized ODS separation medium that achieves the intended purpose can be prepared by simply adding the metal in excess to the silica gel in the step of bonding the metal to the silica gel.

  The immobilized metal may be bonded to the surface of the silica gel. Since silica gel is porous, the surface includes not only the outer surface but also the surface inside the pores. However, the metal need not be contained within the silica gel crystal. The metal concentration shown above is an average metal concentration including the metal incorporated in the inside. The average concentration is not directly related to the properties of the filler.

  The separation medium of Example (1) is prepared by bonding a functional group such as an octadecyl group to the silica gel particles on which the metal is immobilized in this manner by a general method. As a general method for bonding an octadecyl group as a functional group to silica gel particles, for example, triethoxyoctadecylsilane is reacted with silica gel.

  The separation medium of Example (1) obtained in this manner is a metal-immobilized ODS separation medium, which may be hereinafter referred to as “MAqC-ODS”. Symbols such as Al, Fe (II) (ferrous iron), and Fe (III) (ferric iron) are included as specific metals of MAqC indicating metals. For the MAqC-ODS separation medium of the example, an ODS separation medium in which no metal is immobilized is used as a comparative example. The ODS separation medium of the comparative example is different from the separation medium of the example in that it does not undergo a step of bonding metal.

As Example (2), a separation medium suitable for use in a pretreatment column or cartridge was prepared. Weigh about 2 g of silica gel particles. The silica gel particles are not particularly limited, but for example, those having an average particle diameter of 50 μm, an average pore diameter of 12 nm, and an average specific surface area of 350 m ² / g. The silica gel particles have a larger average particle diameter than that of Example (1).

  To this, 200 mL of an aqueous solution of a metal salt (for example, ferric chloride or aluminum chloride) having an appropriate concentration (for example, 0.2% by weight) is added. The solution is bonded for 5 to 10 hours, washed with water and methanol, and filtered to collect silica gel particles. The silica gel particles are dried at about 150 ° C. for about 2 hours.

  In Example (3), a functional group such as an octadecyl group is further bonded to the separation medium of Example (1) to prepare a separation medium.

  This separation medium is also a metal-immobilized ODS separation medium, which may also be referred to as “MAqC-ODS” hereinafter. Symbols such as Al, Fe (II) (ferrous iron), and Fe (III) (ferric iron) are included as specific metals of MAqC indicating metals. For the MAqC-ODS separation medium according to the present invention, an ODS separation medium in which no metal is immobilized is used as a comparative example. The ODS separation medium of the comparative example is different from that according to the present invention in that it does not undergo a step of bonding metal.

  An HPLC column is manufactured by filling an empty column with the separation medium according to the present invention and the separation medium of the comparative example by a conventionally used method.

<Characteristics of HPLC column>
In order to show the retention characteristics of the columns produced by the above method in comparison with the ODS columns packed with the ODS separation medium of the comparative example, these columns were mounted on an HPLC apparatus. FIG. 8 shows a flow path configuration diagram of an example of the HPLC apparatus. On the analysis flow path 68, the liquid feed pump 54, the autosampler 52, the analysis column 16B, and the detector 58 are connected from the upstream side. The liquid feeding pump 54 includes two pumps 54A and 54B. The suction side of the pump 54A is a container containing the mobile phase 62A, and the suction side of the pump 54B is a container containing a mobile phase 62B different from the mobile phase 62A. They are connected via a degasser 64. As the analytical column 16B, the column according to the present invention or the ODS column of the comparative example is used.

  The discharge sides of the pumps 54A and 54B are both connected to the mixer 66. An autosampler 52 is connected to the downstream side of the mixer 66, and a sample is injected into the analysis flow path 68. The analysis column 16B connected to the downstream side of the autosampler 52 is accommodated in a column oven 56 and maintained at a constant temperature, and the sample injected by the autosampler 52 is separated into components. The detector 58 connected to the downstream side of the analysis column 16B performs optical measurement of the liquid that has passed through the analysis column 16B. As the detector 58, an ultraviolet absorbance detector is used.

  The HPLC apparatus shown here is an example, and the liquid chromatograph in which the column of the present invention is used is not limited to that shown in FIG.

  In addition, the separation medium of the present invention can be used as a filler for a trap column for preliminarily processing an injected sample and leading it to an analysis column, and it is naturally possible to adopt a trap injection type apparatus configuration.

  In the HPLC apparatus of FIG. 8, a chromatogram was obtained by analysis under the following conditions. The analysis conditions here are often used in the reverse phase mode.

Column 16B: inner diameter 4.6 mm, length 150 mm
The analysis conditions by HPLC are as follows.
Mobile phase: Liquid A (described later)
B liquid (described later)
Gradient program:
B liquid concentration: 0% (0 minutes) → 100% (12 minutes)
Mobile phase flow rate: 1.0 mL / min Detection: wavelength 280 nm
Column temperature: 40 ° C

Sample: Each of the following 8 components is contained at a concentration of 62.5 μg / mL.
(Neutral compound)
(1) Methylparaben (2) Propylparaben (acidic compound)
(3) Benzoic acid (4) Ketoprofen (basic compound)
(5) Trimethoprim (6) Imipramine (7) Azelastine (8) Donepezil

  Sample injection volume to the HPLC injector: 10 μL

Chromatogram description:
The type of column packing is shown in each figure. The Capcell Pack ODS in the figure is an ODS filler with no metal immobilized thereon and is manufactured by Shiseido Co., Ltd., and the ODS is produced by omitting the metal immobilization step in the method for producing a separation medium according to the present invention. . The chromatograms of Capcell Pack ODS and ODS are shown as comparative examples.

  In each chromatogram, the numbers attached to the peaks represent the above components in the sample. The horizontal axis of each chromatogram represents time, and the maximum (right end) scale is 20 minutes. The vertical axis of the chromatogram is absorbance (mAU), and the full scale of each chromatogram is 200 mAU. What is important in these chromatograms is the order in which the peaks appear, that is, the elution order of the compounds that are the components to be analyzed, and not the absolute values of the retention time and peak height. 9 to 23 are mainly for showing the elution order of the compounds.

The mobile phase A liquid and B liquid which obtained the chromatogram of FIGS. 9-16 are as follows.
9 to 10:
Liquid A: water / acetic acid = 1000/1 (V / V)
Liquid B: acetonitrile / water / acetic acid = 600/400/1 (V / V / V)
The mobile phase component ratio V represents volume, and W represents weight.
FIGS. 11-12:
Liquid A: water / acetic acid = 1000/1 (V / V)
Liquid B: acetonitrile / water / TFA = 600/400/1 (V / V / V)
TFA is trifluoroacetic acid.
13 to 14:
Liquid A: water / phosphoric acid = 1000/1 (V / V)
Liquid B: acetonitrile / water / phosphoric acid = 600/400/1 (V / V / V)
15 to 16:
Liquid A: water / ammonium acetate = 1000 / 0.77 (V / W)
Liquid B: acetonitrile / water / ammonium acetate = 600/400 / 0.77 (V / V / W)

  In the chromatograms of FIGS. 9 to 16, when compared with the chromatograms of the ODS column without immobilized metal and the immobilized ODS column, the retention on the basic compound is selectively observed, depending on the type of mobile phase. There is a tendency to become stronger. This is particularly noticeable when an ODS column in which Fe (III), Fe (II) or Al is immobilized is used. Depending on the mobile phase, the retention of the basic compound may be too strong to be eluted.

  Depending on the type of metal, the tendency for selective retention to be strong against basic compounds may not be significant, but the elution order of the components to be analyzed is still different from the chromatogram of the comparative ODS column. In other words, it can be said that the effect of the immobilized metal on the retention characteristics appears.

  Since the retention behavior varies depending on the metal species to be immobilized in this way, columns having various retention characteristics can be manufactured. This means that the options for obtaining a column suitable for the component to be separated are widened.

The column using the metal-immobilized ODS separation medium according to the present invention exhibits different retention characteristics even when a salt is added to the mobile phase. Next, the effect of the salt in the mobile phase is shown. As the salt to be added, KCl, MgCl ₂ , (NH ₄ ) ₂ SO ₄ , EDTA (ethylenediaminetetraacetic acid: chelating agent), PFPA (perfluoropropionic acid: paired ion reagent) and the like can be used. Of these, the effects of adding KCl and PFPA to the mobile phase are shown in FIGS.

Chromatogram description:
The type of column packing material, the type of salt and the concentration thereof are shown in each figure. ODS-only in the figure is produced by omitting the metal fixing step in the production method of the ODS separation medium according to the present invention, and is shown as a comparative example.

The mobile phase A liquid and B liquid which obtained the chromatogram of FIGS. 17-23 are as follows.
Liquid A: water / acetic acid = 1000/1 (V / V)
Liquid B: acetonitrile / water / acetic acid = 600/400/1 (V / V)
The salt is added in the B liquid, and the concentration is described in the figure.

  The column size, HPLC analysis conditions, and sample are the same as those obtained when the chromatograms of FIGS. 9 to 16 were obtained.

  As is apparent from the results of FIGS. 17 to 23, for example, in the reverse phase mode, a basic compound having a strong retention can be eluted by adding a salt to the mobile phase. That is, it can be seen that the column packed with the metal-immobilized ODS separation medium according to the present invention can control the retention characteristics by adding salt to the mobile phase. For example, components that cannot be sufficiently separated by a normal ODS column can be separated by adding an appropriate salt to the mobile phase by selecting the type of metal to be immobilized. Can contribute to the analysis.

  The retention behavior of the analytical column packed with this separation medium has both a reverse phase mode and a cation exchange mode, and the retention behavior varies depending on the metal species to be immobilized as described above. It can respond to various requests for It can be estimated from FIG. 17 that the cation exchange mode is exhibited. When “Column: ODS-Only, KCl = 0 mM” and “Column: ODS-Only, KCl = 10 mM” are compared, there is almost no change in both chromatograms, whereas “Column: Fe (III) -ODS”. , KCl = 0 mM ”and“ Column :, Fe (III) -ODS, KCl = 10 mM ”, a basic compound that was not eluted at KCl = 0 mM was eluted by adding a salt at KCl = 10 mM. doing. This result shows that the cation exchange mode is working.

  As described above, the column using the metal-immobilized ODS separation medium according to the present invention as a packing material has been described based on the configuration provided in the liquid chromatograph apparatus. However, the separation medium having such characteristics can be used offline. It is. When used off-line, it is preferable to adjust the particle diameter to be slightly larger than when the separation medium according to the present invention is used as a column packing material and use it as a cartridge. If the particle diameter is increased, a large pressure for passing the liquid through the cartridge is not necessary, and pretreatment by manual operation of a syringe or the like is also possible. By selecting and using a cartridge that exhibits a strong holding force for a certain component, it can be used as a pretreatment cartridge that removes that component from the analysis target. Since it is off-line, it is not necessary to rearrange the flow path, and the condition study becomes easy.

2 Cartridge 14 and 20 Separation medium 16 Column 16A Pretreatment column 16B and 32 Analysis column 30 Six-way valve 34 and 58 Detector 35 Flow path for supplying pretreatment solvent 40 Injector 52 Autosampler 68 Analysis flow path

Claims (9)

A carrier that is silica gel or a carrier comprising silica gel as a component;
See containing and a more metals 50ppm bound to the carrier to provide hydrophobicity to said carrier,
The metal is at least one selected from the group consisting of Al, Fe, Zn, Mn, Ni, Mg, and Ag,
A functional group which is one type selected from the group consisting of an octadecylsilyl group, an octyl group, a butyl group and an aminopropyl group is provided on the carrier, in addition to the metal, which gives an affinity different from that of silica gel to components to be analyzed. As a group,
The separation medium is a separation medium that performs both a hydrophobic interaction and a cation exchange interaction with a component to be analyzed.   The separation medium according to claim 1, wherein the metal is bonded to the carrier at a concentration at which the metal is saturated. A column in which the separation medium according to claim 1 or 2 is packed in a tube. The column according to claim 3 , wherein the column is filled with the separation medium according to claim 1 or 2 and has a cartridge shape having a spout and an outlet. A pretreatment column comprising the column according to claim 4 , wherein the separation medium according to claim 1 or 2 is packed in a column body and configured in a column shape having connection ports to flow paths at both ends of the column body;
A first solvent supply channel that supplies the pretreatment column with a first solvent that holds one or more components to be analyzed in the sample;
A second solvent for eluting the analysis target component held in the pretreatment column, and supplying a solvent different from the first solvent to the pretreatment column;
An analytical column for separating an analysis target component connected to a flow path through which an eluate from the pretreatment column flows;
A detector that is connected downstream of the analytical column and detects an analyte component separated by the analytical column;
A first flow path that connects the first solvent supply flow path to the pretreatment column, and a second flow path that connects the second solvent supply flow path to the pretreatment column and an eluent flow path from the pretreatment column to the analysis column A flow path switching mechanism for switching between the second flow path connected to
A liquid chromatograph equipped with The method to manufacture the separation medium of Claim 1 or 2 including the following processes (A) and (B).
(A) A step of drying the carrier after immersing 100 parts by mass of a carrier that is silica gel or a carrier containing silica gel as a component in 0.2 to 20 parts by mass of a metal salt aqueous solution of 0.01 to 1% by mass. ) Then, the process of heat-processing at the temperature of the range of 400-1200 degreeC. The method for producing a separation medium according to claim 6 , wherein in the step (A), a metal is bonded to silica gel in a saturated state. The method for producing a separation medium according to claim 6 or 7 , wherein the metal is at least one selected from the group consisting of Al, Fe, Zn, Mn, Ni, Mg, and Ag. Further, at least either before or after the step (A) or after the step (B), the support is provided with a functional group selected from the group consisting of an octadecylsilyl group, an octyl group, a butyl group, and an aminopropyl group. The method for manufacturing a separation medium according to any one of claims 6 to 8 , further comprising a step of bonding.

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