LIQUID CHROMATOGRAPHY FILLER, LIQUID CHROMATOGRAPHY COLUMN AND METHOD FOR ANALYZING AMINE COMPOUND

Technical Field

This invention relates to liquid chromatography filler, liquid phase chromatographic column and amine compounds of the analytical method.

This application is based on Japanese application in 29 August 2016 2016 - 167038 of claim priority to, its content to the invoked in this text.

Background Art

Amine compound is medical, food, fertilizer, industrial materials such as important material contained in the compound. In addition, amine compounds are used in various industrial in research and development, quality tests such as into the analysis object in the compound. In addition, in the field of environment, in tap water to the human body comprising of the adverse effect caused by the amine compounds widely into the analysis object.

As the amine compound analysis method, the general use of the liquid chromatography analysis method. As the liquid chromatography of the amine compound of the analytical method, widely known for example, using the ODS column of the reversed phase chromatography (RP) mode of analysis method (for example, referring to Patent document 1). ODS column is the amine compound for inhibiting adsorption capping processing filling the octadecyl silica gel (ODS) such as filler material of silica gel system.

However, the amine compound of the RP mode in the analytical method, sometimes the column in is not fully retaining hydrophilic high-amine compound. For example, the use of the method in the ODS column RP, hydrophilic high amine compound is not column reservations. Therefore, in order to make the amine compound fully retained in the column, sometimes need to add 1 - pentane sulfonic acid sodium plasma to the reagent. If adding ion pair reagent such as this, the service life of the causes the column is sometimes significant deterioration.

In addition, in recent years, also generally known by hydrophilic interaction chromatography (HILIC) separating mode analysis method, its use has been filled is introduced as amphoteric ion-based sulfo beet base silica gel filler of the column. However, employing the HILIC separation modes in the method of analysis, but the existence of the hydrophobic amine compound in the column is not fully reserved in such a subject. Then, employing the HILIC separation modes in the method of analysis, the existence of the following topics: because in the RP or HILIC for silica gel filling agent for the residual silanol group, amine compound as the alkaline substance in the adsorption column, to the analysis result such as the adverse impact.

The prior technical literature

Patent literature

Patent document 1: Japanese extra Kaiping 6 - 174708 Bulletin

Generic literature

Generic literature 1: Kyoritsu Publishing, high-speed liquid , 1986 years, p93

Content of the invention

Subject to be solved by the invention

The invention is made in view of such a situation of, its purpose is to provide a more accurate, and more convenient to carry out the analysis of the amine compound in the liquid phase chromatography filler, fills the liquid chromatography filler liquid chromatography column, and the liquid phase of the chromatographic column using the amine compound of the analytical method.

The subject method is used to solve the

The inventors in order to solve the above problem repeatedly and in-depth study, and found that, through the use of each 1 g filling agent is introduced 1.50 mmol of the above carboxyl, and will be in accordance with the provisions of the HILIC separation mode analysis method to control the surface pH of the sexual and hydrophilic polymer particles used for filler of the column, thereby not only HILIC separation mode, but also in combination with the ion exchange chromatography RP mixed mode also come into play, so as to obtain the performance amine compound for the purposes of separation analysis of the performance of the full column, have completed the present invention.

Namely, the invention relates to the following matters.

[1]Aliquid-phase chromatography filler, is having a carboxyl group of the polymer filler, characterized in that each 1 g of the above polymer filler is introduced 1.50 mmol of the above-mentioned carboxyl,

Based on hydrophilic interaction chromatography (HILIC) of, represents the above-mentioned polymer filler surface pH of gender index of 1.30 above, and based on hydrophilic interaction chromatography (HILIC) of, represents the above-mentioned polymer filler surface hydrophilic index of 1.00 or more and 1.30 the following. Represents the above-mentioned polymer filler surface pH of the gender of the index to the theobromine (Tb) with the theophylline (Tp) to HILIC separation mode liquid chromatography determination of separation when a coefficient α 1 (Tb/Tp) definition. In addition, represents the above-mentioned polymer filler surface hydrophilic index to the uridine (U) with the 2' - deoxy uridine (2 dU) to HILIC separation mode liquid chromatography determination of separation when a coefficient α 2 (U/2 dU) definition. Each separating the coefficient α 1, α 2 for their respective to the retention of the coefficient of the ratio of the k, retain the coefficient k are in toluene elution time is set t₀ , Each substance elution time is set under the condition that the t, to k=(t - t₀ )/T₀ The definition. Wherein the HILIC separation mode liquid chromatography assay conditions is: column temperature is 30 °C, the mobile phase is (acetonitrile): (added acetic acid 10mmol/L and ammonium 10mmol/L water)=90/10 (before mixing volume) of the mixed liquid.

[2] According to said liquid phase chromatography filling agent, the polymer filling agent contains a carboxyl group introduced into polyvinyl alcohol resin or poly hydroxy (meth) acrylate resin.

[3]Aliquid phase chromatographic column, it possesses: or said liquid phase chromatography filler; and filling the above-mentioned liquid chromatography filler housing for liquid chromatography.

[4]Acompound of the analysis method, wherein the use of the the liquid phase chromatographic column, adopting the above-mentioned liquid phase chromatographic column separation of the mode have been used for the water-soluble organic solvent and water mixed solvent as a mobile HILIC separating mode, or a combination of the reversed phase chromatography (RP) and ion exchange chromatography of mixed-mode.

[5] According to the compound of the analysis method, the water-soluble organic solvent is selected from acetonitrile, methanol, ethanol, isopropanol, acetone, butanone and in tetrahydrofuran at least 1 kind of.

[6] According to the compound of the analysis method, the water-soluble organic solvent is acetonitrile.

The effect of this invention

According to the present invention, it is possible to provide more accurate, and more easily analysis of amine compound liquid chromatography filler, fills the liquid chromatography filler liquid chromatography column, and the liquid phase of the chromatographic column using the amine compound of the analytical method.

Description of drawings

Figure 1 is the chromatogram of measuring embodiment 1 polymer filler separation coefficient α 1 (Tb/Tp) and separation coefficient α 2 (U/2 dU) time.

Figure 2 is the chromatogram of embodiment 1 liquid chromatography column in the assessment of the uracil with the adrenergic.

Mode of execution

The following, the application of the invention is liquid chromatography filler, fills the liquid chromatography filler liquid chromatography column, and the liquid phase of the chromatographic column using the amine compound of the analysis method of the description of the embodiment.

In addition, this embodiment is to better understand the invention the purpose of the specific description, as long as not particularly specified, it does not limit the final version of the invention.

[Liquid chromatography filler]

The invention liquid chromatography filling agent is a polymer having a carboxyl group of the filler. Characterized in that each 1 g of the above polymer filler is introduced 1.50 mmol the above-mentioned carboxyl. In addition, based on hydrophilic interaction chromatography (HILIC) of, represents the above-mentioned polymer filler surface pH of gender index of 1.30 or more, and represents the above-mentioned polymer filler surface hydrophilic index of 1.00 or more and 1.30 the following.

(Expressed pH gender index, said hydrophilic index definition)

Here, to the invention relates to said polymer filler surface pH of gender index and said polymer filler surface hydrophilic index description.

The surface of the polymer filler, said gender pH of index and said hydrophilic index is 2011 year by the pool and the others proposed HILIC column of performance evaluation index, is based on the general can be applied by the technical evaluation of the value defined by the law (Journal of Chromatography A, 1218 of, 2011 years, 5903 - 5919 page). These index by using the commercially available liquid chromatography analysis device (for example, Nexera - i (commodity name, Kabushiki Kaisha Shimadzu Seisakusho systems), Agilent 1260 Infinity ( · Corporation systems) and the like), so as to whether manufacturers, machine the species can reproduce good access to. However, these index to HILIC separation mode determination. The HILIC separation mode liquid chromatography assay conditions is, as the detector using the ultraviolet detector (UV, wavelength: 254 nm), column temperature is 30 °C, the mobile phase is (acetonitrile): (added acetic acid 10mmol/L and ammonium 10mmol/L water)=90/10 (before mixing volume) of the mixed liquid.

In the invention of the said polymer filler surface pH of gender index and said polymer filler surface hydrophilic index as follows find: the polymer filler filling to the inner diameter of 2.0 mm, length 150 mm or inner diameter 4.6 mm, length 150 mm cylindrical liquid chromatography used in the outer cover, making liquid phase chromatographic column, using the liquid phase chromatographic column, through the above-mentioned determination condition is determined. In addition, based on the literature of rice and others, will at this moment of the fixed linear velocity of the mobile phase of the 3.3 cm/min (in the use of internal diameter 2.0 mm under the condition of the casing, the flow of the mobile phase is 0.11 ml/min. In use the inner diameter 4.6 mm under the condition of the casing, the flow of the mobile phase is 0.55 ml/min. ).

Said gender index as pH with acid-base interaction comprising ionic interaction (ion exchange) corresponding to that of the surface of the filling agent gender pH indicators, to the theobromine (Tb) with the theophylline (Tp) to HILIC separation mode liquid chromatography determination of separation when a coefficient α 1 (Tb/Tp) definition. If the pH of the index is that gender 1.00 or more, due to polymer filler display anionic, therefore performance cation exchange function. On the other hand, if the pH of said gender index is less than 1.00, then the polymer filler display cationic, therefore performance anion exchange function. Therefore, said gender index with pH 1.00 of large difference vietnam, the greater can be the polymer filler performance high ion exchangeability.

Said hydrophilic index as the representation is based on the surface of the thickness of the filling agent hydration of the selective index, to the uridine (U) with the 2' - deoxy uridine (2 dU) to HILIC separation mode liquid chromatography determination of separation when a coefficient α 2 (U/2 dU) definition. If said hydrophilic index of 1.00 or more, the performance of the polymer filler is based on hydrophilic interaction.

Here, separation coefficient α 1, α 2 is respectively to the retention of the coefficient of the ratio of the k. For HILIC, retain the coefficient k are in will not be retained in the column while the eluting out in toluene elution time is set t₀ , Each substance elution time is set under the condition that the t, to k=(t - t₀ )/T₀ The definition. That is, separation coefficient α 1 (Tb/Tp)=k1/k2, k1 is theobromine reservation coefficient, k2 coefficient for the theophylline reservations. In addition, separation coefficient α 2 (U/2 dU)=k3/k4, k3 is uridine reservation coefficient, k4 is 2' - deoxy uridine reservations coefficient.

In addition is also assumed, in HILIC separation mode, because the introduced into the polymer filler carboxyl performance and ion exchange, but generally for HILIC separation mode, often as also includes ion exchange separation method of processing. Therefore, in the specification, also in order to HILIC separation mode of note names.

(Polymer filler)

As the invention relates to polymer filler, the use of the carboxyl group is introduced into at least one hydrophilic polymer, but can be used in addition to the carboxyl outside, also introduced into the carboxyl alkyl, sulfo, sulfo alkyl group or the like of the hydrophilic polymer.

As introduced into the carboxyl polymer filler, preferably by chemical modification or coated is introduced into the carboxyl group of the polymer filler. As such have introduced into the carboxyl polymer filler, preferably polyvinyl alcohol resin or poly hydroxy (meth) acrylate resin.

The carboxyl groups of the polymer filler in the heating device of the polymer filler can be chemical beautification quantity or to adjust. The invention liquid chromatography filler, in order to obtain separation modes based on HILIC, or combination of the reversed phase chromatography (RP) and ion exchange chromatography of a hybrid mode of the full analysis performance, polymer filler in each of the carboxyl groups of the heating device 1 g and the filling agent is 1.50 mmol or more, preferably 1.60 mmol or more, more preferably 1.80 mmol of the above. If the polymer filler in each of the carboxyl groups of the heating device 1 g of the filler is less than 1.50 mmol, performing the HILIC separation mode liquid chromatography determination that the gender of the pH of the index becomes less than 1.30, sometimes cannot fully ion exchange performance, and therefore could not make the amine compound to keep the column.

Said polymer filler surface pH of gender index of 1.30 or more, more preferably 1.30 or more and 1.60 the following, further preferably 1.40 or more and 1.60 the following. If the pH of said gender index less than 1.30, is sometimes polymer filler does not exhibit sufficient ion exchange, therefore in the HILIC separation mode, or in combination with the ion exchange chromatography of RP hybrid mode, amine compounds are not fully retain the column by elution. In addition, if the pH of the index is that gender 1.60 following, then the polymer filler and ion exchange will not be too strong, amine compound is fully retained in the column.

Said polymer filler surface hydrophilic index of 1.00 or more and 1.30 following, is preferably 1.05 or more and 1.25 following, more preferably 1.10 or more and 1.20 the following. If said hydrophilic index is less than 1.00, the uridine and 2' - deoxy uridine based on hydrophilic interaction, demonstrate the amine compound does not wash out, damaged as the HILIC column for liquid chromatography filler used by nature. In addition, if the said hydrophilic index more than 1.30, the hydrophilicity of the filler is too high, and therefore sometimes in combination with the ion exchange chromatography PR mixed mode inoperative.

In order to have sufficient separation performance and high sensitivity, obtain can tolerate 1 mpa - 30 mpa pressure strength, the shape of the polymer filler is preferably the volume average particle diameter is 1 μm - 30 μm of the spherical, more preferably the volume average particle size of 3 μm - 10 μm spherical.

In this invention the use of the volume average particle diameter of a grand counting method as follows determination. Namely, as the use of sophisticated measuring device for measuring the particle size distribution measuring device (trade name: Multisizer 4, · Corporation systems), in the polymer filler 0.2 g (diluent) added in 25 ml, applied 3 minutes after dispersing the ultrasonic and, to about 1000 for determining a number of measuring volume average particle diameter. In order to the polymer filler volume average particle diameter of control in the preferred range, the use of wind power classification, screening of the classification, the use of the precipitation hierarchical.

As in the invention of a carboxyl group into the polymer filler manufacturing method, can be cited for example, Japan 2007 - 191581 Bulletin method and recorded. In the method, in the polyvinyl alcohol resin through the substrate or poly hydroxy (meth) acrylate resin substrate to a carboxyl group-containing water-soluble polymer has the covering, so that the carboxyl group-containing water-soluble high-molecular cross-linking, to to the above-mentioned resin base material is introduced into the carboxyl group. In addition, the carboxyl group of the heating device can be determined by titration.

According to the present invention liquid chromatography filler, per 1 g of the filler into the 1.50 mmol of the above carboxyl polymer filling agent, on the basis of the HILIC, said polymer filler surface pH of gender index of 1.30 or more, and said polymer filler surface hydrophilic index of 1.00 or more and 1.30 following, and therefore can more accurately, and more easily analysis of amine compound.

[Liquid phase chromatographic column]

The invention liquid chromatography column can be cited for example, possesses the cylindrical such as the outer cover for liquid chromatography (the following, referred to as "shell". ), And is filled in the inside of this outer cover of a filler of the column. The invention of the liquid phase in the chromatographic column, as filler, the use of the liquid chromatography filler.

(Shell)

The material and the size of the housing is not particularly limited. As the shell, it is preferred to use the general as the housing for liquid chromatography of stainless steel or polyetheretherketone (PEEK) resin and a cylindrical shell.

In the case of the outer shell of the cylinder, in order to obtain sufficient separation performance and high detection sensitivity, its internal diameter is preferably 1.0 mm - 8.0 mm, more preferably 2.0 mm - 6.0 mm, further preferably 2.0 mm - 4.6 mm. If the inner diameter of the cylindrical housing is 1.0 mm or more, may be within the housing easily and fully filled with liquid chromatography filler. On the other hand, if the inner diameter of the cylindrical housing is 8.0 mm the following, in high-speed analysis also can sufficiently obtain the column performance. In addition, column pressure will not be too high, can make the determination of the flow rate of the mobile phase can be wide.

The length of the outer shell is preferably 35 mm - 300 mm, more preferably 35 mm - 250 mm, further preferably 50 mm - 250 mm. If the shell of a length of 35 mm or more, can make amine compound sufficiently retain the column, as a result, the amine compound can be separated. On the other hand, if the length of the shell is 300 mm the following, is column pressure will not be too high, can make the amine compound fully from the column to elute out.

According to the present invention liquid chromatography column, the invention liquid chromatography filling agent is filled in the housing, and therefore can more accurately, and more easily analysis of amine compound.

[Amine compound analysis method]

The invention of the amine compound of the analysis method is the following analytical method: the use of the present invention liquid chromatography column, using the liquid phase chromatographic column separation modes is to use water-soluble organic solvent and water as the mobile phase of the mixed solvent HILIC separating mode, or a combination of the reversed phase chromatography (RP) and ion exchange chromatography of mixed-mode. Here, as the amine compound, can be examples of the hydrogen atom of ammonia of a part or all of the organic radical substituted hydrocarbyl such as for the structure of the compound.

(The mobile phase)

As the compound of the present invention in the method of analysis of the mobile phase, the use of water-soluble organic solvent mixed with water solvent.

As the water-soluble organic solvent, can be cited acetonitrile, methanol, ethanol, isopropyl alcohol and the like alcohol, acetone, butanone and other ketones, ethers such as tetrahydrofuran and the like. Wherein as the water-soluble organic solvent, is preferably selected from acetonitrile, methanol, ethanol, isopropanol, acetone, butanone and in tetrahydrofuran at least 1 kind of, more preferably acetonitrile. The amine compound of this invention in the analytical method, can be used alone these water-soluble organic solvent 1 for, can also be combined using 2 or more.

(Separating mode)

The amine compound of this invention in the analytical method, the liquid phase of the chromatographic column separation mode is HILIC separation mode, or in combination with the ion exchange chromatography RP mixed mode, so by adjusting the as a mobile phase in the mixed solvent of water-soluble organic solvent and water composition ratio, can be selectively HILIC separation mode with the performance of the mixed-mode.

The mobile phase can be according to the analysis object (amine compound) of appropriate selection of the category. Usually, in the mixed solvent of water-soluble organic solvent of the composition ratio (before the mixing ) in 50% -70% with boundaries. If the composition ratio of the water-soluble organic solvent (before the mixing ) than the low threshold, then the performance in combination with the ion exchange chromatography RP mixed mode. On the other hand, if the composition ratio of the water-soluble organic solvent (before the mixing ) than the high threshold, then the performance HILIC separation mode. Namely, according to the analysis of the kind of the amine compound, confirms and wants of the performance of separating mode, according to the separation mode, determining in the mixed solvent of water-soluble organic solvent of the composition ratio (before the mixing ).

In the mixed solvent of water-soluble organic solvent with water of composition ratio (before the mixing ) usually is preferably a water-soluble organic solvent: water=1:99 - 99:1, more preferably 5:95 - 95:5.

In HILIC separation mode, mixed in a solvent with the water of the water-soluble organic solvent composition ratio (before the mixing ) usually is preferably a water-soluble organic solvent: water=50:50 - 99:1, more preferably 60:40 - 99:1, further preferably 70:30 - 95:5, especially preferably 75:25 - 95:5.

RP has combined with ion exchange chromatography in the mixed mode, mixed in a solvent with the water of the water-soluble organic solvent composition ratio (before the mixing ) usually is preferably a water-soluble organic solvent: water=1:99 - 70:30, more preferably 1:99 - 50:50, further preferably 5:95 - 50:50, especially preferably 10:90 - 50:50.

In addition, the amine compound of this invention in the analytical method, for adjusting the hydrophilic level and the purpose of the retention time of the sample, the mobile phase can be added in any proportion of formic acid, acetic acid, and nitric acid, or ammonium formate, ammonium salt and the like.

The amine compound of this invention in the analytical method, analysis of the column flow rate is not particularly limited. On the column flow rate, namely, in the analysis of the amine compound, of the present invention flows in the liquid chromatographic column comprising amine compounds of the mobile phase of the linear flow rate is preferably 1.5 cm/min - 15.0 cm/min (for example, the outer cover and the inner diameter of 2.0 mm of circumstances, flow rate is 0.05 ml/min - 0.50 ml/min), more preferably 3.0 cm/min - 9.0 cm/min (for example, the outer cover and the inner diameter of 2.0 mm of circumstances, flow rate is 0.10 ml/min - 0.30 ml/min).

Amine compounds according to the present invention analysis method, the use of the present invention liquid chromatography column, using the liquid phase chromatographic column separation modes is to use water-soluble organic solvent mixed with water as a mobile phase solvent HILIC separation modes of, or in combination with the ion exchange chromatography RP mixed mode, therefore according to the kind of the amine compound, to the appropriate selection of HILIC separation mode with the mixed mode, can more accurately, and more easily analysis of amine compound.

If the last-mentioned embodiment as shown in, through each 1 g filling agent is introduced into the 1.50 mmol of the above carboxyl, thus cation exchange role fully-functioning shown RP mode and ion exchange chromatography of mixed mode, so can make the relying solely on RP mode of retaining the amines are fully preserved. At this time, for example, RP indicators to uracil as the substance of the adrenergic reservations coefficient is preferably 2.0 or more, more preferably 2.5 or more. In the realization of the reservation coefficient, can be regarded as the combination of the reversed phase chromatography (RP) and ion exchange chromatography of mixed-mode play a role.

The amine compound of this invention in the analytical method, can be 1 or more amine compounds separation and analysis, especially if adopting the above-mentioned mixed mode, can also be through the 1 analysis for determining the RP mode at the same time easy to retain of aromatic compounds such as compounds, and only in the RP mode hydrophilic too strong and not retained by ionic substances such as amines.

Embodiment

The following, by embodiments and comparative example further specifically to this invention by way of illustration, but the invention is not limited to the following embodiment.

(Expressed pH gender index and said hydrophilic index is appropriate evaluation)

On the basis of the HILIC, said gender pH of index and said hydrophilic index, in accordance with the above non-franchised literature (Journal of Chromatography A, 1218 of, 2011 years, 5903 - 5919 page) of the literature data on the appropriateness of the confirmation.

As the column, the use of the above-mentioned non-franchised adsorption ZIC recorded (registered trademark) HILIC (inner diameter 2.0 mm, length 150 mm, Corporation systems). The above non-franchised literature record that the gender of the pH of the index of 1.20, said hydrophilic index is 2.03.

As the liquid chromatography analysis device, using Nexera - i (commodity name, Shimadzu Corporation). The assay conditions in accordance with the above non-franchised literature, use the ultraviolet detector (UV, wavelength: 254 nm) as the detector, column temperature is 30 °C, the mobile phase (acetonitrile) is made up of: (added acetic acid 10mmol/L and ammonium 10mmol/L water)=90/10 ratio (before mixing volume) of the mixed liquid. In addition, the flow rate of the mobile phase is 0.11 ml/min (linear flow rate: 3.3 cm/min), in sequence into the toluene 1000 μg/mL, tea alkali 100 μg/mL, theobromine 100 μg/mL, uridine 100 μg/mL, 2 - deoxyuridine 100 μg/mL (dissolved in the mobile phase) of the test sample 1 μl and recognizes the elution time, then calculates the pH of said index and said gender hydrophilic index.

As a result, the uniformity of the index is that pH 1.24, said hydrophilic index is 1.99, can be recognized can reproduce the recorded value of the non-franchised adsorption.

In the following embodiment and example in comparison, through the apparatus and measuring conditions, find the pH of said index and said gender hydrophilic index. However, only more example 2 using the internal diameter is 4.6 mm column, so the flow rate of the mobile phase is 0.55 ml/min (linear flow rate: 3.3 cm/min), so that each test sample of the sample 4 μl.

[Embodiment 1]

(Gel and the column of production)

Filler, in accordance with the Japanese 2007 - 191581 Bulletin method recorded, to the polyvinyl alcohol resin (particle size of 5.0 μm) substrate, relative to the each 1 g introduced into the substrate 2.09 mmol carboxyl, making the implementation of the example 1 polymer filler (introduced into the carboxyl polymer filler).

In addition, the polyvinyl alcohol resin base material introduced into the carboxyl group quantity by titration quantitatively.

The polymer filler 0.35 g added to the 0.5 mol/L formic acid aqueous solution 1.2 ml after, using ultrasonic wave bath decentralized processing, in the formic acid aqueous solution is uniformly dispersed into the carboxyl of a filler, modulating a slurry solution.

Use the resulting slurry solution, of the cylindrical, and the internal diameter 2.0 mm, length 150 mm of the shell made of PEEK (Corporation Palestinian the manufacture makes) is filled with the polymeric filler.

Namely, through the outer shell is injected into the slurry solution mentioned above, of the shell from the inlet side toward the outlet side , thus the slurry solution to 29 mpa (constant-pressure) pressure 20 minutes, formed in the housing comprising a polymer filler of the filling layer, to obtain the embodiment 1 liquid chromatography column.

In accordance with the above-mentioned method, find the embodiment 1 that the gender of the filler of the pH of the index and said hydrophilic index, results can be recognized, said gender pH of index (theobromine with theophylline separation coefficient α 1 (Tb/Tp)) is 1.44, said hydrophilic index (uridine and 2 '- deoxy uridine separation coefficient α 2 (U/2 dU)) is 1.09, uridine in the 2' - deoxy uridine was later eluting, it has as a filler for HILIC performance.

The implementation of the example 1 polymer filler separation coefficient α 1 (Tb/Tp) and separation coefficient α 2 (U/2 dU) for determination of chromatography is shown in the Figure 1 in. In addition, in fig. 1 in, symbol 1 said toluene peak, symbol 2 (Tp) of said theophylline peak, symbol 3 said 2' - deoxy uridine (2 dU) peak, symbol 4 expressed uridine (U) of the peak, symbol 5 theobromine (Tb) of that peak.

(Evaluation of the column)

As the liquid chromatography analysis device, use Nexera - i (commodity name, Kabushiki Kaisha Shimadzu Seisakusho systems), as a detector, use the ultraviolet detector (UV, wavelength: 275 nm), carried out embodiment 1 of evaluation from a liquid chromatography column.

As the mobile phase, the use of the acetonitrile and 50mmol/L formic acid aqueous solution in order to acetonitrile: 50mmol/L formic acid aqueous solution=20:80 (before the mixing ) obtained by the mixing ratio of the mixed solution.

Causes the column temperature is 40 °C, make the column in the mobile phase of the flow rate is 0.2 ml/min (linear flow rate: 6.0 cm/min), is injected into the uracil (t RP mode₀ Index material) 50 μg/mL and as amine compounds of the adrenergic 1000 μg/mL of mixing the test sample 2 μl, to uracil with the adrenergic carries out separate analysis.

Results of the evaluation of the column is displayed on table 1 in, the embodiment 1 of the column in the assessment of the chromatogram is shown in the Figure 2 in. In addition, in fig. 2 in, symbol 1 said uracil peak, symbol 2 expressed adrenergic peak.

By the table 1 of the result can be recognized: adrenergic elution time is 9.06 minutes, adrenergic reservations coefficient (by k=(- t t adrenergic elution time₀ Indicators substance elution time)/t₀ Indicators substance elution time to define) is 4.10, embodiment 1 liquid chromatography column also fully with the RP as a combination with the ion exchange chromatography of a mixed-mode performance.

[Embodiment 2]

(Gel and the column of production)

The use of embodiment 1 of the same polyvinyl alcohol resin base material, and with the embodiment 1 the same method, the manufacture of each 1 g introduced into the substrate 1.76 mmol of carboxyl, embodiment 2 of the polymer filler (introduced into the carboxyl polymer filler).

The polymer filler with the embodiment 1 to fill the same with the method of the embodiment 1 of the same in the housing, has obtained the embodiment 2 liquid chromatography column.

In accordance with the above-mentioned method, find the embodiment 2 of the pH of the polymer filler that gender index and said hydrophilic index, the result can be recognized, said gender pH index of 1.37, said hydrophilic index is 1.19, HILIC has as a filler for performance.

(Evaluation of the column)

The use of embodiment 1 with the same device, and with the embodiment 1 the same assay conditions, injection uracil 50 μg/mL with the adrenergic 1000 μg/mL of mixing the test sample of 2 μl, the uracil with adrenergic carries out separate analysis.

Results of the evaluation of the column is displayed on table 1 in.

By the table 1 of the result can be recognized, adrenergic elution time is 6.56 minutes, adrenergic reservations coefficient is 2.63, although with the embodiment 1 can see the adrenalin compared with the retention strength is reduced, but also fully with the RP as a combination with the ion exchange chromatography of a mixed-mode performance.

[Embodiment 3]

(Gel and the column of production)

The use of embodiment 1 of the same polyvinyl alcohol resin base material, and with the embodiment 1 the same method, each 1 g substrate introduced into the 1.62 mmol carboxyl, manufactured embodiment 3 of polymer filler (introduced into the carboxyl polymer filler).

The polymer filler with the embodiment 1 to fill the same with the method of the embodiment 1 of the same in the housing, has obtained the embodiment 3 liquid chromatography column.

In accordance with the above-mentioned method, find the embodiment 3 of the pH of the polymer filler that sexual index and said hydrophilic index, results can be recognized, said gender pH index of 1.33, said hydrophilic index is 1.19, HILIC has as a filler for performance.

(Evaluation of the column)

The use of embodiment 1 with the same device, and with the embodiment 1 the same assay conditions, injection uracil 50 μg/mL with the adrenergic 1000 μg/mL of mixing the test sample 2 μl, to uracil with the adrenergic carries out separate analysis.

Results of the evaluation of the column is displayed on table 1 in.

By the table 1 of the result can be recognized, adrenergic elution time is 6.44 minutes, adrenergic reservations coefficient is 2.57, with embodiment 1 can see the adrenalin compared with the retention strength is reduced, but also fully with the RP as a combination with the ion exchange chromatography of a mixed-mode performance.

[Comparative example 1]

(Gel and the column of production)

The use of embodiment 1 of the same polyvinyl alcohol resin base material, and with the embodiment 1 the same method, the manufacture of each 1 g introduced into the substrate 1.38 mmol of carboxyl group, comparison example 1 polymer filler (introduced into the carboxyl polymer filler).

The polymer filler with the embodiment 1 to fill the same with the method of the embodiment 1 in the same housing, have obtained the more example 1 liquid chromatography column.

In accordance with the above-mentioned method, find the comparison example 1 polymer filler that gender of the pH of the index and said hydrophilic index, the result can be recognized, that the pH of the index for the gender 1.22, said hydrophilic index is 1.36, HILIC has as a filler for performance.

(Evaluation of the column)

The use of embodiment 1 with the same device, and with the embodiment 1 the same assay conditions, injection uracil 50 μg/mL with the adrenergic 1000 μg/mL of mixing the test sample 2 μl, to uracil with the adrenergic carries out separate analysis.

Results of the evaluation of the column is displayed on table 1 in.

From table 1 the results of the, adrenergic with uracil is not separated from the washing. It can be considered that this is because, as the pH of said gender index is low, therefore in particular does not show ion exchange, thereby not fully with the RP as a combination with the ion exchange chromatography of a mixed-mode performance.

[Comparative example 2]

As a commercially available column, using filled with not introduced into the carboxyl group of the inducts glycol HILIC column for filler (tradenames: HILIC - 1, the inner diameter 4.6 mm, length 150 mm, Corporation systems).

In accordance with the above-mentioned method, find the comparison example 2 of glycol that inducts filler of the pH of the index and said gender hydrophilic index, the result can be recognized, said gender pH index of 1.00, said hydrophilic index is 0.89, although the method for the HILIC, but said hydrophilic index less than 1.00.

(Evaluation of the column)

The use of embodiment 1 with the same device, make the column in the mobile phase of the flow rate is 1.0 ml/min (linear flow rate: 6.0 cm/min), in addition, with the embodiment 1 the same assay conditions, injection uracil 50 μg/mL with the adrenergic 1000 μg/mL of mixing the test sample of 10 μl, to uracil with adrenergic carries out separate analysis.

The results of the evaluation of the column is displayed on table 1 in.

According to table 1 the results of, adrenergic in uracil before eluting out. It can be considered that this is because, pH of said gender index is low, therefore in particular the performance of the ion exchange role, thereby not fully with the RP as a combination with the ion exchange chromatography of a mixed-mode performance.

[Comparative example 3]

As a commercially available column, using filled with not introduced into the carboxyl amide-based inducts HILIC column for a bulking agent (trade name: Amide - 80, and the inner diameter of 2.0 mm, length 150 mm, Corporation systems).

In accordance with the above-mentioned method, find the comparison example 3 amide-based inducts said filler of the gender of the pH of the index and said hydrophilic index, the result can be recognized, said gender index pH of 1.31, said hydrophilic index of 1.65, HILIC has as a filler for performance.

(Evaluation of the column)

The use of embodiment 1 with the same device, and with the embodiment 1 the same assay conditions, injection uracil 50 μg/mL with the adrenergic 1000 μg/mL of mixing the test sample 2 μl, to uracil with the adrenergic carries out separate analysis.

Results of the evaluation of the column is displayed on table 1 in.

According to table 1 the results of, adrenergic elution time is 3.34 minutes, adrenergic reservations coefficient is 0.67, although slightly retains the adrenergic, however with the embodiment 1 can be see adrenergic reservations compared with the reduce the strength is large. In addition, because the said hydrophilic index is high, therefore in particular the role of the RP does not exhibit, thus not fully with the RP as a combination with the ion exchange chromatography of a mixed-mode performance.

[Comparative example 4]

As a commercially available column, using said index and said pH sex of hydrophilic index the feasibility of the use of the evaluation, ZIC HILIC (registered trademark) (inner diameter 2.0 mm, length 150 mm, Corporation systems).

As mentioned above, pH can be recognized that the gender of the index of 1.24, said hydrophilic index is 1.99, HILIC has as a filler for performance.

(Evaluation of the column)

The use of embodiment 1 with the same device, and with the embodiment 1 the same assay conditions, injection uracil 50 μg/mL with the adrenergic 1000 μg/mL of mixing the test sample 2 μl, to uracil with the adrenergic carries out separate analysis.

Results of the evaluation of the column is displayed on table 1 in.

According to table 1 the results of, adrenergic elution time is 2.94 minutes, adrenergic reservations coefficient is 0.38, although slightly retains the adrenergic, however with the embodiment 1 can be see adrenergic reservations compared with the reduce the strength is large. In addition, pH sex of said index is low, and said hydrophilic index is high, and therefore do not fully with the RP as a combination with the ion exchange chromatography of a mixed-mode performance.

Industrial availability

The present invention relates to have introduced into the carboxyl polymer filling agent of a filler, the filler is filled with the liquid phase of the chromatographic column, and the liquid phase of the chromatographic column using the amine compound of the analysis method, the filling agent is used to analyze amine compound liquid chromatography filling agent, through the HILIC separation mode, or in combination with the ion exchange chromatography RP mixed mode, can make various amine compounds with good retention, separation. Therefore, the invention in various industrial in research and development, quality testing, and environmental analysis and the like, to as medical, food, fertilizer, industrial materials such as important compound material contained in the amine compound analysis becomes useful.