Preparation method of chromatographic stationary phase with mixed mode of hydrophilic interaction chromatography and ion exchange chromatography

Abstract

The invention relates to a preparation method of a hydrophilic interaction-anion exchange mixed mode chromatographic stationary phase, which comprises the following steps: by taking silica gel microspheres as a matrix, reacting silicon hydroxyl on the surface of the silica gel microspheres with a first modified compound, and bonding a layer of epoxy compound; then, by taking BF3 as a catalyst, carrying out ring-opening reaction on hydroxyl on the second modified compound and an epoxy group of the first modified compound under an anhydrous condition so as to graft the second modified compound onto the surface of the silicon ball; the second modification compound comprises one or more mixtures containing a plurality of hydroxyl groups or / and amino groups. The surface of the obtained stationary phase has a large number of hydroxyl groups and amino groups at the same time, and a mixed mode chromatographic separation performance of hydrophilic interaction chromatography and anion exchange chromatography can be provided, so that special separation selectivity is provided for separation of some hydrophilic compounds and ionic compounds.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical analysis and detection, and particularly relates to a preparation method of a stationary phase for a mixed-mode chromatography combining hydrophilic interaction chromatography and ion exchange chromatography. Background Art

[0002] Although the reversed phase liquid chromatography (RPLC) separation technology has been widely used in the field of analysis, there are still great challenges in separating some hydrophilic compounds and polar compounds by reversed phase chromatography, because these compounds are difficult to be retained by the hydrophobic reversed phase chromatography stationary phase, thus achieving effective separation. In many applications, hydrophilic interaction chromatography (HILIC) has well solved this problem. HILIC uses a packing with a polar surface as the stationary phase and utilizes the interaction between it and polar compounds to enhance the retention of these polar compounds and achieve effective separation. At the same time, HILIC retains the same mobile phase system as RPLC, ensuring compatibility with mass spectrometry. However, in some practical applications, due to the complexity of samples, this traditional chromatographic technology with a single separation mode still has certain limitations.

[0003] Mixed-mode chromatography (MMC) is a new type of chromatographic analysis method developed in recent years. It utilizes multiple interaction forces between the sample and the stationary phase to achieve the combined action of two or more separation mechanisms in a single separation process, thus showing different separation selectivities from single chromatographic separation modes. Currently reported MMC mainly includes reversed phase chromatography (RP) / ion exchange chromatography (IEX), HILIC / IEX, RP / HILIC, RP / HILIC / IEX, RP / WAX / SCX, and HILIC / WCX / SAX and other modes. It can be seen that it is mainly different combinations of RP, HILIC, and IEX.

[0004] Conventional ion-exchange chromatography stationary phases have good hydrophilicity due to the presence of ionic groups. Under appropriate conditions, this stationary phase shows a mixed separation mode of HILIC and IEX (Xuefang Dong et al., Carbohydrate Research, 361 (2012) 195 - 199). However, since it has only one type of chromatographic functional group (ion-exchange group), it is generally not considered a mixed-mode chromatography stationary phase. A HILIC / IEX mixed-mode chromatography stationary phase must simultaneously have hydrophilic interaction groups for HILIC and ion-exchange groups for IEX. For example, a chromatographic stationary phase with two or more ligands such as amino or quaternary ammonium ionic groups and amide groups simultaneously bonded to silica gel (CN 103877748B), where the amino or quaternary ammonium ionic groups provide chromatographic mechanisms for weak anion or strong anion exchange respectively, and the amide groups provide a hydrophilic interaction chromatographic mechanism due to their hydrophilicity. This stationary phase has been successfully applied to the separation and analysis of some polar compounds, such as polysaccharide compounds. Another method is to bond a ligand with both ion-exchange groups (such as amino / amine / quaternary ammonium ions) and hydrophilic interaction groups (such as zwitterions or ionic liquids) on silica gel to form a HILIC / IEX mixed chromatographic stationary phase (CN 106552610 B).

[0005] However, these methods all require the synthesis of some complex ligands to obtain the functions of hydrophilic interaction and ion exchange. Moreover, these methods cannot flexibly control the contributions of hydrophilic interaction and ion exchange during the entire separation process. For example, in the commonly used method of using two independent ligands, increasing the proportion of the hydrophilic interaction ligand in the synthesis correspondingly reduces the amount and contribution of the ion-exchange ligand. In another method, concentrating the two functional groups on the same ligand fixes the contributions of these two separation modes and cannot be effectively adjusted. Summary of the Invention

[0006] To overcome the above technical deficiencies, the present invention provides a method for preparing a HILIC and ion-exchange chromatography mixed-mode chromatography stationary phase. The prepared mixed-mode stationary phase has a large number of hydroxyl and amino groups on its surface, which can provide the mixed-mode chromatographic separation performance of hydrophilic interaction chromatography and anion-exchange chromatography, thereby providing special separation selectivity for the separation of some hydrophilic compounds and ionic compounds.

[0007] To achieve the purpose of this invention, the present invention adopts the following technical solutions: A method for preparing a HILIC and ion-exchange chromatography mixed-mode chromatography stationary phase, the mixed-mode chromatography stationary phase includes a silica microsphere matrix and functional groups with both hydrophilic interaction chromatography and ion-exchange chromatography functions bonded to the surface of the silica microspheres; the preparation method includes the following steps:

[0008] 1) React the silanol groups on the surface of silica microspheres with an epoxy group compound as the first modification compound, thereby bonding epoxy groups to the surface of silica microspheres to obtain silica microspheres with epoxy groups.

[0009] 2) React the silica microspheres with epoxy groups with a second modification compound, and the second modification compound is a mixture of one or more compounds containing multiple hydroxyl groups or / and amino groups; after the reaction, hydroxyl groups and amino groups are simultaneously bonded to the surface of silica microspheres to obtain the mixed-mode chromatographic stationary phase with hydrophilic interaction chromatography and ion exchange chromatography.

[0010] Preferably, the first modification compound is an alkoxysilane compound with an epoxy group.

[0011] Preferably, the first modification compound includes one or more of the compounds having the structural characteristics shown in formula (i):

[0012]

[0013] In formula (i), R1, R2, and R3 are each independently a C1-C5 alkyl group or a C1-C5 alkoxy group, and at least one is a C1-C5 alkoxy group; n is 1-5.

[0014] Preferably, in the first modification compound, R1, R2, and R3 are each independently methyl, methoxy, or ethoxy, and at least one is methoxy or ethoxy, and n is 1.

[0015] Preferably, the particle size of the silica microspheres used in step 1) is 1 μm - 50 μm; the pore size is More preferably, the particle size of the silica microspheres used in step 1) is 1.5 μm - 10 μm; the pore size is

[0016] Preferably, the second modification compound includes one or more polyhydric alcohols and polyhydroxy-substituted compounds of amino groups.

[0017] Preferably, the polyhydric alcohols include ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, glycerol, diglycerol, and polyglycerol; the polyhydroxy-substituted compounds of amino groups include ethanolamine, diethanolamine, N-methyldiethanolamine, N-ethyldiethanolamine, triethanolamine, isopropanolamine, diisopropanolamine, N-methyldiisopropanolamine, N-ethyldiisopropanolamine, and triisopropanolamine.

[0018] Preferably, the polyhydric alcohols include ethylene glycol and glycerol; the polyhydroxy-substituted compounds of amino groups include diethanolamine and triethanolamine.

[0019] Preferably, the mass ratio between the polyol and the polyhydroxy-substituted compound of amine group in the second modifying compound is (1-10):(10-1).

[0020] Preferably, in step 2), the mass ratio between the silica microspheres with epoxy groups and the second modifying compound is 1:(0.1-5).

[0021] Preferably, a reaction solvent is further added in step 2), and the reaction solvent is one or more of tetrahydrofuran, 1,4-dioxane, toluene and xylene.

[0022] Preferably, in step 2), the reaction is carried out in the presence of a catalyst, and the catalyst is boron trifluoride diethyl ether. The dosage of the catalyst is 1%-10% of the mass of the silica microspheres.

[0023] The present invention also provides a mixed-mode chromatographic stationary phase having hydrophilic interaction chromatography and ion exchange chromatography, and the stationary phase is prepared by the preparation method according to any one of claims 1 to 9.

[0024] The present invention also provides a chromatographic column, and the stationary phase packing of the chromatographic column is the mixed-mode chromatographic stationary phase having hydrophilic interaction chromatography and ion exchange chromatography according to any one of claims 1 to 9.

[0025] The chromatographic column is used for analyzing hydrophilic compounds, polar compounds and ionic compounds.

[0026] Compared with the prior art, the present invention has the following beneficial effects: 1) The stationary phase prepared by the present invention has a hydrophilic retention effect on polar substances; 2) The stationary phase prepared by the present invention is positively charged and has an anion exchange chromatography function; 3) The stationary phase prepared by the present invention can well separate nucleotides, choline compounds and N-oligosaccharides due to the combined action of hydrophilic interaction chromatography and anion exchange chromatography; 4) The present invention can adjust the hydrophilicity and ion exchange ability of the final stationary phase by controlling two or more components and their ratios in the second modifying compound; 5) The present invention has a special selectivity for the separation of some hydrophilic compounds and ionic compounds.

[0027] The present invention uses silica microspheres as a matrix, reacts with a first modifying compound using the silanol groups on its surface to first bond a layer of epoxy compounds; then, using BF3 as a catalyst, under anhydrous conditions, the hydroxyl groups on the second modifying compound react with the epoxy groups of the first modifying compound to carry out a ring-opening reaction, thereby grafting the second modifying compound onto the surface of the silica spheres; the second modifying compound includes one or more mixtures containing multiple hydroxyl groups and / or amino groups. The resulting stationary phase surface has a large number of hydroxyl and amino groups at the same time, which can provide the chromatographic separation performance of a mixed mode of hydrophilic interaction chromatography and anion exchange chromatography, thereby providing special separation selectivity for the separation of some hydrophilic compounds and ionic compounds. Description of the Drawings

[0028] Figure 1 It is the test result of the retention behavior of different substances by the mixed-mode chromatographic stationary phase 5A provided in Example 1 under different organic phases;

[0029] Figure 2 It is the test result of the retention behavior of different substances by the mixed-mode chromatographic stationary phase 5A provided in Example 1 under different salt concentrations;

[0030] Figure 3 It is the test result of the retention behavior of different substances by the mixed-mode chromatographic stationary phase 5A provided in Example 1 under different pH values;

[0031] Figure 4 It is the comparison result of the hydrophilic interaction chromatography capabilities of the mixed-mode chromatographic stationary phases 5A - 5D provided in Examples 1 - 4;

[0032] Figure 5 It is the comparison result of the ion exchange capabilities of the mixed-mode chromatographic stationary phases 5A - 5D provided in Examples 1 - 4;

[0033] Figure 6 It is a comparison chart of the separation of 5 nucleotides by the mixed-mode chromatographic stationary phase 5B provided in Example 2 and the hydrophilic interaction chromatography packing material of Comparative Example 1; in the figure, peak 1: CMP, peak 2: AMP, peak 3: UMP, peak 4: IMP, peak 5: GMP;

[0034] Figure 7 It is a comparison chart of the separation of choline compounds by the mixed-mode chromatographic stationary phase 5B provided in Example 2 and the hydrophilic interaction chromatography packing material of Comparative Example 1; in the figure, peak 1: choline, peak 2: betaine, peak 3: Cl - ;

[0035] Figure 8It is a separation comparison diagram of the mixed-mode chromatographic stationary phase 5C provided in Example 3 and the hydrophilic interaction chromatography packing material in Comparative Example 1 for the N-oligosaccharide sample; in the figure, peak 1: neutral N-oligosaccharide, peak 2: mono-sialic acid N-oligosaccharide, peak 3: di-sialic acid N-oligosaccharide, peak 4: tri-sialic acid N-oligosaccharide, peak 5: tetra-sialic acid N-oligosaccharide. Detailed implementation mode

[0036] The technical solution of the present invention will be further described below through specific implementation modes. Those skilled in the art should understand that the described embodiments are only for helping to understand the present invention and should not be regarded as specific limitations to the present invention.

[0037] A preparation method of a mixed-mode chromatographic stationary phase of hydrophilic interaction chromatography and ion exchange chromatography includes the following steps:

[0038] 1) React the silanol groups on the surface of the silica microspheres with an epoxy group compound as the first modification compound, so as to bond epoxy groups on the surface of the silica microspheres to obtain silica microspheres with epoxy groups;

[0039] 2) React the silica microspheres with epoxy groups with a second modification compound, and the second modification compound is a mixture of one or more compounds containing multiple hydroxyl groups or / and amine groups; after the reaction, hydroxyl groups and amine groups are simultaneously bonded to the surface of the silica microspheres to obtain the mixed-mode chromatographic stationary phase with hydrophilic interaction chromatography and ion exchange chromatography.

[0040] As shown in formula ii, that is, reacting the silica microspheres with the first modification compound (1) in formula ii to obtain an intermediate (2), that is, silica microspheres with epoxy groups; then adding a polyol (3) and a polyhydroxy-substituted compound (4) of an amine group, reacting to obtain silica microspheres (5) simultaneously bonded with hydroxyl groups and amine groups, that is, the mixed-mode chromatographic stationary phase with hydrophilic interaction chromatography and ion exchange chromatography of the present invention.

[0041]

[0042] Example 1

[0043] This example is a mixed-mode chromatographic stationary phase with hydrophilic interaction chromatography and weak anion exchange chromatography, and the stationary phase 5A is obtained by the following preparation method:

[0044] (1) Preparation of silica microspheres with epoxy groups: Under the protection of dry nitrogen, disperse 80 grams of (2,3-epoxypropoxy)propyltrimethoxysilane and 80 grams of silica into 500 milliliters of toluene. After stirring at 100 °C for 24 hours, filter the reaction mixture, wash it successively with toluene, dioxane and acetone, and then dry it in vacuo at 50 °C for 8 hours to obtain an intermediate: silica microspheres with epoxy groups;

[0045] (2) Preparation of HILIC-WAX mixed-mode chromatographic stationary phase: Under the protection of dry nitrogen, 30 g of ethylene glycol, 30 g of triethanolamine and 80 g of intermediate were dispersed in 500 ml of tetrahydrofuran. After stirring at room temperature for 15 minutes, 3 ml of boron trifluoride diethyl ether was added to the reaction mixture, and then the reaction mixture was heated to the reflux temperature and stirred continuously for 8 hours. After the reaction was completed, the reaction mixture was filtered and washed successively with acetone, deionized water and acetone; the filter cake was dried in vacuo at 50 °C for 8 hours to obtain the mixed-mode chromatographic stationary phase 5A.

[0046] Example 2:

[0047] The difference from Example 1 is that the amounts of ethylene glycol and triethanolamine in step (2) are 20 g and 40 g respectively, to obtain the mixed-mode chromatographic stationary phase 5B.

[0048] Example 3:

[0049] The difference from Example 1 is that the amounts of ethylene glycol and triethanolamine in step (2) are 15 g and 45 g respectively, to obtain the mixed-mode chromatographic stationary phase 5C.

[0050] Example 4:

[0051] The difference from Example 1 is that the amounts of ethylene glycol and triethanolamine in step (2) are 12 g and 48 g respectively, to obtain the mixed-mode chromatographic stationary phase 5D.

[0052] Comparative Example 1:

[0053] ChromCore HILIC-Diol chromatographic stationary phase was used as the comparative example. This hydrophilic interaction chromatographic stationary phase has a large number of hydroxyl groups on its surface but no amino groups, so it has no anion exchange function.

[0054] Example 5

[0055] Performance testing was carried out on the mixed-mode chromatographic stationary phase 5A provided in Example 1. The mixed-mode chromatographic stationary phase 5A to be tested was filled into a 4.6x150 mm stainless steel column using traditional high-pressure slurry technology, and then tested as follows:

[0056] Test the retention behavior of the stationary phase for hydrophobic compounds, hydrophilic compounds, inorganic anions and cations under mobile phase conditions with different organic phase ratios.

[0057] Test conditions: Isocratic elution, the mobile phase contains 10 mM ammonium acetate solution (pH 5.0), and the acetonitrile content in the organic phase is 0, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90% respectively. The flow rate is 1.0 mL / min; the temperature is 30 °C; the injection volume is 5 μL. When the samples are naphthalene (0.05 mg / mL) and cytosine (0.1 mg / mL), the UV detection wavelength is 245 nm; when the samples are NaCl (2 mg / mL) and mannitol (2 mg / mL), the detector is ELSD.

[0058] Figure 1 These are the test results of the stationary phase 5A provided in Example 1. As Figure 1 shown, for the hydrophobic substance naphthalene, as the proportion of the organic phase increases, the peak elution time weakens to no retention; for the polar substances cytosine, Na+ and mannitol, there is basically no retention at 0 - 40% acetonitrile, and then the retention increases as the proportion of the organic phase increases; for Cl-, the retention gradually decreases before 80% acetonitrile and increases when the acetonitrile content rises to 90%, indicating that this stationary phase has a hydrophilic retention effect on polar substances under the condition that the acetonitrile content in the mobile phase is greater than 40%.

[0059] Example 6

[0060] Test the retention behavior of the stationary phase for neutral hydrophilic compounds, anionic and cationic organic compounds, inorganic anions and cations under the conditions of mobile phases with different buffer concentrations.

[0061] Test conditions: Isocratic elution, the mobile phase contains 10% acetonitrile, but the concentration of ammonium acetate solution (pH 5.0) is 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM and 90 mM respectively. The flow rate is 1.0 mL / min; the temperature is 30 °C; the injection volume is 5 μL. When the samples are benzyltrimethylammonium chloride (1 mg / mL), acetanilide (0.1 mg / mL) and sodium p-toluenesulfonate (1 mg / mL), the UV detection wavelength is 245 nm; when the samples are NaCl (2 mg / mL) and mannitol (2 mg / mL), the detector is ELSD.

[0062] Figure 2 These are the test results of the stationary phase 5A provided in Example 1. As Figure 2 shown, the peak elution times of the neutral substances mannitol and acetanilide are not affected by the buffer salt concentration. For the cations Na+ and benzyltrimethylammonium chloride, the peak elution times increase slightly as the buffer salt concentration increases. For the anions Cl- and p-toluenesulfonic acid, the peak elution times advance as the buffer salt concentration increases, indicating that this stationary phase is positively charged under pH 5.0 conditions and has an anion exchange chromatography function.

[0063] Example 7

[0064] Test the retention behaviors of neutral compounds, anionic and cationic organic compounds, inorganic anions, and cations by the stationary phase under the conditions that the mobile phase contains different pH buffer solutions.

[0065] Test conditions: isocratic elution, the mobile phase contains 10% acetonitrile and 10 mM ammonium acetate buffer solution, and the pH values of the buffer solution are 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, and 5.4 respectively. The flow rate is 1.0 mL / min; the temperature is 30 °C; the injection volume is 5 μL. When the samples are benzyltrimethylammonium chloride (1 mg / mL), acetanilide (0.1 mg / mL), and sodium p-toluenesulfonate (1 mg / mL), the UV detection wavelength is 245 nm; when the samples are NaCl (2 mg / mL) and mannitol (2 mg / mL), the detector is ELSD.

[0066] Figure 3 are the test results of the stationary phase 5A provided in Example 1. As Figure 3 shown, the elution times of the neutral substances mannitol and acetanilide are not affected by pH. For the cations Na+ and benzyltrimethylammonium chloride, the elution times increase slightly with the increase of pH. For the anions Cl- and p-toluenesulfonic acid, the elution times advance with the increase of pH, indicating that this HILIC-WAX mixed-mode chromatographic stationary phase is positively charged in the range of pH 3.8 - 5.4, has anionic exchange function, and its anionic exchange ability gradually weakens with the increase of pH.

[0067] Example 8

[0068] Compare the chromatographic performances of the mixed-mode chromatographic stationary phases 5A - 5D provided in Examples 1 - 4 under hydrophilic interaction chromatography conditions. Test the retention behaviors of hydrophobic compounds, hydrophilic compounds, inorganic anions, and cations by each stationary phase under the condition that the mobile phase contains 90% acetonitrile.

[0069] Test conditions: isocratic elution, the mobile phase is 90 / 10 (v / v) acetonitrile / 100 mM ammonium acetate solution (pH 5.0). The flow rate is 1.0 mL / min; the temperature is 30 °C; the injection volume is 5 μL. When the samples are naphthalene (0.05 mg / mL) and cytosine (0.1 mg / mL), the UV detection wavelength is 245 nm; when the samples are NaCl (2 mg / mL) and mannitol (2 mg / mL), the detector is ELSD.

[0070] Figure 4The retention times of various compounds or ions on four mixed-mode chromatographic stationary phases 5A - 5D were compared under the condition of 90% acetonitrile mobile phase. As can be seen from Figure 4 what is shown, the hydrophobic substance naphthalene has no retention on the four HILIC-WAX mixed-mode chromatographic stationary phases; for the polar substances cytosine and mannitol, their retention increases successively with the increase in the amount of triethanolamine used during bonding on the four stationary phases. This indicates that during the bonding process in step 2), with the increase in the amount of triethanolamine, the hydrophilic interaction of the stationary phase is enhanced. This is because the bonding of triethanolamine can bring more hydroxyl groups to the surface of the stationary phase. At the same time, it also introduces amino groups to the surface of the stationary phase, which carry a positive charge under this test condition (pH 5.0), making the stationary phase have anionic exchange ability. Therefore, with the increase in the bonded amount of triethanolamine, its anionic exchange ability also increases. The retention behavior of the inorganic ions Na+ and Cl- is the result of the combined action of two mechanisms: hydrophilic interaction chromatography and anionic exchange chromatography. The cation Na+ carries the same charge as the surface of the stationary phase and there is an electrostatic repulsion between them. Although the hydrophilic interaction of the stationary phase is enhanced with the increase in the bonded amount of triethanolamine, the increase in the positive charge on the surface of the stationary phase also increases the repulsion force on the cation. It can be seen that the retention time of the cation Na+ is the longest on the chromatographic column of stationary phase 5B and the shortest on 5A. For the anion Cl-, with the increase in the bonded amount of triethanolamine, both the hydrophilic interaction and the anionic exchange interaction are enhanced, resulting in a significant increase in its retention time.

[0071] Example 9

[0072] The ion exchange chromatographic performance of the mixed-mode chromatographic stationary phases 5A - 5D provided in Comparative Examples 1 - 4 was compared under low organic phase conditions. The retention capabilities of the stationary phases for neutral hydrophilic compounds, anionic and cationic organic compounds, inorganic anions, and cations were tested under the condition of a mobile phase containing 10% acetonitrile and 20 mM ammonium acetate solution (pH 5.0).

[0073] Test conditions: Isocratic elution, the mobile phase is 10% acetonitrile / 20 mM ammonium acetate solution (pH 5.0). The flow rate is 1.0 mL / min; the temperature is 30 °C; the injection volume is 5 μL. When the samples are benzyltrimethylammonium chloride (1 mg / mL), acetanilide (0.1 mg / mL), and sodium p-toluenesulfonate (1 mg / mL), the UV detection wavelength is 245 nm; when the samples are NaCl (2 mg / mL) and mannitol (2 mg / mL), the detector is ELSD.

[0074] Figure 5 The retention times of various compounds or ions on four mixed-mode chromatographic stationary phases 5A - 5D were compared under the condition of 10% acetonitrile / 20 mM ammonium acetate solution (pH 5.0) mobile phase. According to Figure 1As a result, under this condition, the stationary phase does not have an obvious hydrophilic interaction chromatography function and mainly exhibits anion exchange chromatography. Therefore, from Figure 5 It can be seen that the elution times of neutral substances mannitol and acetanilide on the four different stationary phases are basically the same; the elution times of cations Na+ and benzyltrimethylammonium chloride on the stationary phases from 5A to 5D decrease in turn, while the elution times of anions Cl- and p-toluenesulfonic acid on each stationary phase increase in turn. This is because as the bonding amount of triethanolamine increases, the anion exchange ability of the stationary phase increases, and at the same time, the repulsive effect on cations also gradually increases.

[0075] Example 10

[0076] Compare the separation of the mixed-mode chromatography stationary phase provided in the comparative example and the HILIC-Diol hydrophilic interaction chromatography column in Comparative Example 1 in some applications.

[0077] Separation of 5 nucleotides

[0078] Test conditions: isocratic elution, mobile phase is 50 / 20 / 30 v / v / v acetonitrile / water / 10 mM KH2PO4, pH 3.0. Flow rate is 1.0 mL / min; temperature is 30 °C; injection volume is 2 μL. UV detection wavelength is 260 nm. The sample is a mixture of 5 nucleotides: CMP (cytidine monophosphate), AMP (adenosine monophosphate), UMP (uridine monophosphate), IMP (inosine monophosphate), GMP (guanosine monophosphate) (0.1 mg / mL).

[0079] Figure 6 Compare the separation of the mixed-mode chromatography stationary phase 5B provided in Example 2 and the HILIC-Diol chromatography column in Comparative Example 1 for 5 nucleotides under this mobile phase condition. From Figure 6 As shown, the HILIC-Diol chromatography column in the comparative example has almost no retention for all nucleotides under this condition and cannot perform effective separation. However, the HILIC-WAX mixed-mode stationary phase 5B of the present invention can well separate 5 nucleotides by the combined action of hydrophilic interaction chromatography and anion exchange chromatography.

[0080] Separation of choline compounds

[0081] Test conditions: isocratic elution, mobile phase is 90 / 10 v / v acetonitrile / 100 mM ammonium formate solution, pH 3.0. Flow rate is 1.0 mL / min; temperature is 30 °C; injection volume is 5 μL. The detector is ELSD. The sample is a mixed sample of choline chloride and betaine (0.1 mg / mL).

[0082] Figure 7The separation of choline compounds by the mixed-mode chromatographic stationary phase 5B provided in Example 2 and the HILIC-Diol chromatographic column in Comparative Example 1 under these mobile phase conditions was compared. From Figure 7 As shown, the HILIC-Diol chromatographic column in the comparative example could effectively separate choline and betaine, but chloride ions and betaine were not separated. However, the HILIC-WAX mixed-mode stationary phase 5B of the present invention could well separate choline, betaine, and chloride ions by the combined action of hydrophilic interaction chromatography and anion exchange chromatography.

[0083] Separation of N-oligosaccharides

[0084] The mixed-mode chromatographic stationary phase 5C to be tested and the stationary phase in Comparative Example 1 were filled into a 2.1 x 150 mm stainless steel column using the traditional high-pressure slurry technique.

[0085] Test conditions: Mobile phase A was 50 mM ammonium formate solution, pH 4.5; mobile phase B was acetonitrile. Gradient elution: First, equilibrate the chromatographic column with 25% A and then inject the sample; 0 - 3 minutes, 25% A; 3 - 53 minutes, 25 - 60% A; 53 - 56 minutes, 60% A; 56 - 59 minutes, 60 - 25% A; 59 - 75 minutes, 25% A. The flow rate was 0.4 mL / min; the temperature was 60 °C; the injection volume was 1 μL. A fluorescence detector was used: excitation wavelength 265 nm; emission wavelength 425 nm. The sample was a 2-AB-labeled N-oligosaccharide sample.

[0086] Figure 8 The mixed-mode chromatographic stationary phase 5C provided in Example 3 and the HILIC-Diol chromatographic column in Comparative Example 1 were compared under the same conditions to test this N-oligosaccharide standard. From Figure 8 As shown, this sample contained N-oligosaccharide components modified with different numbers of sialic acids: 0 (neutral), 1, 2, 3, and 4. Since each sialic acid carries a negative charge, these components respectively carry different negative charges: 0, -1, -2, -3, and -4. The separation of these components on the HILIC-Diol chromatographic column was very poor. However, on the HILIC-WAX mixed-mode chromatographic column 5C of the present invention, these components were well separated according to the different numbers of sialic acids (i.e., the amount of negative charge carried). In each group containing the same number of sialic acids, the individual components were further separated by hydrophilic interaction chromatography according to their polarity. This fully demonstrated the chromatographic separation ability of the mixed mechanism of hydrophilic interaction chromatography and anion exchange chromatography. Currently, only the HILIC-WAX mixed-mode chromatographic column of the present invention can obtain such a chromatogram with separation selectivity.

[0087] As can be seen from the above embodiments, the present invention has the following technical effects: 1) The stationary phase prepared by the present invention has a hydrophilic retention effect on polar substances, especially on polar substances under the condition that the acetonitrile content in the mobile phase is greater than 40%; 2) The stationary phase prepared by the present invention is positively charged under conventional application conditions, including within the tested pH range, and has an anion exchange function; 3) With the increase in the dosage of the polyhydroxy-substituted compound of the amine group in the reaction, the hydrophilicity of the prepared stationary phase is enhanced, and the anion exchange ability is also enhanced accordingly; it fully shows that the present invention can adjust the hydrophilicity and ion exchange ability of the final stationary phase by controlling two or more components and their ratios in the second modifier compound, and further shows that the preparation method of the present invention can control the contributions of the hydrophilic effect and the ion exchange effect in the whole separation process; 4) The stationary phase prepared by the present invention can well separate nucleotides; 5) The stationary phase prepared by the present invention can well separate choline compounds and chloride ions; 6) The stationary phase prepared by the present invention can well separate N-oligosaccharides according to the number of sialic acid residues; the above points 4), 5), and 6) also further show the special selectivity of the present invention for the separation of some hydrophilic compounds and ionic compounds.

[0088] The present invention uses silica microspheres as the matrix, and the surface of the obtained stationary phase simultaneously has a large number of hydroxyl and amine groups, which can provide the mixed-mode chromatographic separation performance of hydrophilic interaction chromatography and anion exchange chromatography, thereby providing special separation selectivity for the separation of some hydrophilic compounds and ionic compounds.

[0089] The above-described embodiments are only some preferred solutions of the present invention, but they are not intended to limit the present invention. Those of ordinary skill in the relevant technical fields can still make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all technical solutions obtained by adopting equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A preparation method of a mixed-mode chromatographic stationary phase with hydrophilic interaction chromatography and ion exchange chromatography, characterized in that, The mixed-mode chromatographic stationary phase includes a silica microsphere matrix and functional groups bonded to the surface of the silica microspheres that have both hydrophilic interaction chromatography and ion exchange chromatography functions; The preparation method includes the following steps: 1) React the silanol groups on the surface of the silica microspheres with an epoxy compound as the first modifying compound, thereby bonding epoxy groups to the surface of the silica microspheres to obtain silica microspheres with epoxy groups; 2) React the silica microspheres with epoxy groups with a second modifying compound, where the second modifying compound is a mixture of one or more compounds containing multiple hydroxyl groups or / and amino groups; after the reaction, hydroxyl and amino groups are simultaneously bonded to the surface of the silica microspheres to obtain the mixed-mode chromatographic stationary phase with hydrophilic interaction chromatography and ion exchange chromatography.

2. The preparation method of the mixed-mode chromatographic stationary phase with hydrophilic interaction chromatography and ion exchange chromatography according to claim 1, characterized in that, The first modifying compound is an alkoxysilane compound with an epoxy group.

3. The preparation method of the mixed-mode chromatographic stationary phase with hydrophilic interaction chromatography and ion exchange chromatography according to claim 1, characterized in that, The first modifying compound includes one or more of the compounds having the structural characteristics shown in formula (i): In formula (i), R1, R2, and R3 are each independently a C1-C5 alkyl group or a C1-C5 alkoxy group, and at least one is a C1-C5 alkoxy group; n is 1-5.

4. The preparation method of the mixed-mode chromatographic stationary phase with hydrophilic interaction chromatography and ion exchange chromatography according to claim 3, characterized in that, In the first modifying compound, R1, R2, and R3 in formula (i) are each independently a methyl group or a methoxy group or an ethoxy group, and at least one is a methoxy group or an ethoxy group, and n is 1.

5. The preparation method of the mixed-mode chromatographic stationary phase with hydrophilic interaction chromatography and ion exchange chromatography according to claim 1, characterized in that, The particle size of the silica microspheres used in step 1) is 1 μm - 50 μm; the pore size is 6. The preparation method of the mixed-mode chromatographic stationary phase with hydrophilic interaction chromatography and ion exchange chromatography according to claim 1, characterized in that, The second modifying compound includes one or more polyhydric alcohols and polyhydroxy-substituted compounds of amino groups respectively.

7. The preparation method of the mixed-mode chromatographic stationary phase with hydrophilic interaction chromatography and ion exchange chromatography according to claim 6, characterized in that, The polyhydric alcohols include ethylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, glycerol, diglycerol, and polyglycerol; the polyhydroxy-substituted compounds of amino groups include ethanolamine, diethanolamine, N-methyldiethanolamine, N-ethyldiethanolamine, triethanolamine, isopropanolamine, diisopropanolamine, N-methyldiisopropanolamine, N-ethyldiisopropanolamine, and triisopropanolamine.

8. The preparation method of the mixed-mode chromatographic stationary phase with hydrophilic interaction chromatography and ion exchange chromatography according to claim 6, characterized in that, The mass ratio of the polyhydric alcohol to the polyhydroxy-substituted compound of amino group in the second modifying compound is (1-10):(10-1).

9. The preparation method of the mixed-mode chromatographic stationary phase with hydrophilic interaction chromatography and ion exchange chromatography according to claim 1, characterized in that, The reaction conditions in step 2) include one or more of the following characteristics: The mass ratio of the silica microspheres with epoxy groups to the second modifying compound is 1:(0.1-5); In step 2), a reaction solvent is further added, and the reaction solvent is one or more of tetrahydrofuran, 1,4-dioxane, toluene, and xylene; The reaction in step 2) is carried out in the presence of a catalyst, and the catalyst is boron trifluoride diethyl ether; The dosage of the catalyst is 1%-10% of the mass of the silica microspheres.

10. A chromatographic column, characterized in that, The stationary phase packing of the chromatographic column is the mixed-mode chromatographic stationary phase with hydrophilic interaction chromatography and ion exchange chromatography according to any one of claims 1-9.

11. The chromatographic column according to claim 10, characterized in that, The chromatographic column is used to analyze hydrophilic compounds, polar compounds, and ionic compounds.

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