CN114133571A

CN114133571A - PMO (SLLTP-POSS) hydrophilic microsphere and preparation method and application thereof

Info

Publication number: CN114133571A
Application number: CN202111210304.7A
Authority: CN
Inventors: 陈桐; 李丙祥; 徐亮; 刘涛; 徐何辰; 肖震; 吴艳玲; 杨敏
Original assignee: Zhenjiang Customs Comprehensive Technology Center
Current assignee: Zhenjiang Customs Comprehensive Technology Center
Priority date: 2021-10-18
Filing date: 2021-10-18
Publication date: 2022-03-04
Anticipated expiration: 2041-10-18
Also published as: CN114133571B

Abstract

The invention discloses a PMO (SLLTP-POSS) hydrophilic microsphere and a preparation method and application thereof. The PMO (SLLTP-POSS) hydrophilic microspheres are used as a hydrophilic stationary phase and have the characteristics of HILIC and PALC, and similar to HILIC, PALC can also retain polar compounds. And the acid and alkali resistance of the PMO (SLLTP-POSS) stationary phase is greatly improved. The stationary phase can separate organic acids, sugar alcohol and amino acid mixture, sweetener and other polar compounds, and has great separation degree and high selectivity. The immobilization is less retention of relatively non-polar and less polar compounds than the common C18 column, and more retention of more polar compounds. From the perspective of developing green color spectrum and the harm of ACN to the environment, PALC as a green color spectrum mode is expected to become an alternative mode of HILIC and a complementary mode of RPLC.

Description

PMO (SLLTP-POSS) hydrophilic microsphere and preparation method and application thereof

Technical Field

The invention belongs to the field of new materials, and particularly relates to a PMO (SLLTP-POSS) hydrophilic microsphere as well as a preparation method and application thereof.

Background

Food safety is always a big concern all over the world. The main separation method of polar compounds in food additives is Hydrophilic interaction chromatography (HILIC) [1,2 ]. At present, a plurality of hydrophilic chromatographic stationary phases are developed on the market, and most of the stationary phases are prepared by bonding hydrophilic groups on the surface of silica gel. When they are used in the HILIC mode, there are three problems:

(1) the mobile phase needs to use a high percentage of organic solvents, such as Acetonitrile (ACN) and the like [3], and the waste liquid generated after a large amount of use, such as random discharge, can cause serious pollution to the environment.

(2) Although HILIC chromatography columns have a strong ability to separate polar compounds, the column efficiency is usually not high [4 ].

(3) Such chromatographic columns are not very acid-base resistant and, when used under strong acid or strong base conditions for a long time, the separation capacity and selectivity of the chromatographic column are greatly reduced [5 ].

Aiming at the problems of low column efficiency and general lack of acid and alkali resistance of HILIC chromatographic columns, the organic-inorganic hybrid material-ordered mesoporous organic silicon oxide (P)eriodic meso organisilicas, PMO) (R' O)₃Si-R-Si(OR')₃[6]And the solution can be achieved. PMO can completely incorporate organic groups into the mesoporous framework of the material, rendering the material to exhibit special properties such as greater hydrothermal stability and mechanical stability. More interestingly, the PMO shows good chemical stability in alkaline medium, and is very beneficial to solving the problem that the alkaline compound is easy to form a tailing peak on a silica gel matrix. Meanwhile, various chemical reactions can be carried out through the embedded organic group, so that the purpose of functionally modifying the material is achieved. As the proportion of organic and inorganic components can be regulated and controlled by precursors, the materials show great advantages in the aspects of organic functional group density and high guarantee of column efficiency [7]. Therefore, PMO has been applied to preparative chromatography and high performance liquid chromatography as a new class of chromatographic packing. Li et al [8 ]]1, 2-di (triethoxysilyl) ethane and 3-aminopropyl triethoxy silane are copolymerized to prepare the amino-functionalized ethylene bridged hybrid silica gel filler. After the chromatographic column is prepared, the stability of the hybridized amino column in an alkaline medium is obviously superior to that of a bonded amino column. Under the conditions of pH 1.6 and pH 11.8, some release type compounds are separated, and good peak shape and column effect are obtained.

Meanwhile, in a water-rich chromatography (PALC) mode [9], a high percentage of water is usually used as a mobile phase, and the stationary phase is applied to the field of food detection, is expected to become a substitute of HILIC and a supplement of reversed phase chromatography, and can also relieve the problem that a large amount of organic solvent is required to be used for the mobile phase in the HILIC mode. Currently, the types of commercially available PMO liquid chromatography stationary phases are limited. Therefore, the method has important significance for the preparation and development of the stationary phase, and opens up a new path for developing a new stationary phase with excellent performance.

Reference to the literature

1.Walker S.H.,Carlisle B.C.,Muddiman D.C.Systematic comparison of reverse phase and hydrophilic interaction liquid chromatography platforms for the analysis of N-Linked glycans. Analytical Chemistry,2012,84(19):8198-8206.

2.Shen Q.,Wu H.M.,Wang H.H.,Zhao Q.L.,Xue J.,Ma J.F.,Wang H.X.Monodisperse microsphere-based immobilized metal affinity chromatography approach for preparing Antarctic krill phospholipids followed by HILIC-MS analysis.Food Chemistry,2021,344: 128585-128593.

3.Costa P.P.K.G.,Mendes T.D.,Salum T.F.C.,Pacheco T.F.,Rodrigues C.M.Development and validation of HILIC-UHPLC-ELSD methods for determination of sugar alcohols stereoisomers and its application for bioconversion processes of crude glycerin.Journal of Chromatography A, 2019,1589:56-64.

4.LiangT.,Fu Q.,Shen A.J.,Wang H.,Jin Y.,Xin H.X.,Ke Y.X.,Guo Z.M.,Liang X.M.Preparation and chromatographic evaluation of a newly designedsteviol glycoside modified-silica stationary phase in hydrophilicinteraction liquid chromatography and reversed phase liquidchromatography.Journal of Chromatography A,2015,1388:110–118.

5.Spicer V.,Krokhin O.V.Peptide retention time prediction in hydrophilic interaction liquid chromatography and comparison of separation selectivity between bare silica and bonded stationary phases.Journal of Chromatography A,2018,1534:75-84.

6.Huang X.,Zhang M.N.,Wang M.J.,Li W.,Wang C.,Hou X.J.,Luan S.,Wang Q. Gold/periodic mesoporous organosilicas with controllable mesostructure by using compressedCO2.Langmuir,2018,34:3642-3653.

7.Kaczmarek A.M.,Maegawa Y.,Abalymov A.,Skirtach A.G.,Inagaki S.,Voort P.V.D. Lanthanide-grafted bipyridine periodic mesoporous organosilicas(BPy-PMOs)physiological range and wide temperature range luminescence thermometry.ACS Applied Materials& Interfaces,2020,12:13540-13550.

8.Li C.,Di B.,Hao W.,Yan F.,Su M.Aminopropyl-functionalized ethane-bridged periodic mesoporous organosilica spheres:preparation and application in liquid chromatography.Journal of Chromatography A,2016,1218(3):408-415.

9.Dembek M.,Bocian S.Pure water as a mobile phase in liquid chromatography techniques. Trends in Analytical Chemistry,2020,123:115793-115806.

Disclosure of Invention

The invention aims to provide PMO (SLLTP-POSS) hydrophilic microspheres and a preparation method thereof.

The PMO (SLLTP-POSS) hydrophilic microsphere provided by the invention is prepared by the following steps:

1) synthesis of SLLTP-bridged silane (SLLTPBS)

a. Dissolving lily polysaccharide (LLTP) in ionic liquid, then dropwise adding chlorosulfonic acid diluted by anhydrous pyridine, stirring at constant temperature of 30-70 ℃ for 0.5-3h, and adjusting to neutrality by using an alkali solution to obtain sulfated and modified lily polysaccharide (SLLTP);

b. under the protection of nitrogen, stirring and reacting the N, N-Dimethylformamide (DMF) solution of SLLTP with the THF solution of chloromethyl trimethoxy silane (CMTMS) at the constant temperature of 40-80 ℃ for 3-10h to obtain SLLTP bridged silane (SLLTPBS);

2) in the presence of NaOH, using C18TACL as template agent to make POSS [ C ]₂H₄Si(OEt)₃]₈SLLTPBS, C18TACL in a mixed solvent of water and THF; filtering after the reaction is finished, washing the product with ethanol, deionized water and methanol in sequence, and drying in vacuum; then, a solvent extraction method is adopted to remove the template agent, and the PMO (SLLTP-POSS) hydrophilic microspheres are obtained.

In the step a of step 1) of the method, the ionic liquid is an imidazole ionic liquid, and specifically may be any one of the following: 1-butyl-3-methylimidazolium chloride salt, 1-butylimidazolium chloride salt, and 1, 3-dimethylimidazolium chloride salt.

In the step a of step 1) of the method, the ratio of the LLTP to the ionic liquid may be 500 mg: 10mL-500 mg: 50 mL; specifically, the content is 500 mg: 30 mL.

In the step a of the step 1), the molar amount of the chlorosulfonic acid is 40 to 80 percent, specifically 60 percent, of the molar number of the hydroxyl groups on the LLTP;

in the step a of the step 1), the volume ratio of the chlorosulfonic acid to the anhydrous pyridine is 1: 10-1: 20.

in step 1) a of the above method, the mixture may be stirred at a constant temperature of 50 ℃ for 1 hour.

In step 1), the alkali may be potassium hydroxide or ammonia.

In the step a of the step 1), after the solution is adjusted to be neutral by the sodium hydroxide solution, the method further comprises the following steps: loading the system adjusted to neutral into dialysis bag with cut-off of 10KDa, dialyzing in ultrapure water for 24-72 hr (specifically 48 hr), concentrating, precipitating with ethanol, and freeze drying at-20 deg.C to obtain SLLTP.

In step b) of step 1) of the above process, the molar ratio of SLLTP to chloromethyltrimethoxysilane is from 1:2 to 1: 4.

In step b) of step 1) of the above process, the volume ratio of chloromethyltrimethoxysilane to THF is from 1:3 to 1:6, specifically 1: 4.

In the step b) of step 1), the N, N-dimethylformamide is anhydrous N, N-dimethylformamide; the THF is anhydrous THF.

In the step b) of step 1), the method further comprises the following steps after the reaction is finished: loading the reaction solution into a dialysis bag with cut-off of 10KDa, dialyzing in ultrapure water for 24-72 hr (specifically 48 hr), concentrating, precipitating with ethanol, and freeze-drying at-20 deg.C to obtain SLLTPBS.

In the step 2) of the above method, the POSS [ C ]₂H₄Si(OEt)₃]₈And SLLTPBS at a molar ratio of 0.5:1 to 1.5:1, preferably at a molar ratio of 0.75:1 to 1.5: 1.

In step 2) of the method, the mass ratio of the C18TACl to the SLLTPBS is 1: 17.

in step 2) of the method, the mass ratio of NaOH to SLLTPBS is 1: 37.5.

in the step 2) of the method, the volume ratio of water to THF in the mixed solvent of water and THF may be 30: 30-60: 30, preferably in a volume ratio of 40: 30-50: 30.

in step 2) of the above method, the reaction temperature of the reaction may be 50 ℃ to 90 ℃, preferably 60 ℃ to 70 ℃.

In step 2) of the above method, the reaction time of the reaction may be 10 to 20 hours, specifically 15 hours.

In the step 2) of the method, the reaction is carried out in a miniature high-pressure reaction kettle.

In step 2) of the above method, the temperature of the vacuum drying may be 65 ℃.

In the step 2), the specific method for removing the template agent by using the solvent extraction method comprises the following steps: adding the dried solid into 150mL of concentrated hydrochloric acid (mass fraction is 36% -38%)/deionized water/ethanol (v/v ═ 5/45/50) solution per gram, heating and refluxing for 8h to extract the template, repeating twice, filtering, washing and drying.

The method further comprises the step of reaming the obtained PMO (SLLTP-POSS) hydrophilic microspheres, and the specific method comprises the following steps: and (3) reacting the PMO (SLLTP-POSS) hydrophilic microspheres, the DMDA and the DDA in ultrapure water, filtering after the reaction is finished, washing with ultrapure water and methanol in sequence, and drying at 60 ℃ in vacuum to obtain the PMO (SLLTP-POSS) hydrophilic microspheres after pore expansion.

The reaming method further comprises the following steps: and (3) removing the DMDA and the DDA from the pore-enlarged PMO (SLLTP-POSS) hydrophilic microspheres by adopting a solvent extraction method.

Wherein the mass ratio of the PMO (SLLTP-POSS) hydrophilic microspheres to the DMDA to the DDA can be 4.0: (3.0-6.0): (0.5-1.5), specifically 4.0:5.0: 1.2.

The reaction condition of the reaction is that the mixture is kept stand for 24 to 72 hours at the temperature of 110 ℃.

The invention also provides application of the PMO (SLLTP-POSS) hydrophilic microsphere.

The application of the PMO (SLLTP-POSS) hydrophilic microsphere provided by the invention is the application of the PMO hydrophilic microsphere in preparation of a hydrophilic chromatographic stationary phase.

The invention also provides a hydrophilic chromatographic column.

The stationary phase of the hydrophilic chromatographic column is the PMO (SLLTP-POSS) hydrophilic microsphere.

The invention also protects the application of the hydrophilic chromatographic column in separating and/or detecting polar substances.

Specifically, the polar substance is selected from at least one of organic acids, sugar alcohol, amino acid, and sweetener (including artificial and natural sweetener);

the organic acid is at least one selected from oxalic acid, tartaric acid, quinic acid, malic acid, shikimic acid, ascorbic acid, acetic acid and maleic acid;

the sweetener is selected from saccharin sodium, sucralose, sodium cyclamate, aspartame, acesulfame potassium, alitame, neotame, glycyrrhizic acid, glycyrrhetinic acid, stevioside, and steviolbioside.

In the separation or detection method, the sample to be detected can be food; more specifically, it may be a food containing or suspected of containing the above-mentioned organic acids, monosaccharides, sugar alcohols, amino acids or sweeteners.

The PMO (SLLTP-POSS) filler is prepared as a hydrophilic stationary phase, is characterized by utilizing infrared spectroscopy, elemental analysis, a scanning electron microscope and the like, and is researched for chromatographic behavior. The novel stationary phase has the characteristics of HILIC and PALC. Similar to HILIC, PALC can also retain polar compounds, and the acid and alkali resistance is greatly improved. The stationary phase can separate organic acids, sugar alcohol and amino acid mixture, sweetener and other polar compounds, and has great separation degree and high selectivity. The immobilization is less retention of relatively non-polar and less polar compounds than the common C18 column, and more retention of more polar compounds. From the perspective of developing green color spectrum and the harm of ACN to the environment, PALC as a green color spectrum mode is expected to become an alternative mode of HILIC and a complementary mode of RPLC.

Drawings

FIG. 1 is a flow chart of the synthetic reaction of PMO (SLLTP-POSS) hydrophilic microspheres.

FIG. 2 is an infrared spectrum of PMO (SLLTP-POSS) hydrophilic microsphere at each stage of synthesis; (A) LLTP; (B) SLLTP; (C) SLLTPBS; (D) non-templated PMO (SLLTP-POSS); (E) except for PMO (SLLTP-POSS) of the template.

FIG. 3 is a scanning electron micrograph of PMO (SLLTP-POSS) microspheres obtained under different conditions. A and B correspond to experiment 3 in Table 1; c, D and E correspond to

experiments

8,9 and 10 in table 1, respectively.

FIG. 4 is a thermogravimetric plot of PMO (SLLTP-POSS) microspheres.

FIG. 5 is a low angle XRD pattern of PMO (SLLTP-POSS) microspheres.

FIG. 6 is a comparison of column pressure/flow rate performance of PMO (SLLTP-POSS) columns prepared at different POSS [ C2H4Si (OEt)3]8/SLLTPBS molar ratios with commercial C18 columns.

Fig. 7 is a graph of the change in retention factor (a) and tray number (B) for a PMO (SLLTP-POSS) column versus a commercial C18 column at pH 11. (mobile phase: aqueous triethylamine solution/ACN 95:5, flow rate: 1.0 mL/min.); PMO (SLLTP-POSS) columns retain changes in factor (C) and number of trays (D) from commercial C18 columns at pH 1.0. (mobile phase: 1% TFA/ACN 92:8, flow rate: 1.0mL/min.)

FIG. 8 is a separation chromatogram of eight organic acids in the PLAC mode. (A) The chromatogram was obtained by column separation using PMO (SLLTP-POSS), (B) was obtained by column chromatography using C18, (C) was obtained by column chromatography using PMO (SLLTP-POSS) after column washing for 30 days, and (D) was obtained by column chromatography using C18 after column washing for 30 days. Chromatographic peak: (1) oxalic acid; (2) tartaric acid; (3) quinic acid; (4) malic acid; (5) (ii) shikimic acid; (6) ascorbic acid; (7) acetic acid; (8) maleic acid.

Detailed Description

The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified.

The experimental materials used in the following examples are as follows:

the lily polysaccharide is purchased from Shaanxi blue grass Biotech limited. 1-butyl-3-methyl chloride, chlorosulfonic acid, chloromethyltrimethoxysilane, Octadecyltrimethylammonium chloride (C18 TACL), octakis (triethoxysilylethyl) oligomeric silsesquioxane (POSS [ C ] S₂H₄Si(OEt)₃]₈) N, N-dimethyldecylamine (DMDA) and Dodecylamine (DDA) were purchased from Alfa Aesar, Germany. Pyridine, tetrahydrofuran, N-dimethylformamide, methanol and ethanol were all purchased fromIn Nanjing chemical reagents, Inc. (Nanjing, China).

The compounds adenine, caffeine, clenbuterol, salicylic acid, potassium sorbate, fumaric acid, thiourea, tartrazine, sunset yellow, brilliant blue, new red, vitamin B2(VB2) and vitamin B6(VB6) for hydrophilic and water-rich chromatographic pattern evaluation were purchased from alatin (beijing, china). 8 organic acid compounds including oxalic acid, tartaric acid, quinic acid, malic acid, shikimic acid, ascorbic acid, acetic acid, maleic acid; ribose, mannitol, sucrose, maltitol, Raynaud's sugar, melezitose, phenylalanine, methionine, glutamic acid and histidine were purchased from sigma.

The ultrapure Water used in the experimental procedure was obtained from a BWT ultrapure Water system (Best Water Technology, Germany). The mobile phase of the HPLC apparatus was a mixture of acetonitrile (chromatographic grade, merck, Germany) and ultrapure water, and both the mobile phase and the test compound were filtered using a 0.22 μm filter before use.

Example 1 preparation of PMO (SLLTP-POSS) hydrophilic microspheres

The process is divided into three steps, and the specific content is as follows:

1. synthesis of SLLTP-bridged silane (SLLTPBS)

Firstly, SLLTP is prepared by adopting a green synthesis technology and an ionic liquid-pyridine chlorosulfonate method. 500mg of lily polysaccharide (LLTP) was dissolved in 30mL of 1-butyl-3-methylimidazolium chloride ([ C4mim ] Cl), and then 5mL of anhydrous pyridine-diluted chlorosulfonic acid (the content of chlorosulfonic acid is 60% of the number of moles of hydroxyl groups on LLTP) was added dropwise thereto, followed by stirring at 50 ℃ for 1 hour at a constant temperature and then adjusting to neutrality with a sodium hydroxide solution. Dialyzing with 10kDa dialysis bag in ultrapure water for 48 hr, concentrating, precipitating with ethanol, and freeze-drying at-20 deg.C to obtain SLLTP.

Then, the SLLTP was dissolved in 30mL of anhydrous N, N-Dimethylformamide (DMF). After 2mL of Chloromethyltrimethoxysilane (CMTMS) was dissolved in 8mL of anhydrous THF, the above DMF solution was added dropwise. Stirring at constant temperature of 60 ℃ for 5h under the protection of nitrogen. After the reaction is finished, the reaction solution is filled into a dialysis bag with the cut-off quantity of 10KDa, dialyzed in ultrapure water for 48 hours, concentrated, precipitated by ethanol, frozen and dried at the temperature of minus 20 ℃ to obtain SLLTPBS.

2. Preparation of PMO (SLLTP-POSS) hydrophilic microsphere

A certain amount of C18TACL was weighed into a flask, THF and ultrapure water were sequentially added thereto, and the mixture was sufficiently stirred. Then, NaOH was added and dissolved. Reacting POSS [ C ]₂H₄Si(OEt)₃]₈And SLLTPBS were dissolved in THF and an aqueous solution, respectively, and added to the above reaction system. Stirring for 5h at room temperature, and heating in a miniature high-pressure reaction kettle for reaction. After the reaction is finished, filtering, washing the product with ethanol, deionized water and methanol in sequence, and drying in vacuum at 65 ℃.

The optimum reaction conditions were determined by examining the variation of the various reaction parameters in the system, the reaction parameters being set up as shown in table 1. Then, a solvent extraction method is adopted to remove the template agent. Adding the dried solid into 150mL concentrated hydrochloric acid (mass fraction is 36% -38%)/deionized water/ethanol (v/v ═ 5/45/50) solution per gram, heating and refluxing for 8h to extract the template, and repeating twice. After filtration, washing and drying, the PMO (SLLTP-POSS) hydrophilic microspheres are finally obtained, and the synthetic flow chart is shown in figure 1.

TABLE 1 gradient of reaction parameters

Wherein the mass ratio of the C18TACL to the SLLTPBS is 1: 17.

3. reaming of PMO (SLLTP-POSS) hydrophilic microspheres

4.0g of PMO (SLLTP-POSS) hydrophilic microspheres and 5.0g of DMDA and 1.2g of DDA were weighed into a flask, and 120mL of ultrapure water was added thereto and stirred at room temperature for 1 hour. Placing into a miniature high-pressure reaction kettle, and standing in an electric heating thermostat at 110 deg.C for 48 h. And filtering after the reaction is finished, washing with ultrapure water and methanol in sequence, and drying in vacuum at 60 ℃ to obtain the PMO (SLLTP-POSS) hydrophilic microspheres after pore expansion. Then solvent extraction is adopted to remove the template agents DMDA and DDA. The procedure for removing DMDA and DDA was the same as for removing the templating agent in 2 above.

Example 2 characterization of PMO (SLLTP-POSS) hydrophilic microspheres

The chemical structural changes of the surface of the PMO (SLLTP-POSS) hydrophilic microspheres were analyzed by Nicolet is model 10 Fourier Infrared Spectroscopy (Thermo Fisher, USA). C. The results of the variation of the contents of H and S elements were obtained by a Vario EL type element analyzer (Elementar Co., Germany). The thermal stability and order of the materials were analyzed on a thermo gravimetric analyzer model STA 409PC (NETZSCH, Germany) and X-ray diffractometer model Ultimate IV (XRD), respectively (Rigaku, Japan). JSM-6360LV scanning electron microscope (Japan) observes the surface topography of hydrophilic microspheres and determines particle size. A BI-200SM DLS type dynamic light scattering instrument (Brookhaven, USA) examined the particle size distribution of PMO (SLLTP-POSS) hydrophilic microspheres prepared under different conditions. Changes in specific surface area, pore diameter and pore volume of PMO (SLLTP-POSS) hydrophilic microspheres were examined using a model ASAP-2460 nitrogen adsorption specific surface area analyzer (Micromeritics Instruments Corporation, USA).

The results are shown below.

1. First, the results of IR spectroscopy and elemental analysis of LLTP, SLLTP, SLLTPBS and PMO (SLLTP-POSS) hydrophilic microspheres are shown in FIG. 2 and Table 2. LLTP is 3400cm^-1The broad peak at (A) is O-H stretching vibration, 2910cm^-1The absorption peak is C-H stretching vibration, 1380cm^-11020cm of flexural vibration having an absorption peak of C-H^-1The absorption peaks at (A) are C-O stretching vibrations, which are characteristic peaks of polysaccharides (FIG. 2A). Compared with LLTP, SLLTP has two new characteristic peaks, one is 1198cm^-1The position is an asymmetric S-O stretching vibration peak, and the other is 820cm^-1The peak is a symmetric C-O-S stretching vibration peak (figure 2B). Elemental analysis of table 2 showed that element S (9.02%) was found in SLLTP due to the introduction of sulfonic acid groups. The above results all indicate that the synthesis of the sulfated polysaccharide SLLTP was successful.

FIG. 2C is an IR spectrum of SLLTPBS. As can be seen from the figure, at 1080cm^-1A new absorption peak appears, which is a Si-O-Si stretching vibration peak and indicates the existence of a silicon oxygen group; and a C-O stretching vibration peak (1020 cm) with LLTP^-1) Substantially coincident and become broader and larger absorption peaks. 3400cm^-1The absorption peak is significantly reduced because of the LLTPMost of the hydroxyl groups have been substituted with sulfonic acid groups and silanes. Elemental analysis results showed that the incorporation of silane resulted in a slight decrease in C, H and S content of SLLTPBS compared to SLLTP. Compared with SLLTPBS, the PMO (SLLTP-POSS) hydrophilic microspheres without template removal after POSS introduction are at 1080cm^-1The stretching vibration peak of Si-O-Si is obviously enhanced; at the same time, the absorption peaks at 2920 and 2818 cm-1 were correspondingly enhanced, mainly due to the introduction of the templating agent C18TACL (FIG. 2D). FIG. 2E is an IR spectrum of PMO (SLLTP-POSS) after purification. As can be seen, the template C18TACL (2920 and 2818 cm)^-1) The intensity of the infrared absorption peak of (C18 TACl) was greatly diminished, indicating that most of the C18TACl was removed by solvent extraction. In addition, the infrared spectrum has little change, and the basic chemical structure of the PMO (SLLTP-POSS) hydrophilic microsphere is unchanged after the template is removed. Table 2 shows that the C and H content of the de-templated PMO (SLLTP-POSS) was significantly reduced, the S content was slightly increased, and it was also demonstrated that most of the C18TACL was removed, compared to the non-de-templated PMO (SLLTP-POSS) hydrophilic microspheres.

TABLE 2 results of elemental analysis at various stages of stationary phase synthesis

2. Table 3 and FIG. 3 show the particle size distribution and morphology of PMO (SLLTP-POSS) microspheres obtained under different conditions, respectively. By adopting a controlled variable method, under the condition of no change of other synthesis conditions, POSS [ C ]₂H₄Si(OEt)₃]₈When the mol ratio of/SLLTPBS is changed from 0.5:1 to 1.5:1, the PMO (SLLTP-POSS) microspheres can keep spherical shape, and the average particle size is between 4.4 and 5.1 mu m. It is shown that the average particle size of the microspheres does not change much when the molar ratio is within this range. FIGS. 3A and B show POSS [ C ]₂H₄Si(OEt)₃]₈SEM results at a 0.75:1 molar ratio of/SLLTPBS showed that the microspheres obtained had smooth surfaces, an average particle size of about 5 μm and a uniform particle size distribution.

3. In order to explore the influence of the change of the NaOH content on the morphology of the synthesized material, the NaOH content was from 50mg to 400mg under the same conditions, and the results are shown in Table 3. It can be seen that the average particle size of the microspheres is only about 2.1 μm when the NaOH content is 50 mg. The particle size of the microspheres gradually increases with the increase of the NaOH content, and when the NaOH content is increased to 400mg, the average particle size of the finally obtained microspheres reaches about 10.2 mu m.

4. The hydrolysis rate of the silicon source is also greatly affected by the water content, and with a slight increase in the amount of water, the hydrolysis rate of the silicon source is significantly increased. The process of silica particle formation is a complex competing process of hydrolysis, nucleation and particle growth, wherein hydrolysis is the key to the overall reaction, and thus the change in water content is also one of the main factors affecting the morphology of the final particles. When H is present₂When O/THF is 30/30, the resulting material has an irregular morphology (fig. 3C); with H₂The volume ratio of O/THF is gradually increased (40/30 and 50/30), the morphology of the material gradually tends to be regular spherical (FIGS. 3A, B and D), the average particle size is 4.2 μm and 4.9 μm respectively (Table 3), and the particle size distribution of the microspheres is relatively uniform; when the volume ratio was further increased to 60/30, the average particle size of the microspheres became smaller and the particle size distribution became broader (fig. 3E). The influence of the water content on the morphology of the material is mainly related to the hydrolytic polycondensation rate of the silicon source. When the water content is low, the relative content of NaOH becomes high, at the moment, the silicon source polycondensation rate is increased and is far greater than the hydrolysis rate, and the obtained material is thick and irregular; with the gradual increase of the water content, the hydrolytic polycondensation rate of the silicon source gradually tends to be balanced, so that the shape of the material gradually tends to be regular and uniform; when the water content is further increased, the hydrolysis rate of the silicon source becomes higher, and the nucleation rate is faster than the growth rate, so that new and old crystal nuclei grow together, resulting in uneven size of finally obtained particles.

5. The reaction temperature also has a great influence on the formation of the mesoporous hybrid silica material morphology. The changes in particle size of the microspheres at 50 deg.C, 60 deg.C, 70 deg.C, 80 deg.C and 90 deg.C, respectively, were investigated and are shown in Table 3. It can be seen that PMO (SLLTP-POSS) microspheres having an average particle size of about 5.9 μm were obtained at a reaction temperature of 50 ℃. The particle size of the microspheres tends to decrease gradually with increasing temperature. When the temperature reaches 90 ℃, the average grain diameter of the microspheres is reduced to about 1.3 μm.

TABLE 3 particle size distribution of PMO (SLLTP-POSS) microspheres obtained under different conditions

6. Physical parameters such as specific surface area and pore size of non-expanded PMO (SLLTP-POSS) and expanded PMO (SLLTP-POSS) microspheres are shown in Table 4. The results showed that the specific surface area, pore diameter and pore volume of the non-pore-enlarged PMO (SLLTP-POSS) were 517m, respectively²G, 3.7nm and 0.74cm³(ii) in terms of/g. The pore diameter of the reamed PMO (SLLTP-POSS) is 9.6nm, which is far larger than that of the non-reamed PMO (SLLTP-POSS), and the specific surface area is slightly increased. Thus, the reamed PMO (SLLTP-POSS) has a high surface area, large pore size and spherical morphology and is suitable as an HPLC packing material.

TABLE 4 specific surface area and pore Structure parameters of microspheres at different reaction stages

7. From an application point of view, the thermal stability of PMO (SLLTP-POSS) microspheres is an important aspect, and its thermogravimetric analysis curve in air is shown in fig. 4. As can be seen from FIG. 4, from 50 ℃ to 320 ℃, there is almost no loss of weight of the PMO (SLLTP-POSS) microspheres, which ensures a satisfactory thermal stability of the material. The temperature for starting pyrolysis is about 330 ℃, the decomposition and combustion of organic components in the hybrid microsphere skeleton are attributed, and finally the inorganic residue of the PMO (SLLTP-POSS) microsphere (mainly SiO crosslinked by silane)₂) 46 percent, and ensures the stronger mechanical strength of the microsphere. The higher thermal stability of PMO (SLLTP-POSS) microspheres is mainly due to the fact that POSS has a regular cage-like structure, and the inorganic silica framework structure of the POSS has obvious increase on the thermal stability and the mechanical strength of the microspheresHas strong effect.

8. The structural order of the organic-inorganic hybrid microspheres was evaluated by XRD. FIG. 5 shows the XRD pattern of PMO (SLLTP-POSS) microspheres. In the low-angle spectrum, the main peak observed at 2 θ ═ 0.74 ° can be attributed to the diffraction peak of the (100) plane, and in addition, the two small peaks observed at 2 θ ═ 1.63 ° and 2.02 ° are attributed to the diffraction peaks of the (110) and (200) planes, respectively, which is the structure of a typical hexagonal mesoporous material, indicating that PMO (SLLTP-POSS) microspheres have a highly ordered characteristic.

Example 3 chromatographic evaluation of PMO (SLLTP-POSS) hydrophilic microspheres as a hydrophilic stationary phase

The chromatographic analysis was performed on an agilent 1260 high performance liquid chromatograph (usa).

1. Preparation of PMO (SLLTP-POSS) hydrophilic chromatographic column

A PMO (SLLTP-POSS) hydrophilic chromatographic column is prepared by a homogenization method. Using isopropanol/trichloromethane (1: 3) (v/v) as homogenate, adding 4.0g of PMO (SLLTP-POSS) hydrophilic microspheres into the homogenate, performing ultrasonic treatment for 10min to uniformly disperse the microspheres, and pouring the mixture into a homogenate tank. Methanol was used as a displacement liquid, and the displacement liquid was packed in a stainless steel column tube (150 mm. times.4.6 mm) under a pressure of 370bar, to obtain a novel hydrophilic column.

2. Investigation of mechanical Strength of PMO (SLLTP-POSS) hydrophilic column

In the PALC mode, whether the mechanical strength of the microspheres meets the packing requirement of a chromatographic column is judged by examining the relationship between the flow rate and the pressure drop of the PMO (SLLTP-POSS) chromatographic column. The column pressure was measured with the mobile phase of 100% methanol and the column temperature at room temperature while changing the flow rate, i.e., 0.25mL/min, 0.5mL/min, 0.75mL/min, 1.0mL/min, 1.25mL/min, 1.5mL/min, 1.75 mL/min, 2.0mL/min, 2.5mL/min, 3.0mL/min, 3.5mL/min and 4.0 mL/min.

The mechanical strength of the chromatographic column is very important for HPLC and UPLC separations. Whether the column pressure is in direct proportion to the flow rate can be observed, and if the column pressure is in direct proportion to the flow rate, the column pressure has better mechanical strength. The PMO (SLLTP-POSS) packings prepared under reaction conditions Nos. 1 to 4 in Table 1 were labeled PMO (SLLTP-POSS) -1, PMO (SLLTP-POSS) -2, PMO (SLLTP-POSS) -3 and PMO (SLLTP-POSS) -4, respectively, and packed into a column. Column pressures were measured at different flow rates and a commercial, same-specification C18 column (5 μm,150 mm. times.4.6 mm) was selected for comparison. The results are shown in FIG. 6. As can be seen from FIG. 6, the molar ratios of POSS [ C2H4Si (OEt)3]8/SLLTPBS of 1.5:1, 1:1 and 0.75:1 exhibited good linear relationships between the flow rate and the column pressure for PMO (SLLTP-POSS) -1, PMO (SLLTP-POSS) -2 and PMO (SLLTP-POSS) -3. Under the condition that the flow rate reaches 4mL/min, the relation between the column pressure and the flow rate still does not deviate from linearity, which shows that the three hybrid fillers have good mechanical stability and the chromatographic column is well filled. However, at a molar ratio of 0.5:1, corresponding to PMO (SLLTP-POSS) -4, good linearity was exhibited at flow rates from 0.2 to 2.5 mL/min. However, when the flow rate exceeds 2.5mL/min, the column pressure rapidly increases, and the deviation from linearity is severe. It is probably because the POSS content is less, the rigidity of the material is poor, and partial microsphere particles collapse when the pressure of a chromatographic column is too high.

Thus, PMO (SLLTP-POSS) -1, PMO (SLLTP-POSS) -2 and PMO (SLLTP-POSS) -3 can be used as chromatographic stationary phases. However, PMO (SLLTP-POSS) -3 was selected as a subject of subsequent studies, considering that the higher the organic content, the stronger the separation ability of the column.

3. Investigation of acid and alkali resistance and stability of PMO (SLLTP-POSS) hydrophilic chromatographic column

And (4) investigating acid and alkali resistance of the chromatographic column. Maintaining the flow rate at 1.0mL/min, and adjusting the pH value to 1.0 by adopting ACN and trifluoroacetic acid aqueous solution with the mass fraction of 1% as an acid-proof test mobile phase under the condition that the column temperature is room temperature; the alkali-resistant test mobile phase adopts ACN/50 mmol/L triethylamine aqueous solution, and the pH value is adjusted to 11.0. Fumaric acid and thiourea were used as test probes, and the injection was performed every 8h, for 15 times, for a total of 120 h. The acid and base resistance stability of the column was judged by the remaining percentage of fumaric acid and thiourea retention, respectively, relative to the respective initial retention.

3.1 alkaline stability

For silica gel matrix fillers, the generally accepted failure mechanism at high pH is that the silica gel particles will dissolve under base catalysis. The base stability of the column was investigated by successive washings of the column under high pH conditions. FIGS. 7A and B show the retention factor and theory for thioureaThe percentage of plate number remaining relative to the initial value is plotted against the flush time. The retention factor and number of plates of thiourea on the column were 93.8% and 95.2% of the initial values, respectively, and remained above 90% within 120h, although there was a decrease, but not significant. Compared with C18-SiO₂The column, at the same time, showed a faster drop in retention factor and number of plates, 69.2% and 60.1% of the initial values, respectively, probably due to a severe drop in separation capacity caused by partial dissolution of the silica gel particles. Therefore, the PMO (SLLTP-POSS) stationary phase has good alkaline stability.

3.2 acid stability

Under acidic conditions, the degradation mechanism of the silica gel bonded stationary phase is the hydrolysis of the Si-O-Si type bonded phase under acid catalysis, resulting in a decrease in the retention capacity of the chromatographic column. To improve the stability of the packing under acidic conditions, a PMO (SLLTP-POSS) column was used to overcome this difficulty. The stability of this column was tested by continuous washing under acidic conditions. As shown in fig. 7C and D, the retention factor and the number of plates of fumaric acid were plotted against the percentage remaining from the initial value versus the rinsing time. The results show that after 120h of continuous rinsing, the commercial C18-SiO₂On the column, the retention factor of fumaric acid and the number of plates were reduced by 17% and 21%, respectively, compared to the initial values, indicating that the C18 bound phase was significantly lost during the flushing of the mobile phase at pH 1.0. On a PMO (SLLTP-POSS) chromatographic column, the retention factor and the number of the fumaric acid plates are respectively 94.2 percent and 97.7 percent of the initial values, and the reduction is less than 5 percent, which shows that the acidity stability of the newly prepared stationary phase is obviously improved. The above results demonstrate that this hybrid chromatography column has good acid resistance.

And (5) examining the stability in water. Under the conditions of mobile phase water/ACN (90: 10) (v/v), detection wavelength of 260nm and flow rate of 1.0mL/min, a mixture of VB2, VB6, adenine and caffeine (each substance concentration is 30mg/mL) is separated on a chromatographic column, samples are injected for 1 time every other day, the column is continuously flushed by using a mobile phase for three months, and the change of a fixed phase relative to the retention factor and the peak area of the compound is examined, so that the corresponding RSD is respectively calculated, and the stability of the filler under the condition of a long-time water-rich mobile phase is verified.

The results show that the retention factor RSD values of the four compounds are respectively 3.7%, 3.2%, 2.3% and 4.1%, the peak area RSD values are respectively 3.5%, 3.9%, 2.3% and 3.9%, and are all less than 5%, and the chromatographic column is proved to be very stable after being used for three months in the PALC mode.

5. Chromatographic column process repeatability investigation

20 batches of PMO (SLLTP-POSS) -3 packing (product prepared under reaction conditions No. 3 in Table 1) were synthesized as in example 1 and packed into a chromatographic column. 7 of these were randomly sampled and a mixture of four synthetic pigments, lemon yellow, sunset yellow, brilliant blue and new red, was isolated (each substance concentration was 30 mg/mL). The reproducibility of the synthesis process and the column packing technique of the column packing was evaluated using the Relative Standard Deviation (RSD) of five chromatographic parameters of retention time, peak area, peak width, peak asymmetry and retention factor in the case of a mobile phase of water/ACN 80:20(v/v), a flow rate of 1.0mL/min and a detection wavelength of 254 nm. As can be seen from Table 5, the chromatographic columns prepared from 7 batches of the packing have the retention time, the peak width and the RSD of the peak asymmetry of less than 7%, the peak area RSD is between 5% and 8%, and the retention factor RSD is between 6% and 9.2%, so that the separation capability and the column efficiency of 7 chromatographic columns are basically consistent, and the stability of the synthetic process and the column packing technology of the packing is better.

TABLE 57 comparison of RSD values of 5 chromatographic parameters for separation of 4 synthetic pigments by PMO (SLLTP-POSS) hydrophilic stationary phase batches

rt:retention time；pa:peak area；pw:peak width；paf:peak asymmetry factor；rf:retention factor.

6. Reproducibility test

Using VB₂And VB₆To test the probes, the reproducibility of the columns was evaluated by the RSD of the day and day retention factors and peak areas. In the flowing ofVB was added at a flow rate of 1.0mL/min and a detection wavelength of 260nm for water/ACN of 90:10(v/v)₂And VB₆The mixture of (2) (each substance concentration is 30mg/mL) was injected repeatedly 10 times a day for 6 consecutive days, and the daily and daytime RSD values were calculated, respectively. The results show that VB₂And VB₆The RSD of the retention factors in the day is respectively 2.1 percent and 2.7 percent, the RSD of the peak area is respectively 1.9 percent and 3.1 percent, the RSD of the retention factors in the day is respectively 2.8 percent and 3.5 percent, the RSD of the peak area is respectively 3.9 percent and 4.3 percent, and both are less than 5 percent, which indicates that the prepared chromatographic column has good reproducibility.

Example 4 separation of eight organic acids by PMO (SLLTP-POSS) hydrophilic column

Eight organic acids, namely oxalic acid, tartaric acid, quinic acid, malic acid, shikimic acid, ascorbic acid, acetic acid and maleic acid, are taken as research objects, and the separation capability of a PMO (SLLTP-POSS) hydrophilic stationary phase (PMO (SLLTP-POSS) -3) under the PALC condition is examined.

The mobile phase is composed of I: disodium hydrogen phosphate-phosphate buffer (0.01mol/L, pH 2.2) and ii: and (3) ACN. Gradient elution procedure: 0-2min, 95% I → 90% I; 2.1-7min, 90% I → 70% I, 7.1-13min, 70% I → 95% I, 13.1-15min, 95% I. At room temperature, the flow rate was 1.0mL/min, and the detection wavelength was 210 nm.

Preparation of PMO (SLLTP-POSS) -3 hydrophilic column preparation was carried out as under 1 in example 3. A C18 column (150 mm. times.4.6 mm, 5 μm) was used for comparison. Then, under the above mobile phase conditions, the two columns were washed continuously for 30 days, and the eight organic acids were again separated to evaluate the retention behavior.

When organic acids are separated, because of their high polarity, the ionization of the acids often occurs when the proportion of water in the mobile phase is high, thereby affecting the separation effect. It is common practice to select acidic conditions at low pH to reduce the ionization of the target compound, which can improve the peak shape and retention of the acidic compound. As shown in fig. 8A, under the condition of mobile phase pH 2.2, the mixture of eight organic acids on the new stationary phase obtained good separation within 10min, with higher separation degree. Compared to the C18 column, within 12min, oxalic acid, tartaric acid, quinic acid, acetic acid and maleic acid reached baseline separation, but the malic, shikimic and ascorbic acid chromatographic peaks partially overlapped and did not reach baseline separation (fig. 8B). Then, after the column was continuously flushed for 30 days, as shown in FIG. 8C, the PMO (SLLTP-POSS) column showed no significant change in the separation of organic acids. However, on the C18 column, the separation efficiency of the eight organic acids was significantly decreased, the chromatographic peaks of tartaric acid and quinic acid partially overlapped, and the chromatographic peaks of malic acid, shikimic acid and ascorbic acid were completely stacked together and could not be separated (fig. 8D). This is mainly because too low a pH value can seriously affect the lifetime of the C18 column. Therefore, the PMO (SLLTP-POSS) chromatographic column has better acid resistance, and has obvious advantages in separating organic acid.

Example 5 separation of monosaccharide, sugar alcohol and amino acid mixtures by PMO (SLLTP-POSS) hydrophilic chromatography column

A mixture of 10 monosaccharides, sugar alcohols and amino acids was selected as a test probe, including ribose, mannitol, sucrose, maltitol, Raynaud's sugar, melezitose, phenylalanine, methionine, glutamic acid and histidine, and their separation ability by PMO (SLLTP-POSS) hydrophilic stationary phase (PMO (SLLTP-POSS) -3) was studied. Preparation of PMO (SLLTP-POSS) -3 hydrophilic column preparation was carried out as under 1 in example 3.

Meanwhile, C18 column and HILIC column were selected as control, and the specifications were (150 mm. times.4.6 mm, 5 μm). Mobile phase, i: ammonium formate (200mM) and II: and (3) ACN. Optimal separation conditions for PMO (SLLTP-POSS) chromatography columns (gradient elution procedure): 0-2min, 98% I → 90% I; 2.1-6min, 90% I → 65% I, 6.1-11min, 65% I, 11.1-12min, 65% I → 98% I, 12.1-14min, 98% I; optimal separation conditions for a HILIC column: 0-1min, 5% of I; 1.1-10min, 25% I, 10.1-12min, 25% I → 5% I, 12.1-14min, 5% I; optimal separation conditions for C18 column: 0-2min, 90% I; 2.1-8min, 90% I → 75% I, 8.1-12min, 75% I, 12.1-13min, 75% I → 90% I, 13.1-15min, 90% I. The column temperature was room temperature, the flow rate was 1.0mL/min, the drift tube temperatures of the evaporative light scattering detectors were set to 55 ℃ respectively, and the flow rate of high purity nitrogen was 2.2L/min.

10 polar compounds of ribose, sucrose, reynolds sugar, melezitose, maltitol, mannitol, phenylalanine, methionine, glutamic acid and histidine are used as research objects, and PMO (SLLTP-POSS) chromatographic columns, HILIC chromatographic columns and C18 chromatographic columns are compared, so that the separation capacities of the three chromatographic columns on test compounds are respectively and optimally separated.

On a PMO (SLLTP-POSS) chromatographic column, the 10 polar compounds can be subjected to baseline separation within 18min, which is probably because the polysaccharide structure can provide a large number of hydrophilic action sites and ion exchange sites, the column efficiency of the chromatographic column can be greatly improved, and the retention and separation capacity of the polar compounds is strong. On the HILIC column, the chromatographic peaks of ribose and phenylalanine partially overlapped, and the remaining compounds substantially reached baseline separation. The theoretical plate number of the PMO (SLLTP-POSS) column is significantly higher than that of the HILIC column for the above 10 polar compounds. On the C18 chromatographic column, all peaks of 10 compounds appear within 10min, but chromatographic peaks of five compounds of histidine, maltitol, sucrose, mannitol and methionine are stacked together and cannot be separated; at the same time, the chromatographic peaks for melezitose and reynolds sugar did not achieve baseline separation, indicating that the commercial C18 column had poor retention and separation of the test probe. In addition, in consideration of the influence of green chromatography and ACN on the environment, PALC can separate a mixture of the above 10 sugars, sugar alcohols and amino acids instead of the HILIC method.

Claims

A preparation method of PMO (SLLTP-POSS) hydrophilic microspheres comprises the following steps:

1) synthesis of SLLTP-bridged silanes

a. Dissolving lily polysaccharide in ionic liquid, then dropwise adding chlorosulfonic acid diluted by anhydrous pyridine, stirring at constant temperature of 30-70 ℃ for 0.5-3h, and adjusting to neutrality by using an alkali solution to obtain sulfated and modified lily polysaccharide, which is marked as SLLTP;

b. under the protection of nitrogen, stirring and reacting an N, N-dimethylformamide solution of SLLTP and a THF solution of chloromethyl trimethoxy silane at constant temperature of 40-80 ℃ for 3-10h to obtain SLLTP bridged silane which is marked as SLLTPBS;

2)in the presence of NaOH, using C18TACL as template agent to make POSS [ C ]₂H₄Si(OEt)₃]₈SLLTPBS, C18TACL in a mixed solvent of water and THF; filtering after the reaction is finished, washing the product with ethanol, deionized water and methanol in sequence, and drying in vacuum; then, a solvent extraction method is adopted to remove the template agent, and the PMO (SLLTP-POSS) hydrophilic microspheres are obtained.
2. The method of claim 1, wherein:

in the step 1), the ionic liquid is imidazole ionic liquid, and is specifically selected from any one of the following: 1-butyl-3-methylimidazolium chloride salt, 1-butylimidazolium chloride salt, 1, 3-dimethylimidazolium chloride salt;

in the step 1), the ratio of the lily polysaccharide to the ionic liquid is 500 mg: 10mL-500 mg: 50 mL;

in the step 1), the molar usage amount of the chlorosulfonic acid is 40-80% of the molar number of hydroxyl groups in the lily polysaccharide;

in the step 1), the volume ratio of the chlorosulfonic acid to the anhydrous pyridine is 1: 10-1: 20;

in the step 1), the alkali is potassium hydroxide or ammonia water;

in the step 1), after the solution is adjusted to be neutral by sodium hydroxide solution, the method further comprises the following steps: loading the system adjusted to neutral into dialysis bag with cut-off of 10KDa, dialyzing in ultrapure water for 24-72h, concentrating, precipitating with ethanol, and freeze-drying at-20 deg.C to obtain SLLTP.
3. The method according to claim 1 or 2, characterized in that:

in the step 1) b, the molar ratio of SLLTP to chloromethyltrimethoxysilane is 1:2-1: 4;

in the step 1) b, the volume ratio of the chloromethyltrimethoxysilane to the THF is 1:3-1: 6;

in the step 1) b, the N, N-dimethylformamide is anhydrous N, N-dimethylformamide; the THF is anhydrous THF;

in the step 1), b, the method further comprises the following steps after the reaction is finished: putting the reaction solution into a dialysis bag with cut-off amount of 10KDa, dialyzing in deionized water for 48h, concentrating, precipitating with ethanol, and freeze-drying at-20 deg.C to obtain SLLTPBS.
4. The method according to any one of claims 1-3, wherein:

in the step 2), the POSS [ C ]₂H₄Si(OEt)₃]₈And SLLTPBS at a molar ratio of 0.5:1 to 1.5:1, preferably at a molar ratio of 0.75:1 to 1.5: 1;

in the step 2), the mass ratio of the C18TACl to the SLLTPBS is 1: 17;

in the step 2), the mass ratio of NaOH to SLLTPBS is 1: 37.5;

in the step 2), the volume ratio of water to THF in the mixed solvent of water and THF is 30: 30-60: 30, preferably in a volume ratio of 40: 30-50: 30, of a nitrogen-containing gas;

in the step 2), the reaction temperature of the reaction is 50-90 ℃, and preferably 60-70 ℃;

in the step 2), the reaction time is 10-20 h;

in the step 2), the specific method for removing the template agent by adopting a solvent extraction method comprises the following steps: adding the dried solid into 150mL of mixed solution of concentrated hydrochloric acid/deionized water/ethanol per gram, heating and refluxing for 8h to extract the template agent, repeating twice, filtering, washing and drying; wherein the volume ratio of the concentrated hydrochloric acid to the deionized water to the ethanol in the mixed solution is 5: 45: 50; the mass fraction of the concentrated hydrochloric acid is 36-38%.
5. The method according to any one of claims 1-4, wherein: the method also comprises a step of reaming the obtained PMO (SLLTP-POSS) hydrophilic microspheres, and the specific method comprises the following steps: reacting the PMO (SLLTP-POSS) hydrophilic microspheres, the DMDA and the DDA in ultrapure water, filtering after the reaction is finished, washing with ultrapure water and methanol in sequence, and drying at 60 ℃ in vacuum to obtain the PMO (SLLTP-POSS) hydrophilic microspheres after pore expansion;

the reaming method further comprises the following steps: and (3) removing the DMDA and the DDA from the pore-enlarged PMO (SLLTP-POSS) hydrophilic microspheres by adopting a solvent extraction method.
6. PMO (SLLTP-POSS) hydrophilic microspheres prepared by the method of any one of claims 1 to 5.
7. Use of PMO (SLLTP-POSS) hydrophilic microspheres as claimed in claim 6 for the preparation of hydrophilic chromatographic stationary phase.
8. A hydrophilic chromatographic column, wherein the stationary phase of the hydrophilic chromatographic column is PMO (SLLTP-POSS) hydrophilic microspheres as described in claim 6.
9. Use of a hydrophilic chromatography column according to claim 8 for separating and/or detecting polar substances.
10. Use according to claim 9, characterized in that: the polar substance is at least one of organic acids, sugar alcohol, amino acid and sweetener;

the organic acid is at least one selected from oxalic acid, tartaric acid, quinic acid, malic acid, shikimic acid, ascorbic acid, acetic acid and maleic acid;

the biogenic amines are specifically selected from at least one of tryptamine, beta-phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine and spermine.

The sweetener is selected from saccharin sodium, sucralose, sodium cyclamate, aspartame, acesulfame potassium, alitame, neotame, glycyrrhizic acid, glycyrrhetinic acid, stevioside and steviolbioside.