Mixed mode chromatographic stationary phase based on carbon quantum dot bonded silica gel, preparation method and application

Abstract

The invention discloses a mixed mode chromatographic stationary phase based on carbon quantum dot bonded silica gel, a preparation method and application, the mixed mode chromatographic stationary phase is chloropropyl modified silica gel bonded with carbon quantum dots, and the mass ratio of the carbon quantum dots to the chloropropyl modified silica gel is (0.9-1.1): (3.9-4.1); the preparation method comprises the following steps: modifying silica gel by using 3-chloropropyltriethoxysilane to obtain chloropropyl modified silica gel, synthesizing functionalized carbon quantum dots with amino, carboxyl and long-chain alkyl by using mercaptopropionic acid and 1, 12-diaminododecane as raw materials, and bonding the carbon quantum dots and the chloropropyl modified silica gel together to prepare the mixed-mode chromatographic stationary phase. The chromatographic column stationary phase has two separation modes of RPLC and HILIC, can effectively separate hydrophobic and hydrophilic compounds, and is simple to prepare, good in separation performance and high in analysis speed.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of high-performance liquid chromatography separation and analysis, and in particular to a mixed-mode chromatographic stationary phase based on carbon quantum dot bonded silica gel, a preparation method and application. BACKGROUND

[0002] High-performance liquid chromatography has the advantages of high accuracy, high sensitivity and good reproducibility, is a commonly used method for compound separation and analysis, and is widely applied to the fields of environment, food and biomedicine, wherein a chromatographic column stationary phase is the core for guaranteeing good separation performance. A single-mode chromatographic column has limited separation effect and relatively narrow application range, and can only realize high-efficiency separation of a specific type of compound. With the increasing complexity of modern analysis requirements and the increasing complexity and diversity of sample components, a single-mode chromatographic column has been difficult to meet the separation requirements of complex analytes, and therefore mixed-mode chromatographic stationary phases (MMC) having two or more separation mechanisms have attracted wide attention. The MMC has higher sample loading capacity and can realize effective separation of different analytes by adjusting chromatographic conditions, thereby meeting the separation requirements of complex samples. Currently developed MMC stationary phases mainly include reversed-phase liquid chromatography / hydrophilic interaction liquid chromatography (RPLC / HILIC), reversed-phase liquid chromatography / ionic exchange chromatography (RPLC / IEC), hydrophilic interaction liquid chromatography / ionic exchange chromatography (HILIC / IEC), reversed-phase liquid chromatography / hydrophilic interaction liquid chromatography / ionic exchange chromatography (RPLC / HILIC / IEC) and the like. Therefore, it is crucial to further find more promising stationary phase ligands and prepare MMC stationary phases with stronger separation performance and wider application range.

[0003] As a new type of carbon nanomaterial, carbon quantum dots (CQDs) have the advantages of nanoscale size, high specific surface area, good stability and excellent biocompatibility, which makes them have broad application prospects and great development and application potential in the fields of biological imaging, cancer treatment and environment. In view of the many excellent properties, the CQDs are more and more used in the preparation of chromatographic column stationary phases, and researches have shown that the CQDs have excellent separation performance. Therefore, the present research develops a new type of mixed-mode chromatographic column stationary phase based on carbon quantum dots, and provides a new solution for the separation and analysis of complex samples.

[0004] CN 117504838 A discloses a silica gel mixed-mode chromatographic stationary phase and a preparation method and application thereof. The stationary phase is a mercaptopropyl-modified silica gel bonded with carbon quantum dots, and the stationary phase has the problem of pH instability. A mixed-mode chromatographic stationary phase based on carbon quantum dot bonded silica gel, a preparation method and application thereof are proposed. SUMMARY

[0005] In order to make up for the defects of single mode chromatographic column in separating complex samples, the application provides synthesis and application of a chromatographic stationary phase of carbon quantum dot bonded silica gel with multiple functional groups on the surface.

[0006] According to a first aspect of the application, a mixed mode chromatographic stationary phase based on carbon quantum dot bonded silica gel is provided, the mixed mode chromatographic stationary phase is chloropropyl modified silica gel bonded with carbon quantum dots, wherein the mass ratio of carbon quantum dots to chloropropyl modified silica gel is 0.9-1.1:3.9-4.1.

[0007] Further, the mass ratio of chloropropyl silane to silica gel in the chloropropyl modified silica gel is 0.9-1.1:2.3-2.4; The chloropropyl silane is 3-chloropropyl triethoxysilane; The silica gel is a commercial grade chromatographic column packing.

[0008] Further, the mixed mode chromatographic stationary phase has amino, carboxyl and long chain alkyl groups, has hydrophobic and hydrophilic separation performance, and has good stability at pH 3-8.

[0009] According to a second aspect of the application, a preparation method of a mixed mode chromatographic stationary phase based on carbon quantum dot bonded silica gel is provided, the preparation method comprises the following steps: Step 1, preparation of carbon quantum dots: mix and disperse mercaptopropionic acid and 1,12-diaminododecane in ethanol, heat the mixed solution at 200-220 DEG C for 6-8 h, centrifuge the obtained product, discard the precipitate, filter and freeze-dry the supernatant to obtain solid carbon quantum dots; Step 2, preparation of chloropropyl modified silica gel: react silica gel with concentrated hydrochloric acid at 100-120 DEG C for 8-10 h, wash the product with deionized water, suction filter and dry to obtain activated silica gel; disperse the activated silica gel in toluene, add 3-chloropropyl triethoxysilane, stir at 100-120 DEG C for 24-28 h, filter the product, wash with toluene, ethanol and methanol, and dry to obtain chloropropyl modified silica gel.

[0010] Step 3, preparation of mixed mode chromatographic stationary phase: disperse the chloropropyl modified silica gel obtained in step 2 in methanol, add the carbon quantum dots prepared in step 1 and triethylamine, stir at 80-100 DEG C under nitrogen protection for 24-28 h, wash the product with methanol and water for multiple times, filter and dry to obtain the mixed mode chromatographic stationary phase.

[0011] Further, the container for the heating reaction in step 1 is a 100 ml polytetrafluoroethylene autoclave, and the heating reaction is carried out in an oven. The mass mixing ratio of the mercaptopropionic acid to 1,12-diaminododecane in step 1 is 1.0~1.1:2.0~2.1. The concentration of the mercaptopropionic acid in the mixed solution in step 1 is 98%~100%. The centrifugal speed of the product in step 1 is 4000~5000 rpm, and the centrifugal time is 10~15 min. The filter membrane with a pore size of 0.22 μm is used for filtering the supernatant in step 1.

[0012] Further, the silica gel in step 2 is selected from at least one of a pore size of 100 Å and a particle size of 5 μm, a pore size of 120 Å and a particle size of 5 μm. The mass ratio of the silica gel to concentrated hydrochloric acid in step 2 is 0.9~1.1:14~15. The mass ratio of the activated silica gel, toluene and 3-chloropropyltriethoxysilane in the reaction in step 2 is 3.4~3.6:86~88:1.4~1.6.

[0013] Further, the reaction of the chloropropyl-modified silica gel and the carbon quantum dots in step 3 is carried out under nitrogen protection; and the product is filtered by using a G5 sand core funnel. The mass ratio of the chloropropyl-modified silica gel, the carbon quantum dots and triethylamine in step 3 is 3.8~4.2:0.8~1.2:0.7~0.8.

[0014] According to the third aspect of the present application, there is provided an application of a mixed mode chromatography stationary phase based on carbon quantum dot bonded silica gel in compound separation, wherein the mixed mode chromatography stationary phase is used as a filler to separate alkylbenzene compounds, polycyclic aromatic hydrocarbon compounds, aniline compounds, nucleoside base compounds, thiourea compounds and sulfonamide compounds.

[0015] Further, the alkylbenzene compounds are toluene, ethylbenzene, propylbenzene, butylbenzene and pentylbenzene; the polycyclic aromatic hydrocarbon compounds are naphthalene, anthracene, acenaphthene, p-triphenyl and benzanthracene; the aniline compounds are o-phenylenediamine, aniline, 2,4-dinitroaniline, 2,4-dichloroaniline, 2,6-dinitroaniline and diphenylamine; the nucleoside base compounds are uridine, cytidine, adenosine, adenine and cytosine; the thiourea compounds are thiourea, N-ethylthiourea and N,N-diethylthiourea; and the sulfonamide compounds are sulfacetamide, sulfamerazine, sulfathiazole, sulfisomidine and sulfadimoxine.

[0016] Compared with the prior art, the present application has the following beneficial effects: (1) The application provides a novel mixed mode chromatography stationary phase based on carbon quantum dots, wherein silica gel is modified by 3-chloropropyltriethoxysilane, carbon quantum dots are synthesized by mercaptopropionic acid and 1,12-diaminododecane, and the carbon quantum dots are bonded to the chloropropyl-modified silica gel.

[0017] (2) The mixed mode chromatography stationary phase based on carbon quantum dots has amino groups, carboxyl groups and long-chain alkyl groups, has two separation modes of RPLC and HILIC, and can effectively separate hydrophobic and hydrophilic compounds.

[0018] (3) The mixed mode chromatography stationary phase provided by the application can maintain stability under extreme pH, and has the advantages of simple preparation process, good separation performance and fast analysis speed. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 SEM characterization graphs of the chromatographic column stationary phase filler Sil-CQDs and silica gel obtained in Embodiment 1 of the application (wherein a. silica gel, b. Sil-CQDs); Figure 2 FT-IR characterization graphs of the chromatographic column stationary phase filler Sil-CQDs, Sil-Cl and silica gel obtained in Embodiment 1 of the application.

[0020] Figure 3 BET characterization graphs of the chromatographic column stationary phase filler Sil-CQDs, Sil-Cl and silica gel obtained in Embodiment 1 (wherein a. silica gel, b. Sil-Cl, c. Sil-CQDs); Figure 4 XPS characterization graphs of the chromatographic column stationary phase filler Sil-CQDs obtained in Embodiment 1 of the application (a. total spectrum, b. C1s spectrum, c. N1s spectrum, d. O1s spectrum); Figure 5 TGA characterization graphs of the chromatographic column stationary phase filler Sil-CQDs and silica gel obtained in Embodiment 1 of the application (wherein a. silica gel, b. Sil-CQDs); Figure 6 Chromatograms obtained by separating alkylbenzene compounds, polycyclic aromatic hydrocarbon compounds and aniline compounds in the chromatographic column in Embodiment 2 of the application (wherein a. alkylbenzene compounds, b. polycyclic aromatic hydrocarbon compounds, c. aniline compounds); Figure 7The line graph of the influence of different methanol, acetonitrile content on alkylbenzene compounds, polycyclic aromatic hydrocarbon compounds and aniline compounds in Example 3 of the present application (wherein a. the change of log k of alkylbenzene compounds separated by different methanol content, b. the change of log k of polycyclic aromatic hydrocarbon compounds separated by different acetonitrile content, c. the change of log k of aniline compounds separated by different acetonitrile content); Figure 8 The chromatogram obtained by separating nucleoside base compounds, thiourea compounds and sulfonamide compounds by the chromatographic column described in Example 4 of the present application (wherein a. nucleoside base compounds, b. thiourea compounds, c. sulfonamide compounds); Figure 9 The line graph of the influence of different acetonitrile content on nucleoside base compounds in Example 5 of the present application; Figure 10 The result graph of the "U" type curve detection of sulfonamide compounds in Example 5 of the present application; Figure 11 The chromatogram of the influence of buffer salt on the separation of sulfonamide compounds by the chromatographic column in Example 6 of the present application; Figure 12 The chromatogram obtained by continuously injecting 10 times of alkylbenzene compounds, polycyclic aromatic hydrocarbon compounds, nucleoside base compounds and thiourea compounds in Example 8 of the present application (a. alkylbenzene compounds, b. polycyclic aromatic hydrocarbon compounds, c. nucleoside base compounds, d. thiourea compounds); Figure 13 The line graph obtained by eluting three sulfonamide compounds at pH 3 and 8 in Example 8 of the present application; Figure 14 The chromatogram obtained by comparing the separation performance of the Sil-CQDs fixed phase of the chromatographic column prepared in Example 2 with Sil-Cl described in Comparative Example 1 (wherein a. alkylbenzene compounds, b. polycyclic aromatic hydrocarbon compounds, c. aniline compounds, d. nucleoside base compounds, e. sulfonamide compounds). DETAILED DESCRIPTION

[0021] The present application is further described below in conjunction with specific examples, but in no way limits the present application.

[0022] The test methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0023] A preparation method of a mixed mode chromatographic fixed phase based on carbon quantum dot bonded silica gel comprises the following steps: Step 1, preparation of carbon quantum dots: thiolpropanoic acid and 1,12-diaminododecane are mixed and dispersed in ethanol, the mixed solution is heated at 200-220℃ for 6-8h, the obtained product is centrifuged, the precipitate is discarded, the supernatant is filtered and freeze-dried to obtain carbon quantum dots; Step 2, preparation of chloropropyl-modified silica gel: silica gel is reacted with concentrated hydrochloric acid at 100-120℃ for 8-10h, the product is washed with deionized water, suction filtered and dried to obtain activated silica gel; the activated silica gel is dispersed in toluene, 3-chloropropyltriethoxysilane is added, and the mixture is stirred at 100-120℃ for 24-28h, the product is filtered and washed with toluene, ethanol and methanol, and dried to obtain chloropropyl-modified silica gel.

[0024] Step 3, preparation of mixed-mode chromatographic stationary phase: the chloropropyl-modified silica gel obtained in step 2 is dispersed in methanol, the carbon quantum dots prepared in step 1 and triethylamine are added, and the mixture is stirred at 80-100℃ for 24-28h under nitrogen protection, the product is washed with methanol and water for several times, filtered and dried to obtain a mixed-mode chromatographic stationary phase.

[0025] In the above preparation method, the container for heating the mixed solution in step 1 at 200-220℃ is a 100ml polytetrafluoroethylene autoclave, and the reaction is carried out in an oven.

[0026] In the above preparation method, the mixing ratio of thiolpropanoic acid to 1,12-diaminododecane in step 1 is 1.0-1.1:2.0-2.1.

[0027] In the above preparation method, the concentration of thiolpropanoic acid in the mixed solution in step 1 is 98-100%.

[0028] In the above preparation method, the centrifugation speed of the product in step 1 is 4000-5000rpm, and the centrifugation time is 10-15min.

[0029] In the above preparation method, the filter membrane used for filtering the supernatant in step 1 is 0.22μm.

[0030] In the above preparation method, the silica gel used in step 2 is selected from at least one of silica gel with a pore size of 100Å and a particle size of 5μm, and silica gel with a pore size of 120Å and a particle size of 5μm.

[0031] In the above preparation method, the mass ratio of silica gel to concentrated hydrochloric acid in step 2 is 0.9-1.1:14-15.

[0032] In the above preparation method, the mass ratio of activated silica gel, toluene and 3-chloropropyltriethoxysilane used in the reaction in step 2 is 3.4-3.6:86-88:1.4-1.6.

[0033] In the preparation method, the reaction between the chloropropyl-modified silica gel and the carbon quantum dots in step 3 is carried out under nitrogen protection; the product is filtered by using a G5 sand core funnel; and the silica gel is a commercial chromatographic column filler.

[0034] In the preparation method, the carbon quantum dots in step 3 are concentrated by vacuum distillation, 20 ml of water is added, and solid carbon quantum dots are obtained by freeze-drying.

[0035] In the preparation method, the mass ratio of the chloropropyl-modified silica gel, the carbon quantum dots and triethylamine in step 3 is 3.8-4.2:0.8-1.2:0.7-0.8.

[0036] In the preparation method, the organic solvent is a chromatographic grade solvent, and the water is deionized water.

[0037] The application further provides a mixed mode chromatographic column, which is filled with the mixed mode chromatographic stationary phase bonded with the carbon quantum dots.

[0038] The application further provides the separation and detection of hydrophobic and hydrophilic compounds by using the chromatographic column filled with the mixed mode chromatographic stationary phase.

[0039] The application further provides a separation method of hydrophobic alkylbenzene compounds by using the chromatographic column filled with the mixed mode chromatographic stationary phase, and the chromatographic conditions are as follows: (A) methanol (B) water as mobile phase for isocratic elution (elution program: 0-15 min, 53% A), a flow rate of 0.8 ml / min, a column temperature of 25 DEG C, and a DAD detector wavelength of 210 nm.

[0040] The application further provides a separation method of hydrophobic polycyclic aromatic hydrocarbon compounds by using the chromatographic column filled with the mixed mode chromatographic stationary phase, and the chromatographic conditions are as follows: (A) acetonitrile (B) water as mobile phase for isocratic elution (elution program: 0-15 min, 52% A), a flow rate of 0.8 ml / min, a column temperature of 25 DEG C, and a DAD detector wavelength of 210 nm.

[0041] The application further provides a separation method of hydrophobic aniline compounds by using the chromatographic column filled with the mixed mode chromatographic stationary phase, and the chromatographic conditions are as follows: (A) acetonitrile (B) water as mobile phase for isocratic elution (elution program: 0-15 min, 25% A), a flow rate of 0.8 ml / min, a column temperature of 25 DEG C, and a DAD detector wavelength of 254 nm.

[0042] The application further provides a separation method of hydrophilic nucleoside base compounds by using a chromatographic column packed with a mixed mode chromatographic stationary phase, and the chromatographic conditions are isocratic elution with (A) acetonitrile and (B) water as mobile phases (the elution program is 0-15 min, 90% A), a flow rate of 0.8 ml / min, a column temperature of 25 DEG C, and a DAD detector wavelength of 254 nm.

[0043] The application further provides a separation method of sulfonamide compounds by using a chromatographic column packed with a mixed mode chromatographic stationary phase, and the chromatographic conditions are isocratic elution with (A) acetonitrile and (B) ammonium acetate (20 mM) as mobile phases (the elution program is 0-30 min, 7% A), a flow rate of 0.8 ml / min, a column temperature of 25 DEG C, and a DAD detector wavelength of 254 nm.

[0044] The application further provides a separation method of hydrophilic thiourea compounds by using a chromatographic column packed with a mixed mode chromatographic stationary phase, and the chromatographic conditions are isocratic elution with (A) acetonitrile and (B) water as mobile phases (the elution program is 0-15 min, 97% A), a flow rate of 0.8 ml / min, a column temperature of 25 DEG C, and a DAD detector wavelength of 254 nm.

[0045] In the embodiments of the application, the alkylbenzene compounds are toluene, ethylbenzene, propylbenzene, butylbenzene and pentylbenzene; the polycyclic aromatic hydrocarbon compounds are naphthalene, anthracene, acenaphthene, p-terphenyl and benzanthracene; the aniline compounds are o-phenylenediamine, aniline, 2,4-dinitroaniline, 2,4-dichloroaniline, 2,6-dinitroaniline and diphenylamine; the nucleoside base compounds are uridine, cytidine, adenosine, adenine and cytosine; the sulfonamide compounds are sulfacetamide, sulfamethazine, sulfathiazole, sulfisoxazole and sulfadimoxine; and the thiourea compounds are thiourea, N-ethylthiourea and N,N-diethylthiourea.

[0046] In the embodiments of the present application, the organic solvents used in the experiments are all chromatographic grade, purchased from Tianjin Concord Chemical Reagent Factory; toluene is purchased from Shandong Yuwang Industry Co., Ltd.; ethylbenzene, propylbenzene, butylbenzene, amylbenzene, naphthalene, anthracene, acenaphthene, p-triphenyl, benzanthracene, uridine, cytidine, adenosine, adenine, cytosine, 1-vinylimidazole are purchased from Shanghai Maikelin Biochemical Technology Co., Ltd.; sulfacetamide, sulfamerazine, sulfathiazole, sulfadimethoxine, sulfadimethoxine are purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; o-phenylenediamine, aniline, 2,4-dinitroaniline, 2,4-dichloroaniline, 2,6-dinitroaniline, diphenylamine are purchased from Shanghai Yuan Ye Biological Technology Co., Ltd.; thiourea, N-ethylthiourea, N,N-diethylthiourea, mercaptopropionic acid, 1,12-diaminododecane, 3-chloropropyltriethoxysilane, N,N-dimethylformamide are purchased from Shanghai Adamas Reagent Co., Ltd.; silica gel is purchased from Japan Osaka Soda Co., Ltd.; deionized water is purchased from Hangzhou Wahaha Group Co., Ltd.

[0047] In the present application, the concentration of all analyte standard stock solutions is 1 mg / ml, and the solutions are stored at 4℃.

[0048] Example 1 Step 1, preparation of carbon quantum dots: 1.06 g of mercaptopropionic acid and 2.00 g of 1,12-diaminododecane are mixed in a mass ratio of 1:1.9 and dispersed in 30 ml of ethanol, and then the mixture is transferred to a polytetrafluoroethylene autoclave, heated at 200℃ for 6 h, centrifuged, and the supernatant is filtered to obtain carbon quantum dots. The obtained carbon quantum dots are concentrated and added to 20 ml of water, and then freeze-dried to obtain carbon quantum dots.

[0049] Step 2, preparation of chloropropyl-modified silica gel: 4 g of silica gel (SEM characterization diagram shown in Figure 1 a) is dispersed in 50 ml of concentrated hydrochloric acid, refluxed at 100℃ for 8 h, and then the product is washed, filtered and dried to obtain activated silica gel; 3.5 g of activated silica gel is suspended in 100 ml of toluene, and then 1.5 ml of 3-chloropropyltriethoxysilane is added, and the mixture is stirred at 110℃ for 24 h under nitrogen protection. The obtained product is filtered, washed with toluene, ethanol and methanol, and dried to obtain chloropropyl-modified silica gel (i.e., Sil-Cl).

[0050] Step 3, preparation of mixed-mode chromatographic stationary phase: 4 g of chloropropyl-modified silica gel is dispersed in 75 ml of methanol, and then 1 g of carbon quantum dots and 1 ml of triethylamine are added, and the mixture is stirred at 80℃ for 24 h under nitrogen protection. The obtained product is filtered, washed with methanol and water, and dried to obtain a mixed-mode chromatographic stationary phase (Sil-CQDs). The SEM characterization of the Sil-CQDs is shown in Figure 1b, which shows that the bare silica gel surface is relatively smooth, while the silica gel surface bonded with CQDs is rough, and granular clusters appear, indicating that carbon quantum dots have been bonded to the silica gel surface; the XPS characterization of Sil-CQDs is shown in Figure 4 As shown in the figure, the total spectrum shows that the material contains elements such as O, N, C, Si, S, etc. According to the C1s spectrum, the C peak mainly has C-C, C-S, C-O, O=C-O groups, C-S and O=C-O groups are derived from mercaptopropionic acid in CQDs; according to the N1s spectrum, -NR2, -NH2 belong to 1,12-diaminododecane in CQDs; according to the O1s spectrum, C-OH, C=O, C-O groups in the O peak are mainly derived from the carboxyl group in mercaptopropionic acid, indicating that the chromatographic column stationary phase Sil-CQDs has been successfully prepared.

[0051] The FT-IR characterization of the silica gel, Sil-Cl, Sil-CQDs is shown in Figure 2 As shown in the figure, for Sil-CQDs, 3422.69 cm-1 belongs to the stretching vibration of O-H bond, 1670 cm-1 belongs to the stretching vibration of C=O bond, which belongs to mercaptopropionic acid in CQDs; 1571.86 cm-1 corresponds to the deformation vibration of N-H bond, 2925.08 and 2850.66 cm-1 respectively belong to the asymmetric and symmetric stretching vibration of C-H bond, which belongs to 1,12-diaminododecane; 1104.78 cm-1 belongs to the stretching vibration of Si-O bond. Based on the above analysis, it is shown that the chromatographic stationary phase Sil-CQDs with amino, carboxyl and long chain alkyl groups has been successfully prepared.

[0052] The BET characterization of the silica gel, Sil-Cl, Sil-CQDs is shown in Figure 3 As shown in the figure, compared with the silica gel, the pore volume and specific surface area of Sil-Cl and Sil-CQDs are significantly reduced, indicating that the carbon quantum dots block the pores of the silica gel, indicating that the carbon quantum dots have been successfully bonded to the silica gel.

[0053] The elemental analysis (EA) characterization results of the silica gel, Sil-Cl, Sil-CQDs are shown in Table 1, which shows that compared with the silica gel, the introduction of 3-chloropropyltrimethoxysilane significantly increases the content of C and H elements in Sil-Cl. After modifying the carbon quantum dots with amino, carboxyl and long chain alkyl groups, the content of C, H and N elements in Sil-CQDs is significantly improved, indicating that the carbon quantum dots have been successfully bonded to the silica gel.

[0054] The thermogravimetric analysis (TGA) characterization results of the silica gel, Sil-CQDs are shown in Figure 5As shown in the figure, the results show that the weight damage of Sil-CQDs is very low below 250℃ compared with silica gel, indicating that the thermal stability of Sil-CQDs is good, and the weight of Sil-CQDs decreases when the temperature is between 300℃ and 400℃, indicating that the carbon quantum dots have been successfully bonded on the silica gel.

[0055] Table 1 is the elemental analysis results of silica gel, Sil-Cl and Sil-CQDs in Example 1 of the present application

[0056] Example 2 The hydrophobic performance of the mixed mode chromatographic stationary phase (Sil-CQDs) prepared in Example 1 was tested, that is, 3g of the mixed mode chromatographic stationary phase prepared in Example 1 was dispersed in 30ml of tetrahydrofuran-pyrrolidone (volume ratio of 9:1), methanol was used as the propelling solvent, the slurry was loaded into a stainless steel column under a pressure of 40Mpa, the filter plate at the rear end was blocked to fill the packing and uniformly deposit it in the column tube, and finally the front end filter plate was assembled to obtain the chromatographic column Sil-CQDs.

[0057] In the reversed phase chromatographic retention mode, three types of hydrophobic compounds, alkylbenzenes, polycyclic aromatic hydrocarbons and aniline compounds, were separated by the chromatographic column Sil-CQDs to investigate the hydrophobic performance of the stationary phase Sil-CQDs, wherein the five alkylbenzene compounds were toluene, ethylbenzene, propylbenzene, butylbenzene and amylbenzene, the five polycyclic aromatic hydrocarbon compounds were naphthalene, anthracene, acenaphthene, p-triphenyl and benzanthracene, and the six aniline compounds were o-phenylenediamine, aniline, 2,4-dinitroaniline, 2,4-dichloroaniline, 2,6-dinitroaniline and diphenylamine.

[0058] The five alkylbenzene compounds, polycyclic aromatic hydrocarbon compounds and six aniline compounds were prepared into standard stock solutions with a concentration of 1mg / ml and stored at 4℃.

[0059] The chromatographic conditions for the alkylbenzene compounds were as follows: the mobile phase was (A) methanol 53% (B) water 47%, the flow rate was 0.8ml / min, the column temperature was 25℃, and the wavelength was 210nm.

[0060] The chromatographic conditions for the polycyclic aromatic hydrocarbon compounds were as follows: the mobile phase was (A) acetonitrile 52% (B) water 48%, the flow rate was 0.8ml / min, the column temperature was 25℃, and the wavelength was 210nm.

[0061] The chromatographic conditions for the aniline compounds were as follows: the mobile phase was (A) acetonitrile 25% (B) water 75%, the flow rate was 0.8ml / min, the column temperature was 25℃, and the wavelength was 254nm.

[0062] The chromatographic test results are as follows: Figure 6As shown, five alkylbenzenes, five polycyclic aromatic hydrocarbons and six anilines were effectively separated within 15 min.

[0063] Example 3 In the reversed-phase chromatographic retention mode, the Sil-CQDs mixed-mode column prepared in Example 2 was used to investigate the effect of methanol and acetonitrile content in the organic phase on the separation of alkylbenzenes, polycyclic aromatic hydrocarbons and anilines, which were the same as those in Example 2. The chromatographic conditions were as follows: the content of methanol in the mobile phase was 30%, 40%, 50%, 60% and 70%, the flow rate was 0.8 ml / min, and the column temperature was 25°C; the content of acetonitrile in the mobile phase was 30%, 40%, 50%, 60% and 70%, the flow rate was 0.8 ml / min, and the column temperature was 25°C; the content of acetonitrile in the mobile phase was 15%, 20%, 25%, 30%, 35% and 40%, the flow rate was 0.8 ml / min, and the column temperature was 25°C. The detection results are shown in Table 2. Figure 7 As shown, the log k of alkylbenzenes, polycyclic aromatic hydrocarbons and anilines gradually decreased as the content of methanol increased from 30% (v / v) to 70% (v / v) and the content of acetonitrile increased from 30% (v / v) to 70% (v / v) and from 15% (v / v) to 40% (v / v), indicating that the column had a typical reversed-phase retention mode.

[0064] Example 4 To investigate the hydrophilic performance of the Sil-CQDs mixed-mode column prepared in Example 2, i.e., in the hydrophilic chromatographic retention mode, the column was used to separate two types of hydrophilic compounds, nucleoside bases and thioureas, and one type of sulfonamides, to evaluate the hydrophilic performance of the Sil-CQDs stationary phase. The five nucleoside bases were uridine, cytidine, adenosine, adenine and cytosine; the three thioureas were thiourea, N-ethylthiourea and N,N-diethylthiourea; and the five sulfonamides were sulfacetamide, sulfamethazine, sulfathiazole, sulfisoxazole and sulfadimoxazole.

[0065] The five nucleoside bases, three thioureas and five sulfonamides were prepared into standard stock solutions with a concentration of 1 mg / ml and stored at 4°C.

[0066] The chromatographic conditions for the nucleoside base compounds were as follows: the mobile phase was (A) acetonitrile 90% (B) water 10%, the flow rate was 0.8 ml / min, the column temperature was 25°C, and the wavelength was 254 nm.

[0067] The chromatographic conditions of the thiourea compounds were as follows: the mobile phase was (A) acetonitrile 97% (B) water 3%, the flow rate was 0.8 ml / min, the column temperature was 25°C, and the wavelength was 254 nm.

[0068] The chromatographic conditions of the sulfonamide compounds were as follows: the mobile phase was (A) acetonitrile 7% (B) ammonium acetate solution (20 mM) 93%, the flow rate was 0.8 ml / min, the column temperature was 25°C, and the wavelength was 254 nm.

[0069] The chromatographic test results are shown in Figure 8 The five nucleobases, three thioureas, and five sulfonamides were effectively separated within 15 min.

[0070] Example 5 In the hydrophilic chromatographic retention mode, the mixed-mode chromatographic column Sil-CQDs prepared in Example 2 was used to explore the effect of acetonitrile content in the organic phase on the nucleobase compounds, which were consistent with those in Example 4. The chromatographic conditions were as follows: the acetonitrile content in the mobile phase was 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95%, the flow rate was 0.8 ml / min, and the column temperature was 25°C. The chromatographic detection results are shown in Figure 9 As the acetonitrile content increased from 55% (v / v) to 95% (v / v), the log k of the five nucleobase compounds gradually increased, indicating that the chromatographic column had a typical hydrophilic retention mode.

[0071] To further evaluate the hydrophobic and hydrophilic performance of the chromatographic column, the "U" curve detection of the sulfonamide compounds was performed. The sulfonamide compounds were consistent with those in Example 4. The chromatographic conditions for the "U" curve detection of the sulfonamide compounds were as follows: the mobile phase was (A) acetonitrile 5-95% (B) ammonium acetate solution (20 mM) 95%-5%, the flow rate was 0.8 ml / min, the column temperature was 25°C, and the wavelength was 254 nm. The chromatographic detection results are shown in Figure 10 As the acetonitrile content increased from 5% (v / v) to 80% (v / v), the log k of sulfonamide dimethoxyl gradually decreased, which was consistent with the reverse-phase retention mode. As the acetonitrile content increased from 80% (v / v) to 95% (v / v), the log k gradually increased, which was consistent with the hydrophilic retention mode.

[0072] To further investigate the separation mechanism of the chromatographic column, the peak order of the moderately polar aniline compounds was studied. The aniline compounds were consistent with those in Example 4, and the results are shown in Figure 6As shown in FIG. c, compared with aniline, o-phenylenediamine cannot interact with the stationary phase through hydrogen bonds, and is eluted fastest. 2,4-Dichloroaniline has a strongly acidic aromatic ring and carries a positive charge, which is attracted to -COO- on the chromatographic column, so it is eluted later. 2,6-Dinitroaniline is more acidic than 2,4-dinitroaniline, and carries more positive charges, so it is more strongly attracted to the chromatographic column and is eluted later. Diphenylamine is the most hydrophobic and has the longest retention time, indicating that the chromatographic column has hydrogen bonding, electrostatic attraction, and hydrophobic interaction mechanisms.

[0073] Example 6 The effect of buffer salt concentration on the separation of the mixed-mode chromatographic column Sil-CQDs prepared in Example 2 was investigated, i.e., the separation and detection of sulfonamide compounds under different buffer salt concentrations (10 mM, 20 mM, 30 mM, and 40 mM ammonium acetate solution). The sulfonamide compounds were consistent with those in Example 4, to investigate the effect of buffer salt concentration on the separation of the chromatographic column. The chromatographic conditions for the sulfonamide compounds were: mobile phase (A) acetonitrile 7% (B) different concentrations of ammonium acetate solution 93%, flow rate 0.8 ml / min, column temperature 25°C, and wavelength 254 nm. When the stationary phase surface has ionizable groups or the sample to be tested is an ionic compound, as the buffer salt concentration increases, salt ions will form an ion cloud around the analyte and the ionizable groups on the stationary phase, thereby reducing the electrostatic interaction between them. If the interaction is mainly electrostatic attraction, the ion shielding effect will weaken the retention. If the interaction is mainly electrostatic repulsion, the ion shielding effect will enhance the retention. The chromatographic detection results are shown in FIG. 5. Figure 11 As shown in FIG. 5, as the buffer salt concentration in the mobile phase increases, the retention of the five sulfonamide compounds decreases, indicating that there is electrostatic attraction between the sulfonamide compounds and the chromatographic column.

[0074] Example 7 To explore the effect of temperature on the separation of Sil-CQDs prepared in Example 2, i.e. the separation and detection of alkylbenzene compounds, polycyclic aromatic hydrocarbon compounds, aniline compounds, nucleoside base compounds and sulfonamide compounds at different temperatures (20-50℃) to investigate the effect of temperature on the separation of the chromatographic column. The alkylbenzene compounds, polycyclic aromatic hydrocarbon compounds, aniline compounds are consistent with Example 2; the nucleoside base compounds and sulfonamide compounds are consistent with Example 4; the chromatographic conditions of the alkylbenzene compounds are: mobile phase (A) methanol 53% (B) water 30%, flow rate 0.8 ml / min, column temperature 20-50℃, wavelength 210 nm; the chromatographic conditions of the polycyclic aromatic hydrocarbon compounds are: mobile phase (A) acetonitrile 52% (B) water 48%, flow rate 0.8 ml / min, column temperature 20-50℃, wavelength 210 nm; the chromatographic conditions of the aniline compounds are: mobile phase (A) acetonitrile 25% (B) water 75%, flow rate 0.8 ml / min, column temperature 20-50℃, wavelength 254 nm; the chromatographic conditions of the nucleoside base compounds are: mobile phase (A) acetonitrile 90% (B) water 10%, flow rate 0.8 ml / min, column temperature 20-50℃, wavelength 254 nm; the chromatographic conditions of the sulfonamide compounds are: mobile phase (A) acetonitrile 7% (B) ammonium acetate solution (20 mM) 93%, flow rate 0.8 ml / min, column temperature 20-50℃, wavelength 254 nm.

[0075] The thermodynamic parameters of each analyte were studied and calculated using the Van't Hoff equation (Formula I):

[0076] Formula I; The results are shown in Tables 2-6. Except for o-phenylenediamine, aniline and 2,4-dinitroaniline, the ΔH and ΔS of the analytes are negative, indicating that the transfer of these compounds from the mobile phase to the stationary phase is an exothermic process with reduced disorder, and the retention mechanism is partitioning. The ΔH and ΔS of o-phenylenediamine and aniline are positive, indicating that the separation of the two is an endothermic process with increased disorder, and the retention mechanism is adsorption. The ΔH of 2,4-dinitroaniline is negative and the ΔS is positive, indicating that its separation is an exothermic process with increased disorder, and the retention mechanism is partitioning. Therefore, the retention mechanism of the chromatographic column is determined by the dual action of adsorption and partitioning.

[0077] Table 2 is the chromatographic detection curve of Example 7 on alkylbenzene compounds

[0078] Table 3 is the chromatographic detection curve of polycyclic aromatic compounds in Example 7

[0079] Table 4 is the chromatographic detection curve of aniline compounds in Example 7

[0080] Table 5 is the chromatographic detection curve of nucleoside base compounds in Example 7

[0081] Table 6 is the chromatographic detection curve of sulfonamides in Example 7

[0082] Example 8 The repeatability and stability of the mixed-mode chromatographic column Sil-CQDs prepared in Example 2 were evaluated, i.e. with alkylbenzene compounds, polycyclic aromatic compounds, nucleoside base compounds, and thiourea compounds as analytes, 10 consecutive injection experiments were carried out, the changes of retention time and peak area of the analytes in the chromatogram were observed, and the repeatability of the chromatographic column was evaluated. The alkylbenzene compounds, polycyclic aromatic compounds were consistent with Example 2; the nucleoside base compounds and thiourea compounds were consistent with Example 4; the chromatographic conditions of the alkylbenzene compounds were: mobile phase was (A) methanol 53% (B) water 47%, flow rate was 0.8 ml / min, column temperature was 25°C, and wavelength was 210 nm; the chromatographic conditions of the polycyclic aromatic compounds were: mobile phase was (A) acetonitrile 52% (B) water 48%, flow rate was 0.8 ml / min, column temperature was 25°C, and wavelength was 210 nm; the chromatographic conditions of the nucleoside base compounds were: mobile phase was (A) acetonitrile 90% (B) water 10%, flow rate was 0.8 ml / min, column temperature was 25°C, and wavelength was 254 nm; the chromatographic conditions of the thiourea compounds were: mobile phase was (A) acetonitrile 97% (B) water 3%, flow rate was 0.8 ml / min, column temperature was 25°C, and wavelength was 254 nm; the results of the alkylbenzene compounds, polycyclic aromatic compounds, and thiourea compounds are shown in Table 6, which indicates that the repeatability of the chromatographic column is good. Figure 12

[0083] Three sulfonamide compounds were used as analytes (sulfacetamide, sulfathiazole, and sulfadimethoxine), and the retention time of the analytes was observed under the conditions of elution at pH 3 and 8 to evaluate the stability of the chromatographic column. The chromatographic conditions of the sulfonamide compounds were: mobile phase was (A) acetonitrile 7% (B) ammonium acetate solution (20 mM) 93%, flow rate was 0.8 ml / min, column temperature was 25°C, and wavelength was 254 nm. The chromatographic detection results of the sulfonamide compounds are shown in Table 6.​Figure 13 As shown, the retention times of the three sulfonamide compounds remained stable, indicating that the chromatographic column has good stability.

[0084] Comparative Example 1 The chloropropyl-modified silica gel Sil-Cl prepared in Example 1 was used as a chromatographic column to separate alkylbenzene compounds, polycyclic aromatic hydrocarbons, aniline compounds, nucleoside base compounds, and sulfonamide compounds. The specific chromatographic conditions for the alkylbenzene compounds, polycyclic aromatic hydrocarbons, aniline compounds, and related compounds were consistent with those in Example 2. The specific chromatographic conditions for the nucleoside base compounds, sulfonamide compounds, and related compounds were consistent with those in Example 4. The chromatographic detection results are as follows: Figure 14 As shown, the results indicate that hydrophobic substances on the Sil-CQDs column can be completely separated in a short time, while hydrophobic substances on the Sil-Cl column cannot be separated and are not retained. Only four nucleoside bases were separated on the Sil-Cl column, while only three sulfonamides were separated. Under the same chromatographic conditions, these two types of compounds could not be separated in a short time, but they could be separated quickly on the Sil-CQDs column with good peak shapes. Therefore, this indicates that the long-chain alkyl and hydrophilic groups of carbon quantum dots play an important role, and alkylbenzenes, polycyclic aromatic hydrocarbons, anilines, nucleoside bases, and sulfonamides are well separated on the Sil-CQDs column.

[0085] In summary, the embodiments of this invention have yielded a high-performance liquid chromatography (HPLC) mixed-mode column stationary phase packing material. Specifically, silica gel is modified with 3-chloropropyltriethoxysilane to obtain chloropropyl-modified silica gel. Mercaptopropionic acid and 1,12-diaminododecane are reacted at 200°C for 6 h to obtain carbon quantum dots. The chloropropyl-modified silica gel is then bonded to the carbon quantum dots to obtain the chromatographic stationary phase (Sil-CQDs). This stationary phase possesses amino, carboxyl, and long-chain alkyl groups, effectively separating hydrophobic and hydrophilic compounds and exhibiting excellent separation performance, effectively overcoming the limitations of single-mode chromatographic columns.

[0086] The above description is merely a few embodiments of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any modifications or alterations made by those skilled in the art without departing from the scope of the technical solution of the present invention using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A mixed-mode chromatographic stationary phase based on carbon quantum dot-bonded silica gel, characterized in that, The stationary phase for the mixed-mode chromatography is chloropropyl-modified silica gel with bonded carbon quantum dots, wherein the mass ratio of carbon quantum dots to chloropropyl-modified silica gel is 0.9~1.1:3.9~4.

1.

2. The mixed-mode chromatographic stationary phase according to claim 1, characterized in that, The mass ratio of chloropropylsilane to silica gel in the chloropropyl-modified silica gel is 0.9~1.1:2.3~2.4; The chloropropylsilane is selected from at least one of 3-chloropropyltrimethoxysilane, 3-chloropropyltriethoxysilane, and 3-chloropropylmethyldimethoxysilane; Preferably, the chloropropylsilane is 3-chloropropyltriethoxysilane.

3. The mixed-mode chromatographic stationary phase according to claim 1, characterized in that, The mixed-mode chromatographic stationary phase has amino, carboxyl, and long-chain alkyl groups, exhibiting both hydrophobic and hydrophilic separation properties, and demonstrates good stability at pH 3–8.

4. The method for preparing the mixed-mode chromatographic stationary phase according to any one of claims 1 to 3, characterized in that, The preparation method includes the following steps: Step 1, Preparation of carbon quantum dots: Mercaptopropionic acid and 1,12-diaminododecane were mixed and dispersed in ethanol. The mixed solution was heated at 200℃~220℃ for 6 h~8 h. After centrifuging the obtained product, the precipitate was discarded. The supernatant was then filtered and freeze-dried to obtain solid carbon quantum dots. Step 2, Preparation of chloropropyl modified silica gel: Silica gel is reacted with concentrated hydrochloric acid at 100℃~120℃ for 8 h~10 h. The product is washed with deionized water, filtered, and dried to obtain activated silica gel. The activated silica gel is dispersed in toluene, and 3-chloropropyltriethoxysilane is added. The mixture is stirred and reacted at 100℃~120℃ for 24 h~28 h. The product is filtered, washed with toluene, ethanol, and methanol, and dried to obtain chloropropyl modified silica gel. Step 3, Preparation of mixed-mode chromatographic stationary phase: The chloropropyl modified silica gel obtained in step 2 is dispersed in methanol, and the carbon quantum dots and triethylamine prepared in step 1 are added. The mixture is stirred at 80℃~100℃ for 24 h~28 h under nitrogen protection. The product is washed multiple times with methanol and water, filtered, and dried to obtain the mixed-mode chromatographic stationary phase.

5. The preparation method according to claim 4, characterized in that, The container for the heating reaction in step 1 is a 100ml polytetrafluoroethylene autoclave, and the heating reaction is carried out in an oven; The mass mixing ratio of mercaptopropionic acid and 1,12-diaminododecane in step 1 is 1.0~1.1:2.0~2.1; The concentration of mercaptopropionic acid in the mixed solution described in step 1 is 98%~100%; The product in step 1 is centrifuged at a speed of 4000~5000 rpm for a time of 10~15 min; The supernatant filtration in step 1 uses a 0.22 μm filter membrane.

6. The preparation method according to claim 4, characterized in that, The silica gel mentioned in step 2 is selected from at least one of pore size 100 Å and particle size 5 μm, pore size 120 Å and particle size 5 μm; The mass ratio of silica gel to concentrated hydrochloric acid in step 2 is 0.9~1.1:14~15; In step 2, the mass ratio of activated silica gel, toluene, and 3-chloropropyltriethoxysilane in the reaction is 3.4~3.6:86~88:1.4~1.

6.

7. The preparation method according to claim 4, characterized in that, The reaction of chloropropyl modified silica gel and carbon quantum dots in step 3 is carried out under nitrogen protection; the product is filtered using a G5 sand core funnel. In step 3, the mass ratio of chloropropyl modified silica gel, carbon quantum dots, and triethylamine is 3.8~4.2:0.8~1.2:0.7~0.

8.

8. The application of the mixed-mode chromatographic stationary phase according to any one of claims 1 to 3 and / or the mixed-mode chromatographic stationary phase prepared by any one of claims 4 to 7 in the separation of compounds, characterized in that, Using a mixed-mode chromatographic stationary phase as packing material, alkylbenzene compounds, polycyclic aromatic hydrocarbons, aniline compounds, nucleoside base compounds, thiourea compounds, and sulfonamide compounds are separated.

9. The application according to claim 7, characterized in that, The alkylbenzene compounds are toluene, ethylbenzene, propylbenzene, butylbenzene, and pentabenzene; the polycyclic aromatic hydrocarbon compounds are naphthalene, anthracene, acenaphthene, p-terphenyl, and benzanthracene; the aniline compounds are o-phenylenediamine, aniline, 2,4-dinitroaniline, 2,4-dichloroaniline, 2,6-dinitroaniline, and diphenylamine; the nucleoside base compounds are uridine, cytidine, adenosine, adenine, and cytosine; the thiourea compounds are thiourea, N-ethylthiourea, and N,N-diethylthiourea; and the sulfonamide compounds are sulfacetamide, sulfamethoxypyrimidine, sulfathiazole, sulfadiazine, and sulfadimethoxypyrimidine.

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