A fluorine-containing carbon point bonded silica gel chromatographic stationary phase and its preparation method and application
By using fluorocarbon dot-bonded silica gel stationary phase on the chromatographic column, the problem of poor retention of strong polar compounds in traditional chromatographic columns is solved, and the rapid and effective separation and good retention of these compounds are achieved.
Patent Information
- Application Number
- CN202510361952.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-26
- Publication Date
- 2025-06-06
- Estimated Expiration
- 2045-03-26
AI Technical Summary
Traditional C18 chromatography columns have poor retention effects on strong polar compounds such as nucleosides, saponins, alkaloids, and sulfonamides, making it difficult to meet the separation needs of complex samples.
Fluorocarbon dot bonded silica gel as the chromatographic stationary phase, and SiO2-CDs-F3 chromatographic stationary phase is formed by bonding hexacarbon perfluoropolyether siloxane and fluorocarbon dots on the surface of the bare silica gel.
It has achieved good retention effect on nucleosides, saponins, alkaloids and sulfonamide compounds, has strong selectivity and stability, and is suitable for use under acidic and alkaline conditions.
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Figure CN119869490B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a chromatographic stationary phase and a preparation method and application thereof, and in particular to a fluorine-containing carbon point bonded silica gel chromatographic stationary phase and a preparation method and application thereof, belonging to the technical field of chromatographic stationary phases. Background Art
[0002] High performance liquid chromatography (HPLC) has become the most widely used analytical technology due to its advantages of high efficiency, high sensitivity, and high selectivity. It is widely used in the fields of drug analysis, food analysis, environmental monitoring, etc. Nowadays, the components of separated samples are complex, and liquid chromatography with a single retention mechanism cannot meet the separation requirements. For example, the traditional C18 chromatographic column has a poor retention effect on highly polar compounds such as nucleosides, saponins, alkaloids, and sulfonamides.
[0003] Carbon dots (CDs) are a type of zero-dimensional carbon nanomaterials with significant fluorescent properties, usually less than 10 nm in size. Carbon dots have the characteristics of high hydrophilicity, good stability, rich surface functional groups and moderate adsorption capacity. When bonded with silica gel, they can be evenly dispersed on the silica gel surface to avoid agglomeration, and have broad application prospects in the field of chromatographic separation. Summary of the invention
[0004] In order to overcome the deficiencies of the prior art, the object of the present invention is to provide a fluorine-containing carbon point bonded silica gel chromatographic stationary phase having a good retention effect (strong selectivity) for strongly polar compounds such as nucleosides, saponins, alkaloids, sulfonamides, etc., as well as a preparation method and application of the chromatographic stationary phase.
[0005] In order to achieve the above object, the present invention adopts the following technical solution:
[0006] A fluorine-containing carbon point bonded silica gel chromatographic stationary phase takes bare silica gel as a matrix, and the surface of the bare silica gel is bonded with two substances, one is a directly bonded hexacarbon perfluoropolyether siloxane, and the other is a fluorine-containing carbon point bonded through 3-aminopropyltrimethoxysilane, wherein the bonding amount of the fluorine-containing carbon point is 2%-20%.
[0007] The preparation method of the aforementioned fluorine-containing carbon point bonded silica gel chromatographic stationary phase comprises the following steps:
[0008] (1) Tetrafluoroterephthalic acid and tetraethylenepentamine are heated to synthesize fluorine-containing carbon dots;
[0009] (2) Aminosilane reagent and silica gel are heated to synthesize amino silica gel;
[0010] (3) Fluorine-containing carbon dots are bonded to amino-modified silica gel at room temperature to obtain fluorine-containing carbon dots modified silica gel;
[0011] (4) Fluorine-containing carbon dot-modified silica gel is heated with a fluorosilane reagent to synthesize a fluorine-containing carbon dot-bonded silica gel chromatographic stationary phase.
[0012] Preferably, in step (1), the method for heating tetrafluoroterephthalic acid and tetraethylenepentamine to synthesize fluorine-containing carbon dots is as follows:
[0013] (a) adding tetrafluoroterephthalic acid, ultrapure water and tetraethylenepentamine into a reaction container, and ultrasonicating to obtain a mixed solution;
[0014] (b) pouring the mixed solution into a high pressure reactor and heating it at 180° C. for 24 h to obtain a reaction solution;
[0015] (c) The reactor was naturally cooled to room temperature, the reaction solution was filtered through a 0.22 μm filter membrane, and the filtrate was dialyzed through a 500 Da dialysis bag to obtain a dialysate;
[0016] (d) The dialysate is concentrated by rotary evaporation to obtain a fluorine-containing carbon dot solution.
[0017] Preferably, in step (2), the method for heating the aminosilane reagent and silica gel to synthesize the amino silica gel is as follows:
[0018] (a) dispersing bare silica gel in a nitric acid solution, stirring at room temperature for 2 h, then centrifugally washing and drying to obtain acidified silica gel;
[0019] (b) Anhydrous toluene, 3-aminopropyltrimethoxysilane and acidified silica gel were mixed in a ratio of 20 mL:1.5 mL:3 g and heated to 400 °C under N 2 Under the protection of , the reaction was first carried out at 110°C for 3 hours, and then the heating was stopped for 9 hours and then the reaction was continued at 110°C for 24 hours;
[0020] (c) After the reaction is completed, the product is centrifuged, washed and dried to obtain amino silica gel.
[0021] Preferably, in step (3), the method of bonding the fluorinated carbon dots to the amino-modified silica gel at room temperature to obtain the fluorinated carbon dots modified silica gel is as follows:
[0022] (a) activating the fluorine-containing carbon dots with an activation solution to obtain an activated fluorine-containing carbon dot solution;
[0023] (b) dispersing the activated fluorinated carbon dot solution and amino-modified silica gel in a ratio of 10 mL:1 g into 2-morpholineethanesulfonic acid buffer and reacting at room temperature for 12 h;
[0024] (c) After the reaction is completed, the silica gel modified with fluorinated carbon dots is obtained by centrifugation, washing and drying.
[0025] Preferably, in step (4), the method for synthesizing the fluorinated carbon dot-bonded silica gel chromatographic stationary phase by heating the fluorinated carbon dot-modified silica gel with a fluorosilane reagent is as follows:
[0026] (a) Hexafluoropolyether siloxane, triethylamine, and fluorinated carbon-dot-modified silica gel were dispersed in anhydrous toluene at a ratio of 0.2576 g:445 μL:1 g. 2 Under protection, the reaction was carried out at 105°C for 12 hours. After the reaction was completed, the product A was obtained by centrifugation, washing and drying.
[0027] (b) Disperse hexafluoropolyether siloxane, triethylamine and product A in anhydrous toluene at a ratio of 0.2675 g:445 μL:total amount and 2 Under protection, the reaction was carried out at 105°C for 12 hours. After the reaction was completed, the product B was obtained by centrifugation, washing and drying.
[0028] (c) Trifluoropropyltrichlorosilane, triethylamine and product B were mixed in a ratio of 449.4 μL:445 μL:total amount, and reacted at 105° C. for 12 h. After the reaction, the mixture was centrifuged, washed and dried to obtain a fluorine-containing carbon point bonded silica gel chromatographic stationary phase.
[0029] The aforementioned fluorine-containing carbon point bonded silica gel chromatographic stationary phase is used for separating highly polar compounds, wherein the highly polar compounds include nucleosides, saponins, alkaloids and sulfonamides.
[0030] The present invention is beneficial in that:
[0031] (1) The carbon quantum dots derived from tetrafluoroterephthalic acid and tetraethylenepentamine contain polar groups such as carboxyl, amino, double bond, and fluorine atoms, as well as non-polar groups such as alkyl chains and benzene rings, which makes the chromatographic stationary phase SiO prepared by the present invention 2 -CDs-F3 has both hydrophobic and hydrophilic effects and has RPLC mode, which enriches the types of liquid chromatography stationary phases;
[0032] (2) The chromatographic stationary phase SiO prepared by the present invention 2 -CDs-F3 has strong selectivity for nucleosides, saponins, alkaloids and sulfonamides, and can achieve rapid and effective separation, making up for the poor retention of highly polar compounds by traditional C18 columns;
[0033] (3) The chromatographic stationary phase SiO prepared by the present invention 2 -CDs-F3 has strong stability and is suitable for use under both acidic and alkaline conditions, and has a wide range of applications;
[0034] (3) The chromatographic stationary phase SiO provided by the present invention 2 The raw materials used in the preparation method of -CDs-F3 are widely available and inexpensive, and are suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS
[0035] Figure 1 It is a schematic diagram of the preparation process of the fluorine-containing carbon point bonded silica gel chromatographic stationary phase provided by the present invention;
[0036] Figure 2 It is bare silica gel (SiO 2 )、SiO 2 -CDs、SiO 2 -F3 and SiO 2 - Transmission electron microscopy image of CDs-F3, where A is SiO 2 Transmission electron microscopy image of SiO 2 -Transmission electron microscopy image of CDs, C is SiO 2 - Transmission electron microscopy image of F3, D is SiO 2 - Transmission electron microscopy image of CDs-F3;
[0037] Figure 3 It is bare silica gel (SiO 2 )、SiO 2 -CDs、SiO 2 -F3 and SiO 2 -Scanning electron micrograph of CDs-F3, where E is SiO 2 Scanning electron microscope image of F is SiO 2 -Scanning electron microscopy image of CDs, G is SiO 2 -Scanning electron micrograph of F3, H is SiO 2 - Scanning electron micrograph of CDs-F3;
[0038] Figure 4 It is bare silica gel (SiO 2 )、SiO 2 -CDs、SiO 2 -F3 and SiO 2 -Infrared spectrum of CDs-F3;
[0039] Figure 5 It is bare silica gel (SiO 2 )、SiO 2 -CDs、SiO 2 -F3 and SiO 2 -Elemental analysis of CDs-F3;
[0040] Figure 6 There are five nucleoside compounds in SiO 2 -Separation chromatogram on CDs-F3, wherein 1 is cytidine, 2 is guanosine, 3 is 5'-methyluridine, 4 is thymine, and 5 is β-thymidine;
[0041] Figure 7 There are 7 sulfonamide compounds in SiO 2 -Separation chromatogram on CDs-F3, wherein 1 is sulfonamide, 2 is sulfacetamide, 3 is sulfadiazine, 4 is sulfapyridine, 5 is sulfamerazine, 6 is sulfadimethoxine, and 7 is sulfamethoxazole;
[0042] Figure 8 There are five saponin compounds in SiO 2 -Separation chromatogram on CDs-F3, wherein 1 is ginsenoside Re, 2 is ginsenoside Rh1, 3 is ginsenoside Rb1, 4 is ginsenoside Rb3, and 5 is ginsenoside Rd;
[0043] Fig. 9 There are five alkaloid compounds in SiO 2 -Separation chromatogram on CDs-F3, wherein 1 is lycorine hydrochloride, 2 is tetrahydropalmatine, 3 is berberine hydrochloride, 4 is reserpine, and 5 is gefitinib;
[0044] Fig.10 7 common pesticides in SiO 2 -Separation chromatogram on CDs-F3, wherein 1 is imidacloprid, 2 is pyrimethanil, 3 is difenoconazole, 4 is chlorfenapyr, 5 is hexaflumuron, 6 is chlorpyrifos, and 7 is pyridaben;
[0045] Fig.11 Total ginsenosides in SiO 2 - Gradient elution chromatogram on CDs-F3. DETAILED DESCRIPTION
[0046] The present invention is described in detail below with reference to the accompanying drawings and embodiments.
[0047] 1. Preparation of Fluorinated Carbon-dot Bonded Silica Gel Chromatographic Stationary Phase
[0048] The fluorine-containing carbon point bonded silica gel chromatographic stationary phase provided by the present invention has the following structure: bare silica gel (SiO 2 ) is used as the matrix, and there are two substances bonded on the surface, one is a directly bonded hexacarbon perfluoropolyether siloxane, and the other is a fluorine-containing carbon point bonded through 3-aminopropyltrimethoxysilane, among which the bonding amount of the fluorine-containing carbon point is 2%-20%.
[0049] like Figure 1As shown, the preparation method of the fluorine-containing carbon dot bonded silica gel chromatographic stationary phase provided by the present invention comprises the following four steps: 1. heating tetrafluoroterephthalic acid and tetraethylenepentamine to synthesize fluorine-containing carbon dots; 2. heating aminosilane reagent and silica gel to synthesize amino-modified silica gel; 3. bonding the fluorine-containing carbon dots to amino-modified silica gel at room temperature to obtain fluorine-containing carbon dot-modified silica gel; 4. heating the fluorine-containing carbon dot-modified silica gel with a fluorosilane reagent to synthesize a fluorine-containing carbon dot bonded silica gel chromatographic stationary phase.
[0050] 1. Synthesis of fluorinated carbon dots by heating tetrafluoroterephthalic acid and tetraethylenepentamine
[0051] Add 1.5 g of tetrafluoroterephthalic acid and 60 mL of ultrapure water to a beaker, then add 20 mL of tetraethylenepentamine, and ultrasonicate for 5 min to obtain a mixed solution. Pour the mixed solution into a polytetrafluoroethylene-lined autoclave and heat at 180 ° C for 24 h. After heating, the autoclave is naturally cooled to room temperature. The cooled reaction solution is filtered with a 0.22 μm filter membrane, and then the filtrate is dialyzed in a 500 Da dialysis bag for 48 h. The dialyzate obtained is concentrated to 10 mL by rotary evaporation to obtain a fluorinated carbon dot (F-CDs) solution.
[0052] After testing, the nitrogen and fluorine doping levels of the F-CDs were 7.25% and 19.63%, respectively.
[0053] 2. Synthesis of aminosilane silica gel by heating aminosilane reagent and silica gel
[0054] 3 g bare silica gel (SiO 2 ) were dispersed in 30 mL of 15% (v / v) nitric acid solution and stirred at room temperature for 2 h. After the reaction, the silica gel was centrifuged and washed with deionized water until the pH of the supernatant was neutral, then washed with anhydrous ethanol for 3 times, and dried at 60 °C to obtain acidified silica gel.
[0055] 20 mL of anhydrous toluene, 1.5 mL of 3-aminopropyltrimethoxysilane and 3 g of the acidified silica gel obtained above were mixed evenly and heated under N 2 Under the protection of , the reaction was carried out at 110 ° C for 3 hours, and then the heating was stopped for 9 hours, and then the reaction was carried out at 110 ° C for 24 hours. After the reaction, centrifugation was performed, and toluene (or anhydrous ethanol, 50% (v / v) ethanol, methanol) was used for washing 3 times, and then dried at 60 ° C to obtain amino silica gel (SiO 2 -NH 2 ).
[0056] 3. Fluorine-containing carbon dots are bonded to amino-modified silica gel at room temperature to obtain fluorine-containing carbon dots modified silica gel
[0057] 10 mL of the previously obtained F-CDs solution was activated with 12 mL of activation solution (equal volumes of a 20 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution and a 10 mg / mL N-hydroxysuccinimide solution) at room temperature for 10 min to obtain an activated F-CDs solution.
[0058] 10 mL of the activated F-CDs solution obtained above and 1 g of the SiO 2 -NH 2 Disperse into 45mL of 0.1mM 2-morpholineethanesulfonic acid buffer and react at room temperature for 12h. After the reaction, centrifuge and wash, first wash with pure water 3 times, then wash with anhydrous ethanol 3 times, and finally dry at 60℃ to obtain fluorinated carbon dot modified silica gel (SiO 2 -CDs).
[0059] After testing, the SiO 2 The fluorine-containing carbon point bonding content of -CDs is 9.36%.
[0060] 4. Synthesis of fluorinated carbon-dot bonded silica gel chromatographic stationary phase by heating fluorinated carbon-dot modified silica gel with fluorosilane reagent
[0061] 0.2576 g of hexafluoropolyether siloxane, 445 μL of triethylamine and 1 g of SiO 2 -CDs were uniformly dispersed in 25 mL of anhydrous toluene and heated under N 2 Under protection, the reaction was carried out at 105°C for 12 hours. After the reaction was completed, the product was centrifuged and washed with toluene for 3 times, then with anhydrous ethanol for 3 times, and finally dried at 60°C to obtain product A.
[0062] 0.2675 g of hexafluoropolyether siloxane, 445 μL of triethylamine and the total amount of product A obtained above were uniformly dispersed in 25 mL of anhydrous toluene and stirred at N 2 Under protection, the reaction was carried out at 105°C for 12 hours. After the reaction was completed, the mixture was centrifuged and washed with toluene for 3 times, then with anhydrous ethanol for 3 times, and finally dried at 60°C to obtain product B.
[0063] 449.4 μL trifluoropropyltrichlorosilane, 445 μL triethylamine and the entire amount of product B obtained above were mixed and reacted at 105°C for 12 h. After the reaction, the mixture was centrifuged and washed with toluene 3 times, then with anhydrous ethanol 3 times, and finally dried at 60°C to obtain a fluorinated carbon point bonded silica gel chromatographic stationary phase (SiO 2 -CDs-F3).
[0064] After testing, the SiO 2 The fluorine-containing carbon point bonding content of -CDs-F3 is 9.36%.
[0065] As a control, amino silica gel (SiO 2 -NH 2 ) and fluorosilane reagent to synthesize a silica gel chromatographic stationary phase without carbon dots (SiO 2 -F3), SiO 2 -F3 preparation method and SiO 2 The preparation method of -CDs-F3 is basically the same, except that the SiO 2 -CDs (1g).
[0066] 2. Analysis of the structure of fluorinated carbon point bonded silica gel chromatographic stationary phase
[0067] The unreacted bare silica gel (SiO 2 ) and the SiO 2 -F3、SiO 2 -CDs and SiO 2 -CDs-F3 structure was analyzed.
[0068] 1. Transmission electron microscopy analysis
[0069] SiO 2 、SiO 2 -CDs、SiO 2 -F3 and SiO 2 - Transmission electron microscopy image of CDs-F3 is shown in Figure 2 .
[0070] Through transmission electron microscopy, it can be observed that SiO 2 The surface of SiO 2 -CDs、SiO 2 -F3 and SiO 2 -CDs-F3 has a rough surface, among which SiO 2 -CDs-F3 has the roughest surface.
[0071] This shows that: SiO 2 -CDs、SiO 2 -F3 and SiO 2 -CDs-F3 are bonded with compounds on their surfaces, among which SiO 2 -CDs-F3 has the most compounds bonded to its surface.
[0072] 2. Scanning electron microscopy analysis
[0073] SiO 2 、SiO 2 -CDs、SiO2 -F3 and SiO 2 -Scanning electron microscopy image of CDs-F3 is shown in Figure 3 .
[0074] Scanning electron microscopy revealed that SiO 2 、SiO 2 -CDs、SiO 2 -F3 and SiO 2 -CDs-F3 are all spherical and evenly dispersed, except for SiO 2 The surface is smooth, SiO 2 -CDs、SiO 2 -F3 and SiO 2 -CDs-F3 surfaces show different degrees of roughness, among which SiO 2 -CDs-F3 has the roughest surface.
[0075] This shows that: SiO 2 -CDs、SiO 2 -F3 and SiO 2 -CDs-F3 are bonded with compounds on their surfaces, among which SiO 2 -CDs-F3 has the most compounds bonded to its surface.
[0076] 3. Infrared spectroscopy analysis
[0077] Bare silica gel (SiO 2 )、SiO 2 -CDs、SiO 2 -F3 and SiO 2 -Infrared spectrum of CDs-F3 is shown in Figure 4 .
[0078] It can be observed from the infrared spectrum: SiO 2 、SiO 2 -CDs、SiO 2 -F3 and SiO 2 -CDs-F3 at 3450cm -1 and 1070cm -1 There are absorption peaks at both ends, which are attributed to the vibration of ν (OH / NH) and ν (Si-O) respectively; SiO 2 -CDs、SiO 2 -F3 and SiO 2 -CDs-F3 at 2930cm -1 Different from SiO 2 The absorption peak is attributed to the vibration of ν(CH); SiO 2 -F3 and SiO2 -CDs-F3 at 1540cm -1 、1710cm -1 and 1210cm -1 New absorption peaks appeared at all locations, and these three absorption peaks were attributed to the vibrations of ν (RNC=O), ν (C=O) and ν (CF), respectively.
[0079] This shows that: SiO 2 -CDs successfully bonded to the surface of F-CDs, SiO 2 The surface of -F3 was successfully bonded with hexafluoropolyether siloxane, SiO 2 The surface of -CDs-F3 was successfully bonded with F-CDs and hexafluoropolyether siloxane.
[0080] 4. Quantitative analysis of elements
[0081] The bare silica gel (SiO 2 )、SiO 2 -CDs、SiO 2 -F3 and SiO 2 -CDs-F3 performs quantitative analysis of the three elements C, O, and F.
[0082] Bare silica gel (SiO 2 )、SiO 2 -CDs、SiO 2 -F3 and SiO 2 -The quantitative analysis results of the three elements C, O and F of CDs-F3 are shown in Figure 5 .
[0083] From the element quantitative analysis chart, we can know that SiO 2 The content of F in -CDs-F3 is 40.59%, and the contents of C and O are 7.97% and 40.93% respectively, which indicates that SiO 2 -CDs-F3.
[0084] 3. Testing the Selectivity of Fluorinated Carbon-dot Bonded Silica Gel Chromatographic Stationary Phase
[0085] The SiO prepared by the present invention 2 -CDs-F3 has both hydrophobic and hydrophilic effects due to the presence of polar groups such as carboxyl, amino, double bonds, and fluorine atoms and non-polar groups such as alkyl chains and benzene rings (present in carbon quantum dots). It has a reverse phase chromatography (RPLC) mode. In the RPLC mode, the SiO 2 -CDs-F3 has selectivity for nucleosides, saponins, alkaloids and sulfonamides.
[0086] 1. Selectivity for nucleoside compounds
[0087] Take cytidine, guanosine, 5'-methyluridine, thymine and β-thymidine as examples.
[0088] Chromatographic conditions: mobile phase: 0.4% (v / v) acetic acid solution; detection wavelength: 260nm; flow rate: 0.2mL / min; injection volume 1μL; column temperature: 30℃.
[0089] Detection process: Cytidine, guanosine, 5'-methyluridine, thymine and β-thymidine were prepared into 1000ppm solutions with 0.5% (v / v) methanol solution, 100μL cytidine solution, 100μL guanosine solution, 100μL 5'-methyluridine solution, 150μL thymine solution and 200μL β-thymidine solution were pipetted into mixed standards, and the samples were injected 5 times continuously by high performance liquid chromatography to obtain the compound on SiO 2 -Separation chromatogram on CDs-F3.
[0090] Cytidine, guanosine, 5'-methyluridine, thymine and β-thymidine are five nucleoside compounds that can be used in the treatment of inflammatory cytotoxicity. 2 -Separation chromatogram on CDs-F3 is shown in Figure 6 .
[0091] Depend on Figure 6 It can be seen that SiO 2 -CDs-F3 has a good separation effect on five nucleoside compounds, namely cytidine, guanosine, 5'-methyluridine, thymine and β-thymidine. The plate numbers are 8700N / m, 9120N / m, 11240N / m, 15080N / m and 11620N / m in the order of peak elution, and the separation degrees R are all greater than 1.5.
[0092] 2. Selectivity for sulfonamide compounds
[0093] Take sulfonamide, sulfacetamide, sulfadiazine, sulfapyridine, sulfamethazine, sulfadimethoxine and sulfamethoxazole as examples.
[0094] Chromatographic conditions: mobile phase: methanol and 10 mM acetic acid solution mixed in a volume ratio of 13:87; detection wavelength: 270 nm; flow rate: 0.2 mL / min; injection volume: 1 μL; column temperature: 30°C.
[0095] Detection process: Sulfonamide, sulfacetamide, sulfadiazine, sulfapyridine, sulfamerazine, sulfadimethoxine and sulfamethoxazole were prepared into 1000 ppm solutions with 50% (v / v) acetonitrile solution, and 25 μL of sulfonamide solution, 50 μL of sulfacetamide solution, 100 μL of sulfadiazine solution, 100 μL of sulfapyridine solution, 100 μL of sulfamerazine solution, 150 μL of sulfadimethoxazole solution and 300 μL of sulfamethoxazole solution were pipetted into a mixed standard, and the sample was injected continuously for 5 times by high performance liquid chromatography to obtain the compound on SiO 2 -Separation chromatogram on CDs-F3.
[0096] Sulfonamide, sulfacetamide, sulfadiazine, sulfapyridine, sulfamerazine, sulfadimethoxine and sulfamethoxazole were used as the sulfonamides in the SiO 2 -Separation chromatogram on CDs-F3 is shown in Figure 7 .
[0097] Depend on Figure 7 It can be seen that SiO 2 -CDs-F3 has a good separation effect on seven sulfonamide compounds, namely sulfonamide, sulfacetamide, sulfadiazine, sulfapyridine, sulfamethoxazole, sulfadimethazine and sulfamethoxazole. The plate numbers are 12750N / m, 12700N / m, 11040N / m, 12200N / m, 11950N / m, 13040N / m and 17030N / m in the order of peak elution, and the separation degrees are all greater than 1.5.
[0098] 3. Selectivity for saponin compounds
[0099] Take ginsenosides Re, Rh1, Rb1, Rb3 and Rd as examples.
[0100] Chromatographic conditions: mobile phase: 50% (v / v) methanol solution; detection wavelength: 203 nm; flow rate: 0.2 mL / min; injection volume 1 μL; column temperature: 30°C.
[0101] Detection process: Ginsenoside Re, Rh1, Rb1, Rb3 and Rd were prepared into 10000ppm solutions with 44% (v / v) methanol solution, and 50μL Rh1 solution, 200μL Rb1 solution, 200μL Rb3 solution, 200μL Rd solution and 70μL Re solution were pipetted into mixed standards. The samples were injected 5 times continuously by HPLC to obtain the compound on SiO 2 -Separation chromatogram on CDs-F3.
[0102] Ginsenosides Re, Rh1, Rb1, Rb3 and Rd are five saponin compounds in SiO 2 -Separation chromatogram on CDs-F3 is shown in Figure 8 .
[0103] Depend on Figure 8 It can be seen that SiO 2 -CDs-F3 has a good separation effect on the five saponin compounds, namely ginsenoside Re, Rh1, Rb1, Rb3 and Rd. The plate numbers are 7530N / m, 10460N / m, 11380N / m, 12380N / m and 14630N / m in the order of peak elution, and the separation degrees are all greater than 1.5.
[0104] 4. Selectivity for alkaloid compounds
[0105] Take lycorine hydrochloride, tetrahydropalmatine, berberine hydrochloride, reserpine and gefitinib as examples.
[0106] Chromatographic conditions: mobile phase: methanol and 10 mM acetic acid solution mixed in a volume ratio of 11:9; detection wavelength: 280 nm; flow rate: 0.2 mL / min; injection volume: 1 μL; column temperature: 30°C.
[0107] Detection process: Lycorine hydrochloride, tetrahydropalmatine, berberine hydrochloride, reserpine and gefitinib were prepared into 1000 ppm solutions with 83% (v / v) acetonitrile solution, 100 μL tetrahydropalmatine solution, 100 μL reserpine solution, 100 μL lycorine hydrochloride solution, 100 μL berberine hydrochloride solution and 200 μL gefitinib solution were pipetted into mixed standards, and the samples were injected continuously for 5 times by high performance liquid chromatography to obtain the compound on SiO 2 -Separation chromatogram on CDs-F3.
[0108] Lycorine hydrochloride, tetrahydropalmatine, berberine hydrochloride, reserpine and gefitinib were detected on SiO 2 -Separation chromatogram on CDs-F3 is shown in Fig. 9 .
[0109] Depend on Fig. 9 It can be seen that SiO 2 -CDs-F3 has a good separation effect on five alkaloid compounds, namely lycorine hydrochloride, corydaline, berberine hydrochloride, reserpine and gefitinib. The plate numbers are 11790N / m, 15100N / m, 11950N / m, 12450N / m and 4550N / m in the order of peak elution, and the separation degrees are all greater than 1.5.
[0110] 5. Selectivity for common pesticides
[0111] Take common pesticides such as imidacloprid, pyrimethanil, dimethoate, carbofuran, fluazifop, chlorpyrifos, and chlorpyrifos as examples.
[0112] Chromatographic conditions: mobile phase: 61% (v / v) methanol solution; detection wavelength: 270 nm; flow rate: 0.2 mL / min; injection volume 1 μL; column temperature: 30°C.
[0113] Detection process: imidacloprid, pyrimethanil, difenoconazole, chlorfenapyr, hexaflumuron, chlorpyrifos and pyridaben were prepared into solutions with a concentration of 1000 ppm with methanol, and 10 μL of imidacloprid solution, 10 μL of pyrimethanil solution, 50 μL of chlorpyrifos solution, 70 μL of hexaflumuron solution, 70 μL of chlorpyrifos solution, 200 μL of chlorpyrifos solution and 200 μL of difenoconazole solution were pipetted with a pipette to prepare a mixed standard. The sample was injected continuously for 5 times by high performance liquid chromatography to obtain the compound on SiO 2 -Separation chromatogram on CDs-F3.
[0114] Seven common pesticides, including imidacloprid, pyrimethanil, dimethomorph, chlorpyrifos, hexaflumuron, chlorpyrifos and pyridaben, were detected on SiO 2 -Separation chromatogram on CDs-F3 is shown in Fig.10 .
[0115] Depend on Fig.10 It can be seen that SiO 2 -CDs-F3 has a good separation effect on seven common pesticides, including imidacloprid, pyrimethanil, dimethoate, chlorpyrifos, hexaflumuron, chlorpyrifos and pyridaben. The plate numbers are 7250N / m, 12240N / m, 12510N / m, 18190N / m, 18560N / m, 18750N / m and 18500N / m in the order of peak elution, and the separation degrees are all greater than 1.5.
[0116] 4. Practical Application Cases
[0117] The SiO prepared by the present invention 2 -CDs-F3 was assembled into a chromatographic column, and total ginsenosides were used as the analyte to separate and analyze R1, Rd, Rh2, and Rg3 in total ginsenosides.
[0118] Sample pretreatment: Weigh 1g of total ginsenosides, dissolve it in 20mL of 70% (v / v) methanol solution, ultrasonicate for 30min, and then filter with a 0.22μm filter membrane. Rotary evaporate the filtrate (to remove excess solvent) to obtain a solid. Then dissolve the obtained solid in 5mL of 70% (v / v) methanol solution, and finally filter with a 0.22μm filter membrane to obtain the filtrate to be tested.
[0119] Use a pipette to draw 0.5 mL of the filtrate to be tested for liquid phase separation. Mobile phase: A: 0.1% (v / v) formic acid solution, B: acetonitrile; injection volume: 1 μL; column temperature: 30°C; flow rate: 0.2 mL / min.
[0120] Mobile phase: 0min: 85%A, 15%B; 0-3min: 15%-20%B; 3-4min: 20%B; 4-10min: 20%-30%B; 10-25min: 30%-32.5%B; 25-27min: 32.5%-60%B; 27-39min: 60-95%B; 39-40min: 95%B; 40-40.5min: 95%-15%B; 40.5-41min: 15%B.
[0121] The separation results of R1, Rd, Rh2 and Rg3 in ginsenosides are shown in Fig.11 The results showed that within 40 minutes of rapid separation, R1, Rd, Rh2, and Rg3 in ginsenosides were successfully separated. This proves that the SiO prepared by the present invention 2 -CDs-F3 has good application prospects in the separation and analysis of complex samples.
[0122] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the embodiments here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.
Claims
1. A method for preparing a fluorinated carbon dot bonded silica gel chromatographic stationary phase, wherein the fluorinated carbon dot bonded silica gel chromatographic stationary phase is based on bare silica gel, and the surface of the bare silica gel is bonded with two substances, one is a directly bonded hexacarbon perfluoropolyether siloxane, and the other is a fluorinated carbon dot bonded via 3-aminopropyltrimethoxysilane, wherein: The fluorine-containing carbon point bonding amount is 2%-20%, characterized in that the preparation method of the fluorine-containing carbon point bonded silica gel chromatographic stationary phase comprises the following steps: (1) Tetrafluoroterephthalic acid and tetraethylenepentamine are heated to synthesize fluorine-containing carbon dots; (2) Aminosilane reagent and silica gel are heated to synthesize amino silica gel; (3) Fluorine-containing carbon dots are bonded to amino-modified silica gel at room temperature to obtain fluorine-containing carbon dots modified silica gel; (4) Fluorine-containing carbon dot-modified silica gel is heated with a fluorosilane reagent to synthesize a fluorine-containing carbon dot-bonded silica gel chromatographic stationary phase.
2. The preparation method according to claim 1, characterized in that: In step (1), the method for heating tetrafluoroterephthalic acid and tetraethylenepentamine to synthesize fluorine-containing carbon dots is as follows: (a) adding tetrafluoroterephthalic acid, ultrapure water and tetraethylenepentamine into a reaction container, and ultrasonicating to obtain a mixed solution; (b) pouring the mixed solution into a high pressure reactor and heating it at 180° C. for 24 h to obtain a reaction solution; (c) The reactor was naturally cooled to room temperature, the reaction solution was filtered through a 0.22 μm filter membrane, and the filtrate was dialyzed through a 500 Da dialysis bag to obtain a dialysate; (d) The dialysate is concentrated by rotary evaporation to obtain a fluorine-containing carbon dot solution.
3. The preparation method according to claim 1, characterized in that: In step (2), the method for synthesizing aminosilane silica gel by heating the aminosilane reagent and silica gel is as follows: (a) dispersing bare silica gel in a nitric acid solution, stirring at room temperature for 2 h, then centrifugally washing and drying to obtain acidified silica gel; (b) Anhydrous toluene, 3-aminopropyltrimethoxysilane and acidified silica gel were uniformly mixed in a ratio of 20 mL:1.5 mL:3 g, and reacted at 110°C for 3 h under the protection of N2, and then stopped heating for 9 h and then reacted at 110°C for 24 h; (c) After the reaction is completed, the product is centrifuged, washed and dried to obtain amino silica gel.
4. The preparation method according to claim 1, characterized in that: In step (3), the method of bonding the fluorinated carbon dots to the amino-modified silica gel at room temperature to obtain the fluorinated carbon dots modified silica gel is as follows: (a) activating the fluorine-containing carbon dots with an activation solution to obtain an activated fluorine-containing carbon dot solution; (b) dispersing the activated fluorinated carbon dot solution and amino-modified silica gel in a ratio of 10 mL:1 g into 2-morpholineethanesulfonic acid buffer and reacting at room temperature for 12 h; (c) After the reaction is completed, the silica gel modified with fluorinated carbon dots is obtained by centrifugation, washing and drying.
5. The preparation method according to claim 1, characterized in that: In step (4), the method for synthesizing the fluorinated carbon dot-bonded silica gel chromatographic stationary phase by heating the fluorinated carbon dot-modified silica gel with a fluorosilane reagent is as follows: (a) Dispersing hexafluoropolyether siloxane, triethylamine and silica gel modified with fluorinated carbon dots in anhydrous toluene in a ratio of 0.2576 g:445 μL:1 g, reacting at 105° C. for 12 h under N2 protection, centrifuging, washing and drying after the reaction to obtain product A; (b) dispersing hexafluoropolyether siloxane, triethylamine and product A in anhydrous toluene in a ratio of 0.2675 g:445 μL:total amount, reacting at 105° C. for 12 h under N2 protection, and centrifugally washing and drying after the reaction to obtain product B; (c) Trifluoropropyltrichlorosilane, triethylamine and product B were mixed in a ratio of 449.4 μL:445 μL:total amount, and reacted at 105° C. for 12 h. After the reaction, the mixture was centrifuged, washed and dried to obtain a fluorine-containing carbon point bonded silica gel chromatographic stationary phase.
6. Use of the fluorine-containing carbon point bonded silica gel chromatographic stationary phase prepared by the preparation method of claim 1 in separating highly polar compounds, wherein the highly polar compounds include nucleosides, saponins, alkaloids and sulfonamides.