Preparation method and application of spermine-bonded silica gel hydrophilic interaction chromatography stationary phase

Abstract

The application discloses a preparation method of spermine-bonded silica gel hydrophilic chromatographic stationary phase and application thereof, and belongs to the technical field of hydrophilic chromatography. The preparation steps are as follows: first, epoxy propyl silane modification is carried out on spherical silica gel to prepare epoxy propyl functionalized silica gel; then, spermine is uniformly mixed with the epoxy propyl functionalized silica gel in an organic solvent to carry out reaction, so that spermine-bonded silica gel chromatographic stationary phase Sil-SPM is prepared. It is proved by infrared spectrum and element analysis characterization that spermine has been successfully bonded to the surface of the silica gel. Chromatographic separation experiments show that the hydrophilic chromatographic stationary phase Sil-SPM prepared by the application has good hydrophilic chromatographic selectivity, and has excellent separation selectivity for base nucleosides. Meanwhile, the stationary phase can also be applied to the determination of the contents of uric acid and creatine in urine, and has potential application value in the separation of hydrophilic biological samples.

Description

Technical Field

[0001] The invention belongs to the technical field of hydrophilic chromatography and relates to a preparation method and application of a spermine-bonded silica gel hydrophilic chromatography stationary phase. Background Art

[0002] With the increasing number of application scenarios for separation and analysis of highly polar hydrophilic biological samples, hydrophilic interaction chromatography has received more and more attention due to its great advantages in separating polar and complex samples. The research and preparation of hydrophilic interaction chromatography stationary phases with high separation selectivity has become an important part of research in the field of chromatography.

[0003] The amino stationary phase is an ionizable stationary phase, which can provide hydrophilic partitioning, electrostatic interaction and hydrogen bond interaction for the separation of hydrophilic samples in the hydrophilic chromatography mode, which is very beneficial for the separation of polar or hydrophilic substances (TrAC Trends in Analytical Chemistry 2020,124,115570). Spermine, as a polyamino compound with a long straight chain structure, is expected to become an ideal material for preparing a hydrophilic chromatography stationary phase. Therefore, the present invention bonds spermine to the silica surface to prepare a spermine-bonded silica gel hydrophilic chromatography stationary phase, and uses it for the separation of base nucleosides and the quantitative analysis of uric acid and creatinine in urine. So far, there are no reports and products based on spermine-bonded silica gel hydrophilic chromatography stationary phases. Summary of the Invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a method for preparing a spermine-bonded silica gel hydrophilic chromatography stationary phase;

[0005] Another object of the present invention is to study the chromatographic separation performance of the above-mentioned spermine-bonded silica gel hydrophilic chromatographic stationary phase.

[0006] Technical solution: The preparation method of a spermine-bonded silica gel hydrophilic chromatography stationary phase of the present invention comprises the following specific preparation steps:

[0007] Step (1), preparation of epoxypropyl functionalized silica gel: spherical silica gel and epoxypropyl silane coupling agent are ultrasonically dispersed in an organic solvent, heated at a constant temperature of 50 to 100° C., and mechanically stirred under nitrogen for 6 to 24 hours to obtain epoxypropyl functionalized silica gel;

[0008] Step (2), preparation of spermine bonded silica gel: the epoxypropyl functionalized silica gel prepared in step (1) and spermine are uniformly mixed in an organic solvent, heated at a constant temperature of 40 to 80° C., and mechanically stirred under the protection of nitrogen for 6 to 24 hours. The reaction product is washed with an organic solvent and water, centrifuged, and dried to obtain a spermine bonded silica gel hydrophilic chromatographic stationary phase.

[0009] Furthermore, in step (1), the structure of the epoxypropyl silane coupling agent is:

[0010]

[0011] Wherein, R1 is a methyl group or an ethyl group, R2 is a methoxy group or an ethoxy group, and n is an integer of 0-6 (preferably 1 to 3).

[0012] Furthermore, in step (1), the mass ratio of the spherical silica gel to the epoxypropyl silane coupling agent is 1:1-5:1.

[0013] Furthermore, in step (1), the organic solvent is one of methanol, acetonitrile, tetrahydrofuran, N,N-dimethylformamide, and toluene.

[0014] Furthermore, in step (2), the organic solvent used for the reaction is one of methanol, ethanol, acetonitrile, tetrahydrofuran, N,N-dimethylformamide, and acetone.

[0015] Furthermore, in step (2), the mass ratio of the epoxypropyl functionalized silica gel to spermine is 1:1-5:1.

[0016] Furthermore, in step (2), the organic solvent used for washing is one or more of acetone, methanol, water and ethanol.

[0017] Furthermore, the spermine-bonded silica gel hydrophilic chromatography stationary phase prepared by the method is used in the separation of base nucleosides.

[0018] Furthermore, the spermine-bonded silica gel hydrophilic chromatography stationary phase prepared by the method is used in the separation and quantitative detection of uric acid and creatine in urine.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following characteristics: 1. Novel structure: The present invention proposes to modify the silica gel surface with a long straight-chain polyamino compound spermine as a bonding phase. In addition to providing hydrophilic partitioning, electrostatic interaction and hydrogen bond interaction for the separation of highly polar and hydrophilic samples, the long straight-chain polyamino structure of the stationary phase can further improve its chromatographic separation selectivity, making it very suitable for use as a hydrophilic chromatographic stationary phase; 2. The spermine-bonded silica gel chromatographic stationary phase provided by the present invention has good separation selectivity for base nucleoside substances, and has great application potential in the separation, analysis and purification preparation of various highly polar and hydrophilic samples; 3. The preparation process of the spermine-bonded silica gel chromatographic stationary phase provided by the present invention is simple and reliable, which is conducive to industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1This is an infrared spectrum (FTIR) diagram of the spermine-bonded silica gel hydrophilic chromatography stationary phase of the present invention;

[0021] Figure 2 The chromatogram of the separation of 14 nucleosides on a spermine-bonded silica gel hydrophilic chromatography stationary phase in the embodiment of the present invention is shown; wherein the corresponding analytes are: (1) thymine, (2) uracil, (3) thymidine, (4) 2′-deoxyuridine, (5) 2′-deoxyadenosine, (6) adenine, (7) uridine, (8) adenosine, (9) hypoxanthine, (10) cytosine, (11) 2′-deoxycytidine, (12) inosine, (13) cytidine, and (14) guanosine;

[0022] Figure 3 is a comparison of the chromatograms of spiked and unspiked urine samples in an embodiment of the present invention;

[0023] Figure 4 The standard curve of uric acid and creatine in the examples of the present invention. DETAILED DESCRIPTION

[0024] In order to more clearly illustrate the technical solution of the present invention, the technical solution of the present invention is further described in detail below with reference to the accompanying drawings:

[0025] The present invention provides a method for preparing a spermine-bonded silica gel hydrophilic chromatography stationary phase; the specific preparation steps are as follows:

[0026] 1. Preparation method of spermine bonded silica gel hydrophilic chromatography stationary phase:

[0027] a. Preparation of epoxypropyl functionalized silica gel: Spherical silica gel and epoxypropyl silane coupling agent are ultrasonically dispersed in an organic solvent, heated at a constant temperature of 50-100°C, and mechanically stirred under nitrogen for 6-24 hours to obtain epoxypropyl functionalized silica gel;

[0028] The mass ratio of the spherical silica gel and the epoxypropyl silane coupling agent used is 1:1-5:1;

[0029] The structure of the epoxypropyl silane coupling agent used is:

[0030]

[0031] wherein R1 is a methyl group or an ethyl group, and R2 is a methoxy group or an ethoxy group; n is an integer of 0 to 6 (preferably 1 to 3);

[0032] The organic solvent used is an organic solvent that has good dispersibility for spherical silica gel, including methanol, acetonitrile, tetrahydrofuran, N,N-dimethylformamide, toluene, etc. The amount of organic solvent used is 5mL-30mL of organic solvent per gram of silica gel.

[0033] b. Preparation of a spermine-bonded silica gel hydrophilic chromatographic stationary phase: uniformly mix the glycidyl-functionalized silica gel obtained in step a and spermine in an organic solvent, heat at a constant temperature of 40-80° C., and mechanically stir under nitrogen for 6-24 hours. The reaction product is washed with an organic solvent and water, centrifuged, and dried to obtain a spermine-bonded silica gel hydrophilic chromatographic stationary phase;

[0034] The mass ratio of the added epoxypropyl functionalized silica gel to spermine is 1:1-5:1.

[0035] The organic solvent used for the reaction is an organic solvent that has good dispersibility for the epoxypropyl functionalized silica gel, and can be one of methanol, ethanol, acetonitrile, tetrahydrofuran, N,N-dimethylformamide, and acetone.

[0036] The amount of organic solvent used is 5 mL to 30 mL of organic solvent per gram of epoxypropyl functionalized silica gel.

[0037] The organic solvent used for washing is one or more of water, methanol, acetone and ethanol.

[0038] 2. Structure of spermine bonded silica gel hydrophilic chromatography stationary phase:

[0039] 1. Elemental analysis

[0040] Table 1 shows the elemental analysis results of the spermine-bonded silica gel hydrophilic chromatography stationary phase. The elemental analysis results show that the C, N, and H contents of Sil-SMP are significantly increased compared to bare silica gel, and the presence of N element indicates that spermine has been successfully modified on the surface of the silica gel filler.

[0041] Table 1 Elemental analysis of spermine bonded silica gel hydrophilic chromatography stationary phase

[0042]

[0043] 2. Infrared spectroscopy (FT-IR):

[0044] Figure 1 This is the infrared spectrum (FTIR) of spermine bonded silica gel hydrophilic chromatography stationary phase; in the infrared spectrum, 1636cm -1 The absorption peak at corresponds to the bending vibration peak of the NH bond; the presence of this absorption peak confirms the successful bonding of spermine to the silica gel surface.

[0045] Example 1

[0046] Weigh 2.5g of spherical silica gel and 1.182g of 3-(2,3-epoxypropoxy)propyltrimethoxysilane, ultrasonically disperse them in 20mL of anhydrous toluene, maintain constant temperature at 100℃, and mechanically stir for 24h under nitrogen protection. Place the functionalized silica gel suspension after the reaction in a centrifuge tube, cool it, and spin it at 8000r·min. -1 The silica gel was centrifuged at a speed of 5 min; the functionalized silica gel after centrifugation was taken out and washed three times with anhydrous toluene and anhydrous acetonitrile respectively to obtain 3-(2,3-epoxypropoxy)propyltrimethoxysilane functionalized silica gel; then, the silica gel after centrifugation was placed in a 100 mL round-bottom flask, 20 mL of acetonitrile and 0.986 g of spermine were added, and the mixture was kept at a constant temperature of 60 ° C and mechanically stirred under nitrogen for 24 h; the suspension after reaction was heated at 8000 r·min -1 The mixture was centrifuged at a speed of 100°C for 5 minutes, the supernatant was removed, and the mixture was washed three times by centrifugation with anhydrous acetonitrile. The mixture was then washed several times with water, methanol, and ethanol in sequence, and then dried in an oven at 60°C overnight to obtain a spermine-bonded silica gel hydrophilic chromatographic stationary phase (Sil-SPM). The obtained chromatographic filler was loaded into a 150 mm × 4.6 mm id stainless steel chromatographic column to obtain Sil-SPM chromatographic column A.

[0047] Example 2

[0048] Weigh 3.0g of spherical silica gel and 2.364g of 3-(2,3-epoxypropoxy)propyltriethoxysilane, ultrasonically disperse them in 20mL of anhydrous methanol, maintain constant temperature at 100℃, and mechanically stir for 24h under nitrogen protection. Place the functionalized silica gel suspension after the reaction in a centrifuge tube, cool it, and centrifuge it at 8000r·min. -1 The silica gel was centrifuged at a speed of 5 min; the functionalized silica gel after centrifugation was taken out and washed three times with anhydrous ethanol and anhydrous methanol respectively to obtain 3-(2,3-epoxypropoxy)propyltriethoxysilane functionalized silica gel; then, the silica gel after centrifugation was placed in a 100 mL round-bottom flask, 20 mL of methanol and 1.510 g of spermine were added, and the mixture was kept at a constant temperature of 50 ° C and mechanically stirred under nitrogen for 24 h; the suspension after the reaction was heated at 8000 r·min -1 The mixture was centrifuged at a speed of 100 nm for 5 minutes, the supernatant removed, and the packing material washed several times with methanol, water, ethanol, and acetone, followed by drying in an oven at 60°C overnight to obtain a spermine-bonded silica gel hydrophilic chromatographic stationary phase. The resulting chromatographic packing material was loaded into a 150 mm × 4.6 mm ID stainless steel column to produce Sil-SPM column B. The column efficiency of the resulting column B was comparable to that of column A.

[0049] Example 3

[0050] The difference from Example 1 is that 3-(2,3-epoxypropyloxy)propylmethyldimethoxysilane is used instead of 3-(2,3-epoxypropyloxy)propyltrimethoxysilane; the chromatographic retention ability of the prepared Sil-SPM chromatographic column C is slightly weaker than that of Sil-SPM chromatographic column A.

[0051] Example 4

[0052] The Sil-SPM chromatographic column A obtained in Example 1 was used for the separation and analysis of base nucleosides; Figure 2 The results show that the separation effect of spermine-bonded silica gel hydrophilic chromatographic stationary phase on 14 base nucleosides can be achieved within 25 minutes. The 14 base nucleosides (thymine, uracil, thymidine, 2'-deoxyuridine, 2'-deoxyadenosine, adenine, uridine, adenosine, hypoxanthine, cytosine, 2'-deoxycytidine, inosine, cytidine, guanosine) are completely separated, and the chromatographic peaks are symmetrical.

[0053] Chromatographic conditions were: 90% acetonitrile, 10% ammonium formate aqueous solution (50 mM, pH 6.5); column temperature 35°C, UV detector (UV) 254 nm, flow rate 1.0 mL min -1 .

[0054] Example 5

[0055] The Sil-SPM chromatographic column A obtained in Example 1 was used to determine the contents of uric acid and creatinine in urine;

[0056] Chromatographic conditions: 77% acetonitrile, 8% 50 mM ammonium formate (pH 6.2), column oven temperature 35°C, UV detector (UV) 254 nm, flow rate 1 mL min -1 ;

[0057] (1) Qualitative analysis:

[0058] The peak positions of uric acid and creatine in urine samples were determined by adding standard substances to actual urine samples. The comparison between spiked and unspiked urine samples is shown in the figure below. Figure 3 As shown in the figure, it can be judged from the figure that chromatographic peak 1 is creatine liver and chromatographic peak 2 is uric acid;

[0059] (2) Drawing of standard curve:

[0060] Figure 4 The standard curve of uric acid and creatine liver is shown in Figure 2. By parallel determination of 0.01mmol·L -1 , 0.025mmol·L -1 , 0.1mmol·L -1 , 0.5mmol·L -1 , 2.5mmol·L -1、10mmol·L -1 , 25mmol·L -1 , 50mmol·L -1 、100mmol·L -1 The concentration of uric acid and creatinine standard solution was measured three times, and the corresponding relationship between the mean peak area of ​​creatinine liver and uric acid and the sample concentration was used to perform linear fitting to obtain the standard curve of uric acid and creatinine liver.

[0061] Table 2 shows the regression equation, linear range and correlation coefficient of the analytes. The linear correlation coefficients R 2 They are 0.9999 and 0.9995 respectively.

[0062] Table 2 Linear range and correlation coefficient of regression equation of analytes

[0063]

[0064]

[0065] (3) Quantitative experiment:

[0066] Table 3 shows the average concentrations of uric acid and creatinine in mg / L obtained by diluting urine samples from three different subjects 100 times and measuring them three times in parallel. -1 ) and relative standard deviation RSD;

[0067] Table 3 Results of determination of uric acid and creatine content in urine samples

[0068]

[0069] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions based on the principles of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, various improvements and modifications that do not depart from the principles of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a spermine-bonded silica gel hydrophilic chromatography stationary phase, characterized in that: First, a glycidyl silane coupling agent is bonded to the surface of spherical silica gel through a silanization reaction. Then, the spermine molecule is bonded to the surface of the glycidyl functionalized silica gel through an epoxy ring-opening reaction between the amino group on the spermine molecule and the glycidyl group, thereby preparing a spermine-bonded silica gel hydrophilic chromatographic stationary phase. The purpose of the glycidyl functionalization step is to introduce glycidyl functional groups that are easily reactive with spermine onto the silica gel surface. Then, using the glycidyl group as a bridging group, spermine can be bonded to the silica gel surface. The specific preparation steps are as follows: Step (1), preparing epoxypropyl functionalized silica gel; Step (2), preparing a spermine-bonded silica gel hydrophilic chromatography stationary phase.

2. The method for preparing a spermine bonded silica gel hydrophilic chromatography stationary phase according to claim 1, wherein: In step (1), the preparation method of the epoxypropyl functionalized silica gel is specifically as follows: spherical silica gel and epoxypropyl silane coupling agent are ultrasonically dispersed in an organic solvent, heated at a constant temperature of 50 to 100° C., and mechanically stirred under nitrogen protection for 6 to 24 hours to obtain epoxypropyl functionalized silica gel; In step (2), the preparation method of the spermine-bonded silica gel hydrophilic chromatography stationary phase is specifically as follows: the glycidyl functionalized silica gel obtained in step (1) and spermine are uniformly mixed in an organic solvent, heated at a constant temperature of 40 to 80° C., mechanically stirred for 6 to 24 hours under the protection of nitrogen, and the reaction product is washed with an organic solvent and water, centrifuged, and dried to obtain the spermine-bonded silica gel hydrophilic chromatography stationary phase.

3. The method for preparing a spermine bonded silica gel hydrophilic chromatography stationary phase according to claim 2, characterized in that: In step (1), the structure of the epoxypropyl silane coupling agent is shown in formula (I): Wherein, R1 is one of methyl or ethyl; R2 is one of methoxy or ethoxy; n=an integer from 0 to 6.

4. The method for preparing a spermine bonded silica gel hydrophilic chromatography stationary phase according to claim 2, wherein: In step (1), the mass ratio of the spherical silica gel to the epoxypropyl silane coupling agent is 1:1-5:

1.

5. The method for preparing a spermine bonded silica gel hydrophilic chromatography stationary phase according to claim 2, characterized in that: In step (1), the organic solvent is one of methanol, acetonitrile, tetrahydrofuran, N,N-dimethylformamide, and toluene.

6. The method for preparing a spermine bonded silica gel hydrophilic chromatography stationary phase according to claim 2, wherein: In step (2), the organic solvent used for the reaction is one of methanol, ethanol, acetonitrile, tetrahydrofuran, N,N-dimethylformamide, and acetone.

7. The method for preparing a spermine-bonded silica gel hydrophilic chromatography stationary phase according to claim 2, characterized in that: In step (2), the mass ratio of the epoxypropyl functionalized silica gel to spermine is 1:1-5:

1.

8. The method for preparing a spermine-bonded silica gel hydrophilic chromatography stationary phase according to claim 2, characterized in that: In step (2), the organic solvent used for washing is one or a combination of acetone, methanol, water and ethanol.

9. Use of the spermine bonded silica gel hydrophilic chromatography stationary phase prepared by the preparation method according to any one of claims 1 to 8 in the separation of base nucleosides.

10. Use of the spermine bonded silica gel hydrophilic chromatography stationary phase prepared by the preparation method according to any one of claims 1 to 8 in the separation and quantitative detection of uric acid and creatinine in urine.

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