CN115624960A - Mixed-mode chromatographic stationary phase and preparation method and application thereof - Google Patents
Mixed-mode chromatographic stationary phase and preparation method and application thereof Download PDFInfo
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- CN115624960A CN115624960A CN202211494733.6A CN202211494733A CN115624960A CN 115624960 A CN115624960 A CN 115624960A CN 202211494733 A CN202211494733 A CN 202211494733A CN 115624960 A CN115624960 A CN 115624960A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/282—Porous sorbents
- B01J20/283—Porous sorbents based on silica
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/20—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the sorbent material
- B01D15/206—Packing or coating
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/265—Adsorption chromatography
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
- B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
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- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention discloses a mixed-mode chromatographic stationary phase as well as a preparation method and application thereof, wherein the mixed-mode chromatographic stationary phase is a HILIC/RPLC/IEC mixed-mode chromatographic stationary phase, and the preparation method comprises the following steps: selectively oxidizing microcrystalline cellulose serving as a raw material into dialdehyde cellulose; grafting imidazole ionic liquid on primary hydroxyl of dialdehyde cellulose by using 4-chlorobutyryl chloride to obtain a dialdehyde cellulose ionic liquid derivative DACL; covalently coupling the DACL obtained in the second step on amino-silica gel to obtain ACL-SIL; and reacting residual aldehyde groups on the ACL-SIL with phenylalanine to obtain the mixed-mode chromatographic stationary phase PCL-SIL. The PCL-SIL retains hydrophilic group carboxyl and increases pi-pi action, so that the stationary phase material has certain hydrophobicity. The results of mechanism analysis show that the PCL-SIL and the analyte have various interactions such as hydrophilicity, hydrogen bonds, pi-pi action, ion exchange, hydrophobicity and the like, and have good separation selectivity and quantitative detection capability on complex actual samples.
Description
Technical Field
The invention relates to the field of materials, in particular to a mixed-mode chromatographic stationary phase, and also relates to a preparation method and application of the mixed-mode chromatographic stationary phase.
Background
High performance liquid chromatography is widely used in the material science, environmental science, biological science and other directions, and has strong capability in the aspects of separation, qualitative and quantitative. The chromatographic stationary phase is used as the core of high performance liquid chromatography, and in order to meet the requirements of high selectivity and high sensitivity when complex samples are analyzed, the development of a novel chromatographic stationary phase with high separation efficiency and good selectivity has become a hotspot of research in the separation science field.
In a liquid chromatographic column, a C18 reversed-phase chromatographic column has the widest application range and is generally applied to separation and analysis of nonpolar substances or weakly polar substances, a strongly polar compound is weaker to be retained in a reversed-phase chromatographic fixed phase, the separation effect is poor, and the acting force of the C18 reversed-phase chromatographic column is single. HILIC can separate hydrophilic and polar substances well, has certain advantages and is a good supplement for RPLC. However, as the species of substances in the sample to be analyzed become more and more complex, the requirement for the separation capability of the chromatographic column is gradually increased, and the chromatographic stationary phase in a single mode is difficult to meet the separation requirement of a complex sample system.
Mixed Mode Chromatography (MMC) is a method in which two or more different interactions exist between a stationary phase and an analyte, separation can be performed according to different characteristics of an analyte, separation selectivity is improved, better separation selectivity and higher sample loading capacity are provided, and the MMC is suitable for separation and analysis of complex samples, and is one of the hot spots in the current chromatographic stationary phase research. At present, besides the two modes of the mixed mode stationary phase, three and more modes of the mixed mode stationary phase development also appear successively, such as RP/HILIC/IEC.
Cellulose is the most abundant renewable resource in nature, not only has rich source and low price, but also has the advantages of renewability, microbial degradation, safety, no toxicity and the like, accords with the green development concept, is widely concerned all the time, is applied to various fields and shows excellent performance. Dialdehyde cellulose is obtained by oxidizing low-cost cellulose, has the advantages of green safety and degradability, is an excellent intermediate, contains abundant hydroxyl groups and has strong hydrophilicity. However, no reports are provided for preparing HILIC/RPLC/IEC mixed mode chromatography stationary phase materials by using hydrophilic cellulose as raw materials.
Disclosure of Invention
In view of the above, the first objective of the present invention is to provide a mixed mode chromatography stationary phase.
In order to achieve the purpose, the invention adopts the following technical scheme:
the mixed-mode chromatographic stationary phase is a HILIC/RPLC/IEC mixed-mode chromatographic stationary phase, and the structural formula of the mixed-mode chromatographic stationary phase is as follows:
wherein the HILIC mode is a hydrophilic interaction chromatography mode; the RPLC mode is a reversed phase chromatography mode; the IEC mode is an ion exchange chromatography mode.
The invention also provides a method for preparing the mixed-mode chromatographic stationary phase, which comprises the following steps:
the first step, selectively oxidizing microcrystalline cellulose as a raw material to prepare dialdehyde cellulose;
secondly, grafting imidazole ionic liquid on primary hydroxyl of the dialdehyde cellulose by using 4-chlorobutyryl chloride to obtain a dialdehyde cellulose ionic liquid derivative DACL;
step three, covalently coupling the DACL obtained in the step two on amino silica gel by using Schiff base reaction to obtain dialdehyde cellulose derivative bonded silica gel ACL-SIL;
and reacting residual aldehyde groups on the dialdehyde cellulose derivative bonded silica gel ACL-SIL with phenylalanine to obtain the mixed-mode chromatographic stationary phase PCL-SIL.
In the present invention, the first step specifically includes the following: and (2) selectively oxidizing the microcrystalline cellulose by using sodium periodate as an oxidizing agent under a dark condition, quenching the reaction by using glycol after the reaction is finished, and then sequentially separating, washing and drying to obtain the dialdehyde cellulose.
In the invention, the second step is a two-step reaction, which specifically comprises the following steps: firstly, carrying out esterification reaction on 4-chlorobutyryl chloride and primary hydroxyl of dialdehyde cellulose in DMF (dimethyl formamide); and carrying out amidation reaction on the intermediate product obtained by esterification and N-methylimidazole in DMF to obtain the dialdehyde cellulose ionic liquid derivative.
The invention also provides application of the mixed-mode chromatographic stationary phase as a HILIC/RPLC/IEC mixed-mode chromatographic stationary phase material in high performance liquid chromatography. The concrete application is as follows:
the mixed mode chromatographic stationary phase material PCL-SIL has hydrophilic performance, and can be used for selectively separating nucleoside bases including 4, 6-dichloropyrimidine, thymine, uracil, 5-methylurea, uridine, adenosine and the like in a hydrophilic chromatographic mode.
The benzene ring in the mixed-mode chromatographic stationary phase material PCL-SIL and the benzene ring in an analyte have pi-pi action, and the selective separation of benzene ring substances (such as anilines, monosubstituted benzene, phenol substances and the like) can be realized in a reverse phase mode. Among them, the aniline species include, but are not limited to, p-phenylenediamine, o-phenylenediamine, p-methylaniline, acetanilide, p-bromoaniline, p-nitroaniline, and 1-naphthylamine;
monosubstituted benzenes include, but are not limited to, benzyl alcohol, anisole, chlorobenzene, bromobenzene, and iodobenzene;
phenolics include, but are not limited to, p-aminophenol, phenol, m-cresol, and resorcinol.
The mixed mode chromatography stationary phase material PCL-SIL has the function of ion exchange,selective separation of inorganic anions can be achieved in ion exchange chromatography mode. Wherein the inorganic anion includes but is not limited to BrO 3 - 、NO 2 - 、NO 3 - 、Br - 、I - And SCN - 。
The mixed-mode chromatographic stationary phase material PCL-SIL can realize the selective separation of amino acid in HILIC/RPLC/IEC mode. Amino acids include, but are not limited to, tyrosine, phenylalanine, and tryptophan.
Compared with the prior art, the invention has the following advantages:
the cellulose has the advantages of rich source, low price, reproducibility, microbial degradation, safety, no toxicity and the like, and is a widely applied natural polymer material. The invention takes cellulose as raw material, adopts ionic liquid to modify macromolecular cellulose, and is fixed on silicon amide spheres, thereby not only having the mechanical strength of silica gel filler, but also increasing the binding sites on the material; the ionic liquid modified macromolecular cellulose fixed on the amino acid silicon spheres introduces phenylalanine on an ACL-SIL material through Schiff base reaction, maintains hydrophilic group carboxyl, and increases the pi-pi action of a stationary phase material, so that the stationary phase material has certain hydrophobicity.
The PCL-SIL has a HILIC/RPLC/IEC mixed separation mode, and the results of mechanism analysis show that the PCL-SIL and an analyte have various interactions such as hydrophilic interaction, hydrogen bond interaction, pi-pi action, ion exchange interaction, hydrophobic interaction and the like, and have good separation selectivity and quantitative detection capability on complex practical samples.
The mixed-mode chromatographic stationary phase material PCL-SIL has the advantages of high separation speed, high separation selectivity and the like, can make up for the defects of single-mode chromatography in complex sample analysis, and improves the separation efficiency. Specifically, the method comprises the following steps: the PCL-SIL prepared by the method can realize the separation of hydrophilic compounds such as nucleoside base substances and the like, can realize the good separation of hydrophobic compounds such as aniline substances, phenol substances, monosubstituted benzene and the like, can realize the selective separation of inorganic anions, can realize the separation of amino acid in three chromatographic modes, and has good separation effect.
The mobile phase of the invention is preferably acetonitrile/water, potassium chloride or methanol water and the like, and the mobile phase is low in toxicity or non-toxic, so that a strong toxic organic reagent required by normal phase chromatography is avoided, and the harm to the body of a detector is reduced.
Drawings
FIG. 1 is a structural formula of a mixed mode chromatography stationary phase material according to the present invention.
FIG. 2A is a synthetic pathway diagram of a mixed mode chromatography stationary phase material according to the invention.
FIG. 2B is the synthesis diagram of mixed mode chromatographic stationary phase material PCL-SIL and hydrophilic interaction chromatographic stationary phase material PCL-SIL.
FIG. 3 shows NH in the present invention 2 SEM scans of SIL and PCL-SIL.
FIG. 4 shows NH in the present invention 2 TEM-EDS characterization of SIL and PCL-SIL.
In fig. 4: a is NH 2 -TEM images of SIL silicon spheres; B. c and D are TEM representation images of PCL-SIL under different multiples respectively; e is an element surface distribution map of the PCL-SIL silicon spheres; f is EDS spectrum of PCL-SIL silicon ball.
FIG. 5 shows NH in the present invention 2 -infrared spectra of SIL and PCL-SIL.
FIG. 6 shows a solid nuclear magnetic spectrum of PCL-SIL in the present invention.
FIG. 7 is a thermogravimetric analysis of PCL-SIL in the present invention.
FIG. 8 shows the contact angle of PCL-SIL in the present invention.
FIG. 9 is a chromatogram obtained by separating six kinds of nucleoside base substances in a hydrophilic interaction chromatography mode by using a PCL-SIL column of the present invention.
FIG. 10 is a chromatogram for separating seven anilines in a reversed phase chromatography mode on a PCL-SIL column of the present invention.
FIG. 11 is a chromatogram of a PCL-SIL column of the present invention in reversed phase chromatography mode for the separation of five mono-substituted benzene species.
FIG. 12 is a chromatogram of a PCL-SIL column of the present invention in reversed phase chromatography mode for the separation of four phenols.
FIG. 13 is a selective separation chromatogram of a PCL-SIL column of the present invention for six inorganic anions in ion exchange chromatography separation mode.
FIG. 14 is a chromatogram for separating three amino acids on a PCL-SIL column of the present invention in ion exchange chromatography separation mode.
FIG. 15 shows retention factors for three amino acids in example 8 of the present inventionkWith acetonitrile concentration in the mobile phaseC ACN A graph of the relationship (c).
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings, which illustrate embodiments of the present invention, but the present invention is not limited to the embodiments.
It is emphasized that, unless otherwise specified, all reagents used in the present invention are commercially available reagents; the pure water is secondary distilled water, the acetonitrile/water is prepared according to the volume ratio, and the methanol/water is prepared according to the volume ratio.
Example 1 mixed mode chromatography stationary phase according to the invention
Preparation of carboxylated dialdehyde cellulose grafted imidazole ion liquid type hydrophilic interaction chromatographic stationary phase
The preparation process of the invention comprises the steps of firstly oxidizing cellulose, then grafting ionic liquid, then fixing on amino microspheres, and finally covalently coupling residual aldehyde groups on the cellulose with phenylalanine to obtain the phenylalanine-derivatized dialdehyde cellulose-grafted imidazole ionic liquid type mixed mode chromatographic stationary phase material PCL-SIL. Wherein the structural formula of the mixed mode chromatographic stationary phase material PCL-SIL is shown in figure 1.
The synthetic route of the phenylalanine-derivatized dialdehyde cellulose grafted imidazole ion liquid mixed mode chromatography stationary phase material PCL-SIL is shown in figure 2A, and specifically comprises the following details:
step one, adding 1 g of microcrystalline cellulose (MCC) and 1.283 g of sodium periodate into pure water, and stirring for 5 days at room temperature under the condition of keeping out of the sun; adding 20 mL of ethylene glycol to quench for 1 h;8000 r/min, centrifuging for 8 min to obtain precipitate, washing with pure water twice, washing with anhydrous ethanol, centrifuging, filtering, grinding with mortar, air drying the obtained solid product, and vacuum drying at 40 deg.C for 12 h to obtain solid product dialdehyde cellulose DAC;
secondly, adding 0.5 g of dialdehyde cellulose into 35 mL of DMF, stirring at 0 ℃ under the protection of nitrogen to ensure that the temperature of a mixed system is between 0 and 4 ℃, slowly dropwise adding 500 mu L of 4-chlorobutyryl chloride, and continuously reacting at low temperature for 30 min after dropwise adding; then reacting for 24 hours at room temperature; after the reaction is finished, adding 100 mL of pure water, standing for 1 h, filtering, washing for 2-3 times by using absolute ethyl alcohol, and drying the washed precipitate in vacuum at 40 ℃ for 12 h to obtain an esterification product DAC-Cl of 4-chlorobutyryl chloride and dialdehyde cellulose, wherein the structural formula is as follows:
adding 0.5 g of DAC-Cl into 35 mL of DMF, heating to 60 ℃, dropwise adding 1.0mL of N-methylimidazole after the DAC-Cl is dissolved, and reacting for 24 hours at 60 ℃; introducing the reaction solution into a beaker filled with 200 mL of water, and violently stirring for 5 min; filtering; washing with absolute ethyl alcohol; vacuum drying at 40 ℃ for 12 h to obtain solid powder of the dialdehyde cellulose ionic liquid derivative DACL, wherein the structural formula is as follows:
thirdly, 0.2 g of dialdehyde cellulose ionic liquid derivative DACL is added into 50 mL of pyridine, stirred under the protection of nitrogen and heated to 90 ℃; then adding 1 g of amino silica gel, stirring and reacting for 24 h at 90 ℃ to ensure that aldehyde group of dialdehyde cellulose and amino of the amino silica gel are covalently coupled through Schiff base reaction; filtering with a G4 sand core funnel when the reaction is finished, and then washing for 3 times with hot pyridine, pure water and methanol in sequence; vacuum drying at 60 ℃ for 12 h to obtain dialdehyde cellulose derivative bonded silica gel ACL-SIL with the structural formula:
dissolving 0.5 g of phenylalanine into 60 mL of methanol solution, adding 3 g of ACL-SIL, and reacting for 12 h at 60 ℃ by mechanical stirring; and after the reaction is finished, filtering by using a G4 sand core funnel, washing the product for 3 times by using methanol, pure water and methanol in sequence, removing unreacted phenylalanine, and finally drying in vacuum at 60 ℃ for 12 hours to obtain the mixed-mode chromatographic stationary phase PCL-SIL with the structural formula shown in figure 1.
2. Characterizing the prepared mixed mode chromatographic stationary phase material
1. Using SEM to separately align PCL-SIL and silicon amide spheres (i.e., NH) 2 SIL) were performed and the results are shown in fig. 3. Wherein, A in FIG. 3 is NH 2 SEM scan of SIL, NH 2 SIL has a size of 5 μm and is a smooth surfaced aminosilicone sphere; FIG. 3 is SEM scanning picture of PCL-SIL, which shows that the surface of PCL-SIL is rough, and proves the success of PCL-SIL preparation of mixed mode chromatogram fixed phase material.
2. PCL-SIL was characterized by TEM-EDS and the results are shown in FIG. 4. In FIG. 4A is NH 2 Transmission electron microscopy characterization of SIL, B-D transmission electron microscopy characterization of PCL-SIL at different magnifications. A distinct bonding layer is seen at the silicon ball edge as seen by B in fig. 4.
In FIG. 4, E is the distribution diagram of elements of PCL-SIL silicon spheres, and it is evident that C, N, O and Cl elements are uniformly and densely distributed on the silicon spheres; in addition, the existence of Cl element also proves that DACL is successfully bonded on NH 2 -on the SIL; FIG. 4, wherein F is the spectrum distribution of PCL-SIL silicon spheres, shows that the presence of Cl was also successfully detected on PCL-SIL, further proving that DACL is successfully bonded on NH 2 -on SIL.
3. ACL-SIL and PCL-SIL were characterized by FT-IR spectroscopy, and the results are shown in FIG. 6. From FIG. 6, with NH 2 PCL-SIL at 1640 cm, comparable to SIL -1 C = N stretching vibration peak of imine bond, 1480 cm -1 And 1560 cm -1 It is the characteristic absorption peak of benzene ring, which proves the success of phenylalanine bonding.
4. PCL-SIL was characterized by solid nuclear magnetic resonance and the results are shown in FIG. 7. As can be seen from FIG. 7, the peaks at 10 ppm and 20 ppm in PCL-SIL are assigned to methyl and methylene groups of PCL-SIL, the peak at 37 ppm is assigned to the displacement of methyl carbon in methyl imidazole, the peak at 128 ppm is assigned to the displacement of carbon in imidazole ring in ionic liquid, and the peak at 172 ppm is assigned to the displacement of carbon in carboxyl group, indicating that dialdehyde cellulose is successfully bonded to the surface of silicon spheres, and successfully obtaining the hydrophilic interaction chromatographic stationary phase material.
5. PCL-SIL was analyzed using an elemental analyzer, and the elemental analysis data are shown in Table 1. As can be seen from Table 1, the C content in PCL-SIL increased from 7.96% to 16.17% compared to the aminated silica gel, indicating that the bonding of phenylalanine to ACL-SIL was successful.
TABLE 1 elemental analysis Table
6. Thermal stability analysis was performed on the PCL-SIL prepared according to the present invention using a thermogravimetric analyzer, and the results are shown in fig. 7. A first weight drop is observed at 20-100 ℃, which is caused by evaporation of water adsorbed on the stationary phase; heating to 200 deg.C or above to degrade the organic component of PCL-SIL; when the temperature is increased to 800 ℃, the total weight loss of PCL-SIL is 16.91 percent, which shows that dialdehyde cellulose and phenylalanine are successfully bonded to the surface of the silicon ball and have good thermal stability, and the PCL-SIL can be used as a separation material of a chromatographic column.
7. The contact angle measuring instrument is used for investigating the hydrophilicity and hydrophobicity of the surface of the hydrophilic interaction chromatography stationary phase material, and the result is shown in figure 8. As can be seen from FIG. 8, the contact angle of the hydrophilic interaction chromatography stationary phase material PCL-SIL is 21 degrees, which indicates that the PCL-SIL has good hydrophilicity.
EXAMPLE 2 preparation of PCL-SIL Mixed mode chromatography column
Dispersing 3 g of the mixed-mode chromatographic material PCL-SIL prepared in the example 1 into 60 mL of methanol solution for activation for 12 h, and replacing the supernatant every 4 h;
loading the chromatographic stationary phase (PCL-SIL of the invention) into a stainless steel chromatographic column (15 cm multiplied by 4.6 mm) under 40 MPa, taking methanol as a uniform mixing liquid and a displacement liquid, keeping the pressure at 40 MPa for 30 min, gradually reducing the pressure, and respectively keeping the pressure at 30 MPa and 20 MPa for 10 min; respectively adding a micron-sized sieve plate at two ends of the column, and sealing to obtain a PCL-SIL chromatographic column;
marking the direction, name and date of the prepared PCL-SIL chromatographic column; when the PCL-SIL chromatographic column is used, the PCL-SIL chromatographic column is firstly arranged on a high performance liquid chromatography analyzer, and is washed by acetonitrile (the initial flow rate is 0.2 mL/min) until the base line and the column pressure are stable, and then the flow rate is gradually increased to 1 mL/min until the column pressure is stable.
Example 3 Selective separation of nucleoside bases on a PCL-SIL column in hydrophilic working chromatography mode
Six kinds of nucleoside bases, 4, 6-dichloropyrimidine, thymine, uracil, 5-methylurea, uridine and adenosine, were separated in a hydrophilic mode by using the PCL-SIL column prepared in example 2. In addition, the inventor used the earlier synthesized hydrophilic interaction chromatography stationary phase CCL-SIL (the preparation steps are different from the PCL-SIL of the invention only in that the last step is the carboxylation of the residual aldehyde group in the dialdehyde cellulose derivative bonded silica gel ACL-SIL by sodium hypochlorite, and the specific synthetic route is shown in figure 2B) for the separation of the six nucleoside bases. The chromatographic separation conditions are as follows: mobile phase: acetonitrile/water = 90/10, flow rate: 1 mL/min, detection wavelength: 254 nm, see FIG. 9.
In FIG. 9, the retention characteristics of the CCL-SIL column and the PCL-SIL column are the same, and the substances corresponding to 1 to 6 in the figure are 4, 6-dichloropyrimidine, thymine, uracil, 5-methylurea, uridine and adenosine, respectively. As can be seen from FIG. 9, compared with the separation results of the CCL-SIL column, the PCL-SIL column of the present invention can completely realize the baseline separation of six nucleobases in the hydrophilic mode, which indicates that the present invention also has good hydrophilic performance and good separation selectivity.
EXAMPLE 4 use of a PCL-SIL column for selective separation of anilines
P-phenylenediamine, o-phenylenediamine, p-methylaniline, acetanilide, p-bromoaniline, p-nitroaniline and 1-naphthylamine were separated in reversed phase mode using the PCL-SIL column of example 2; the chromatographic conditions are as follows: mobile phase: methanol/water =15/85, flow rate: 1 mL/min; the detection wavelength was 254 nm, and the results are shown in FIG. 10.
The substances corresponding to the isolated peaks 1 to 7 in FIG. 10 are phenylenediamine, o-phenylenediamine, p-methylaniline, acetanilide, p-bromoaniline, p-nitroaniline and 1-naphthylamine, respectively. As shown in FIG. 10, the PCL-SIL chromatographic column of the present invention realizes the separation of the seven anilines in a reversed phase mode, which is attributed to the pi-pi action between the benzene ring in the PCL-SIL stationary phase and the benzene ring in the substance to be separated.
Example 5 application of the PCL-SIL column of the present invention in the selective separation of monosubstituted benzenes
Using the PCL-SIL chromatographic column in the example 2 to separate benzyl alcohol, anisole, chlorobenzene, bromobenzene and iodobenzene in a reversed phase mode; the chromatographic conditions are as follows: mobile phase: methanol/water =25/75, 20/80, 15/85, and 10/90, flow rate: 1 mL/min; the detection wavelength was 254 nm, and the results are shown in FIG. 11.
The substances corresponding to the separation peaks 1 to 5 in FIG. 11 are benzyl alcohol, anisole, chlorobenzene, bromobenzene and iodobenzene, respectively. As can be seen from FIG. 11, under the condition of mobile phases with different volume ratios, the PCL-SIL chromatographic column can realize the selective separation of benzyl alcohol, anisole, chlorobenzene, bromobenzene and iodobenzene; in addition, when the PCL-SIL chromatographic column prepared by the invention is used for separating the five kinds of monosubstituted benzene, the retention time of the monosubstituted benzene is reduced along with the increase of the methanol content in the mobile phase, and the separation capability of the PCL-SIL in a reversed-phase separation mode is further verified.
Example 6 Selective separation of phenols on a PCL-SIL column in reverse phase mode
P-aminophenol, phenol, m-cresol and resorcinol were separated in reversed phase mode using a PCL-SIL column of example 2; the chromatographic conditions are as follows: mobile phase: methanol/water = 10/90, flow rate: 1 mL/min; the detection wavelength was 254 nm, and the results are shown in FIG. 12.
The substances corresponding to the separation peaks 1-4 in FIG. 12 are aminophenol, phenol, m-cresol and resorcinol, respectively. As can be seen from FIG. 12, the PCL-SIL chromatographic column prepared by the invention realizes good separation of the four phenols in the reversed phase mode, and further verifies the separation capability of the PCL-SIL in the reversed phase separation mode.
EXAMPLE 7 use of a PCL-SIL chromatography column of the invention for the selective separation of inorganic anions
Six inorganic anions were separated by ion exchange using the PCL-SIL column prepared in example 2 under the following chromatographic conditions: the mobile phase was 75 mM potassium chloride, the flow rate was 1 mL/min, the detection wavelength was 210 nm, and the chromatographic separation result is shown in FIG. 13.
In FIG. 13, the species corresponding to separation peaks 1 to 6 are BrO 3 - 、NO 2 - 、NO 3 - 、Br - 、I - And SCN - . As can be seen from fig. 13, the mixed-mode chromatography stationary phase prepared by the present invention can completely realize the selective separation of the above six inorganic anions in the ion exchange mode, and has high selectivity.
Example 8 separation Performance of Mixed-mode PCL-SIL column of the present invention on amino acids in IEC, RPLC and HILIC modes
The PCL-SIL column prepared in example 2 was used for chromatographic separation in IEC mode under the following chromatographic conditions: mobile phase: 100 mM aqueous potassium chloride solution, flow rate: 1 mL/min; detection wavelength: 254 The nm, IEC mode separation spectrum is shown in FIG. 14.
Compared with the PCL-SIL chromatographic column, the PCL-SIL chromatographic column disclosed by the invention realizes baseline separation on three amino acids in an IEC mode, and has better separation performance.
The PCL-SIL column prepared in example 2 was used for chromatographic separation in three modes, RPLC and HILIC, under the following chromatographic conditions: mobile phase: acetonitrile/water, flow rate: 1 mL/min; detection wavelength: 254 And (5) nm. Then with retention factorkAs ordinate, in the concentration of acetonitrile in the mobile phaseC ACN (i.e., volume concentration) is plotted on the abscissa with retention factorkAndC ACN the relationship of (2) is shown in FIG. 15.
As can be seen from the graph of figure 15,C ACN the retention factors k of the three amino acids are completely different at the same concentration when the concentration of acetonitrile in the mobile phase is less than or equal to 0.3 (the corresponding chromatographic mode is an RPLC mode), which indicates that the PCL-SIL chromatography is performedThe column can realize the separation of amino acid in an RPLC mode;C ACN >0.6 (the concentration of acetonitrile in the mobile phase is higher, and the chromatographic separation mode is HILIC mode), the retention factors of the three amino acids under the same concentrationkThe complete difference indicates that the PCL-SIL chromatographic column can completely realize the separation of amino acid in HILIC mode.
In summary, it can be seen from the above examples 3-8 that when the mixed-mode chromatography stationary phase PCL-SIL of the present invention is used as a high performance liquid chromatography stationary phase material, it can perform reverse phase/hydrophilic/ion exchange functions, respectively, perform a good separation function on corresponding substances, and can realize selective separation of amino acids in IEC, RPLC and HILIC modes.
Claims (6)
1. A mixed-mode chromatography stationary phase, characterized by: the mixed-mode chromatographic stationary phase is a HILIC/RPLC/IEC mixed-mode chromatographic stationary phase, and the structural formula is as follows:
2. a method of preparing a mixed mode chromatography stationary phase as defined in claim 1, comprising the steps of:
the first step, selectively oxidizing microcrystalline cellulose as a raw material to prepare dialdehyde cellulose;
secondly, grafting imidazole ionic liquid on primary hydroxyl of the dialdehyde cellulose by using 4-chlorobutyryl chloride to obtain a dialdehyde cellulose ionic liquid derivative DACL;
step three, covalently coupling the DACL obtained in the step two on amino silica gel by using Schiff base reaction to obtain dialdehyde cellulose derivative bonded silica gel ACL-SIL;
and (3) reacting residual aldehyde group on the dialdehyde cellulose derivative bonded silica gel ACL-SIL with phenylalanine to obtain the mixed-mode chromatographic stationary phase PCL-SIL.
3. A process for preparing a mixed mode chromatography stationary phase according to claim 2, characterized in that: the first step specifically comprises the following steps: and (2) selectively oxidizing the microcrystalline cellulose by using sodium periodate as an oxidizing agent under a dark condition, quenching the reaction by using glycol after the reaction is finished, and separating, washing and drying to obtain the dialdehyde cellulose.
4. The method for preparing a mixed mode chromatography stationary phase according to claim 2, wherein: the second step is a two-step reaction, and specifically comprises the following steps: firstly, carrying out esterification reaction on 4-chlorobutyryl chloride and primary hydroxyl of dialdehyde cellulose in DMF (dimethyl formamide); and carrying out amidation reaction on the intermediate product obtained by esterification and N-methylimidazole in DMF to obtain the dialdehyde cellulose ionic liquid derivative.
5. Use of the mixed mode chromatography stationary phase of claim 1 as HILIC/RPLC/IEC mixed mode chromatography stationary phase material in high performance liquid chromatography.
6. Use according to claim 5, characterized in that: the high performance liquid chromatography separation column prepared from the mixed mode chromatography stationary phase selectively separates amino acid under HILIC, RPLC and IEC modes; selectively separating inorganic anions in an ion exchange chromatography mode; selectively separating the nucleobases in a hydrophilic interaction chromatography mode; selectively separating benzene ring substances in a reversed phase chromatography mode.
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