Preparation and application of quinine functionalized polyvinylimidazole modified silica gel stationary phase for hydrophilic interaction chromatography
Zhou Xing, Chen Jia, Zhang Yingshan, Zhao Liang, Qiu Hongdeng*
Chromatography, 2020, 38 (4): 438-444
DOI: 10.3724/SP.J.1123.2019.06008
abstract
A novel quinine functionalized polyvinylimidazole modified silica gel stationary phase (Si PIM QN) was prepared by surface free radical chain transfer polymerization and nucleophilic substitution reaction. The stationary phase was characterized by elemental analysis and infrared spectroscopy, and its chromatographic performance was evaluated by HILIC mode. The results showed that the immobilization was more selective than 5 amino acids, 9 sulfonamides and 10 nucleotides. The effects of acetonitrile volume fraction in mobile phase and ammonium acetate concentration in water phase on the retention behavior of separated substances were investigated. The repeatability of stationary phase separation was further investigated. The RSD of retention time was 0.08% ~ 2.30% (n = 10). The method is simple and has excellent separation performance. It is expected to be used in the separation and analysis of hydrophilic substances in sulfonamides and biological samples.
As the core of chromatographic separation technology, chromatographic stationary phase is the basis of promoting the development of chromatographic separation technology. In 1990, Alpert established the hydrophilic interaction chromatography (HILIC). Under the separation mode of hydrophilic interaction, the substances with strong polarity and strong hydrophilicity can be separated and detected, which makes up for the shortcomings of reversed-phase chromatography, and makes HILIC rapidly popularized and applied. The mobile phase of HILIC consists of a certain salt concentration of aqueous solution and a high proportion of organic solvent. The stationary phase is a strong polar adsorbent, and the analyte is usually a polar compound. Therefore, the more hydrophilic the packing is, the stronger the polarity of the solvent is, and the stronger the retention of solute on the column is. With the increase of the proportion of water system, the solute is gradually eluted, so that the substance to be separated can be separated. In recent years, a large number of literatures have reported that the organic small molecules of polar functional groups and hydrophilic carbon nanomaterials are bonded to the surface of silica gel, which can be used for the separation and detection of hydrophilic substances such as nucleosides, amino acids and natural drugs in HILIC mode.
Ionic liquids (ILS) are a kind of non molecular substances composed of organic cations and anions, which are generally liquid at room temperature and atmospheric pressure, so they are also called room temperature molten salts. Because two nitrogen atoms in imidazole ring can provide active sites for post modification, imidazole cationic liquids with different functionalities can be easily obtained. At present, the work of imidazole stationary phases has been widely reported. On the basis of a lot of research work, Zhang et al. Reviewed the method of further modifying imidazole ring by nucleophilic substitution reaction at N3 position in 1H imidazole and polyvinylimidazole, which provided a broad idea for the development of different types of imidazole modified silica gel stationary phase.
Quinine, commonly known as cinchona alkaloid, is the main alkaloid in the bark of cinchona and its homologous plants. Quinine has benzopyridine, tertiary ammonium positive center, multiple chiral sites, and hydroxyl and terminal olefin functional groups which are easy to modify. It is suitable for chromatographic separation and analysis as a modified small molecule bonded on the surface of silica gel or as a monomer polymerized into a whole material. At present, most of the reported stationary phases of quinine are reversed phase and strong anion exchange mode, which mainly use the π - π interaction between benzopyridine ring of quinine and the compounds to be separated and the tertiary ammonium cation structure of quinine. In this study, the modified quinine was applied to HILIC mode. Two main reasons were considered. In HILIC mode, quinine stationary phase with tertiary amine cation structure can provide electrostatic interaction for the separation of ionic analytes; (2) The methoxy and pyridine rings in quinine, as well as the amide and ester bonds formed by isocyanate bridging, will significantly increase the polarity of the stationary phase, and provide abundant sites for hydrogen bond interaction with analytes, so as to improve the separation performance of the chromatographic column in the hydrophilic interaction mode.
In this work, a quinine functionalized polyvinylimidazole modified silica gel stationary phase (Si PIM QN) was prepared by combining imidazole ionic liquid with quinine. Firstly, vinyl imidazole was polymerized on the surface of silica gel by free radical chain transfer polymerization, then quinine modified by 2-chloroethyl isocyanate was grafted on the N3 position of polyvinylimidazole to prepare Si PIM QN, and the separation ability of hydrophilic chromatography was investigated.
01
Preparation of SIL PIM QN
Preparation of low eutectic solvent (DES)
CHCl and EG were added into a round bottom flask according to the ratio of 1:3 substance, and then put into an oil bath at 60 ℃ and stirred to a clear and transparent solution by magnetic force at a speed of 300 R / min. the low eutectic solvent needed for this experiment was obtained.
Preparation of thiopropyl modified silica gel (Si MPS)
Weigh 6.0 g of silica gel for ultrasonic dispersion, place it in a three port round bottom flask containing 50 ml DES, add 2.9 ml of 3-mercaptopropyltriethoxysilane into the three port flask one by one with flow injection pump, then place the three port flask in an oil bath at 60 ℃ and mechanically stir it for 24 hours at 250 R / min; after the reaction is cooled to room temperature, wash the product with ethanol, ultrapure water and ethanol for three times respectively, The final silmps was dried in an oven at 60 ℃.
Preparation of polyvinylimidazole modified silica gel (SIL PIM)
3.0 g of thiopropyl silica gel was ultrasonically dispersed in a three port round bottom flask containing 30 ml des by surface free radical chain transfer polymerization. 0.02 g of AIBN was added first, and then 2.0 ml of vinyl imidazole was added to the three port round bottom flask dropwise under mechanical agitation. The polymerization was carried out in an oil bath at 60 ℃ under the protection of nitrogen for 20 h. After the reaction was cooled to room temperature, the reaction was carried out, The product was washed by centrifugation with ethanol, ultrapure water and ethanol for three times respectively and dried in an oven at 60 ℃ to obtain silpim.
Preparation of SIL PIM QN
Firstly, 2.3 g of quinine was dissolved in a three port flask containing 50 ml of anhydrous dichloromethane (water removed by molecular sieve) and placed in an ice water bath; 0.6 ml of 2-chloroethyl isocyanate was added to 10 ml of anhydrous dichloromethane, and then added to quinine solution drop by drop through a flow injection pump, and the mixed solution was reacted at room temperature for 16 h. After the reaction, the mixture was concentrated with a rotary evaporator, 2-chloroethyl isocyanate-modified quinine (CI QN) was obtained and dissolved in 50 ml ethanol. 2.8 g Si PIM was added under the assistance of ultrasound. The reaction was carried out under the protection of nitrogen at 85 ℃ for 48 hours. After the reaction, the product was centrifuged with ethanol, ultrapure water and ethanol for three times respectively and dried in an oven at 60 ℃. Finally, Si PIM QN was obtained. Fig. 1 is a schematic diagram of the preparation process.
Fig. 1 Preparation of SIL PIM QN
Fig. 1 Schematic diagram for the preparation of Sil-PIm-Qn
Sil-PIm-Qn: quinine-functionalized poly(1-vinylimidazole)-modified hydrophilic silica stationary phase; Sil-MPS: γ-mercaptopropyltriethoxysilane-modified silica; DES: deep eutectic solvents.
02
Chromatographic conditions
The gas flow and temperature of the detector are 1.5 L / min and 60 ℃, respectively, and the detection wavelength of the detector is 254 nm; the column is Si PIM QN; the column temperature is 30 ℃; the flow rate is 1 ml / min; the sample volume is 10 μ L.
Separation of amino acids: mobile phase was acetonitrile-0.10 mol / L ammonium acetate aqueous solution (85:15, V / V); isoelution. Separation of sulfonamides: mobile phase was (a) acetonitrile and (b) 0.02 mol / L ammonium acetate aqueous solution; gradient elution conditions were 0-12 min, 95% a; 12-30 min, 87% a. The mobile phase was (a) acetonitrile and (b) 0.10 mol / L ammonium acetate aqueous solution; gradient elution conditions were 0-10 min, 93% a; 10-13 min, 93% a-83% a; 13-30 min, 83% a.
conclusion
In this study, quinine functionalized polyvinylimidazole modified silica gel was prepared by surface free radical chain transfer polymerization and nucleophilic substitution reaction. The fixed relative hydrophilic material has excellent separation selectivity and good separation repeatability, and has a broad application prospect in the separation and analysis of hydrophilic materials such as base nucleosides in sulfonamides and biological samples.
China Chemical Society:
http://www.ccspublishing.org.cn/article/doi/10.3724/SP.J.1123.2019.06008
China HowNet:
http://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CJFD&filename=SPZZ202004011
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