“一锅法”制备聚(苯乙烯-丙烯酸)共聚物改性的硅基固定相及其在混合模式液相色谱中的应用

doi:10.3724/SP.J.1123.2023.01005

摘要/Abstract

摘要：

聚合物作为一种来源广泛、功能基团丰富、生物相容性良好的修饰配体,被广泛用作硅基色谱固定相。本工作以苯乙烯和丙烯酸为聚合单体,乙烯基三甲氧基硅烷为硅烷偶联剂,通过自由基聚合反应一锅法制备了聚(苯乙烯-丙烯酸)共聚物改性的硅基固定相(SiO₂@P(St-b-AA))。傅里叶红外光谱、热重分析、扫描电镜、N₂吸附-脱附、Zeta电势分析等表征手段证明了该固定相已成功合成,且保持了良好的介孔球形结构。在固定相性能评价中,分别采用疏水性分析物、极性分析物和离子型化合物作为探针,对固定相的分离性能和保留机理进行了考察。其中,由于苯环提供的疏水和π-π相互作用,烷基苯和多环芳香烃在固定相上的保留随着流动相中甲醇含量的增加而减弱;由于羧酸基团可以为固定相与核苷碱基等极性溶质之间提供亲水相互作用,随着流动相中乙腈含量的增加,极性分析物的保留逐渐增强;与自制的C18和Amide固定相相比,SiO₂@P(St-b-AA)固定相在反相和亲水模式下均表现出良好的分离性能。此外,通过探究流动相pH对离子型化合物保留的影响,证明了该固定相还具有弱阳离子交换能力。多种分离模式表明SiO₂@P(St-b-AA)固定相可提供多种相互作用,并在不同极性组分分析物的分离中具有良好的应用前景。通过考察制备方法的重复性,证明了该方法具有良好的制备批次稳定性,简便的“一锅法”为新型聚合物改性硅基固定相的发展提供了一种新思路。

关键词: 聚合物改性固定相, 一锅法, 混合模式液相色谱

Abstract:

As modified ligands with a wide range of sources, abundant functional groups, and good biocompatibility, polymers have been widely used in the development of silica-based chromatographic stationary phases. In this study, a poly(styrene-acrylic acid) copolymer-modified silica stationary phase (SiO₂@P(St-b-AA)) was prepared via one-pot free-radical polymerization. In this stationary phase, styrene and acrylic acid were used as functional repeating units for polymerization and vinyltrimethoxylsilane (VTMS) was used as a silane coupling agent to link the copolymer and silica. Various characterization methods, such as Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric analysis (TGA), scanning electron microscopy (SEM), N₂ adsorption-desorption analysis, and Zeta potential analysis, confirmed the successful preparation of the SiO₂@P(St-b-AA) stationary phase, which had a well-maintained uniform spherical and mesoporous structure. The retention mechanisms and separation performance of the SiO₂@P(St-b-AA) stationary phase in multiple separation modes were then evaluated. Hydrophobic and hydrophilic analytes as well as ionic compounds were selected as probes for different separation modes, and changes in the retention of the analytes under various chromatographic conditions, including different methanol or acetonitrile contents and buffer pH values, were investigated. In reversed-phase liquid chromatography (RPLC) mode, the retention factors of alkyl benzenes and polycyclic aromatic hydrocarbons (PAHs) on the stationary phase decreased with increasing methanol content in the mobile phase. This finding could be attributed to the hydrophobic and π-π interactions between the benzene ring and analytes. The retention changes of alkyl benzenes and PAHs revealed that the SiO₂@P(St-b-AA) stationary phase, similar to the C18 stationary phase, exhibited a typical reversed-phase retention behavior. In hydrophilic interaction liquid chromatography (HILIC) mode, as the acetonitrile content increased, the retention factors of hydrophilic analytes gradually increased, and a typical hydrophilic interaction retention mechanism was inferred. In addition to hydrophilic interaction, the stationary phase also demonstrated hydrogen-bonding and electrostatic interactions with the analytes. Compared with the C18 and Amide stationary phases prepared by our groups, the SiO₂@P(St-b-AA) stationary phase exhibited excellent separation performance for the model analytes in the RPLC and HILIC modes. Owing to the presence of charged carboxylic acid groups in the SiO₂@P(St-b-AA) stationary phase, exploring its retention mechanism in ionic exchange chromatography (IEC) mode is of great importance. The effect of the mobile phase pH on the retention time of organic bases and acids was further studied to explore the electrostatic interaction between the stationary phase and charged analytes. The results revealed that the stationary phase has weak cation exchange ability toward organic bases and electrostatically repels organic acids. Moreover, the retention of organic bases and acids on the stationary phase was influenced by the analyte structure and mobile phase. Thus, the SiO₂@P(St-b-AA) stationary phase could provide multiple interactions, as demonstrated by the separation modes described above. The SiO₂@P(St-b-AA) stationary phase showed excellent performance and reproducibility in the separation of mixed samples with different polar components, indicating that it has promising application potential in mixed-mode liquid chromatography. Further investigation of the proposed method confirmed its repeatability and stability. In summary, this study not only described a novel stationary phase that could be used in RPLC, HILIC, and IEC modes but also presented a facile “one-pot” preparation approach that could provide a new route for the development of novel polymer-modified silica stationary phases.

Key words: polymer modified stationary phases, one-pot synthesis, mixed-mode liquid chromatography

中图分类号:

O658

王晓庆, 崔健, 顾一鸣, 王硕, 周谨, 王树东. “一锅法”制备聚(苯乙烯-丙烯酸)共聚物改性的硅基固定相及其在混合模式液相色谱中的应用[J]. 色谱, 2023, 41(7): 562-571.

WANG Xiaoqing, CUI Jian, GU Yiming, WANG Shuo, ZHOU Jin, WANG Shudong. One-pot synthesis of a poly(styrene-acrylic acid) copolymer-modified silica stationary phase and its applications in mixed-mode liquid chromatography[J]. Chinese Journal of Chromatography, 2023, 41(7): 562-571.

图/表 10

图 1 “一锅法”制备SiO2@P(St-b-AA)

Fig. 1 One-pot synthesis of the poly(styrene-acrylic acid) copolymer-modified silica stationary phase (SiO2@P(St-b-AA)) VTMS: vinyltrimethoxylsilane; St: styrene; AA: acrylic acid; AIBN: 2,2-azobis(2-methylpropionitrile).

图 2 SiO2和SiO2@P(St-b-AA)的(a)红外光谱和(b)热重分析曲线

Fig. 2 (a) Fourier transform infrared spectra and (b) thermogravimetric analysis curves of SiO2 and SiO2@P(St-b-AA)

图 3 (a)SiO2, (b)SiO2@P(St-b-AA), 50 MPa压力下(c)装填1次及(d)装填3次后SiO2@P(St-b-AA)的扫描电镜图

Fig. 3 Scanning electron microscopes of (a) SiO2, (b) SiO2@P(St-b-AA), (c) SiO2@P(St-b-AA) after packing one time and (d) three times under 50 MPa pressure

图 4 SiO2和SiO2@P(St-b-AA)的(a)N2吸附-脱附等温线及(b)BJH孔径分布图

Fig. 4 (a) N2 adsorption-desorption isotherms and (b) pore size distributions via BJH method of SiO2 and SiO2@P(St-b-AA)

图 5 疏水性分析物在反相模式下的保留及分离评价

Fig. 5 Evaluation of retention and separation performance of hydrophobic analytes in RPLC mode a, c. effects of MeOH content in mobile phase on the retention factors of (a) alkyl benzenes and (c) PAHs; b, d. separation chromatograms of (b) alkyl benzenes and (d) PAHs on different columns. Mobile phase: MeOH-H2O (50∶50, v/v) for PSA (stationary phase: SiO2@P(St-b-AA)) and Amide columns in (b) and (d), MeOH-H2O (75∶25, v/v) for C18 column in (b) and MeOH-H2O (60∶40, v/v) for C18 column in (d). Peak identifications: (1) toluene, (2) ethylbenzene, (3) n-propylbenzene, (4) n-butylbenzene, (5) pentylbenzene in (b); (1) benzene, (2) naphthalene, (3) acenaphthylene, (4) fluorene in (d).

图 6 亲水性分析物在亲水模式下的保留及分离评价

Fig. 6 Evaluation of retention and separation performance of hydrophilic analytes in hydrophilic interaction liquid chromatography mode a. effect of ACN content in mobile phase on the retention factors of hydrophilic analytes; b. separation chromatograms of hydrophilic analytes on different columns. Mobile phase: ACN-100 mmol/L NH4FA (pH=6.2) (90∶10, v/v) for all columns in (b). Peak identifications: 1. 5-fluorouracil; 2. uracil; 3. uridine; 4. orotic acid; 5. 5-fluorocytosine; 6. cytidine; 7. cytosine.

图 7 4种有机碱和4种有机酸的结构式及pKa

Fig. 7 Structure formulas and pKa values of four organic bases and four organic acids

图 8 4种有机碱、4种有机酸保留时间随流动相pH的变化及其在PSA色谱柱上的分离色谱图

Fig. 8 Retention time changes of four organic bases and four organic acids with the mobile phase pH and the separation chromatograms on the PSA column The effect of pH in the mobile phase on the retention times of (a) organic bases and (c) organic acids. Separation chromatograms of (b) organic bases and (d) organic acids on the PSA column. Mobile phase: ACN-100 mmol/L NH4FA (85∶15, v/v) in (a) and (c) (solid symbol); ACN-H2O (85∶15, v/v) in (c) (hollow symbol); ACN-100 mmol/L NH4FA (pH=6.2) (97∶3, v/v) in (d). Peak identifications: (1) theophylline, (2) theobromine, (3) dyphylline, (4) ribavirin in (b); (1) 4-aminobenzoic acid, (2) 4-hydroxybenzoic acid, (3) benzoic acid, (4) acetylsalicylic acid in (d).

图 9 (a)PSA色谱柱的进样稳定性及(b)SiO2@P(St-b-AA)合成过程的批次重复性

Fig. 9 (a) Injection stability of the PSA column and (b) repeatability of the synthesis procedure for SiO2@P(St-b-AA) Mobile phase: ACN-100 mmol/L NH4FA (pH=6.2) (85∶15, v/v). Peak identifications: 1. toluene; 2. benzoic acid; 3. uridine; 4. ribavirin; 5. cytidine.

表1 混合样品在PSA色谱柱上的连续进样稳定性和日间稳定性

Table 1 Intra-day stability and inter-day stability of mixed samples on the PSA column

No.	Analyte	RSDs/%
No.	Analyte	Intra-day stability (n=6)	Inter-day stability (n=4)
1	toluene	0.10	0.39
2	benzoic acid	0.92	0.85
3	uridine	0.75	0.48
4	ribavirin	0.95	0.18
5	cytidine	1.0	0.33

参考文献

[1]	Kumar B R. J Pharm Anal, 2017, 7(6): 349
[2]	Chai P J, Song Z H, Liu W H, et al. Chinese Journal of Chromatography, 2021, 39(8): 816
[2]	柴佩君, 宋志花, 刘万卉, 等. 色谱, 2021, 39(8): 816
[3]	Erkmen C, Gebrehiwot H W, Uslu B. Curr Pharm Anal, 2021, 17(3): 316
[4]	Deisenhofer R L, Ballschmiter K. Fresenius' J Anal Chem, 1998, 360(7): 763
[5]	Xiong W Q, Peng B, Duan A H, et al. Chinese Journal of Chromatography, 2021, 39(6): 607
[5]	熊婉淇, 彭博, 段爱红, 等. 色谱, 2021, 39(6): 607
[6]	Aslani S, Wahab M F, Kenari M E, et al. Anal Chim Acta, 2022, 1200: 339608
[7]	Dai X, Zhou J, Yang H, et al. Microchem J, 2022, 181: 107840
[8]	Zeng L, Jiang L J, Yao X D, et al. Chinese Journal of Chromatography, 2022, 40(6): 547
[8]	曾磊, 姜利娟, 姚兴东, 等. 色谱, 2022, 40(6): 547
[9]	Xie W B, Xia L, Li H, et al. Chinese Journal of Chromatography, 2022, 40(3): 234
[9]	谢文博, 夏璐, 李浩, 等. 色谱, 2022, 40(3): 234
[10]	Qiao L, Lv W, Chang M, et al. J Chromatogr A, 2018, 1559: 141
[11]	Cheng X D, Li Y P, He Y J, et al. Chinese Journal of Chromatography, 2019, 37(7): 683
[11]	成晓东, 李云萍, 贺银菊, 等. 色谱, 2019, 37(7): 683
[12]	Ciogli A, Buonsenso F, Proietti N, et al. J Chromatogr A, 2022, 1675: 463173
[13]	Wang J, Liu H, Wu D, et al. Chinese Journal of Chromatography, 2020, 38(4): 424
[13]	王婕, 刘宏, 吴丹, 等. 色谱, 2020, 38(4): 424
[14]	Wang J Z, Wang J S, Ning X H, et al. Talanta, 2021, 225: 122084
[15]	Wang L, Wei W, Xia Z, et al. TrAC-Trends Anal Chem, 2016, 80: 495
[16]	Wang X H. Journal of Instrumental Analysis, 2018, 37(12): 1425
[16]	王晓欢. 分析测试学报. 2018, 37(12): 1425
[17]	Fan F, Lu X, Wang S, et al. Talanta, 2021, 233: 122548
[18]	Xiao Z, Zhou W Q, Guo Y H, et al. Modern Chemical Industry, 2022, 42(S2): 407
[18]	肖震, 周卫强, 郭元亨, 等. 现代化工, 2022, 42(S2): 407
[19]	Kotoni D, D’Acquarica I, Ciogli A, et al. J Chromatogr A, 2012, 1232: 196
[20]	Han H, Zhang Y, Lu R, et al. J Chromatogr A, 2019, 1593: 24
[21]	Hosseini E S, Heydar K T. Talanta, 2021, 221: 121445
[22]	El-Debs R, Marechal A, Dugas V, et al. J Chromatogr A, 2014, 1326: 89
[23]	Ali A, Sun G, Kim J S, et al. J Chromatogr A, 2019, 1594: 72
[24]	Wu C, Xu P, Wang X, et al. Anal Methods, 2019, 11(28): 3590
[25]	Geng H, Wang Z, Zhang F, et al. J Chromatogr A, 2022, 1670: 462946
[26]	Lou X H, Zuo H Y, Wang Y, et al. Chinese Journal of Chromatography, 2020, 38(4): 430
[26]	娄旭华, 左慧颖, 王媛, 等. 色谱, 2020, 38(4): 430
[27]	Fan C, Chen J, Li H, et al. J Chromatogr A, 2022, 1667: 462912
[28]	Ni R, Peng J, Wen M, et al. Anal Methods, 2020, 12(3): 324
[29]	Yu D, Shen A, Guo Z, et al. Chem Commun, 2015, 51(79): 14778
[30]	Yang B, Cai T, Li Z, et al. Talanta, 2017, 175: 256
[31]	Jin G, Guo Z, Zhang F, et al. Talanta, 2008, 76(3): 522