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Fabrication and application of zwitterionic phosphorylcholine functionalized monoliths with different hydrophilic crosslinkers in hydrophilic interaction chromatography
Analytica Chimica Acta xxx (xxxx) xxx
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Fabrication and application of zwitterionic phosphorylcholine functionalized monoliths with different hydrophilic crosslinkers in hydrophilic interaction chromatography Qiqin Wang a, b, c, 1, Qiaoxuan Zhang d, 1, Hao Huang a, Pengwei Zhao e, Lingjue Sun a, Kun Peng a, Xiao Liu a, Meng Ruan a, Huikai Shao a, Jacques Crommen a, f, Pei Yu a, b, c, **, Zhengjin Jiang a, b, c, * a
Institute of Pharmaceutical Analysis, College of Pharmacy, Jinan University, Guangzhou, 510632, China International Cooperative Laboratory of Traditional Chinese Medicine Modernization, Innovative Drug Development of Chinese Ministry of Education (MOE), School of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou, 510632, China c Department of Pharmacy, Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine & New Drug Research, Jinan University, Guangzhou, 510632, China d Department of Laboratory Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, China e Traditional Chinese Medicine College, Guangdong Pharmaceutical University, Guangzhou, 510006, China f Laboratory of Analytical Pharmaceutical Chemistry, Department of Pharmaceutical Sciences, CIRM, University of Liege, CHU B36, B-4000, Liege, Belgium b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
The polarity of zwitterionic monolithic materials is related to the polarity of the crosslinker, which further affects column selectivity and efficiency. The novel poly(MPC-co-MMPC) monolith exhibited high column efficiency, selectivity for polar analytes as well as good enrichment potential for N-glycopeptides. MMPC has a great potential as a new and alternative hydrophilic crosslinker for the development of zwitterionic monolithic materials.
a r t i c l e i n f o Article history: Received 25 September 2019 Received in revised form 30 November 2019 Accepted 6 December 2019 Available online xxx
Keywords: Zwitterionic polymeric monolith
a b s t r a c t In this study, a series of zwitterionic phosphorylcholine functionalized monolithic columns were fabricated via the thermally initiated co-polymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC) and different hydrophilic crosslinkers, including 1,4-bis(acryloyl)piperazine (PDA), N,N0 -methylenebisacrylamide (MBA) and 2-(methacryloyloxy)ethyl-[N-(2-methacryloyloxy)ethyl]phosphorylcholine (MMPC). The physicochemical and chromatographic properties of these MPC functionalized monoliths, including column morphology, pore size distribution, permeability, column efficiency, retention mechanism and z-potential analysis, were systematically compared. Furthermore, the influence of the crosslinker on the chromatographic performance of these MPC functionalized monoliths was evaluated. The chromatographic results indicate that the polarity of MPC functionalized monoliths may be related
* Corresponding author. Institute of Pharmaceutical Analysis, College of Pharmacy, Jinan University, Guangzhou, 510632, China. ** Corresponding author. Department of Pharmacy, Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine & New Drug Research, Jinan University, Guangzhou, 510632, China. E-mail addresses: [email protected] (P. Yu), [email protected] (Z. Jiang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.aca.2019.12.016 0003-2670/© 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Q. Wang et al., Fabrication and application of zwitterionic phosphorylcholine functionalized monoliths with different hydrophilic crosslinkers in hydrophilic interaction chromatography, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.016
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Hydrophilic interaction chromatography Phosphorylcholine Hydrophilic crosslinker Separation science
to the polarity of the crosslinker, which further affects the column selectivity and efficiency. A particularly high column efficiency (88,000 plates/m) was obtained on the novel poly(MPC-co-MMPC) monolith at optimum linear velocity using thiourea as test analyte. Compared to the poly(MPC-co-MBA) and poly(MPC-co-PDA) monoliths, the poly(MPC-co-MMPC) monolith exhibited higher separation selectivity for polar analytes, including nucleobases, nucleosides and benzoic acid derivatives. Moreover, 24 Nglycopeptides could be detected after enrichment with the poly(MPC-co-MMPC) versus 19 and 10 Nglycopeptides with the poly(MPC-co-MBA) and poly(MPC-co-PDA) monoliths, and no N-glycopeptide without enrichment. Therefore, MMPC has a great potential as a new and alternative hydrophilic crosslinker for the development of zwitterionic polymeric monoliths. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Zwitterionic stationary phases with positively and negatively charged moieties, have attracted increasing attention and have been widely employed for the separation of various polar compounds in hydrophilic interaction chromatography (HILIC) mode [1e3]. Their superior performance was mainly attributed to the fact that they possess advantage of having weak dipole-dipole interactions between charged compounds and zwitterionic functional groups combined with the high efficiency and selectivity of hydrophilic interactions [4]. To date, some silica-based zwitterionic stationary phases, such as sulfobetaine-based ZIC-HILIC, ZIC-pHILIC, and phosphorylcholine-based ZIC-cHILIC, were commercialized by SeQuant [5e8]. Although these silica-based zwitterionic stationary phases have achieved great successes in separation science, the preparation method with high technical requirements limited their further applications involving self-lab preparation or chemical modification [8,9]. Lots of efforts were oriented to develop novel zwitterionic HILIC stationary phases. In recent years, zwitterionic monolithic stationary phases were developed due to their distinct advantages over silica-based zwitterionic stationary phases, such as fast and simple preparation, high permeability and stability, and wide selection of monomers available with different functional groups [10e13]. For example, Jiang et al. reported the preparation of an N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)ammonium betaine functionalized monolith for the separation of neutral, basic and acidic polar analytes with good selectivity [8]. Subsequently, in order to improve the hydrophilicity and chromatographic properties of zwitterionic HILIC monoliths, various kinds of hydrophilic functional monomers (Fig. S1), including sulfobetaine [8,14e16], phosphorylcholine (PC) [17], and choline phosphate [11], were employed to fabricate zwitterionic monolithic stationary phases. However, a single change of the hydrophilic functional monomer could not significantly improve the hydrophilicity of the zwitterionic monolithic stationary phase [8,17]. The polarity and size of both the functional monomer and the crosslinker, and their respective concentrations in the polymerization solution, affect the early stages of the polymerization, where the phase separation and formation of the first crosslinked polymeric nuclei occur [18]. Therefore, the type and polarity of the crosslinker could influence the chromatographic performance of the zwitterionic monolithic stationary phase, such as the polymer backbone hydrophilicity as well as its selectivity [19]. However, only few studies have been reported about the comparative investigation of the influence of the crosslinker on the chromatographic performance of zwitterionic monolithic stationary phases [18e20]. Urban et al. [18] synthesized seven different polymethacrylate monolithic stationary phases using N,Ndimethyl-N-methacryloyloxyethyl-N-(3-sulfopropyl) ammonium betaine and various oligo-methylene and oligo-oxyethylene
crosslinkers differing in polarity and size. All monoliths showed a dual retention mechanism and could be applied to the separation of low molecular weight analytes such as phenolic acids in the HILIC mode using ACN-rich mobile phases or in the reverse-phase mode using mobile phases with higher concentrations of water. Furthermore, the efficiency of these monolithic stationary phases for polar low molecular weight analytes in HILIC mode depends on the polarity, rather than on the size of the crosslinkers. Jiang et al. [19] fabricated three sulfoalkylbetaine functionalized monolithic stationary phases using 1,4-bis(acryloyl)piperazine (PDA), ethylene dimethacrylate (EDMA) and N,N0 -methylenebisacrylamide (MBA) as crosslinkers, respectively. Mechanism studies have shown that the polarity of crosslinker can significantly influence the hydrophilicity of the resulting monoliths, while electrostatic interactions seem more dependent on the properties of the zwitterionic functional monomer. However, the use of these conventional crosslinkers will inevitably decrease the proportion of functional monomer in the polymerization system, resulting in the reduction of the density of functional moieties in the monolithic stationary phase. Unlike conventional crosslinkers, 2-(methacryloyloxy)ethyl-[N(2-methacryloyloxy)ethyl] phosphorylcholine (MMPC) possesses both high hydrophilicity and a specific PC group, which could further enrich the functionality of the resulting monolithic stationary phase [21]. Therefore, in this study, a series of zwitterionic MPC functionalized monoliths were fabricated via one-step in situ co-polymerization of MPC and the crosslinker (MMPC, MBA, or PDA) within 100 mm I.D. capillaries. The physicochemical and chromatographic properties of these zwitterionic monoliths, including permeability, column efficiency, retention mechanism and column performance, were systematically evaluated by scanning electron microscopy (SEM), energy-dispersive X-ray spectrometry (EDS), mercury intrusion porosimetry and micro-HPLC. The potential applications of these monoliths were also evaluated using a series of polar analytes and tryptic digests of human IgG. Finally, the effects of the type of crosslinker on the chromatographic performance of the resulting monoliths were also discussed. 2. Materials and methods 2.1. Chemicals and reagents The hydrophilic monomer MPC was from Sigma-Aldrich (St. Louis, MO, USA). The crosslinkers MBA and PDA were from Aladdin Chemicals (Shanghai, China). The fused-silica capillaries (375 mm O.D. 100 mm I.D.) were from Ruifeng Chromatography Ltd. (Yongnian, Hebei, China). 3-(Trimethoxysilyl)-propyl methacrylate (g-MAPS), methanol (MeOH), tetrahydrofuran (THF), isopropanol (IPA), dimethyl sulfoxide (DMSO), 2,20 -azobisisobutyronitrile (AIBN), acetonitrile (ACN), toluene, thiourea, acrylamide, benzoic
Please cite this article as: Q. Wang et al., Fabrication and application of zwitterionic phosphorylcholine functionalized monoliths with different hydrophilic crosslinkers in hydrophilic interaction chromatography, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.016
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acid (B), 2,4-dihydroxybenzoic acid (2,4-DHB), 3,4dihydroxybenzoic acid (3,4-DHB), 3,5-dihydroxybenzoic acid (3,5DHB), 3,4,5-trihydroxybenzoic acid (3,4,5-THB), formic acid, ammonium formate, 2,5-dihydroxybenzoic acid, thymine, adenine, adenosine, cytidine, cytosine, ammonium bicarbonate (NH4HCO3), urea and trifluoroacetic acid (TFA) were from Aladdin Chemicals (Shanghai, China). Human IgG and trypsin (from bovine pancreas, TPCK-treated) were from Sigma-Aldrich (Shanghai, China). Dithiothreitol and iodoacetamide were from Energy Chemical (Shanghai, China). The tryptic digestion of human IgG [22] and the preparation of MMPC were performed following previously published methods without any modification [21]. Distilled water was filtered through a 0.22-mm membrane before use. 2.2. Instrumentation The thermally initiated free-radical polymerization of the mixture composed of MPC, crosslinker and AIBN was carried out using a Jinghong DKS22 water bath (Shanghai, China). SEM measurements were performed on a Zeiss ULTRA 55 field emission scanning electron microscope (Oberkochen, Germany) at an acceleration voltage of 3 or 5 kV. EDS experiments were performed on a Zeiss LEO 1530 VP Field Emission Scanning Electron Microscope equipped with an Oxford INCA 400 energy dispersive X-ray microanalysis system (Oberkochen, Germany) at an acceleration voltage of 5 kV. The pore size distribution was determined using a Quantachrome PoreMaster 60 GT Mercury Intrusion Porosimeter (Boynton Beach, FL, USA). The z-potential values of monolithic materials were measured with a Zetasizer Nano-ZS z-potential meter (Malvern Panalytical, Malvern, UK). FT-IR spectra of the monoliths prepared as KBr pellets were recorded at room temperature in the range from 4000 to 400 cm1 with a Gilson-306 FTIR Spectrometer (Villiers-le-Bel, France). The pH values were € ttingen, Germany). measured using a Sartorius PB-10 pH meter (Go All micro-HPLC experiments were performed on a self-assembled HPLC system that consisted of a DiNa nano gradient pump (Tokyo, Japan), a Valco four-port injection valve with 20 nL internal loop (Houston, TX, USA), and a Shimadzu SPD-15C UV detector (Kyoto, Japan) with a lab-made on-column detection system. The column efficiency (N) was calculated by HW-2000 chromatographic working station based on the peak width at half height. Data acquisition and handling were carried out using an Unimicro TrisepTM Workstation 2003 (Shanghai, China) and all chromatograms were redrawn using Microcal Origin 8.0. The calculated logP values (cLogP) of different crosslinkers were obtained using ACD/ Labs v.12.0 (Advanced Chemistry Development, Toronto, Ontario, Canada). 2.3. Fabrication of zwitterionic MPC functionalized monoliths To fabricate the zwitterionic MPC functionalized monoliths with different hydrophilic crosslinkers, the fused-silica capillaries (20 cm) were first pretreated with g-MAPS to provide anchoring sites for the bulk polymer on the inner wall during polymerization [11,23,24]. Various proportions of the functional monomer (MPC), the crosslinker (MMPC, PDA or MBA), the initiator (AIBN, ~1% w/w with respect to the monomers) and the porogens (v/v, the mixture of THF with MeOH (IPA or DMSO)) were accurately weighed and mixed ultrasonically into a 2-mL vial (Fig. 1, Table 1, S1 and S2). After ultrasonic degassing for 5 min to remove any dissolved air, the resultant solution was introduced into capillaries pretreated with the g-MAPS. Both ends of the capillaries were then sealed with GC septa, and the capillaries were submerged into a 60 C water bath for 12 h. After the polymerization, these monoliths were thoroughly flushed with MeOH (0.5 h) to wash out the residual reagents
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using a high-pressure pump. A 2e3 mm detection window was then created at a distance of 2e3 cm from the end of the columns using a thermal wire stripper for the self-assembled micro-HPLC system. A 1 cm length of monolith was also cut from each column for SEM and EDS analysis. 2.4. Chromatographic conditions The mobile phases were prepared by mixing appropriate volume of buffer solution or H2O to ACN. The stock buffer solutions of ammonium formate were prepared by dissolving an appropriate amount of the salt in deionized H2O and adjusted to the desired pH with formic acid. Unless otherwise stated, the mobile phase pH values mentioned in this research refer to the aqueous portion only. The stock sample solutions were prepared by dissolving appropriate amounts of samples into MeOH to give a concentration around 1 mg/mL. Both mobile phases and sample solutions were subjected to a filtration through 0.22 mm membrane prior to the micro-HPLC experiments. Chromatograms were recorded at a wavelength of 214 or 254 nm. All separations were performed at room temperature. 2.5. N-Glycopeptide enrichment The on-column N-glycopeptide enrichment using zwitterionic MPC functionalized monolithic columns was performed at a constant flow rate (1 mL/min) with a Longer Precision Pump with TS2A/L0107-2A Syringe Pump Controller (Baoding, Hebei, China). Firstly, the monolithic column (140 mm 100 mm I.D., about 1 mg monolithic polymer) was washed with the loading buffer (9% H2O and 1% TFA in ACN, v/v) for 1 h. The lyophilized tryptic digests of human IgG were re-dissolved in loading buffer at a concentration of 1 mg/mL, then 10 mL samples were pumped through the monolith column at a flow rate of 1 mL/min. Non-glycopeptides were removed with loading buffer for 200 min at same flow rate. Then, the captured N-glycopeptides were eluted with the elution buffer (30% ACN and 1% TFA in H2O, v/v) for 10 min. 2.6. Calculations The permeability (K) of the zwitterionic MPC functionalized monoliths was calculated according to Darcy’s Law by using the following equation (1) [25]:
K ¼
uLh DP
(1)
where u (m/s), h (Pa∙s), L (m) and DP (Pa) refer to the linear velocity, the viscosity of mobile phase, the length of the monolithic column and the pressure drop across the monolithic column, respectively. 3. Results and discussion 3.1. Fabrication and optimization of the zwitterionic MPC functionalized monoliths with different hydrophilic crosslinkers Because the composition of the polymerization mixture significantly affects the permeability, morphology, efficiency and selectivity of the zwitterionic monoliths [11], several key parameters including the composition and the weight content of the porogens, as well as the weight content of the crosslinker, were systematically optimized. The physicochemical properties and morphological characteristics of the resulting monoliths were evaluated using micro-HPLC, SEM, and EDS. Firstly, based on our previous study
Please cite this article as: Q. Wang et al., Fabrication and application of zwitterionic phosphorylcholine functionalized monoliths with different hydrophilic crosslinkers in hydrophilic interaction chromatography, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.016
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Fig. 1. Fabrication of zwitterionic MPC functionalized monoliths with different hydrophilic crosslinkers.
Table 1 Composition of the polymerization mixtures for the fabrication of poly(MPC-co-MMPC) monoliths. Column
Monomer mixture (%, w/w)
Porogen mixture (%, w/w)
MPC
MMPC
MeOH
THF
D1 D2 D3 D4 D5 D6 D7 D8
50 50 50 50 45 55 50 50
50 50 50 50 55 45 50 50
35 35 35 35 35 35 40 30
65 65 65 65 65 65 60 70
Monomers: Porogens (%, w/w)
Backpressure (MPa)
Theoretical plates (plates/m)
20:80 30:70 35:65 40:60 35:65 35:65 35:65 35:65
0.1 0.4 1.2 / 1.8 0.8 4.4 0.3
6100 28500 88000 / 55000 34800 48400 26900
Experimental conditions: column dimensions, 140 mm 100 mm I.D.; column temperature: room temperature; UV detection wavelength, 254 nm; mobile phase, H2O/ACN (5/ 95, v/v); flow rate: 800 nL/min; injection volume, 20 nL; sample: thiourea.
[24], a binary porogenic system including IPA and THF was employed to prepare the poly(MPC-co-MBA) monolith with some modification (Table S1). Therefore, the IPA/THF system was chosen initially as binary porogen for the preparation of the poly(MPC-coPDA) and poly(MPC-co-MMPC) monoliths. However, the solubility of PDA and MMPC was not satisfactory in the IPA/THF system. To improve their solubility, a binary porogenic system including DMSO and THF was selected to prepare the poly(MPC-co-PDA) monolith (Table S2), while a binary porogenic system consisting of MeOH and THF was employed to fabricate the poly(MPC-co-MMPC) monolith (Table 1). Then, the composition of the polymerization mixture for the poly(MPC-co-MMPC) monolith was further optimized. The influence of the total content of the monomer mixture (MPC and MMPC) on the column performance was first investigated. When the weight content of the monomer mixture was increased from 20% (column D1) to 40% (column D4, it was too hard to pump the mobile phase through it), the column backpressure and efficiency were found to raise significantly. Therefore, column D3 was selected for further optimization due to its satisfactory backpressure (1.2 MPa) and column efficiency (88,000 plates/m for thiourea). Since the content of the functional monomer (MPC) has a significant effect on the column properties, the weight ratio of MPC in the monomer mixture was then varied from 45% (column D5) to 55% (column D6). By increasing the weight ratio of MPC, the backpressure decreased moderately from 1.8 MPa (column D5) to 0.8 MPa (column D6), whereas either a decrease (column D5) or an increase (column D6) of the amount of MPC results in a drop of column efficiency. Thus, the conditions for preparing column D3 were chosen for further optimization. The effect of the porogen mixture composition was then investigated by varying the weight ratio of MeOH and THF (w/w) from 30/70 (column D8) to 40/60
(column D7). This minor change of the porogen composition had a significant influence on the column backpressure and efficiency. The backpressure significantly increased from 0.3 (column D8) to 4.4 (column D7) MPa, while column D3 exhibited the highest column efficiency and acceptable backpressure. Therefore, the polymerization conditions used to produce column D3 were selected for all further studies. Finally, the preparation of the poly(MPC-coMBA) and poly(MPC-co-PDA) monolithic columns were also optimized according to the above methods (Tables S1 and S2). 3.2. Characterization of the zwitterionic MPC functionalized monoliths To verify the success of the preparation process, these zwitterionic MPC functionalized monoliths were comparatively evaluated by FT-IR, SEM, and EDS. As shown in Fig. S2, all characteristic peaks of the PC moieties in the poly(MPC-co-MMPC) monolith for the P] O, PeO-alkyl and Nþ(CH3)3 stretches could be identified as ~1222.65, ~1078.98 and ~968.09 cm1, respectively. Similar characteristic peaks were also detected on the poly(MPC-co-MBA) and poly(MPC-co-PDA) monoliths (Figs. S3 and S4). SEM images (Fig. 2) show that these monoliths possess a porous structure and are well attached to the inner wall of the capillary (except for the poly(MPCco-PDA) monolith). EDS results revealed the existence of the P element on the polymer surface, which indicates that MPC was incorporated into the monolithic matrix (Fig. S5). 3.3. Comparison of the physicochemical properties of the zwitterionic MPC functionalized monoliths To evaluate the influence of the crosslinkers on the physicochemical and chromatographic properties of the zwitterionic MPC
Please cite this article as: Q. Wang et al., Fabrication and application of zwitterionic phosphorylcholine functionalized monoliths with different hydrophilic crosslinkers in hydrophilic interaction chromatography, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.016
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Fig. 3. Van Deemter plots for zwitterionic MPC functionalized monoliths. Experimental conditions: monolithic columns: 170 mm 100 mm I.D.; mobile phase: H2O/ ACN (10/90, v/v); column temperature: room temperature; UV detection wavelength: 254 nm; injection: 20 nL; sample: thiourea.
Fig. 2. SEM results of (a) poly(MPC-co-MMPC), (b) poly(MPC-co-MBA), and (c) poly (MPC-co-PDA) monoliths.
functionalized monoliths, several key factors (including permeability, column efficiency, hydrophilic retention mechanism, and zpotential analysis) of the three MPC functionalized monoliths, i.e. poly(MPC-co-PDA), poly(MPC-co-MBA), and poly(MPC-co-MMPC), were systematically compared. According to Darcy’s law [25], the calculated permeabilities (K values) for the poly(MPC-co-MMPC), poly(MPC-co-MBA) and poly(MPC-co-PDA) monoliths were 6.49 1014, 3.20 1014 and 2.86 1014 m2, respectively, using H2O/ACN (5/95, v/v) as mobile phase. No significant difference on the permeability was found between these zwitterionic MPC functionalized monoliths. Good permeability of these monoliths may be attributed to that all three monolithic materials contain large amounts of macropores (>1 mm) and possess acceptable total porosities (>60%), which permits rapid flow through the column under low backpressure. The effects of the type of crosslinker on the efficiency of the zwitterionic MPC functionalized monolithic columns were then evaluated. A column efficiency as high as 88,000 plates/m was obtained for thiourea on the poly(MPC-co-MMPC) monolith at the optimum linear velocity, while column efficiencies corresponding to 76,000 plates/m and 50,000 plates/m were observed on the poly(MPC-co-PDA) and poly(MPC-co-MBA) monoliths, respectively. Moreover, van Deemter plots for these zwitterionic MPC functionalized monoliths were also compared. As depicted in Fig. 3, the poly(MPC-co-MMPC) monolith exhibited a lower theoretical plate height (H z 11 mm) for thiourea at the optimum linear velocity compared to the poly(MPC-co-PDA) monolith (H z 13 mm) and the poly(MPC-co-MBA) monolith (H z 20 mm). It is worth noting that the efficiency of these zwitterionic MPC functionalized monolithic columns is much higher than that of the poly(MPC-co-EDMA) monolith (Nthiourea ¼ 36000 plates/m [17]). 3.4. Comparison of the retention mechanisms of the zwitterionic MPC functionalized monoliths In the previous HILIC studies, the change of retention
mechanism from a HILIC mode to an apparent RP mode has been observed on hydrophilic stationary phases, and the critical mobile phase composition corresponding to this turning point can be employed to evaluate the polarity of hydrophilic stationary phases [19,26]. To systematically evaluate the influence of the type of crosslinker on the hydrophilicity of zwitterionic MPC functionalized monoliths, the effect of ACN content (ranging from 95% to 10%) on the retention of three test solutes (toluene, acrylamide and thiourea) were investigated. As depicted in Fig. 4, a typical HILIC retention mechanism was observed on these zwitterionic MPC functionalized monoliths. Furthermore, no critical mobile phase composition could be determined for the poly(MPC-co-MBA) monolith. However, the critical mobile phase compositions were around 20% ACN, 30% ACN and 60% ACN for the poly(MPC-co-PDA), poly(MPC-co-MMPC) and poly(MPC-co-EDMA) monoliths [17], respectively. Therefore, the hydrophilicity of the poly(MPC-coMBA) monolith can be considered as higher than that of the poly(MPC-co-PDA) and poly(MPC-co-EDMA) monoliths, which is in accordance with the polarity order of the three crosslinkers (MBA, clogP ¼ 1.44; PDA, clogP ¼ 1.22; EDMA, clogP ¼ 2.99). This observation is consistent with the previous study [19]. In addition, although MBA, MMPC (clogP ¼ 1.38), and PDA have similar polarities, the hydrophilicity of the poly(MPC-co-MMPC) monolith is lower than that of the poly(MPC-co-MBA) and poly(MPC-co-PDA) monoliths, which indicates that more complex interactions could exist in the poly(MPC-co-MMPC) monolithic stationary phase. Although PC has both a positively charged quaternary ammonium group and a negatively charged phosphoric acid group, it has been found that the materials with PC groups have a negative surface charge over a wide pH range [17]. Therefore, the electrostatic interactions could contribute to the retention of charged analytes on the zwitterionic MPC functionalized monoliths. To evaluate the effect of the crosslinker on the electrostatic interactions, the z-potential values for these zwitterionic MPC functionalized monoliths were measured in 5 mM ammonium formate at different pH values (2.0, 3.0, 4.0, 5.0, 6.4, 7.0, and 8.0). As shown in Fig. 5, similar z-potential values were achieved for the poly(MPCco-PDA) and poly(MPC-co-MBA) monoliths due to the fact that both the crosslinkers MBA and PDA were neutral and thus the zwitterionic MPC was responsible for the surface charge of these monolithic materials. However, z-potential measurements revealed that
Please cite this article as: Q. Wang et al., Fabrication and application of zwitterionic phosphorylcholine functionalized monoliths with different hydrophilic crosslinkers in hydrophilic interaction chromatography, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.016
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Fig. 4. Influence of ACN concentration on retention: (a) poly(MPC-co-MMPC) monolith, (b) poly(MPC-co-PDA) monolith, (c) poly (MPC-co-MBA) monolith. Experimental conditions: monolithic columns, 140 mm 100 mm I.D.; column temperature: room temperature; mobile phase: mixture of H2O and ACN; detection wavelength: 254 nm; injection: 20 nL; sample: toluene, acrylamide, thiourea.
Fig. 5. z-potential values of the zwitterionic MPC functionalized monoliths.
the surface of the poly(MPC-co-MMPC) monolith was negatively charged over the whole studied pH range and the absolute z-potential value was found to increase with increasing buffer pH from 2.0 to 8.0, and to be higher than that of the poly(MPC-co-PDA) and poly(MPC-co-MBA) monoliths. This may be due to the fact that MMPC as crosslinker could further enrich the density of specific PC groups on the resulting monolithic stationary phase, which is not the case for the conventional hydrophilic crosslinkers (MBA and PDA). These observed features of the poly(MPC-co-MMPC) monolith seem to be in favor of its applications in bioanalysis. 3.5. Comparison of the chromatographic performance of the zwitterionic MPC functionalized monoliths 3.5.1. Separation of small molecules To compare the separation selectivity of the zwitterionic MPC functionalized monoliths, a test mixture containing nucleobases, nucleosides, and toluene, was selected. As can be seen in Fig. 6, the most hydrophilic compound cytidine exhibited the highest retention on the poly(MPC-co-MMPC) monolith, an intermediate retention on the poly(MPC-co-MBA) monolith, and the lowest retention on the poly(MPC-co-PDA) monolith. Furthermore, an excellent separation of all compounds was achieved on the poly(MPC-co-MMPC) monolith, while adenine and adenosine could not be baseline separated on the poly(MPC-co-MBA) and poly(MPC-co-PDA) monoliths. Clearly, the poly(MPC-co-MMPC) monolith exhibited higher separation selectivity for these compounds than the other two monoliths.
Fig. 6. Separation of nucleobases and nucleosides on the zwitterionic MPC functionalized monoliths. Conditions: monolithic columns, 140 mm 100 mm I.D.; column temperature: room temperature; mobile phase: H2O/ACN (10/90, v/v); flow rate: 1000 nL/min (linear velocity: 1.8 mm/s); UV Wavelength, 254 nm; samples: (0) toluene, (1) thymine, (2) adenine, (3) adenosine, (4) cytosine, (5) cytidine; injection volume: 20 nL.
The chromatographic separation of a series of benzoic acid derivatives was then investigated on these zwitterionic MPC functionalized monoliths using 10 mM ammonium formate (pH 3.0)/ ACN (40/60, v/v) as mobile phase. As depicted in Fig. 7, the baseline separation of the five benzoic acid derivatives was achieved on the poly(MPC-co-MMPC) and poly(MPC-co-MBA) monoliths. Moreover, B (pKa 4.20, logD3.0 ¼ 1.87) eluted first, followed by 2,4-DHB (pKa 3.32, logD3.0 ¼ 1.27), 3,4-DHB (pKa 4.45, logD3.0 ¼ 1.14), 3,5-DHB (pKa 3.96, logD3.0 ¼ 1.07) and 3,4,5-THB (pKa 4.33, logD3.0 ¼ 0.89), successively, which is in accordance with the polarity order of analytes. These results indicate that zwitterionic MPC functionalized monoliths with different hydrophilic crosslinkers have a great potential for the separation of bioactive molecules. 3.5.2. N-Glycopeptide enrichment Recently, a zwitterionic hydrophilic material exhibited excellent enrichment efficiency for N-glycopeptides in human IgG tryptic digest, which will benefit to eliminate interference from nonglycopeptides and to improve the sensitivity of N-glycopeptide detection [27,28]. As shown in Fig. S6, no glycopeptides could be directly detected in human IgG tryptic digest without sample pretreatment due to the severe interference and ion suppression from the highly abundant non-glycopeptides, even when using a high concentration of human IgG digest (1.0 mg/mL). However, after enrichment with the poly(MPC-co-MMPC) monolith, the
Please cite this article as: Q. Wang et al., Fabrication and application of zwitterionic phosphorylcholine functionalized monoliths with different hydrophilic crosslinkers in hydrophilic interaction chromatography, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.016
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signals of the nonglycosylated peptides were significantly eliminated and 24 N-glycopeptides were clearly detected with much enhanced MS intensity (Fig. 8 and Table S3). For comparison, only 19 and 10 N-glycopeptides could be detected with inferior MS intensity after enrichment with the poly(MPC-co-MBA) and poly(MPC-co-PDA) monoliths (Tables S4 and S5), respectively. Moreover, the results obtained with the poly(MPC-co-MMPC) monolith are also better than those previously reported with the commercial ZIC-HILIC material (21 glycopeptides) [22] or ZIC-HILIC polymer particles poly(MBAAm-co-MAA)@L-Cys (17 glycopeptides) [29] for human IgG tryptic digest enrichment. 4. Conclusion
Fig. 7. Separation of benzoic acid derivatives on the zwitterionic MPC functionalized monoliths. Conditions: monolithic columns, 140 mm 100 mm I.D.; column temperature: room temperature; mobile phase: 10 mM ammonium formate (pH 3.0)/ACN (40/60, v/v); flow rate: 800 nL/min (linear velocity: 0.6 mm/s); UV wavelength, 214 nm; samples: (1) B, (2) 2,4-DHB, (3) 3,4-DHB, (4) 3,5-DHB, (5) 3,4,5-THB; injection: 20 nL.
In this study, to evaluate the influence of the type of crosslinker (PDA, MBA or MMPC) on the chromatographic properties of the corresponding monolithic stationary phases, a series of zwitterionic MPC functionalized monoliths were fabricated in 100 mm I.D. capillaries. Under the optimized polymerization conditions, the highest column efficiency (88,000 plates/m) was achieved for thiourea on the novel poly(MPC-co-MMPC) monolithic column. Mechanism studies indicated that the type and polarity of crosslinker could influence the hydrophilicity and electrostatic interactions of the zwitterionic MPC functionalized monoliths. Higher separation selectivity was achieved for small polar analytes, such as nucleobases, nucleosides, and benzoic acid derivatives, on the poly(MPC-co-MMPC) monolith compared to that obtained on the other two MPC functionalized monoliths. Furthermore, the poly(MPC-co-MMPC) monolith exhibited good potential for the enrichment of hydrophilic N-glycopeptides in complex matrices, which makes it a promising analytical tool in glycomics and glycoproteomics. Overall, MMPC not only enriches the selection of hydrophilic crosslinkers, but also opens up interesting possibilities for the fabrication of novel HILIC stationary phases in separation science. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant: 81703460, 81673391). There are no conflicts of interest to declare. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.aca.2019.12.016. Author contribution statement
Fig. 8. (a) MALDI-TOF MS spectra of the standard hIgG digestion solution after enrichment on the zwitterionic MPC functionalized monoliths. (b) N-glycopeptides after enrichment on the poly(MPC-co-MMPC) monolith. Conditions: monolithic columns, 140 mm 100 mm I.D.; loading buffer: ACN/H2O/TFA (90:9:1, v/v/v); eluting buffer: ACN/H2O/TFA (30:69:1, v/v/v); injection: 10 mL. Red stars: N-glycopeptides. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Zhengjin Jiang and Pei Yu designed the project and provided overall guidance. Qiqin Wang and Qiaoxuan Zhang carried out the experiments, analyzed data and wrote the paper. Hao Huang, Pengwei Zhao, Lingjue Sun, Kun Peng, Xiao Liu, Meng Ruan, Huikai Shao carried out some experiments and analyzed the related data. Jacques Crommen edited the article. All authors reviewed the manuscript.
Please cite this article as: Q. Wang et al., Fabrication and application of zwitterionic phosphorylcholine functionalized monoliths with different hydrophilic crosslinkers in hydrophilic interaction chromatography, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.016
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References [1] S.D. Palma, P.J. Boersema, A.J.R. Heck, S. Mohammed, Zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC and ZIC-cHILIC) provide high resolution separation and increase sensitivity in proteome analysis, Anal. Chem. 83 (2011) 3440e3447. [2] L.Z. Qiao, X.Z. Shi, G.W. Xu, Recent advances in development and characterization of stationary phases for hydrophilic interaction chromatography, Trends Anal. Chem. 81 (2016) 23e33. s, Recent advances in stationary phases and understanding [3] P. Jandera, P. Jana of retention in hydrophilic interaction chromatography. A review, Anal. Chim. Acta 967 (2017) 12e32. [4] N.P. Dinh, T. Jonsson, K. Irgum, Probing the interaction mode in hydrophilic interaction chromatography, J. Chromatogr. A 1218 (2011) 5880e5891. [5] W. Jiang, K. Irgum, Synthesis and evaluation of polymer-based zwitterionic stationary phases for separation of ionic species, Anal. Chem. 73 (2001) 1993e2003. €gren, K. Irgum, Chromatographic interactions between pro[6] C. Viklund, A. Sjo teins and sulfoalkylbetaine-based zwitterionic copolymers in fully aqueous low-salt buffers, Anal. Chem. 73 (2001) 444e452. [7] W. Jiang, K. Irgum, Tentacle-type zwitterionic stationary phase prepared by surface-initiated graft polymerization of 3-[N,N-dimethyl-N-(methacryloyloxyethyl)- ammonium] propanesulfonate through peroxide groups tethered on porous silica, Anal. Chem. 74 (2002) 4682e4687. [8] Z.J. Jiang, N.W. Smith, P.D. Ferguson, M.R. Taylor, Hydrophilic interaction chromatography using methacrylate-based monolithic capillary column for the separation of polar analytes, Anal. Chem. 79 (2007) 1243e1250. [9] Y.N. Chou, F. Sun, H.C. Hung, P. Jain, A. Sinclair, P. Zhang, T. Bai, Y. Chang, T.C. Wen, Q.M. Yu, S.Y. Jiang, Ultra-low fouling and high antibody loading zwitterionic hydrogel coatings for sensing and detection in complex media, Acta Biomater. 40 (2016) 31e37. [10] Q.Q. Wang, K. Peng, W.J. Chen, Z. Cao, P.J. Zhu, Y.M. Zhao, Y.Q. Wang, H.B. Zhou, Z.J. Jiang, Development of double chain phosphatidylcholine functionalized polymeric monoliths for immobilized artificial membrane chromatography, J. Chromatogr. A 1479 (2017) 97e106. [11] Q.Q. Wang, H.H. Wu, K. Peng, H.Y. Jin, H.K. Shao, Y.Q. Wang, J. Crommen, Z.J. Jiang, Hydrophilic polymeric monoliths containing choline phosphate for separation science applications, Anal. Chim. Acta 999 (2018) 184e189. ríkov [12] V. Ske a, P. Jandera, Effects of the operation parameters on hydrophilic interaction liquid chromatography separation of phenolic acids on zwitterionic monolithic capillary columns, J. Chromatogr. A 1217 (2010) 7981e7989. [13] F. Svec, Y.Q. Lv, Advances and recent trends in the field of monolithic columns for chromatography, Anal. Chem. 87 (2015) 250e273. [14] Z.J. Jiang, N.W. Smith, P.D. Ferguson, M.R. Taylor, Novel highly hydrophilic zwitterionic monolithic column for hydrophilic interaction chromatography, J. Sep. Sci. 32 (2009) 2544e2555. [15] Z.H. Liu, Y.B. Peng, T.T. Wang, G.X. Yuan, Q.X. Zhang, J.L. Guo, Z.J. Jiang, Preparation and application of novel zwitterionic monolithic column for
hydrophilic interaction chromatography, J. Sep. Sci. 36 (2013) 262e269. [16] G.X. Yuan, Y.B. Peng, Z.H. Liu, J.Y. Hong, Y. Xiao, J.L. Guo, N.W. Smith, J. Crommen, Z.J. Jiang, A facile and efficient strategy to enhance hydrophilicity of zwitterionic sulfoalkylbetaine type monoliths, J. Chromatogr. A 1301 (2013) 88e97. [17] Z.J. Jiang, J. Reilly, B. Everatt, N.W. Smith, Novel zwitterionic polyphosphorylcholine monolithic column for hydrophilic interaction chromatography, J. Chromatogr. A 1216 (2009) 2439e2448. ríkova kov , J. Urban, Cross-linker effects on the [18] M. Stan a, P. Jandera, V. Ske separation efficiency on (poly)methacrylate capillary monolithic columns. Part II. Aqueous normal-phase liquid chromatography, J. Chromatogr. A 1289 (2013) 47e57. [19] C.S. Liu, W.J. Chen, G.X. Yuan, Y. Xiao, J. Crommen, S.H. Xu, Z.J. Jiang, Influence of the crosslinker type on the chromatographic properties of hydrophilic sulfoalkylbetaine-type monolithic columns, J. Chromatogr. A 1373 (2014) 73e80. [20] F. Li, D.Y. Qiu, J.W. Kang, Preparation of a sulfoalkylbetaine-based zwitterionic monolith with enhanced hydrophilicity for capillary electrochromatography separation applications, Chromatographia 80 (2017) 975e981. [21] Y. Kiritoshi, K. Ishihara, Synthesis of hydrophilic cross-linker having phosphorylcholine-like linkage for improvement of hydrogel properties, Polymer 45 (2004) 7499e7504. [22] C.S. Xia, F.L. Jiao, F.Y. Gao, H.P. Wang, Y.Y. Lv, Y.H. Shen, Y.J. Zhang, X.H. Qian, Two-dimensional MoS2-based zwitterionic hydrophilic interaction liquid chromatography material for the specific enrichment of glycopeptides, Anal. Chem. 90 (2018) 6651e6659. [23] X.Y. Wang, D.H. Xia, H. Han, K. Peng, P.J. Zhu, J. Crommen, Q.Q. Wang, Z.J. Jiang, Biomimetic small peptide functionalized affinity monoliths for monoclonal antibody purification, Anal. Chim. Acta 1017 (2018) 57e65. [24] Q.Q. Wang, H.Y. Jin, D.H. Xia, H.K. Shao, K. Peng, X. Liu, H. Huang, Q.X. Zhang, J.L. Guo, Y.Q. Wang, J. Crommen, N. Gan, Z.J. Jiang, Biomimetic polymer-based method for selective capture of C-reactive protein in biological fluids, ACS Appl. Mater. Interfaces 10 (2018) 41999e42008. [25] P.A. Bristow, J.H. Knox, Standardization of test conditions for high performance liquid chromatography columns, Chromatographia 10 (1977) 279e289. [26] J.J. Pesek, M.T. Matyska, S. Larrabee, HPLC retention behavior on hydridebased stationary phases, J. Sep. Sci. 30 (2007) 637e647. [27] Y. Yang, V. Franc, A.J.R. Heck, Glycoproteomics: a balance between highthroughput and in-depth analysis, Trends Biotechnol. 35 (2017) 598e609. [28] Y. Zhang, Y. Peng, L.J. Yang, H.J. Lu, Advances in sample preparation strategies for MS-based qualitative and quantitative N-glycomics, Trends Anal. Chem. 99 (2018) 34e46. [29] J.X. Liu, K.G. Yang, W.Y. Shao, S.W. Li, Q. Wu, S. Zhang, Y.Y. Qu, L.H. Zhang, Y.K. Zhang, Synthesis of zwitterionic polymer particles via combined distillation precipitation polymerization and click chemistry for highly efficient enrichment of glycopeptide, ACS Appl. Mater. Interfaces 8 (2016) 22018e22024.
Please cite this article as: Q. Wang et al., Fabrication and application of zwitterionic phosphorylcholine functionalized monoliths with different hydrophilic crosslinkers in hydrophilic interaction chromatography, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.016
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Fabrication and application of zwitterionic phosphorylcholine functionalized monoliths with different hydrophilic crosslinkers in hydrophilic interaction chromatography Qiqin Wang a, b, c, 1, Qiaoxuan Zhang d, 1, Hao Huang a, Pengwei Zhao e, Lingjue Sun a, Kun Peng a, Xiao Liu a, Meng Ruan a, Huikai Shao a, Jacques Crommen a, f, Pei Yu a, b, c, **, Zhengjin Jiang a, b, c, * a
Institute of Pharmaceutical Analysis, College of Pharmacy, Jinan University, Guangzhou, 510632, China International Cooperative Laboratory of Traditional Chinese Medicine Modernization, Innovative Drug Development of Chinese Ministry of Education (MOE), School of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou, 510632, China c Department of Pharmacy, Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine & New Drug Research, Jinan University, Guangzhou, 510632, China d Department of Laboratory Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, China e Traditional Chinese Medicine College, Guangdong Pharmaceutical University, Guangzhou, 510006, China f Laboratory of Analytical Pharmaceutical Chemistry, Department of Pharmaceutical Sciences, CIRM, University of Liege, CHU B36, B-4000, Liege, Belgium b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
The polarity of zwitterionic monolithic materials is related to the polarity of the crosslinker, which further affects column selectivity and efficiency. The novel poly(MPC-co-MMPC) monolith exhibited high column efficiency, selectivity for polar analytes as well as good enrichment potential for N-glycopeptides. MMPC has a great potential as a new and alternative hydrophilic crosslinker for the development of zwitterionic monolithic materials.
a r t i c l e i n f o Article history: Received 25 September 2019 Received in revised form 30 November 2019 Accepted 6 December 2019 Available online xxx
Keywords: Zwitterionic polymeric monolith
a b s t r a c t In this study, a series of zwitterionic phosphorylcholine functionalized monolithic columns were fabricated via the thermally initiated co-polymerization of 2-methacryloyloxyethyl phosphorylcholine (MPC) and different hydrophilic crosslinkers, including 1,4-bis(acryloyl)piperazine (PDA), N,N0 -methylenebisacrylamide (MBA) and 2-(methacryloyloxy)ethyl-[N-(2-methacryloyloxy)ethyl]phosphorylcholine (MMPC). The physicochemical and chromatographic properties of these MPC functionalized monoliths, including column morphology, pore size distribution, permeability, column efficiency, retention mechanism and z-potential analysis, were systematically compared. Furthermore, the influence of the crosslinker on the chromatographic performance of these MPC functionalized monoliths was evaluated. The chromatographic results indicate that the polarity of MPC functionalized monoliths may be related
* Corresponding author. Institute of Pharmaceutical Analysis, College of Pharmacy, Jinan University, Guangzhou, 510632, China. ** Corresponding author. Department of Pharmacy, Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine & New Drug Research, Jinan University, Guangzhou, 510632, China. E-mail addresses: [email protected] (P. Yu), [email protected] (Z. Jiang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.aca.2019.12.016 0003-2670/© 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Q. Wang et al., Fabrication and application of zwitterionic phosphorylcholine functionalized monoliths with different hydrophilic crosslinkers in hydrophilic interaction chromatography, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.016
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Hydrophilic interaction chromatography Phosphorylcholine Hydrophilic crosslinker Separation science
to the polarity of the crosslinker, which further affects the column selectivity and efficiency. A particularly high column efficiency (88,000 plates/m) was obtained on the novel poly(MPC-co-MMPC) monolith at optimum linear velocity using thiourea as test analyte. Compared to the poly(MPC-co-MBA) and poly(MPC-co-PDA) monoliths, the poly(MPC-co-MMPC) monolith exhibited higher separation selectivity for polar analytes, including nucleobases, nucleosides and benzoic acid derivatives. Moreover, 24 Nglycopeptides could be detected after enrichment with the poly(MPC-co-MMPC) versus 19 and 10 Nglycopeptides with the poly(MPC-co-MBA) and poly(MPC-co-PDA) monoliths, and no N-glycopeptide without enrichment. Therefore, MMPC has a great potential as a new and alternative hydrophilic crosslinker for the development of zwitterionic polymeric monoliths. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Zwitterionic stationary phases with positively and negatively charged moieties, have attracted increasing attention and have been widely employed for the separation of various polar compounds in hydrophilic interaction chromatography (HILIC) mode [1e3]. Their superior performance was mainly attributed to the fact that they possess advantage of having weak dipole-dipole interactions between charged compounds and zwitterionic functional groups combined with the high efficiency and selectivity of hydrophilic interactions [4]. To date, some silica-based zwitterionic stationary phases, such as sulfobetaine-based ZIC-HILIC, ZIC-pHILIC, and phosphorylcholine-based ZIC-cHILIC, were commercialized by SeQuant [5e8]. Although these silica-based zwitterionic stationary phases have achieved great successes in separation science, the preparation method with high technical requirements limited their further applications involving self-lab preparation or chemical modification [8,9]. Lots of efforts were oriented to develop novel zwitterionic HILIC stationary phases. In recent years, zwitterionic monolithic stationary phases were developed due to their distinct advantages over silica-based zwitterionic stationary phases, such as fast and simple preparation, high permeability and stability, and wide selection of monomers available with different functional groups [10e13]. For example, Jiang et al. reported the preparation of an N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)ammonium betaine functionalized monolith for the separation of neutral, basic and acidic polar analytes with good selectivity [8]. Subsequently, in order to improve the hydrophilicity and chromatographic properties of zwitterionic HILIC monoliths, various kinds of hydrophilic functional monomers (Fig. S1), including sulfobetaine [8,14e16], phosphorylcholine (PC) [17], and choline phosphate [11], were employed to fabricate zwitterionic monolithic stationary phases. However, a single change of the hydrophilic functional monomer could not significantly improve the hydrophilicity of the zwitterionic monolithic stationary phase [8,17]. The polarity and size of both the functional monomer and the crosslinker, and their respective concentrations in the polymerization solution, affect the early stages of the polymerization, where the phase separation and formation of the first crosslinked polymeric nuclei occur [18]. Therefore, the type and polarity of the crosslinker could influence the chromatographic performance of the zwitterionic monolithic stationary phase, such as the polymer backbone hydrophilicity as well as its selectivity [19]. However, only few studies have been reported about the comparative investigation of the influence of the crosslinker on the chromatographic performance of zwitterionic monolithic stationary phases [18e20]. Urban et al. [18] synthesized seven different polymethacrylate monolithic stationary phases using N,Ndimethyl-N-methacryloyloxyethyl-N-(3-sulfopropyl) ammonium betaine and various oligo-methylene and oligo-oxyethylene
crosslinkers differing in polarity and size. All monoliths showed a dual retention mechanism and could be applied to the separation of low molecular weight analytes such as phenolic acids in the HILIC mode using ACN-rich mobile phases or in the reverse-phase mode using mobile phases with higher concentrations of water. Furthermore, the efficiency of these monolithic stationary phases for polar low molecular weight analytes in HILIC mode depends on the polarity, rather than on the size of the crosslinkers. Jiang et al. [19] fabricated three sulfoalkylbetaine functionalized monolithic stationary phases using 1,4-bis(acryloyl)piperazine (PDA), ethylene dimethacrylate (EDMA) and N,N0 -methylenebisacrylamide (MBA) as crosslinkers, respectively. Mechanism studies have shown that the polarity of crosslinker can significantly influence the hydrophilicity of the resulting monoliths, while electrostatic interactions seem more dependent on the properties of the zwitterionic functional monomer. However, the use of these conventional crosslinkers will inevitably decrease the proportion of functional monomer in the polymerization system, resulting in the reduction of the density of functional moieties in the monolithic stationary phase. Unlike conventional crosslinkers, 2-(methacryloyloxy)ethyl-[N(2-methacryloyloxy)ethyl] phosphorylcholine (MMPC) possesses both high hydrophilicity and a specific PC group, which could further enrich the functionality of the resulting monolithic stationary phase [21]. Therefore, in this study, a series of zwitterionic MPC functionalized monoliths were fabricated via one-step in situ co-polymerization of MPC and the crosslinker (MMPC, MBA, or PDA) within 100 mm I.D. capillaries. The physicochemical and chromatographic properties of these zwitterionic monoliths, including permeability, column efficiency, retention mechanism and column performance, were systematically evaluated by scanning electron microscopy (SEM), energy-dispersive X-ray spectrometry (EDS), mercury intrusion porosimetry and micro-HPLC. The potential applications of these monoliths were also evaluated using a series of polar analytes and tryptic digests of human IgG. Finally, the effects of the type of crosslinker on the chromatographic performance of the resulting monoliths were also discussed. 2. Materials and methods 2.1. Chemicals and reagents The hydrophilic monomer MPC was from Sigma-Aldrich (St. Louis, MO, USA). The crosslinkers MBA and PDA were from Aladdin Chemicals (Shanghai, China). The fused-silica capillaries (375 mm O.D. 100 mm I.D.) were from Ruifeng Chromatography Ltd. (Yongnian, Hebei, China). 3-(Trimethoxysilyl)-propyl methacrylate (g-MAPS), methanol (MeOH), tetrahydrofuran (THF), isopropanol (IPA), dimethyl sulfoxide (DMSO), 2,20 -azobisisobutyronitrile (AIBN), acetonitrile (ACN), toluene, thiourea, acrylamide, benzoic
Please cite this article as: Q. Wang et al., Fabrication and application of zwitterionic phosphorylcholine functionalized monoliths with different hydrophilic crosslinkers in hydrophilic interaction chromatography, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.016
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acid (B), 2,4-dihydroxybenzoic acid (2,4-DHB), 3,4dihydroxybenzoic acid (3,4-DHB), 3,5-dihydroxybenzoic acid (3,5DHB), 3,4,5-trihydroxybenzoic acid (3,4,5-THB), formic acid, ammonium formate, 2,5-dihydroxybenzoic acid, thymine, adenine, adenosine, cytidine, cytosine, ammonium bicarbonate (NH4HCO3), urea and trifluoroacetic acid (TFA) were from Aladdin Chemicals (Shanghai, China). Human IgG and trypsin (from bovine pancreas, TPCK-treated) were from Sigma-Aldrich (Shanghai, China). Dithiothreitol and iodoacetamide were from Energy Chemical (Shanghai, China). The tryptic digestion of human IgG [22] and the preparation of MMPC were performed following previously published methods without any modification [21]. Distilled water was filtered through a 0.22-mm membrane before use. 2.2. Instrumentation The thermally initiated free-radical polymerization of the mixture composed of MPC, crosslinker and AIBN was carried out using a Jinghong DKS22 water bath (Shanghai, China). SEM measurements were performed on a Zeiss ULTRA 55 field emission scanning electron microscope (Oberkochen, Germany) at an acceleration voltage of 3 or 5 kV. EDS experiments were performed on a Zeiss LEO 1530 VP Field Emission Scanning Electron Microscope equipped with an Oxford INCA 400 energy dispersive X-ray microanalysis system (Oberkochen, Germany) at an acceleration voltage of 5 kV. The pore size distribution was determined using a Quantachrome PoreMaster 60 GT Mercury Intrusion Porosimeter (Boynton Beach, FL, USA). The z-potential values of monolithic materials were measured with a Zetasizer Nano-ZS z-potential meter (Malvern Panalytical, Malvern, UK). FT-IR spectra of the monoliths prepared as KBr pellets were recorded at room temperature in the range from 4000 to 400 cm1 with a Gilson-306 FTIR Spectrometer (Villiers-le-Bel, France). The pH values were € ttingen, Germany). measured using a Sartorius PB-10 pH meter (Go All micro-HPLC experiments were performed on a self-assembled HPLC system that consisted of a DiNa nano gradient pump (Tokyo, Japan), a Valco four-port injection valve with 20 nL internal loop (Houston, TX, USA), and a Shimadzu SPD-15C UV detector (Kyoto, Japan) with a lab-made on-column detection system. The column efficiency (N) was calculated by HW-2000 chromatographic working station based on the peak width at half height. Data acquisition and handling were carried out using an Unimicro TrisepTM Workstation 2003 (Shanghai, China) and all chromatograms were redrawn using Microcal Origin 8.0. The calculated logP values (cLogP) of different crosslinkers were obtained using ACD/ Labs v.12.0 (Advanced Chemistry Development, Toronto, Ontario, Canada). 2.3. Fabrication of zwitterionic MPC functionalized monoliths To fabricate the zwitterionic MPC functionalized monoliths with different hydrophilic crosslinkers, the fused-silica capillaries (20 cm) were first pretreated with g-MAPS to provide anchoring sites for the bulk polymer on the inner wall during polymerization [11,23,24]. Various proportions of the functional monomer (MPC), the crosslinker (MMPC, PDA or MBA), the initiator (AIBN, ~1% w/w with respect to the monomers) and the porogens (v/v, the mixture of THF with MeOH (IPA or DMSO)) were accurately weighed and mixed ultrasonically into a 2-mL vial (Fig. 1, Table 1, S1 and S2). After ultrasonic degassing for 5 min to remove any dissolved air, the resultant solution was introduced into capillaries pretreated with the g-MAPS. Both ends of the capillaries were then sealed with GC septa, and the capillaries were submerged into a 60 C water bath for 12 h. After the polymerization, these monoliths were thoroughly flushed with MeOH (0.5 h) to wash out the residual reagents
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using a high-pressure pump. A 2e3 mm detection window was then created at a distance of 2e3 cm from the end of the columns using a thermal wire stripper for the self-assembled micro-HPLC system. A 1 cm length of monolith was also cut from each column for SEM and EDS analysis. 2.4. Chromatographic conditions The mobile phases were prepared by mixing appropriate volume of buffer solution or H2O to ACN. The stock buffer solutions of ammonium formate were prepared by dissolving an appropriate amount of the salt in deionized H2O and adjusted to the desired pH with formic acid. Unless otherwise stated, the mobile phase pH values mentioned in this research refer to the aqueous portion only. The stock sample solutions were prepared by dissolving appropriate amounts of samples into MeOH to give a concentration around 1 mg/mL. Both mobile phases and sample solutions were subjected to a filtration through 0.22 mm membrane prior to the micro-HPLC experiments. Chromatograms were recorded at a wavelength of 214 or 254 nm. All separations were performed at room temperature. 2.5. N-Glycopeptide enrichment The on-column N-glycopeptide enrichment using zwitterionic MPC functionalized monolithic columns was performed at a constant flow rate (1 mL/min) with a Longer Precision Pump with TS2A/L0107-2A Syringe Pump Controller (Baoding, Hebei, China). Firstly, the monolithic column (140 mm 100 mm I.D., about 1 mg monolithic polymer) was washed with the loading buffer (9% H2O and 1% TFA in ACN, v/v) for 1 h. The lyophilized tryptic digests of human IgG were re-dissolved in loading buffer at a concentration of 1 mg/mL, then 10 mL samples were pumped through the monolith column at a flow rate of 1 mL/min. Non-glycopeptides were removed with loading buffer for 200 min at same flow rate. Then, the captured N-glycopeptides were eluted with the elution buffer (30% ACN and 1% TFA in H2O, v/v) for 10 min. 2.6. Calculations The permeability (K) of the zwitterionic MPC functionalized monoliths was calculated according to Darcy’s Law by using the following equation (1) [25]:
K ¼
uLh DP
(1)
where u (m/s), h (Pa∙s), L (m) and DP (Pa) refer to the linear velocity, the viscosity of mobile phase, the length of the monolithic column and the pressure drop across the monolithic column, respectively. 3. Results and discussion 3.1. Fabrication and optimization of the zwitterionic MPC functionalized monoliths with different hydrophilic crosslinkers Because the composition of the polymerization mixture significantly affects the permeability, morphology, efficiency and selectivity of the zwitterionic monoliths [11], several key parameters including the composition and the weight content of the porogens, as well as the weight content of the crosslinker, were systematically optimized. The physicochemical properties and morphological characteristics of the resulting monoliths were evaluated using micro-HPLC, SEM, and EDS. Firstly, based on our previous study
Please cite this article as: Q. Wang et al., Fabrication and application of zwitterionic phosphorylcholine functionalized monoliths with different hydrophilic crosslinkers in hydrophilic interaction chromatography, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.016
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Fig. 1. Fabrication of zwitterionic MPC functionalized monoliths with different hydrophilic crosslinkers.
Table 1 Composition of the polymerization mixtures for the fabrication of poly(MPC-co-MMPC) monoliths. Column
Monomer mixture (%, w/w)
Porogen mixture (%, w/w)
MPC
MMPC
MeOH
THF
D1 D2 D3 D4 D5 D6 D7 D8
50 50 50 50 45 55 50 50
50 50 50 50 55 45 50 50
35 35 35 35 35 35 40 30
65 65 65 65 65 65 60 70
Monomers: Porogens (%, w/w)
Backpressure (MPa)
Theoretical plates (plates/m)
20:80 30:70 35:65 40:60 35:65 35:65 35:65 35:65
0.1 0.4 1.2 / 1.8 0.8 4.4 0.3
6100 28500 88000 / 55000 34800 48400 26900
Experimental conditions: column dimensions, 140 mm 100 mm I.D.; column temperature: room temperature; UV detection wavelength, 254 nm; mobile phase, H2O/ACN (5/ 95, v/v); flow rate: 800 nL/min; injection volume, 20 nL; sample: thiourea.
[24], a binary porogenic system including IPA and THF was employed to prepare the poly(MPC-co-MBA) monolith with some modification (Table S1). Therefore, the IPA/THF system was chosen initially as binary porogen for the preparation of the poly(MPC-coPDA) and poly(MPC-co-MMPC) monoliths. However, the solubility of PDA and MMPC was not satisfactory in the IPA/THF system. To improve their solubility, a binary porogenic system including DMSO and THF was selected to prepare the poly(MPC-co-PDA) monolith (Table S2), while a binary porogenic system consisting of MeOH and THF was employed to fabricate the poly(MPC-co-MMPC) monolith (Table 1). Then, the composition of the polymerization mixture for the poly(MPC-co-MMPC) monolith was further optimized. The influence of the total content of the monomer mixture (MPC and MMPC) on the column performance was first investigated. When the weight content of the monomer mixture was increased from 20% (column D1) to 40% (column D4, it was too hard to pump the mobile phase through it), the column backpressure and efficiency were found to raise significantly. Therefore, column D3 was selected for further optimization due to its satisfactory backpressure (1.2 MPa) and column efficiency (88,000 plates/m for thiourea). Since the content of the functional monomer (MPC) has a significant effect on the column properties, the weight ratio of MPC in the monomer mixture was then varied from 45% (column D5) to 55% (column D6). By increasing the weight ratio of MPC, the backpressure decreased moderately from 1.8 MPa (column D5) to 0.8 MPa (column D6), whereas either a decrease (column D5) or an increase (column D6) of the amount of MPC results in a drop of column efficiency. Thus, the conditions for preparing column D3 were chosen for further optimization. The effect of the porogen mixture composition was then investigated by varying the weight ratio of MeOH and THF (w/w) from 30/70 (column D8) to 40/60
(column D7). This minor change of the porogen composition had a significant influence on the column backpressure and efficiency. The backpressure significantly increased from 0.3 (column D8) to 4.4 (column D7) MPa, while column D3 exhibited the highest column efficiency and acceptable backpressure. Therefore, the polymerization conditions used to produce column D3 were selected for all further studies. Finally, the preparation of the poly(MPC-coMBA) and poly(MPC-co-PDA) monolithic columns were also optimized according to the above methods (Tables S1 and S2). 3.2. Characterization of the zwitterionic MPC functionalized monoliths To verify the success of the preparation process, these zwitterionic MPC functionalized monoliths were comparatively evaluated by FT-IR, SEM, and EDS. As shown in Fig. S2, all characteristic peaks of the PC moieties in the poly(MPC-co-MMPC) monolith for the P] O, PeO-alkyl and Nþ(CH3)3 stretches could be identified as ~1222.65, ~1078.98 and ~968.09 cm1, respectively. Similar characteristic peaks were also detected on the poly(MPC-co-MBA) and poly(MPC-co-PDA) monoliths (Figs. S3 and S4). SEM images (Fig. 2) show that these monoliths possess a porous structure and are well attached to the inner wall of the capillary (except for the poly(MPCco-PDA) monolith). EDS results revealed the existence of the P element on the polymer surface, which indicates that MPC was incorporated into the monolithic matrix (Fig. S5). 3.3. Comparison of the physicochemical properties of the zwitterionic MPC functionalized monoliths To evaluate the influence of the crosslinkers on the physicochemical and chromatographic properties of the zwitterionic MPC
Please cite this article as: Q. Wang et al., Fabrication and application of zwitterionic phosphorylcholine functionalized monoliths with different hydrophilic crosslinkers in hydrophilic interaction chromatography, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.016
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Fig. 3. Van Deemter plots for zwitterionic MPC functionalized monoliths. Experimental conditions: monolithic columns: 170 mm 100 mm I.D.; mobile phase: H2O/ ACN (10/90, v/v); column temperature: room temperature; UV detection wavelength: 254 nm; injection: 20 nL; sample: thiourea.
Fig. 2. SEM results of (a) poly(MPC-co-MMPC), (b) poly(MPC-co-MBA), and (c) poly (MPC-co-PDA) monoliths.
functionalized monoliths, several key factors (including permeability, column efficiency, hydrophilic retention mechanism, and zpotential analysis) of the three MPC functionalized monoliths, i.e. poly(MPC-co-PDA), poly(MPC-co-MBA), and poly(MPC-co-MMPC), were systematically compared. According to Darcy’s law [25], the calculated permeabilities (K values) for the poly(MPC-co-MMPC), poly(MPC-co-MBA) and poly(MPC-co-PDA) monoliths were 6.49 1014, 3.20 1014 and 2.86 1014 m2, respectively, using H2O/ACN (5/95, v/v) as mobile phase. No significant difference on the permeability was found between these zwitterionic MPC functionalized monoliths. Good permeability of these monoliths may be attributed to that all three monolithic materials contain large amounts of macropores (>1 mm) and possess acceptable total porosities (>60%), which permits rapid flow through the column under low backpressure. The effects of the type of crosslinker on the efficiency of the zwitterionic MPC functionalized monolithic columns were then evaluated. A column efficiency as high as 88,000 plates/m was obtained for thiourea on the poly(MPC-co-MMPC) monolith at the optimum linear velocity, while column efficiencies corresponding to 76,000 plates/m and 50,000 plates/m were observed on the poly(MPC-co-PDA) and poly(MPC-co-MBA) monoliths, respectively. Moreover, van Deemter plots for these zwitterionic MPC functionalized monoliths were also compared. As depicted in Fig. 3, the poly(MPC-co-MMPC) monolith exhibited a lower theoretical plate height (H z 11 mm) for thiourea at the optimum linear velocity compared to the poly(MPC-co-PDA) monolith (H z 13 mm) and the poly(MPC-co-MBA) monolith (H z 20 mm). It is worth noting that the efficiency of these zwitterionic MPC functionalized monolithic columns is much higher than that of the poly(MPC-co-EDMA) monolith (Nthiourea ¼ 36000 plates/m [17]). 3.4. Comparison of the retention mechanisms of the zwitterionic MPC functionalized monoliths In the previous HILIC studies, the change of retention
mechanism from a HILIC mode to an apparent RP mode has been observed on hydrophilic stationary phases, and the critical mobile phase composition corresponding to this turning point can be employed to evaluate the polarity of hydrophilic stationary phases [19,26]. To systematically evaluate the influence of the type of crosslinker on the hydrophilicity of zwitterionic MPC functionalized monoliths, the effect of ACN content (ranging from 95% to 10%) on the retention of three test solutes (toluene, acrylamide and thiourea) were investigated. As depicted in Fig. 4, a typical HILIC retention mechanism was observed on these zwitterionic MPC functionalized monoliths. Furthermore, no critical mobile phase composition could be determined for the poly(MPC-co-MBA) monolith. However, the critical mobile phase compositions were around 20% ACN, 30% ACN and 60% ACN for the poly(MPC-co-PDA), poly(MPC-co-MMPC) and poly(MPC-co-EDMA) monoliths [17], respectively. Therefore, the hydrophilicity of the poly(MPC-coMBA) monolith can be considered as higher than that of the poly(MPC-co-PDA) and poly(MPC-co-EDMA) monoliths, which is in accordance with the polarity order of the three crosslinkers (MBA, clogP ¼ 1.44; PDA, clogP ¼ 1.22; EDMA, clogP ¼ 2.99). This observation is consistent with the previous study [19]. In addition, although MBA, MMPC (clogP ¼ 1.38), and PDA have similar polarities, the hydrophilicity of the poly(MPC-co-MMPC) monolith is lower than that of the poly(MPC-co-MBA) and poly(MPC-co-PDA) monoliths, which indicates that more complex interactions could exist in the poly(MPC-co-MMPC) monolithic stationary phase. Although PC has both a positively charged quaternary ammonium group and a negatively charged phosphoric acid group, it has been found that the materials with PC groups have a negative surface charge over a wide pH range [17]. Therefore, the electrostatic interactions could contribute to the retention of charged analytes on the zwitterionic MPC functionalized monoliths. To evaluate the effect of the crosslinker on the electrostatic interactions, the z-potential values for these zwitterionic MPC functionalized monoliths were measured in 5 mM ammonium formate at different pH values (2.0, 3.0, 4.0, 5.0, 6.4, 7.0, and 8.0). As shown in Fig. 5, similar z-potential values were achieved for the poly(MPCco-PDA) and poly(MPC-co-MBA) monoliths due to the fact that both the crosslinkers MBA and PDA were neutral and thus the zwitterionic MPC was responsible for the surface charge of these monolithic materials. However, z-potential measurements revealed that
Please cite this article as: Q. Wang et al., Fabrication and application of zwitterionic phosphorylcholine functionalized monoliths with different hydrophilic crosslinkers in hydrophilic interaction chromatography, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.016
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Fig. 4. Influence of ACN concentration on retention: (a) poly(MPC-co-MMPC) monolith, (b) poly(MPC-co-PDA) monolith, (c) poly (MPC-co-MBA) monolith. Experimental conditions: monolithic columns, 140 mm 100 mm I.D.; column temperature: room temperature; mobile phase: mixture of H2O and ACN; detection wavelength: 254 nm; injection: 20 nL; sample: toluene, acrylamide, thiourea.
Fig. 5. z-potential values of the zwitterionic MPC functionalized monoliths.
the surface of the poly(MPC-co-MMPC) monolith was negatively charged over the whole studied pH range and the absolute z-potential value was found to increase with increasing buffer pH from 2.0 to 8.0, and to be higher than that of the poly(MPC-co-PDA) and poly(MPC-co-MBA) monoliths. This may be due to the fact that MMPC as crosslinker could further enrich the density of specific PC groups on the resulting monolithic stationary phase, which is not the case for the conventional hydrophilic crosslinkers (MBA and PDA). These observed features of the poly(MPC-co-MMPC) monolith seem to be in favor of its applications in bioanalysis. 3.5. Comparison of the chromatographic performance of the zwitterionic MPC functionalized monoliths 3.5.1. Separation of small molecules To compare the separation selectivity of the zwitterionic MPC functionalized monoliths, a test mixture containing nucleobases, nucleosides, and toluene, was selected. As can be seen in Fig. 6, the most hydrophilic compound cytidine exhibited the highest retention on the poly(MPC-co-MMPC) monolith, an intermediate retention on the poly(MPC-co-MBA) monolith, and the lowest retention on the poly(MPC-co-PDA) monolith. Furthermore, an excellent separation of all compounds was achieved on the poly(MPC-co-MMPC) monolith, while adenine and adenosine could not be baseline separated on the poly(MPC-co-MBA) and poly(MPC-co-PDA) monoliths. Clearly, the poly(MPC-co-MMPC) monolith exhibited higher separation selectivity for these compounds than the other two monoliths.
Fig. 6. Separation of nucleobases and nucleosides on the zwitterionic MPC functionalized monoliths. Conditions: monolithic columns, 140 mm 100 mm I.D.; column temperature: room temperature; mobile phase: H2O/ACN (10/90, v/v); flow rate: 1000 nL/min (linear velocity: 1.8 mm/s); UV Wavelength, 254 nm; samples: (0) toluene, (1) thymine, (2) adenine, (3) adenosine, (4) cytosine, (5) cytidine; injection volume: 20 nL.
The chromatographic separation of a series of benzoic acid derivatives was then investigated on these zwitterionic MPC functionalized monoliths using 10 mM ammonium formate (pH 3.0)/ ACN (40/60, v/v) as mobile phase. As depicted in Fig. 7, the baseline separation of the five benzoic acid derivatives was achieved on the poly(MPC-co-MMPC) and poly(MPC-co-MBA) monoliths. Moreover, B (pKa 4.20, logD3.0 ¼ 1.87) eluted first, followed by 2,4-DHB (pKa 3.32, logD3.0 ¼ 1.27), 3,4-DHB (pKa 4.45, logD3.0 ¼ 1.14), 3,5-DHB (pKa 3.96, logD3.0 ¼ 1.07) and 3,4,5-THB (pKa 4.33, logD3.0 ¼ 0.89), successively, which is in accordance with the polarity order of analytes. These results indicate that zwitterionic MPC functionalized monoliths with different hydrophilic crosslinkers have a great potential for the separation of bioactive molecules. 3.5.2. N-Glycopeptide enrichment Recently, a zwitterionic hydrophilic material exhibited excellent enrichment efficiency for N-glycopeptides in human IgG tryptic digest, which will benefit to eliminate interference from nonglycopeptides and to improve the sensitivity of N-glycopeptide detection [27,28]. As shown in Fig. S6, no glycopeptides could be directly detected in human IgG tryptic digest without sample pretreatment due to the severe interference and ion suppression from the highly abundant non-glycopeptides, even when using a high concentration of human IgG digest (1.0 mg/mL). However, after enrichment with the poly(MPC-co-MMPC) monolith, the
Please cite this article as: Q. Wang et al., Fabrication and application of zwitterionic phosphorylcholine functionalized monoliths with different hydrophilic crosslinkers in hydrophilic interaction chromatography, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.016
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signals of the nonglycosylated peptides were significantly eliminated and 24 N-glycopeptides were clearly detected with much enhanced MS intensity (Fig. 8 and Table S3). For comparison, only 19 and 10 N-glycopeptides could be detected with inferior MS intensity after enrichment with the poly(MPC-co-MBA) and poly(MPC-co-PDA) monoliths (Tables S4 and S5), respectively. Moreover, the results obtained with the poly(MPC-co-MMPC) monolith are also better than those previously reported with the commercial ZIC-HILIC material (21 glycopeptides) [22] or ZIC-HILIC polymer particles poly(MBAAm-co-MAA)@L-Cys (17 glycopeptides) [29] for human IgG tryptic digest enrichment. 4. Conclusion
Fig. 7. Separation of benzoic acid derivatives on the zwitterionic MPC functionalized monoliths. Conditions: monolithic columns, 140 mm 100 mm I.D.; column temperature: room temperature; mobile phase: 10 mM ammonium formate (pH 3.0)/ACN (40/60, v/v); flow rate: 800 nL/min (linear velocity: 0.6 mm/s); UV wavelength, 214 nm; samples: (1) B, (2) 2,4-DHB, (3) 3,4-DHB, (4) 3,5-DHB, (5) 3,4,5-THB; injection: 20 nL.
In this study, to evaluate the influence of the type of crosslinker (PDA, MBA or MMPC) on the chromatographic properties of the corresponding monolithic stationary phases, a series of zwitterionic MPC functionalized monoliths were fabricated in 100 mm I.D. capillaries. Under the optimized polymerization conditions, the highest column efficiency (88,000 plates/m) was achieved for thiourea on the novel poly(MPC-co-MMPC) monolithic column. Mechanism studies indicated that the type and polarity of crosslinker could influence the hydrophilicity and electrostatic interactions of the zwitterionic MPC functionalized monoliths. Higher separation selectivity was achieved for small polar analytes, such as nucleobases, nucleosides, and benzoic acid derivatives, on the poly(MPC-co-MMPC) monolith compared to that obtained on the other two MPC functionalized monoliths. Furthermore, the poly(MPC-co-MMPC) monolith exhibited good potential for the enrichment of hydrophilic N-glycopeptides in complex matrices, which makes it a promising analytical tool in glycomics and glycoproteomics. Overall, MMPC not only enriches the selection of hydrophilic crosslinkers, but also opens up interesting possibilities for the fabrication of novel HILIC stationary phases in separation science. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant: 81703460, 81673391). There are no conflicts of interest to declare. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.aca.2019.12.016. Author contribution statement
Fig. 8. (a) MALDI-TOF MS spectra of the standard hIgG digestion solution after enrichment on the zwitterionic MPC functionalized monoliths. (b) N-glycopeptides after enrichment on the poly(MPC-co-MMPC) monolith. Conditions: monolithic columns, 140 mm 100 mm I.D.; loading buffer: ACN/H2O/TFA (90:9:1, v/v/v); eluting buffer: ACN/H2O/TFA (30:69:1, v/v/v); injection: 10 mL. Red stars: N-glycopeptides. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Zhengjin Jiang and Pei Yu designed the project and provided overall guidance. Qiqin Wang and Qiaoxuan Zhang carried out the experiments, analyzed data and wrote the paper. Hao Huang, Pengwei Zhao, Lingjue Sun, Kun Peng, Xiao Liu, Meng Ruan, Huikai Shao carried out some experiments and analyzed the related data. Jacques Crommen edited the article. All authors reviewed the manuscript.
Please cite this article as: Q. Wang et al., Fabrication and application of zwitterionic phosphorylcholine functionalized monoliths with different hydrophilic crosslinkers in hydrophilic interaction chromatography, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.016
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References [1] S.D. Palma, P.J. Boersema, A.J.R. Heck, S. Mohammed, Zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC and ZIC-cHILIC) provide high resolution separation and increase sensitivity in proteome analysis, Anal. Chem. 83 (2011) 3440e3447. [2] L.Z. Qiao, X.Z. Shi, G.W. Xu, Recent advances in development and characterization of stationary phases for hydrophilic interaction chromatography, Trends Anal. Chem. 81 (2016) 23e33. s, Recent advances in stationary phases and understanding [3] P. Jandera, P. Jana of retention in hydrophilic interaction chromatography. A review, Anal. Chim. Acta 967 (2017) 12e32. [4] N.P. Dinh, T. Jonsson, K. Irgum, Probing the interaction mode in hydrophilic interaction chromatography, J. Chromatogr. A 1218 (2011) 5880e5891. [5] W. Jiang, K. Irgum, Synthesis and evaluation of polymer-based zwitterionic stationary phases for separation of ionic species, Anal. Chem. 73 (2001) 1993e2003. €gren, K. Irgum, Chromatographic interactions between pro[6] C. Viklund, A. Sjo teins and sulfoalkylbetaine-based zwitterionic copolymers in fully aqueous low-salt buffers, Anal. Chem. 73 (2001) 444e452. [7] W. Jiang, K. Irgum, Tentacle-type zwitterionic stationary phase prepared by surface-initiated graft polymerization of 3-[N,N-dimethyl-N-(methacryloyloxyethyl)- ammonium] propanesulfonate through peroxide groups tethered on porous silica, Anal. Chem. 74 (2002) 4682e4687. [8] Z.J. Jiang, N.W. Smith, P.D. Ferguson, M.R. Taylor, Hydrophilic interaction chromatography using methacrylate-based monolithic capillary column for the separation of polar analytes, Anal. Chem. 79 (2007) 1243e1250. [9] Y.N. Chou, F. Sun, H.C. Hung, P. Jain, A. Sinclair, P. Zhang, T. Bai, Y. Chang, T.C. Wen, Q.M. Yu, S.Y. Jiang, Ultra-low fouling and high antibody loading zwitterionic hydrogel coatings for sensing and detection in complex media, Acta Biomater. 40 (2016) 31e37. [10] Q.Q. Wang, K. Peng, W.J. Chen, Z. Cao, P.J. Zhu, Y.M. Zhao, Y.Q. Wang, H.B. Zhou, Z.J. Jiang, Development of double chain phosphatidylcholine functionalized polymeric monoliths for immobilized artificial membrane chromatography, J. Chromatogr. A 1479 (2017) 97e106. [11] Q.Q. Wang, H.H. Wu, K. Peng, H.Y. Jin, H.K. Shao, Y.Q. Wang, J. Crommen, Z.J. Jiang, Hydrophilic polymeric monoliths containing choline phosphate for separation science applications, Anal. Chim. Acta 999 (2018) 184e189. ríkov [12] V. Ske a, P. Jandera, Effects of the operation parameters on hydrophilic interaction liquid chromatography separation of phenolic acids on zwitterionic monolithic capillary columns, J. Chromatogr. A 1217 (2010) 7981e7989. [13] F. Svec, Y.Q. Lv, Advances and recent trends in the field of monolithic columns for chromatography, Anal. Chem. 87 (2015) 250e273. [14] Z.J. Jiang, N.W. Smith, P.D. Ferguson, M.R. Taylor, Novel highly hydrophilic zwitterionic monolithic column for hydrophilic interaction chromatography, J. Sep. Sci. 32 (2009) 2544e2555. [15] Z.H. Liu, Y.B. Peng, T.T. Wang, G.X. Yuan, Q.X. Zhang, J.L. Guo, Z.J. Jiang, Preparation and application of novel zwitterionic monolithic column for
hydrophilic interaction chromatography, J. Sep. Sci. 36 (2013) 262e269. [16] G.X. Yuan, Y.B. Peng, Z.H. Liu, J.Y. Hong, Y. Xiao, J.L. Guo, N.W. Smith, J. Crommen, Z.J. Jiang, A facile and efficient strategy to enhance hydrophilicity of zwitterionic sulfoalkylbetaine type monoliths, J. Chromatogr. A 1301 (2013) 88e97. [17] Z.J. Jiang, J. Reilly, B. Everatt, N.W. Smith, Novel zwitterionic polyphosphorylcholine monolithic column for hydrophilic interaction chromatography, J. Chromatogr. A 1216 (2009) 2439e2448. ríkova kov , J. Urban, Cross-linker effects on the [18] M. Stan a, P. Jandera, V. Ske separation efficiency on (poly)methacrylate capillary monolithic columns. Part II. Aqueous normal-phase liquid chromatography, J. Chromatogr. A 1289 (2013) 47e57. [19] C.S. Liu, W.J. Chen, G.X. Yuan, Y. Xiao, J. Crommen, S.H. Xu, Z.J. Jiang, Influence of the crosslinker type on the chromatographic properties of hydrophilic sulfoalkylbetaine-type monolithic columns, J. Chromatogr. A 1373 (2014) 73e80. [20] F. Li, D.Y. Qiu, J.W. Kang, Preparation of a sulfoalkylbetaine-based zwitterionic monolith with enhanced hydrophilicity for capillary electrochromatography separation applications, Chromatographia 80 (2017) 975e981. [21] Y. Kiritoshi, K. Ishihara, Synthesis of hydrophilic cross-linker having phosphorylcholine-like linkage for improvement of hydrogel properties, Polymer 45 (2004) 7499e7504. [22] C.S. Xia, F.L. Jiao, F.Y. Gao, H.P. Wang, Y.Y. Lv, Y.H. Shen, Y.J. Zhang, X.H. Qian, Two-dimensional MoS2-based zwitterionic hydrophilic interaction liquid chromatography material for the specific enrichment of glycopeptides, Anal. Chem. 90 (2018) 6651e6659. [23] X.Y. Wang, D.H. Xia, H. Han, K. Peng, P.J. Zhu, J. Crommen, Q.Q. Wang, Z.J. Jiang, Biomimetic small peptide functionalized affinity monoliths for monoclonal antibody purification, Anal. Chim. Acta 1017 (2018) 57e65. [24] Q.Q. Wang, H.Y. Jin, D.H. Xia, H.K. Shao, K. Peng, X. Liu, H. Huang, Q.X. Zhang, J.L. Guo, Y.Q. Wang, J. Crommen, N. Gan, Z.J. Jiang, Biomimetic polymer-based method for selective capture of C-reactive protein in biological fluids, ACS Appl. Mater. Interfaces 10 (2018) 41999e42008. [25] P.A. Bristow, J.H. Knox, Standardization of test conditions for high performance liquid chromatography columns, Chromatographia 10 (1977) 279e289. [26] J.J. Pesek, M.T. Matyska, S. Larrabee, HPLC retention behavior on hydridebased stationary phases, J. Sep. Sci. 30 (2007) 637e647. [27] Y. Yang, V. Franc, A.J.R. Heck, Glycoproteomics: a balance between highthroughput and in-depth analysis, Trends Biotechnol. 35 (2017) 598e609. [28] Y. Zhang, Y. Peng, L.J. Yang, H.J. Lu, Advances in sample preparation strategies for MS-based qualitative and quantitative N-glycomics, Trends Anal. Chem. 99 (2018) 34e46. [29] J.X. Liu, K.G. Yang, W.Y. Shao, S.W. Li, Q. Wu, S. Zhang, Y.Y. Qu, L.H. Zhang, Y.K. Zhang, Synthesis of zwitterionic polymer particles via combined distillation precipitation polymerization and click chemistry for highly efficient enrichment of glycopeptide, ACS Appl. Mater. Interfaces 8 (2016) 22018e22024.
Please cite this article as: Q. Wang et al., Fabrication and application of zwitterionic phosphorylcholine functionalized monoliths with different hydrophilic crosslinkers in hydrophilic interaction chromatography, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.016