Synthesis of polymeric monoliths via thiol-maleimide polymerization reaction for highly efficient chromatographic separation

Accepted Manuscript Title: Synthesis of polymeric monoliths via thiol-maleimide polymerization reaction for highly efficient chromatographic separation Authors: Jingyao Bai, Junjie Ou, Haiyang Zhang, Shujuan Ma, Yehua Shen, Mingliang Ye PII: DOI: Reference:

S0021-9673(17)31086-5 http://dx.doi.org/doi:10.1016/j.chroma.2017.07.070 CHROMA 358717

To appear in:

Journal of Chromatography A

Received date: Revised date: Accepted date:

27-3-2017 16-7-2017 19-7-2017

Please cite this article as: Jingyao Bai, Junjie Ou, Haiyang Zhang, Shujuan Ma, Yehua Shen, Mingliang Ye, Synthesis of polymeric monoliths via thiol-maleimide polymerization reaction for highly efficient chromatographic separation, Journal of Chromatography Ahttp://dx.doi.org/10.1016/j.chroma.2017.07.070 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis of polymeric monoliths via thiol-maleimide polymerization reaction for highly efficient chromatographic separation

Jingyao Baia,b, Junjie Oua,* Haiyang Zhanga,b, Shujuan Maa,b, Yehua Shenb *, and Mingliang Yea *

aCAS

Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of

Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China bKey

Laboratory of Synthetic and Natural Function Molecule Chemistry of Ministry of Education,

College of Chemistry and Materials Science, Northwest University, Shaanxi Alcohol Ether and Biomass Energy Engineering Research Center, Key Laboratory of Yulin Desert Plants Resources Xi’an 710069, China

*

To whom correspondence should be addressed:

Prof. Junjie Ou Tel: +86-411-84379576, Fax: +86-411-84379620, E-mail: [email protected] Prof. Yehua Shen Tel: +86-29-88302635, Fax: +86-29-88302635, E-mail: [email protected] Prof. Mingliang Ye Tel: +86-411-84379610, Fax: +86-411-84379620, E-mail: [email protected]

Highlights 

Thiol-maleimide click reaction was firstly adopted to prepare capillary monoliths.



Ultra-high column efficiency (180,500 N/m) was acquired on poly(BMI-co-3SH). 1



The tryptic digests of BSA and HeLa were positively identified in cLC-MS.

Abstract: One-step thiol-maleimide polymerization reaction was firstly adopted for direct preparation of polymeric monoliths via alkaline-catalyzed reaction of 4,4’-bis(maleimidophenyl)methane

(BMI)

and

trimethylolpropane

tris(3-mercaptopropionate) (3SH) / pentaerythriol tetra(3-mercaptopropionate) (4SH) in the presence of a small amount of triethylamine (TEA). The polymerization could be performed within 3 h, which was faster than thermal-initiated free radical polymerization. Two kinds of monoliths, poly(BMI-co-3SH) (marked as I) and poly(BMI-co-4SH) (marked as II), were characterized with scanning electron microscopy (SEM),

attenuated total

reflection

Fourier-transformed infrared

spectroscopy (ATR-FTIR), thermal gravimetric analysis (TGA) and mercury intrusion porosimetry (MIP). Satisfactory chromatographic separation ability and column efficiency were gained for analysis of small molecular compounds such as alkylbenzenes, polynuclear aromatic hydrocarbons (EPA 610) and phenols in reversed-phase capillary liquid chromatography (cLC). High column efficiency (180,500 N/m) for butylbenzene was acquired on poly(BMI-co-3SH) column I-2, which was higher than those on most reported polymeric monoliths. A retention-independent efficient performance of small molecules was obtained by plotting of plate height (H) of alkylbenzenes versus the linear velocity (u). A term values in van Deemter equation of I-2 (1.72-0.24 μm) and poly(BMI-co-4SH) column II-2 (5.28-4.14 μm) were smaller than those of traditional organic/hybrid monoliths. Finally, as a practical application, 53 and 2184 unique peptides from the tryptic digests of bovine serum albumin (BSA) and HeLa cell proteins were positively identified with poly(BMI-co-3SH) monolith in cLC-MS.

Key words: polymeric monolith, thiol-maleimide, capillary liquid chromatography, cLC-MS 2

1 INTRODUCTION Micro/nano-scale separation techniques such as capillary liquid chromatography (cLC) have received increasing research interests due to the fact that eco-friendly, economical, fast and efficient characters they own. As the “heart” of a cLC microsystem, the construction of capillary column has attracted significant concentrations. There were several excellent reviews focused on various preparation approaches, specific properties as well as applications of monolithic columns.[1-4] Compared to both silica-based monoliths and organic-inorganic hybrid monoliths, polymeric monoliths including polymethacrylate, polyacrylamide and polystyrene monoliths possessed remarkable advantages, for instance, great chemical stability, simple preparation and variety of functions.[5-7] Nevertheless, low column efficiency for small molecules was still a tough problem generally occurred in polymeric monoliths on account of low specific surface area, heterogeneous microporous structure or internal spherical structure caused by traditional free radical polymerization reaction.[8-10] Aiming at unsatisfactory column efficiency for small molecules on polymeric monolithic columns, several researchers attempted to synthesis hypercrosslinked polymeric monolith or introduce nanoparticles into the monolith for enhancing surface area, optimizing porous structure and surface chemistry.[11-14] Thus, it is still necessary to utilize an easier synthetic pathway to achieve satisfactory column efficiency of small molecules in particular on polymeric monolithic columns. [10, 15, 16] ‘‘Click’’ chemistry, which was defined by Sharplessin 2001[17], has witnessed rapid development across multiple fields as diverse as preparation of monolithic columns, bioconjugation, surface modification, polymers and materials science, as well as medicine science.[16, 18-22] The appeal of click chemistry resulted from powerful characteristics such as solvent tolerance, insensitivity of oxygen, quantitative yields, regioselectivity and inoffensive byproducts.[23-25] Classical click reaction includes the following: (i) copper catalyzed azide-alkyne cycloaddition (CuAAC), (ii) 3

Diels-Alder reaction and (iii) thiol-based reaction. A series of thiol-based reactions have been labelled as “click” term in the published papers, for instance, thiol-isocyanate,

thiol-epoxy,

thiol-halogenated

hydrocarbon

and

thiol-maleimide.[26-29] Among them, thiol-maleimide click reaction was very attractive due to high degree of reactivity and specificity of sulfhydryl groups with maleimide to form stable thioether bonds since it has been exploited extensively for fluorescent probe, immobilized protein, functionalization of building copolymers and hydrogel surface.[30-36] Up till now, to our best knowledge, there were no reports on direct preparation of monolithic columns via thiol-maleimide polymerization reaction. In this work, we described a novel approach to produce polymeric monoliths via thiol-maleimide polymerization reaction, using 4,4’-bis(maleimidophenyl)methane (BMI) and trimethylolpropane

tris(3-mercaptopropionate)

(3SH)/pentaerythriol

tetra(3-mercaptopropionate) (4SH) as monomers. The construction process was simple and feasible, which was faster than thermal-initiated free radical polymerization. The obtained monolithic capillary columns exhibited great mechanical strength and thermal stability, and were successfully applied to separate complicated biological samples in cLC-MS.

2 EXPERMENTAL SECTION 2.1 Materials. BMI (95%), 3SH (95%), 4SH (95%), triethylamine (TEA, analytical reagent), dimethylphenylphosphine (DMPP, 99%), 2,5-dihydroxybenzoic acid (DHB, 98%), formic acid (FA, 98%), bovine serum albumin (BSA, 98%) and EPA 610 (including naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene,pyrene,

benzo(a)anthracene,

chrysene,

benzo(b)fluoranthene,benzo(k)fluoranthene, benzo(a)pyrene, dibenzo(a,h)-anthracene, benzo(g,h,i)perylene, indeno(1,2,3-cd)pyrene) were obtained from Sigma-Aldrich (St Louis, Mo, USA). (3-Mercaptopropyl)trimethoxysilane was purchased from J&K 4

Scientific Ltd. (Beijing, China). Ethanol, dimethyl sulfoxide (DMSO), n-propanol, 1,4-butylene glycol, thiourea, benzene, toluene, ethylbenzene, propylbenzene and butylbenzene were analytical reagent and gotten from Tianjin Kermel Chemical Plant (Tianjin,

China).

Phloroglucinol,

pyrogallol,

p-cresol,

p-tert-butylphenol,

2,4-dichlorophenol, 2,4,5-trichlorophenol, caffeine, carbamazepine, o-nitroaniline, 1,2-diphenyl hydrazine and 4-aminobiphenyl were analytical reagent and purchased from Sino-American Biotechnology Corporation (Beijing, China). The fused silica capillaries with inner dimensions of 50 and 75 μm i.d. were purchased from Polymicro Technologies (Phoenix, AZ, USA). HPLC-grade acetonitrile (ACN) was acquired from Yuwang Group (Shandong, China). Deionized water was prepared with a Milli-Q system (Milli-pore, MA, USA). 2.2 Instrumentation Attenuated total reflection Fourier-transformed infrared spectroscopy (ATR-FTIR) was carried out on Thermo Nicolet 380 spectrometer (Nicolet, Wisconsin, USA). The microscopic images and energy-dispersive X-ray spectrum (EDS) of monolithic materials were obtained by scanning electron microscopy (SEM, JEOL JSM-5600, Tokyo, Japan). The elemental analysis was obtained by Horiba Emga-930 (HORIBA Scientific, Japan). Thermal gravimetric analysis (TGA) data were collected on TG 209 F3 Tarsus (NETZSCH-Gerätebau GmbH, Germany). The specific surface area was calculated from nitrogen adsorption/desorption measurements of dry bulk monoliths using a Quadrasorb SI surface area analyzer (Quantachrome, Boynton Beach, USA). Pore size distribution was measured by mercury intrusion porosimetry (MIP) on an AutoPore IV 9520 (Micromeritics instrument Ltd., USA). The cLC experiments were performed on an LC system combining with an Agilent 1100 micropump (Hewlett-Packard, Waldbronn, Germany) and a K-2501 UV detector (Knauer, Berlin, Germany). On-column detection wavelength was set at 214 or 254 nm. A 7725i injector with a 20 μL internal sample loop was connected between the micropump and tee-piece to load the samples with split mode. A tee-piece was used as a splitter. One end of the monolithic column (75 μm i.d.) was connected with the tee-piece, and the other end was connected to a 15 cm long blank capillary (50 μm 5

i.d.). The outlet of the monolithic column was connected with a Teflon tube (1.5 cm) to an empty fused-silica capillary (50 μm i.d.), where a detection window was made by removing a 2 mm length of the polyimide coating in a position of 5.0 cm from the separation monolithic column outlet. The chromatographic data were collected and analyzed with a soft-ware program HW-2000 from Qianpu Software (Shanghai, China). The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) experiments were carried out by a 5800 Proteomics Analyzer (Applied Biosystems, USA) with a pulsed Nd/YAG laser at 355 nm in linear positive ion mode. The mixture of sample solution (0.5 μL) and DHB solution (0.5 μL, 25 mg mL−1 DHB in ACN/H2O/H3PO4, 70/29/1, v/v/v) was spotted on the MALDI plate for MS analysis. 2.3 Methods In the cLC experiments, UV detector wavelength was set at 214 nm. The mobile phase was composed of ACN/H2O. The actual flow rate through monolithic column was calculated by measuring the weight of mobile phase, which was collected with a centrifuge tube for 30 min. The retention factor (k) was defined as (tr-t0)/t0, where tr and t0 represent the retention times of the analytes and the void time marker, respectively. The permeability (B0) can be calculated according to Darcy’s law by the equation B0 = FηL/(πr2ΔP), where F (m3/s) is the flow rate of mobile phase, η is the viscosity of mobile phase (0.38×10-3 Pa s for ACN), L and r (m) are effective length and inner radius of the column, respectively, ΔP (Pa) is the pressure drop across the column. The data of ΔP and F were obtained on an ACQUITY UltraPerformance LC (Waters, USA). The yield of monoliths was calculated by the following equation: Yield (%) = 100× Wd/Wt, where Wd is the dry weight of the washed material and Wt is the theoretical weight of the monolith. The reproducibility of monolithic column was evaluated through the relative standard deviation (RSD) for the retention factor (k) of toluene as the model analyte (thiourea as the void time marker). The peak capacity (P) can be 6

calculated as follows[37]: P=1+tg/(1/n)∑𝑛1 𝑤 , where w is the peak widths in the chromatogram, n is the number of peaks selected for the calculation, and tg is the gradient run time. 2.4 Pretreatment of fused-silica capillary The capillary (75 μm i.d.) was rinsed with 0.1 mol/L NaOH for 3 h, then flushed with water for 0.5 h, followed by 0.1 mol/L HCl for 3 h, water for 0.5 h and methanol for 1 h, successively. After being dried under nitrogen stream, the capillary was adequately filled with (3-mercaptopropyl)trimethoxysilane solution in methanol (50%, v/v), and both ends were sealed with rubbers and sank in a water bath at 50 oC for 12 h. The capillary was washed with methanol to flush out the residual reagents and dried under a stream of nitrogen. Afterwards, the dried capillary was adequately filled with BMI solution in THF (10%, v/v) containing 0.7 mmol/L TEA, and both ends were sealed with rubbers and sank in a water bath at 50 oC for 12 h. Finally, the capillary was washed with methanol and dried under a stream of nitrogen. 2.5 Preparation of polymeric monolithic columns via thiol-maleimine click polymerization reaction A certain amount of BMI was first dissolved in TEA/DMSO solution (0.7 mmol/L), which was assigned as solution A, while 3SH (or 4SH) was mixed with n-propanol to form solution B. Solutions A and B were mixed as the prepolymerization solution in a 1.5 mL centrifuge tube, and degassed by sonication for 15 s, in which DMSO and n-propanol served as porogenic agents. The percentages (w/v) of monomers (mg) and porogens (μL) were presented in Table 1. The resulting solution was introduced into the pretreated fused-silica capillary at a pressure of nitrogen gas, and both ends were sealed with rubbers and sank in a water bath for 3 h. Finally, the capillary was also washed with ethanol. At the same time, the rest of prepolymerization solution was also placed in the water bath for 3 h to form bulk monolithic material. Then the bulk monolith was cut into small pieces, extracted with ethanol overnight in a Soxhlet apparatus and dried in a vacuum for more than 12 h at 60oC. 2.6 Preparation of tryptic digests of proteins and analysis on cLC-MS/MS The tryptic digests of BSA and HeLa cell proteins were prepared as previously 7

described with minor modification.[38, 39] Water containing 0.1% (v/v) formic acid (FA) was used as mobile phase A, and ACN containing 0.1% (v/v) FA was used as mobile phase B. The samples were loaded on a homemade C18-particle-packed trap column (4.0 cm in length × 200 μm i.d.) with mobile phase A (5 μL/min) in 15 min, and then separated on a poly(BMI-co-3SH) (30.0 cm in length × 75 μm i.d.) which was prepared by directly tapering the tip from the outlet of capillary. The experiments were implemented by LTQ Orbitrap Velos MS instrument (Thermo Fisher Scientific, San Jose, CA) with a Surveyor HPLC system (Thermo Fisher Scientific). The flow rate was 95 µL/min before splitting in T-union connector. The gradient for the separation of tryptic digest of HeLa cell proteins was 0-5% B in 2 min, 5-35% B in 93 min, 35-80% B in 8 min and retained 80% B for 12 min. The gradient in the analysis of BSA tryptic digest was 0-5% B in 2 min, 5-35% B in 30 min, 35-80% B in 3 min and retained 80% B for 10 min. The spray voltage between spray tip and MS interface was 2.2 kV, and the normalized collision energy was 35%. One full MS scan ranging from m/z 400 to m/z 2000 was selected in the mass spectrometer. 2.7 Database searching The RAW files were obtained by Xcalibur 2.1 and then converted to *.MGF by Proteome Discoverer (v1.2.0.208, Thermo, San Jose, CA) and searched with MASCOT (version 2.5.1, Matrix Science, London, UK). The database of BSA and human obtained from Uniprot (http://www.uniprot.org/) was used for searching. Mass tolerances were 20 ppm and 1 Da for the parent and fragment ions, respectively. Up to two missed cleavages was allowed, and the false positive rates (FDRs) were controlled to < 1% for identification.

3 Results and Discussion 3.1

Preparation

of

Poly(BMI-co-3SH/4SH)

Monolithic

Columns

via

Thiol-Maleimide Polymerization Reaction As shown in Fig. 1, the polymeric monolith was synthesized via alkaline-catalyzed thiol-maleimide polymerization reaction of BMI and 3SH/4SH with TEA as the 8

alkaline catalyst. Several attempts were examined for purpose of seeking a suitable porogenic system, such as DMF/n-butanol, DMSO/PEG200 and DMSO/1,4-butylene glycol. Taking into account their dissolving capacity and porogenic ability, binary DMSO/n-propanol was finally selected as porogenic system. It is widely believed that the micromorphology and permeability of polymeric monoliths have close relationship with polymerization conditions. In our polymerization system, both reaction temperature and proportion of porogenic solvents required much consideration. Table 1 shows the construction conditions and the permeabilities of resulting columns. Along with a slight increase of the content of “good” solvent (DMSO), the permeabilities of poly(BMI-co-3SH) columns (I-1 and I-2) in Table 1 dramatically reduced from 13.9 to 1.65×10-14 m2. Upon increasing reaction temperature from 40 to 45 oC, the permeabilities of columns I-2 and I-3 continuously decreased from 1.65 to 0.85×10-14 m2. As shown in Fig. S1a and c, it could be revealed that the size of macropores in poly(BMI-co-3SH) was significantly decreased. The results of poly(BMI-co-4SH) columns (II-1, II-2 and II-3) were approximately same to those for poly(BMI-co-3SH) columns. The permeabilities of columns II-1 and II-2 decreased from 15.7 to 1.82×10-14 m2 when the content of DMSO was gently increased. It should be pointed out that although thiol-maleimide polymerization reaction has taken place sporadically without any catalyst, the repeatability was lost through several consecutive attempts. Originally, we chose DMPP as the catalyst, but the prepolymerization solution in the centrifuge tube became a white solid within a few seconds, leading to a failure of introducing prepolymerization solution into a capillary. As a result, weakly basic TEA was selected as catalyst in thiol-maleimide polymerization. As shown in Table 1, trace amount of catalyst (TEA) could not only ensure the rate of polymerization reaction, but also had an effect on the occurrence of phase separation. After optimizing the content of TEA, a 0.7 mmol/L TEA was selected for the synthesis of column I. We tried to use the same concentration (0.7 mmol/L) of TEA to construct column II, but phase separation emerged too fast to fill three parallel columns II. Finally, a 0.3 mmol/L TEA was selected for column II. The polymerization could be performed 9

within 3 h, which was faster than thermal-initiated free radical polymerization. 3.2 Characterization of Organic Monoliths The polymerization of BMI and 3SH/4SH was characterized by ATR-FTIR (Fig. 3) and EDS (Fig. S2). As shown in Fig. 3, the signals of strong peaks of α,β-unsaturated C=O stretching vibrations in BMI (1701 cm-1) and 3SH/4SH monomers (1728 cm-1) were changed to 1704 cm-1 (monoliths I-2 and II-2). Moreover, the characteristic peaks of C=C (1602 cm-1) in BMI and SH group (2568 cm-1) in 3SH/4SH were decreased. These results confirmed that thiol-maleimide polymerization reaction took place to form poly(BMI-co-3SH/4SH). Fig. S2 showed the uniform distribution of sulfur element on the surface of monoliths I-2 and II-2, which also demonstrated the introduction of thiol functional groups. Meanwhile, aiming at the reaction efficiency of thiol-maleimide reaction, the yields of poly(BMI-co-3SH) were 82.0, 82.1 and 84.6%, respectively. As for poly(BMI-co-4SH), the yields were 85.6, 89.4 and 89.1%. High

yields

above

82.0%

were

obtained

with

poly(BMI-co-3SH)

and

poly(BMI-co-4SH) monoliths. Subsequently, the elemental sulfur contents of monoliths I-2 and II-2 were determined. The theoretical and calculated sulfur element contents in I-2 were 10.3 and 8.32%, respectively. As for II-2, the theoretical and calculated sulfur element contents were 10.6 and 8.71%. These differences between theoretical and calculated elements content could be possibly related to the removal of oligomers. The oligomers were characterized by a MALDI-TOF-MS (Fig. S3), which were collected from the eluent flushing out of monoliths I-2 and II-2. It could be seen that the mass difference of repeating units was 358 Da corresponding to BMI molecule in the range of 500-2000 Da. As shown in Fig. S3b, the peak of 1205.60 Da corresponded to the molecular weight of the oligomer generated from two BMI and one 4SH molecules. These results also demonstrated that thiol-maleimide polymerization reaction happened. SEM images displayed the cross-sectional morphologies of polymeric monolithic columns I-2 and II-2 (Fig. 2a, b, d and e). It can be seen that the polymerized 10

products exhibited homogeneous network structure, and contained the narrow framework around 0.5 μm and macropores around 1 μm. This bicontinuous microstructure was obviously favorable for faster mass transfer property and lower backpressure. Moreover, pore size distribution was examined by MIP as shown in Fig. 2c and f. It was observed that the macropores about 1.0 and 0.7 μm existed in I-2 and II-2, respectively, which were consistent with the results of SEM. The total intrusion volumes in I-2 and II-2 were 1.13 and 1.11 mL/g, respectively, and the total pore areas in I-2 and II-2 were only 13.5 and 6.28 m2/g, respectively. During the separation process of small molecules, large pore volume would accelerate mass transfer as the convection is dominant, which would result in lower C-term in van Deemter equation. The specific surface areas of poly(BMI-co-3SH/4SH) monoliths were measured by nitrogen adsorption/desorption measurements, and both of them had low surface areas of 6.22 (I-2) and 6.02 (II-2) m2/g, indicating that neither micropores nor mesopores existed. These phenomena were similar with those of our previous works.[40, 41] The relationship between backpressure drop and linear velocity was measured with ACN as the mobile phase (Fig. S4), both of columns showed good linear correlations (R > 0.9980) with the linear velocities increasing from 0.03 to 0.8 μL/min. Thermal gravimetric analysis (TGA) was used to examine thermal decomposition and weight loss of monolithic material (Fig. S5). The thermal degradation pattern of I-2 was almost the same as that of II-2, and the decomposition temperature was over 300 oC. These results indicated that the resulting polymeric monoliths possessed high hydrodynamic stability and thermal stability. 3.3 Chromatographic Evaluation of Poly(BMI-co-3SH/4SH) Monolithic Columns in cLC To assess the chromatographic separation ability, the resulting polymeric monoliths were applicable to cLC using alkylbenzenes as model analytes in a reversed-phase mode (Fig. 4). As shown in Fig. 4a, alkylbenzenes were well separated on I-2 with the mobile phase of ACN/H2O (60/40, v/v) within 5 min at the flow rate of 423 nL/min, 11

exhibiting good peak shape and fast separation ability. Fig. 4b presents the relationship between the velocity (u) and plate height (H) on I-2. It is worth noting that plate heights for alkylbenzene were lower than 6.87 μm, corresponding to the column efficiency of 145,500 N/m, at a velocity of 0.73 mm/s, and the highest one reached 180,500 N/m for butylbenzene, which was obviously superior to the great mass of reported polymeric monoliths and organic-inorganic monoliths.[14, 15, 42, 43] Compared with the monolithic columns prepared via thiol-methacrylate (highest efficiency ca. 142,000 N/m within 15 min)[44] and thiol-epoxy click polymerization reaction (highest efficiency ca. 132,000 N/m within 10 min)[28], poly(BMI-co-3SH) monolithic columns reduced the analytical time on the premise of high column efficiency (highest efficiency ca. 180,500 N/m within 5 min). Good separation efficiency needs both relative short elution time and satisfactory separation performance.[45] Along with the increase of velocity from 0.92 to 1.87 mm/s, the separation time could decrease from 7.24 to 3.57 min on column I-2. However, the plate heights were slightly increased from 6.85 to 8.80 μm (for butylbenzene). When the velocity reached 1.87 mm/s, column efficiency of butylbenzene still kept over 110,000 N/m. As for column II-2, five peaks were also symmetric and baseline-separated with the mobile phase of ACN/H2O (50/50, v/v) at the flow rate of 444 nL/min. The column efficiencies for five alkylbenzenes reached 100,000-140,000 N/m (corresponding to 7.10-9.29 μm of plate height). The highest column efficiencies was slightly lower than that of column I-2, which still reached 140,000 N/m for butylbenzene at a velocity of 0.33 mm/s (Fig. 4d). In general, high specific surface area and uniform micro-porous structure contribute to improving monolithic column efficiency. Nonetheless, the poly(BMI-co-3SH/4SH) monoliths possessed low specific surface area (6.22 and 6.02 m2/g). It can be assumed that the high separation column efficiency is closely related to the homogeneity of internal pore structure. Comparing to traditional free-radical polymerization, thiol-maleimide polymerization reaction exhibited typical step-growth polymerization mechanism, which could bring about a moderate phase separation process and further 12

form uniform and homogenous reticular microstructure. As shown in Fig. 2, thin skeleton could effectively reduce the eddy diffusion. Large through-pore/skeleton size ratio made convection dominant in the separation process, which was conducive to expedite mass transfer. In order to further explore the cause of good separation efficiency, as listed in Table 2, van Deemter parameters (A, B and C) were acquired by plotting plate height (H) of analytes versus linear velocity (u) of mobile phase and directly analyzed to the true diffusion coefficients according to van Deemter equation: H=A+B/u+C·u. A term (eddy diffusion) relates to the average particle diameter (dp) and homogeneities of the support particles, B term (longitudinal diffusion) increases with the decrease of molecular mass, and the C term accounts for solid-liquid mass transfer resistance.[46] A term values for column I-2 decreased from 1.72 to 0.24 μm with an increase of hydrophobicity of alkylbenzenes. Such small A terms were almost equal to those of columns packed with core-shell (0.9) and totally porous particles (1.5).[47] As for column II-2, A terms ranging from 5.28 to 4.14 μm were smaller than several organic/hybrid monoliths[40, 48], which revealed the limit of peak broadening and homogeneous porous structure. B terms and C terms of alkylbenzenes for column I-2 showed no obvious change. As for column II-2, there was not obvious regularity for A terms. The trends of decrease in both B and C terms from benzene to butylbenzene were observed, which might be related with the pore structures and morphologies. It was also observed from Fig. 4b that the plate height of the weak-retained compound (benzene, 6.87 μm) was only slightly higher than that of strong-retained

compound

(butylbenzene,

5.54

μm)

on

I-2,

revealing

a

retention-independent efficient performance on poly(BMI-co-3SH) column. The same phenomenon could also be observed from Fig. 4d. These results showed that the A/C terms of butylbenzene were lower than those of benzene, which demonstrated a good communication between solid and liquid phase as a result of the lack of micropores. Combined these effects, high column efficiencies of small molecules were gained on poly(BMI-co-3SH/4SH) columns, which were correlated with the morphology and pore size distribution properties of the monoliths. 13

Reproducibilities of poly(BMI-co-3SH/4SH) columns were also detected during separation of alkylbenzenes. Run-to-run RSD% values (n=5) in one day based on retention factor of toluene on monoliths I-2 and II-2 were 0.88 and 1.96%. Batch-to-batch RSD% (n=5) values on monoliths I-2 and II-2 were 2.34 and 15.7%, respectively. Compared to poly(BMI-co-3SH) monolith, poly(BMI-co-4SH) monolith exhibited relatively poor reproducibility. As shown in Fig. S6a and b, the retention factors of alkylbenzenes on monoliths I-2 and II-2 decreased with an increase of ACN content, also demonstrating reversed-phase retention mechanism. The hydrophobicities of polymeric monoliths I-2

and

II-2

could

be

characterized

using

the

following

equation:

logk=nlogαCH2+logβ[49, 50], where n is carbon number of homologous compounds, β is the retention factor surmised to n=0. The methylene selectivity, αCH2, values could be calculated in Fig. S6c and d. The results were summarized in Table S1, indicating that the poly(BMI-co-3SH) exhibited higher methylene selectivity and stronger hydrophobic property than poly(BMI-co-4SH). 3.4 Applications of the Organic Monoliths in cLC Several complicated mixtures of small molecules were applied to evaluate the separation ability of poly(BMI-co-3SH) monolith in cLC. EPA 610 is a toxic, mutagenic,

carcinogenic

and

persistence

organic

contaminant

in

the

environment, which includes 16 types of priority pollutant PAHs. Partly separation of EPA 610 was performed on poly(BMI-co-3SH) monolith, as presented in Fig. S7. Seven compounds (peaks No.9-15) could not be separated. Peak capacity is a powerful tool to determine the quality of a gradient separation, the peak capacity of peak No.7, No.8 and No.16 were 69, 82 and 63, respectively. The results demonstrated that this organic monolith owned a potential in the field of environmental samples analysis. Fig. S8a shows the separation chromatogram of six phenols on column I-2, and all of them were well-separated with column efficiencies ranging from 110,000 to 147,000 N/m. 14

Particularly, two positional isomers, phloroglucinol and pyrogallol were baseline-separated, which demonstrated the potential of poly(BMI-co-3SH) in the separation of some positional isomers. As for the separations of basic compounds, good symmetrical peak shapes and high resolution were obtained, and the elution curves were presented in Fig. S8b. The column efficiencies of five compounds were in the range of 59,000-123,000 N/m which indicated good separation ability to basic analytes. To further evaluate the applicability, poly(BMI-co-3SH) monolith was successfully applied for analysis of complex biological samples in cLC-MS (Fig. 5a and b). The 2 μg tryptic digests of BSA and HeLa cell proteins were well separated with gradient elution. Fig. 5c and d show Venn diagrams of the unique peptides identified in three one-dimensional cLC-MS/MS analyses after controlling the FDR<1% for identification. The 52, 53 and 52 unique peptides from the tryptic digests of BSA were positively identified (protein sequence coverages>75%), respectively. The numbers of unique peptides in the tryptic digests of HeLa cell proteins were 2031, 2097 and 2184, respectively. Fifty common peptides were identified in the tryptic digests of BSA (about 90.9% of total), which displayed a receivable and reproducible identification result. However, 26.3% unique peptides were common in the identification of HeLa cell proteins. The relatively poor result was possible attributed to the complexity of HeLa cell proteins. To sum up, poly(BMI-co-3SH) columns showed the potential to high throughout biosamples separation and proteomics in the future.

4 CONCLUSIONS In general, thiol-maleimide polymerization was successfully applied to polymeric capillary monoliths for the first time. The one-step process has the advantages of simple operation, short reaction time and high yield. Good thermal and chemical stabilities were obtained with resulting polymeric monoliths. Compared to the previous reported organic monoliths and 15

organic-inorganic hybrid monoliths (highest column efficiency ca. 142,000 and 132,000

N/m),

this

work

demonstrated

very

good

chromatographic

performance (highest column efficiency ca. 180,500 N/m) on the premise of fast chromatographic separations. Furthermore, it had a wide range of applications in the separation of EPA 610, phenols, basic compounds, the tryptic digests of BSA and HeLa cell proteins. This method fulfilled the need of green chemistry, proved high yield and mild reaction condition, and could be developed in advanced materials synthesis and multidisciplinary research.

ACKONWLEDGMENTS Financial support is gratefully acknowledged from the China State Key Basic Research Program Grant (2016YFA0501402), the National Science Fund for Distinguished Young Scholars (21525524), the National Natural Sciences Foundation of China (No. 21535008) to M. Ye, the National Natural Sciences Foundation of China (No. 21575141) to J. Ou, as well as the National Natural Sciences Foundation of China (21675125) to Y. Shen.

16

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[50] J. Ou, G.T.T. Gibson, R.D. Oleschuk, Fast preparation of photopolymerized poly(benzyl methacrylate-co-bisphenol A dimethacrylate) monoliths for capillary electrochromatography, J. Chromatogr. A, 1217 (2010) 3628-3634.

21

Figure and Table captions

Fig.1 Synthetic route diagram of polymeric monoliths via alkaline-catalyzed thiol-maleimide click reaction.

Fig.2 (a, b) SEM of I-2 and (d, e) II-2. Pore size distribution of monoliths (c) I-2 and (f) II-2.

22

Fig. 3 ATR-FTIR spectra of BMI, 3SH, 4SH, monoliths I-2 and II-2.

23

Fig. 4 Separation of alkylbenzenes on monoliths (a) I-2 and (c) II-2 by cLC. Elution order: thiourea, benzene, toluene, ethylbenzene, propylbenzene and butylbenzene. Dependence of the plate height (H) of analytes on the linear velocity (u) of mobile phase on (b) I-2 and (d) II-2. Experimental conditions: column dimension, 19.0 cm × 75 μm i.d.; mobile phases, (a, b) ACN/H2O (60/40, v/v), (c, d) ACN/H2O (50/50, v/v); flow rates, (a) 423 nL/min, (b) 26-423 nL/min, (c) 444 nL/min and (d) 30-570 nL/min; detection wavelength, 214 nm.

24

Fig. 5 Separations of 2 μg tryptic digests of (a) BSA and (b) HeLa cell proteins on monolith I-2 by cLC-MS. Venn diagrams of the unique peptides identified from the tryptic digests of (c) BSA and (d) HeLa cell proteins by three one-dimensional cLC-MS analyses. Experimental conditions: column dimension, (a, b) 30.0 cm × 75 μm i.d.; mobile phases, (a, b) mobile phase A, 0.10% FA in water, B, 0.10 % FA in ACN; flow rates, (a, b) 95 μL/min; gradients, (a) 0-5% B in 2 min, 5-35% B in 30 min, 35-80% B in 3 min and retained 80% B for 10 min, (b) 0-5% B in 2 min, 5-35% B in 93 min, 35-80% B in 8 min and retained 80% B for 12 min.

25

Table 1 Detailed composition of polymerization mixtures and the permeability of polymeric monoliths.

DMSOc

n-Propanold

(%, v/v)

(%, v/v)

I-1a

53.3

I-2a

Total monomer Temperature

Permeability

(%, w/v)

(oC)

(10-14 m2)

46.7

20.8

40

13.9

54.2

45.8

20.8

40

1.65

I-3a

54.2

45.8

20.8

45

0.85

II-1b

54.5

45.5

21.7

40

15.7

II-2b

55.4

44.5

21.7

40

1.82

II-3b

55.4

44.5

21.7

45

0.65

Monolith

a

contente

The amounts of BMI and 3SH in prepolymerization mixture were kept at 72 and 54

mg, respectively. b

The amounts of BMI and 4SH in prepolymerization mixture were kept at 72 mg and

50 mg, respectively. c

Concentration of catalyst solutions: I-(0.7 mmol/L), II-(0.3 mmol/L).

d

Volume percentage of n-propanol in the porogenic solvents.

e

The ratio of total monomers (mg) to total porogenic solvents (μL).

26

Table 2 Fitted values of A, B and C terms in van Deemter equation: H=A+B/u+C·u.

Monolith I-2

Monolith II-2

Analytes A/μm

B/(μm2 s-1)

C/ms

A/μm

B/(μm2 s-1)

C/ms

benzene

1.72

2122

4.36

4.10

2417

5.28

toluene

1.08

2191

4.60

3.84

2347

5.19

ethylbenzene

0.67

2163

4.61

4.26

1932

4.60

propylbenzene

0.22

2187

4.69

4.20

1656

4.27

butylbenzene

0.24

2117

4.32

3.98

1455

4.14

27