Amino acid and ionic liquid modified polyhedral oligomeric silsesquioxane-based hybrid monolithic column for high-efficiency capillary liquid chromatography

Accepted Manuscript Title: Amino acid and ionic liquid modified polyhedral oligomeric silsesquioxane-based hybrid monolithic column for high-efficiency capillary liquid chromatography Authors: Manman Han, Wan Li, Rui Chen, Yangyang Han, Xiuhua Liu, Tingting Wang, Huaizhong Guo, Xiaoqiang Qiao PII: DOI: Reference:

S0021-9673(18)31065-3 https://doi.org/10.1016/j.chroma.2018.08.045 CHROMA 359641

To appear in:

Journal of Chromatography A

Received date: Revised date: Accepted date:

21-5-2018 16-8-2018 21-8-2018

Please cite this article as: Han M, Li W, Chen R, Han Y, Liu X, Wang T, Guo H, Qiao X, Amino acid and ionic liquid modified polyhedral oligomeric silsesquioxane-based hybrid monolithic column for high-efficiency capillary liquid chromatography, Journal of Chromatography A (2018), https://doi.org/10.1016/j.chroma.2018.08.045 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.

Amino acid and ionic liquid modified polyhedral oligomeric silsesquioxane-based hybrid monolithic column for high-efficiency capillary liquid chromatography Manman Han a, Wan Li a, Rui Chen a, Yangyang Han a, Xiuhua Liu b, Tingting Wang , Huaizhong Guo a, Xiaoqiang Qiao a,*

a

College of Pharmaceutical Sciences, Key Laboratory of Analytical Science and

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c

Technology of Hebei Province, Key Laboratory of Medicinal Chemistry and

Molecular Diagnosis, Ministry of Education, Hebei University, Baoding 071002,

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School of Materials and Chemical Engineering, Ningbo University of Technology,

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Ningbo 315211, China

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c

College of Life Sciences, Hebei University, Baoding 071002, China

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b

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China

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Corresponding author:

*Professor Dr. Xiaoqiang Qiao, College of Pharmaceutical Sciences, Key Laboratory

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of Analytical Science and Technology of Hebei Province, Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education, Hebei

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University, Baoding 071002, China Tel.: +86-312-5971107 Fax: +86-312-5971107 E-mail: [email protected]; [email protected] 1

Highlights 

• Amino acid and ionic liquid modified POSS-based monolithic column was developed.

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• The column exhibited reduced hydrophobicity with introduction of polar L-cysteine.

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• Polar compounds could be efficiently separated under RPLC mode.

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• Intact proteins exhibited strong retention and good separation selectivity.

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ABSTRACT In this study, a novel amino acid and ionic liquid dual organically functionalized

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reagents modified polyhedral oligomeric silsesquioxane methacryl substituted (POSSMA) based hybrid monolithic column (POSS-VBI-Cys) was designed and reported. With amino acid L-cysteine and ionic liquid 1-vinyl-3-butylimidazolium bromide as

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dual monomers, POSS-MA as the crosslinker, the new POSS-VBI-Cys hybrid

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monolithic column could be facilely fabricated via the “one-pot” free radical

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copolymerization and thiol-ene click reaction. Because of the introduction of polar amino acid L-cysteine, the new POSS-VBI-Cys column exhibited attenuated

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hydrophobicity in reversed-phase liquid chromatography separation. Polar amides,

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nucleosides and nucleic acid bases displayed strong retention on the POSS-VBI-Cys column and could be successfully separated. Furthermore, the new POSS-VBI-Cys

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column displayed good separation selectivity for model glycoproteins and nonglycoproteins mixture and it was also successfully used for the purification and

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separation of TARG1 protein from its originally expressed sample. In the future research, we will further exploit its performances for separation of intact proteins and in-depth proteome applications.

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Keywords: Amino acid; Ionic liquid; Polyhedral oligomeric silsesquioxane methacryl substituted; Hybrid monolithic column; Protein separation 1. Introduction

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Monolithic-based columns have been reputed as the fourth generation highperformance liquid chromatography (HPLC) stationary phases. The merits of low

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backpressure, fast mass transfer, high permeability as well as excellent separation

performances for both small molecules and macromolecules render them attracting much attention [1-5]. In the past few years, monolithic columns with inorganic silica

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[6-11], organic polymer [12-17] or organic-silica hybrid [18-22] materials as the

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matrix have been largely reported.

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Polyhedral oligomeric silsesquioxane (POSS) is a naturally molecular-level hybrid

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particle with cage-like structure and specific nanometer dimension [23,24]. The intrinsic virtues of POSS, such as good temperature/oxidation resistant capacity and

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excellent pH tolerance, render it a good choice for preparation of novel kind of

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monolithic columns [25,26]. Zou and coauthors [27] firstly exploited and used POSS methacryl substituted (POSS-MA) and N-(2-(methacryloyloxy)ethyl)dimethyloctadecylammonium bromide (MDOAB) to construct POSS-based hybrid

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monolithic column. The column indicated excellent mechanical and pH (1-11) stability as well as high column efficiency (50,000 plates/m for thiourea). It was successfully used for the separation of intact proteins and bovine serum albumin (BSA) tryptic digests with good chromatographic performances. Most importantly, 3

POSS-MA based monolithic columns often displayed excellent separation selectivity and good peak shapes compared with the conventional alkoxysilane-based monolithic columns since tailing peaks were often present in alkoxysilane-based monolithic columns due to the residual silanol groups [28,29]. Thereafter, a variety of POSS-MA

acrylate (DPEPA) [28], ethylene dimethacrylate (EDMA), bisphenol A

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based monolithic columns with the introduction of dipentaerythritol penta-/hexa-

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dimethacrylate (BPADMA) [30], lauryl methacrylate (LMA), benzyl methacrylate (BeMA), stearyl methacrylate (SMA) [31], pentadecafluorooctyl methacrylate

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(PDFOMA) [32], 1,4-bis(mercaptoacetoxy) butane (BMAB) [33], methacrylic acid

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(MAA), butyl methacrylate (BuMA) [34], 1-allyl-3-vinylimidazolium bromide [35],

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pentaerythritol tetrakis(3-mercaptopropionate) (PTM), 1,6-hexanedithiol (HDT) and

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trimethylolpropane tris(3-mercaptopropionate) (TPTM) [36] were reported.

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Ionic liquids are the promising organically functionalized reagents for fabrication

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of novel stationary phases with specific selectivity and excellent separation efficiency [37] which have been widely reported for preparation of particle-based silica

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stationary phases [38-40]. In our recent work, ionic liquids were further exploited to prepare POSS-MA based monolithic columns since they could potentially combine

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both the merits of ionic liquids and POSS reagents [26,35]. For example, 1-vinyl-3octylimidazolium bromide modified POSS-MA based hybrid monolithic column was firstly prepared via the facile “one-pot” method in our lab. The new column exhibited with high column efficiency (103,000-124,000 plates/m for alkylbenzenes) and excellent separation selectivity for phenolic isomers. It was also successfully used for 4

the separation of aromatic amines and polycyclic aromatic hydrocarbons [26]. However, to the best of our knowledge, the majority of these POSS-MA based hybrid monolithic columns displayed typical reversed-phase liquid chromatography (RPLC) retention mechanism even though imidazolium-based ionic liquids were introduced as

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the monomers. The probable reason could attribute to the strong hydrophobicity nature of the POSS-MA skeleton [41]. Obviously, the development of new POSS-MA

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based monolithic columns possessing hydrophilic characteristic could expand the application scopes of the POSS-based hybrid monolithic columns.

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Herein, we explore to develop new amino acid and ionic liquid dual-functionalized

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monomers modified POSS-MA based hybrid monolithic column. L-cysteine

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hydrochloride (Cys) and 1-vinyl-3-butylimidazolium bromide (VBIBr) modified POSS-MA based hybrid monolithic column (POSS-VBI-Cys) was firstly prepared via

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“one-pot” free radical copolymerization and thiol-ene click reaction. The strong

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hydrophilicity of Cys renders the new POSS-VBI-Cys column can be used for the separation of polar compounds in RPLC mode. Furthermore, based on the principle of

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"similarity and compatibility", the Cys modified POSS-VBI-Cys column could produce enhanced retention for proteins.

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2. Experimental

2.1. Materials and reagents POSS-MA, N,N-dimethylacetamide, thioacetamide, formamide, lysozyme from chicken egg white, BSA and myoglobin from equine heart were provided by Sigma5

Aldrich (St. Louis, MO, USA). Cys, iodoacetamide were supplied by Alfa Aesar (Shanghai, China). VBIBr was from Beijing HWRK Chemical (Beijing, China). γMethacryloxypropyltrimethoxysilane (γ-MAPS) was provided by Acros Organics (NJ, USA). Horseradish peroxidase and human α-transferrin were supplied by

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California Bioscience (CA, USA). Ribonuclease A from bovine pancreas was ordered from Aladdin Industrial Corporation (Shanghai, China). 2,2-Azoisobutyronitrile

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(AIBN), uracil, cytosine and adenine were from J&K Scientific (Beijing, China). Adenosine and cytidine were provided by Tokyo Chemical Industry (Shanghai,

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China). Potassium iodate and N,N-dimethylformamide were supplied by Tianjin

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Kemiou Chemical Reagent (Tianjin, China). Potassium thiocyanate was from Tianjin

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Fuchen Chemical Reagent (Tianjin, China). Dodecanol, benzene, toluene,

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ethylbenzene, propylbenzene, potassium bromate and butylbenzene were supplied by

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Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). Fused-silica capillary (50 and 100 μm i.d., 365 μm o.d.) were provided by Yongnian Optical Fiber

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Plant (Hebei, China). HPLC-grade methanol and acetonitrile (ACN) were from

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Shanghai Xingke Biochemistry (Shanghai, China). Water was purified via a Millipore Milli-Q system (Molsheim, France).

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2.2. Apparatus

Fourier transform infrared (FT-IR) experiments were carried out on a Bruker

Vertex 70 spectrometer (Bruker Optik GmbH, Ettlingen, Germany). Scanning electron microscopy (SEM) experiments were performed on a Japan Electron Optics Laboratory JSM-7500 SEM system (Tokyo, Japan). Thermogravimetric (TGA) 6

analysis was determined on a Perkin-Elmer TGA/SDTA851E system (Boston, USA). The nitrogen adsorption/desorption experiments were carried out on a Quantachrome Quadrasorb-IQ-MP surface area analyzer (Boynton Beach, FL, USA). Elemental analysis experiments were performed on Thermo Fisher Scientific Flash EA1112

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elemental analyzer (Milano, Italy). HPLC experiments were carried out on a selfconstructed nano-LC system [28] and the details of the system were illustrated in the

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Supporting Information. The samples were usually dissolved in the initial mobile

phase (concentration: 2 mg/mL) and roughly 6 nL of samples were injected into the

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nano-LC system.

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2.3. Preparation of POSS-VBI-Cys hybrid monolithic columns

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For preparation of the POSS-VBI-Cys columns, the bare capillary with 100 μm i.d. was firstly pretreated via sequentially flushing by 1 M sodium hydroxide solution (4

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h), purified water (1 h), 1 M hydrochloric acid solution (3 h), purified water (1 h) and

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dried via nitrogen stream at ambient temperature. Then, 50% γ-MAPS methanol solution was introduced for further functionalizing the inner surface of capillary by

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reaction at 60 °C for 12 h. Finally, the capillary was flushed by methanol, dried via

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nitrogen stream and ready for further modification. The prepolymerization solution, including POSS-MA, VBIBr, Cys, AIBN,

dodecanol and methanol, was firstly mixed and ultrasonicated for 15 min. The formed homogeneous solution was manually pumped into the pretreated capillary via a syringe, followed by further polymerization for 12 h at 60 °C in water bath. Finally, 7

the obtained hybrid monolithic columns were flushed by methanol for 2 h and can be used for subsequent nano-LC separation. Under the same conditions, the bulk hybrid POSS-VBI-Cys materials were also fabricated for further characterization via TGA, FT-IR, elemental analysis and nitrogen adsorption/desorption measurement.

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Simultaneously, the VBIBr modified POSS-based hybrid monolithic columns

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(POSS-VBI) were also fabricated. The polymerization conditions referred to that for fabrication of the POSS-VBI-Cys column and only Cys was absent in the polymerization system.

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2.4. Preparation of TARG1 protein sample

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For preparation of TARG1 protein sample, TARG1 was expressed in Escherichia

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coli BL21 (DE3) cells with the pET-15b plasmid. The cells grown at 37 °C and

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induced with 0.12 mM IPTG for 16 h at 20 °C. Then, the cultures were centrifuged at 3500 rpm for 10 min. The obtained cells were further resuspended in lysis buffer (25

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mM Tris-HCl pH 8.0, 250 mM NaCl, 3 mM β-ME) and disrupted by sonication on

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ice. The soluble proteins containing the TARG1 protein were further used for evaluation the POSS-VBI-Cys column. The purified TARG1 proteins could be obtained via Ni-chelating Sepharose column and gel filtration on Superdex 200

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column (GE Healthcare). 3. Results and discussion POSS-MA is an excellent matrix for fabrication of novel hybrid monolithic columns with excellent stability, higher column efficiency and reduced peak tailing 8

which are especially significant for separation of complex samples [1]. Herein, hydrophilic amino acid Cys and ionic liquid VBIBr were exploited as the dual monomers to fabricate new POSS-based hybrid monolithic column. The introduction of polar Cys and ionic liquid VBIBr could reduce the strong hydrophobicity of POSS

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matrix. Thus, the new column could be potentially expanded for the separation of hydrophilic compounds. Furthermore, we hope the new POSS-VBI-Cys column could

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improve the separation of intact proteins based on the principle of "similarity and

compatibility" with the introduction of amino acid as the monomer. Thus, the new

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POSS-VBI-Cys column was designed and further prepared via the “one-pot” free

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radical copolymerization and thiol-ene click reaction, as shown in Fig. 1.

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3.1. Fabrication of the POSS-VBI-Cys hybrid monolithic column The porogenic system and polymerization temperature are the two most important

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factors for fabrication of the POSS-based monolithic columns. Based on our recent

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research [28], methanol-dodecanol is the suitable porogenic system and they were also selected in the present work. As shown in Table 1, if the volume of dodecanol

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was increased from 55 to 60 µL, the permeability of the monolithic columns increased from 0.79 × 10-14 to 2.60 × 10-14 m2 (columns A-B). If further decreased the volume of

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methanol, the permeability of the monolithic columns further increased to 19.87 × 1014

m2 (columns C-D). Obviously, methanol is a good solution in the porogenic system.

The polymerization temperature also has an important influence on the morphology and permeability of the monolithic columns. When 55, 60 and 65 °C were employed, the permeability of the columns sharply decreased from 42.68 × 10-14 to 0.40 × 10-14 9

m2 (columns E, B and F) (Table 1 and Fig. S1). By consideration of both the morphology and permeability of the columns, the conditions for fabrication of column B were selected for the subsequent experiments, including of 9.2 mg VBIBr, 4.3 mg Cys, 27.2 mg POSS-MA, 145 µL methanol, 60 µL dodecanol as well as 1 mg AIBN.

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The polymerization process was performed at 60 °C for 12 h.

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The representative SEM images of the POSS-VBI-Cys column are illustrated in

Fig. 2. The column exhibited homogeneous and continuous porous structure and the materials were connected with the capillary tightly without any discontinuity (Fig. 2a

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and 2b). As can be seen from Fig. 2c and d, irregularly global units (about 0.2-0.4

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μm) further accumulating into large clusters microstructure were observed from the

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column which was simultaneously dispersed with macropore channels (roughly 1-2.5

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μm).

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3.2. Characterization of the POSS-VBI-Cys monolith

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FT-IR and elemental analysis were firstly used to characterize the POSS-VBI-Cys

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monolith. The FT-IR spectra of POSS-MA, VBIBr, Cys and the prepared POSS-VBICys monolith are shown in Fig. 3. For POSS-MA, the absorption bands at 1635 and 1720 cm-1 induced by the stretching vibration of C=C, C=O bonds of methacryl

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groups. For VBIBr, the presence of typical bands at 1550 and 1570 cm-1 could attribute to the stretching vibration of C=N bonds of the imidazolium skeleton in VBIBr while the bands 2850 and 2925 cm-1 induced by the stretching vibration of CH bonds of methylene/methyl groups. In the spectrum of POSS-VBI-Cys monolith, 10

bands 1631, 2854 and 2926 cm-1 which represented both the characteristics of POSSMA and VBIBr could be simultaneously observed, indicating the successful polymerization of POSS-MA and VBIBr. Elemental analysis was further used to characterize the successful introduction of Cys. The elemental contents of POSS-VBI-

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Cys monolith were C 57.16%, H 9.19%, N 2.47% and S 1.29%. The presence of S

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element provided the proof that Cys was successfully introduced into the monolith. TGA was further used to characterize the thermostability of the POSS-VBI-Cys monolith. The endothermic mass loss of the monolith started at about 200 °C and

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exacerbated at about 250 °C, demonstrating the acceptable thermostability (Fig. S2).

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To study the mechanical stability of the POSS-VBI-Cys column, the linear

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relationship between back pressure and flow rate was also investigated in both acidic (pH 2.1) and basic (10.0) mobile phase system. The correlation coefficients were all

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higher than 0.990 and the pressure tolerances reached 23 MPa (Fig. S3), indicating the

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good mechanical stability of the POSS-VBI-Cys column. Nitrogen adsorption/desorption was further used to characterize the pore

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characteristic of the POSS-VBI-Cys monolith (Fig. S4). The BET specific surface area of the POSS-VBI-Cys monolith reached 528.4 m2 g-1 which is higher than that of

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our recently reported POSS-based monolith (478 m2 g-1) [35] and the conventional hybrid monoliths (273.5 and 3.8 m2 g-1) [19,42]. From the inserted pore size distribution diagram, we can see the monolith mainly displayed narrow micropore distribution (0.6-2 nm). Simultaneously, mesoporous distribution (2-6 nm) was also observed. 11

The reproducibility of the POSS-VBI-Cys columns was further investigated via separation of amides. The relative standard deviations (RSDs) of retention factors of run-to-run, day-to-day, column-to-column and batch-to-batch were 1.06-1.58% (n = 5), 2.13-2.82% (n = 3), 3.14-5.62% (n = 3) and 5.35-7.31% (n = 3), respectively. The

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above results demonstrated the acceptable reproducibility of the POSS-VBI-Cys

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columns. 3.3. Retention mechanisms

The retention mechanisms of the POSS-VBI-Cys column were firstly investigated

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with a series of hydrophobic alkylbenzene compounds. As can be seen from Fig. 4a,

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when the concentration of ACN in mobile phase was increased to 80%, the retention

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of these compounds gradually decreased, indicating RPLC retention mechanism. The retention mechanisms of the POSS-VBI-Cys column were also investigated with a

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series of hydrophilic amides, nucleosides and nucleic acid bases compounds. With the

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increment of the concentration of ACN in mobile phase, the retention of these compounds also gradually decreased (Fig. 4b) which further verified the RPLC

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characteristic of the POSS-VBI-Cys column. From these results, we can conclude that even though strong polar Cys was introduced into the POSS matrix, the new POSS-

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VBI-Cys column still displayed RPLC retention mechanism. Furthermore, the inherent cationic structure of VBI and the zwitterionic structure of

Cys (pK1=1.71 (COOH), pK2=10.78 (NH2)) render the POSS-VBI-Cys column possessing ion-exchange retention characteristic. Inorganic anions IO3-, BrO3-, and 12

NO3- were used to investigate the ion-exchange retention characteristic of the POSSVBI-Cys column. As shown in Fig. 5a, with the increment of the concentration of KCl in the mobile phase, the retention factors of these anions gradually reduced, indicating ion-exchange retention mechanism. Furthermore, the effect of pH of

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mobile phase on the retention of these anions was also investigated. The retention factors of these compounds gradually increased with the decrement of pH of the

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mobile phase (Fig. 5b). At pH 6.0 mobile phase system, the carboxyl groups on the POSS-VBI-Cys column fully dissociated which produced electrostatic repulsion

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interaction and thus relatively weak retention for these anions. With further

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decreasing the pH of mobile phase to 2.0, the carboxyl groups on the POSS-VBI-Cys

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column partially dissociated. Thus, the electrostatic repulsion interaction between the

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column and these anions decreased which produced relatively strong retention for

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these anions.

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3.4. Separation of hydrophobic alkylbenzenes Since the developed POSS-VBI-Cys column exhibited RPLC retention mechanism,

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the separation performances of the column were firstly studied with a series of hydrophobic alkylbenzenes. As shown in Fig. 6a, with ACN/H2O (70:30, v/v) as the

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mobile phase, a preliminary separation of the five alkylbenzenes could be observed. If further reduced the concentration of ACN in the mobile phase to 60%, baseline separation of the five compounds could be achieved (Fig. 6b). The column efficiencies of the POSS-VBI-Cys column reached 13,000-42,000 plates/m. For comparison, VBIBr modified POSS-VBI column was also prepared and used to 13

separate the five alkylbenzenes (Fig. 6c). With ACN/H2O (70:30, v/v) as the mobile phase, the five compounds displayed with strong retention and the column efficiencies reached 94,000-118,000 plates/m. Obviously, compared with the POSS-VBI column, the POSS-VBI-Cys column demonstrated reduced hydrophobic characteristic with the

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introduction of strong polar Cys.

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3.5. Separation of hydrophilic compounds

The new POSS-VBI-Cys column was further exploited for separation of

hydrophilic amides. With ACN/H2O (20:80, v/v) as the mobile phase, five amides,

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including formamide (Log P: -1.07), N,N-dimethylformamide (Log P: -0.6), N,N-

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dimethylacetamide (Log P: -0.49), iodoacetamide (Log P: -0.08) and thioacetamide

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(Log P: -0.40), exhibited strong retention (Fig. 7a). The column efficiencies reached 85,000-220,000 plates/m and the tailing factors were 1.07-1.28. Furthermore, the

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eluent orders of formamide, N,N-dimethylformamide, N,N-dimethylacetamide and

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iodoacetamide were the same as the hydrophobicity of these compounds which also verified the RPLC retention mechanism of the POSS-VBI-Cys column. The unique

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exception is thioacetamide. Although it is more hydrophilic than idoacetamide, it displayed strongest retention among all of the five compounds. The probable reason is

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that strong sulfur-sulfur interaction could be produced between thioacetamide and the POSS-VBI-Cys column. POSS-VBI column was also used to separate the amide compounds. Under the same conditions, all these compounds exhibited weak retention and N,N-dimethylformamide (peak 2), N,N-dimethylacetamide (peak 3) almost overlapped together (Fig. 7b). Even though the concentration of ACN in the mobile 14

phase system was reduced to 5%, the separation selectivity of these compounds via the POSS-VBI column was still poorer than that via the POSS-VBI-Cys column (Fig. 7c). Obviously, the new POSS-VBI-Cys column displayed enhanced separation performances for polar amides compounds.

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The results of the POSS-VBI-Cys column for separation of nucleosides and nucleic

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acid bases are shown in Fig. S5a. Within five minutes, cytosine, cytidine, adenosine, adenine and uracil could be baseline-separated. However, when POSS-VBI column was used, all the peaks stacked together because of their strong hydrophilicity (Fig.

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S5b). Similarly, even though the concentration of ACN in the mobile phase system

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was reduced to 5%, baseline-separation of the five compounds still could not be

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achieved via the POSS-VBI column (Fig. S5c). All these results indicated that the new POSS-VBI-Cys column is a good choice for separation of hydrophilic

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compounds.

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3.6. Separation of glycoprotein and non-glycoprotein mixture

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A six-protein-mixture, including three glycoproteins (ovalbumin, horseradish peroxidase and α-transferrin) and three non-glycoproteins (ribonuclease A, BSA and myoglobin), was further used to evaluate the applicability of the new POSS-VBI-Cys

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column for separation of macromolecules. As shown in Fig. 8a, within 30 min linear gradient elution, the six proteins showed strong retention and were successfully separated with sharp peaks and acceptable symmetry. For comparison, POSS-VBI column was also used to separate the six-protein-mixture under the same conditions. 15

The proteins exhibited weak retention and proteins ribonuclease A (peak 1) and ovalbumin (peak 2) almost overlapped together while the resolution of horseradish peroxidase (peak 3) and α-transferrin (peak 4) was only 1.18 (Fig. 8b). If further reduced the elution gradient, although the proteins exhibited stronger retention,

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baseline separation of the six proteins still could not be achieved (Fig. 8c). The different separation selectivity of the new POSS-VBI-Cys column could probably

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ascribe to the introduced amino acid Cys. The Cys on the POSS-VBI-Cys column

could provide multiple interactions which could produce strong retention and thus

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different separation performances for intact proteins.

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3.7. Separation of TARG1 protein sample

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Adenosine diphosphate (ADP)-ribosylation is a kind of chemical modification of macromolecules implicated in the regulation of a range of cellular processes. TARG1

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is an ADP-ribose protein glycohydrolase. The deficiency of TARG1 could affect the

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regulation of many important biological processes, such as DNA repair, apoptosis, bacterial metabolism and some neurodegenerative disease (for example Alzheimer’s

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disease) [43,44]. Obviously, efficient separation and purification of TARG1 protein is vital for further in-depth understanding of the protein. The POSS-VBI-Cys column

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was further exploited for separation of the TARG1 protein from its originally expressed sample. Fig. 9a is the chromatogram of the purified TARG1 protein that was directly injected into the POSS-VBI-Cys column. The protein displayed strong retention under gradient elution. Fig. 9b is the chromatogram of the expressed TARG1 sample separated via the POSS-VBI-Cys column. The impurities of the 16

sample were mainly present in between 8-14 min which were completely separated with the target TARG1 protein. Thus, the new POSS-VBI-Cys column is of good potential for purification of the expressed TARG1 protein. 4. Conclusion

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In conclusion, amino acid and ionic liquid dual monomers embedded POSS-based

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hybrid monolithic column was firstly exploited and fabricated via a “one-pot” free

radical copolymerization and thiol-ene click reaction. The introduction of the polar amino acid Cys renders the new POSS-VBI-Cys column possessing more hydrophilic

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characteristic which expands its applications for separation of hydrophilic

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compounds. Moreover, the new POSS-VBI-Cys column produced enhanced retention

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for intact proteins and it was successfully used for purification of TARG1 protein from its complex expressed sample. We hope the new POSS-VBI-Cys column could

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Acknowledgements

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be further used for separation and purification of complex proteome samples.

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We are grateful for the financial support from National Natural Science Foundation of China (21675039), Young Talent of Hebei Province, Project funded by China Postdoctoral Science Foundation (2016M591401), Hebei University Science Fund for

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Distinguished Young Scholars (2015JQ06), and Natural Science Foundation of Hebei Province (H2016201221). References

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SC R

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IP T

capillary columns for isocratic and gradient hydrophilic interaction liquid

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N

U

zwitterionic silica-based monolithic capillary columns coupled with tandem mass

A

spectrometry, J. Chromatogr. A 1373 (2014) 90-96.

M

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ED

polysilsesquioxane-based wide-bore monolithic capillary columns for hydrophilic interaction chromatography, J. Chromatogr. A 1479 (2017) 204-209.

PT

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CC E

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A

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reversed-phase and hydrophilic interaction capillary liquid chromatography, J. Chromatogr. A 1217 (2010) 22-33. [15] H. Zhang, J. Ou, Y. Wei, H. Wang, Z. Liu, L. Chen, H. Zou, A novel polymeric monolith prepared with multi-acrylate crosslinker for retention-independent

IP T

efficient separation of small molecules in capillary liquid chromatography, Anal.

SC R

Chim. Acta 883 (2015) 90-98.

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N

U

mass spectrometry profiling of antibodies, intact proteins, oligonucleotides, and

A

peptides, J. Chromatogr. A 1498 (2017) 8-21.

M

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ED

preparation of a poly (pentaerythritol tetraglycidyl ether-co-poly ethylene imine) organic monolithic capillary column and its application in hydrophilic interaction

PT

chromatography for polar molecules, Anal. Chim. Acta 988 (2017) 104-113.

CC E

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A

for two-dimensional liquid chromatography-mass spectrometry, J. Chromatogr. A 1524 (2017) 135-142.

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20

chromatography based on "thiol-ene" click chemistry, J. Chromatogr. A. 1355 (2014) 228-237. [20] C. Wu, Y. Liang, Z. Liang, L. Zhang, Y. Zhang, Ethane-bridged hybrid monoliths with well-defined mesoporosity and great stability for high-

IP T

performance peptide separation, Anal. Chim. Acta 1019 (2018) 128-134.

SC R

[21] H. Zhao, H. Lyu, W. Qin, Z. Xie, Synthesis of boronate-functionalized organicinorganic hybrid monolithic column for the separation of cis-diol containing

U

compounds at low pH, Electrophoresis 39 (2018) 924-932.

N

[22] H. Wang, W. Hu, Q. Zheng, W, Bian, Z. Lin, One-pot preparation of

A

mercaptotetrazole-silica hybrid monoliths by the thiol-ene click reaction for

M

mixed-mode capillary liquid chromatography, J. Sep. Sci. 40 (2017) 2344-2354.

ED

[23] H. Liu, C.H. Hsu, Z. Lin, W. Shan, J. Wang, J. Jiang, M. Huang, B. Lotz, X. Yu, W.B. Zhang, K. Yue, S.Z. Cheng, Two-dimensional nanocrystals of molecular

PT

Janus particles, J. Am. Chem. Soc. 136 (2014) 10691-10699.

CC E

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A

1685-1687.

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21

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IP T

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SC R

silsesquioxane as a cross-linker for preparation of inorganic-organic hybrid monolithic columns, Anal. Chem. 82 (2010) 5447-5454.

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penta-/hexa-acrylate based-highly cross-linked hybrid monolithic column:

A

Preparation and its applications for ultrahigh efficiency separation of proteins,

M

Anal. Chim. Acta 963 (2017) 143-152.

ED

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PT

Acta 931 (2016) 1-24.

CC E

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A

electrochromatography and capillary liquid chromatography, Anal. Chim. Acta 761 (2013) 209-216.

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SC R

for capillary liquid chromatography, Analyst 140 (2015) 265-271.

IP T

oligomeric silsesquioxane based hybrid monoliths by thiol-ene click chemistry

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ED

bridged hybrid monolith for high-efficiency capillary liquid chromatography with enhanced selectivity, J. Sep. Sci. 40 (2017) 1024-1031.

PT

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CC E

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A

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IP T

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SC R

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ED

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PT

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CC E

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A

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SC R

IP T

3529.

U

Fig. 1. Schematic diagram for fabrication of the POSS-VBI-Cys hybrid monolithic

N

column.

M

A

Fig. 2. SEM images of the POSS-VBI-Cys hybrid monolithic column with different magnifications: (a) 1,000×; (b) 5,000×; (c) 10,000×; (d) 30,000×.

ED

Fig. 3. FT-IR spectra of POSS-MA, VBIBr, Cys and POSS-VBI-Cys hybrid monolithic

PT

column.

CC E

Fig. 4. Effect of the content of ACN in mobile phase on the retention factors (k) of benzene, toluene, ethylbenzene, propylbenzene, butylbenzene (a) and formamide, adenine, N,N-dimethylformamide, N,N-dimethylacetamide, adenosine,

A

iodoacetamide, thioacetamide (b) on the prepared POSS-VBI-Cys hybrid monolithic column. Experimental conditions: effective length, 300 mm × 100 μm i.d.; mobile phase, ACN/H2O with corresponding volume fractions; flow-rate, 358 nL/min (after split); detection wavelength: 214 nm. 25

Fig. 5. Effect of KCl concentration (a) and pH of the mobile phase (b) on the retention factors (k) of iodate, bromate and nitrate on the prepared POSS-VBI-Cys hybrid monolithic column. Chromatographic conditions: effective length, 300 mm × 100 μm i.d.; mobile phase, (a) pH 4 KCl solution with different concentration, (b), 250 mM

IP T

KCl solution with different pH; flow-rate, 358 nL/min (after split); detection wavelength: 210 nm.

SC R

Fig. 6. Separation chromatograms of alkylbenzenes on the prepared POSS-VBI-Cys (a,

b) and POSS-VBI (c) hybrid monolithic columns. Experimental conditions: mobile

U

phase, ACN/H2O (70/30, v/v) for (a, c), ACN/H2O (60/40, v/v) for (b); flow-rate, 358

N

nL/min (after split); detection wavelength, 214 nm. Analytes: 1, benzene; 2, toluene; 3,

M

A

ethylbenzene; 4, propylbenzene; 5, butylbenzene.

Fig. 7. Separation chromatograms of amides on the prepared POSS-VBI-Cys (a) and

ED

POSS-VBI (b, c) hybrid monolithic columns. Experimental conditions: effective

PT

length, 300 mm × 100 μm i.d.; mobile phase, ACN/H2O (20/80, v/v) for (a, b), ACN/H2O (5/95, v/v) for (c); flow-rate, 358 nL/min (after split); detection

CC E

wavelength, 214 nm. Analytes: 1, formamide; 2, N,N-dimethylformamide; 3, N,Ndimethylacetamide; 4, iodoacetamide; 5, thioacetamide.

A

Fig. 8. Separation chromatograms of glycoproteins and non-glycoproteins on the prepared POSS-VBI-Cys (a) and POSS-VBI (b, c) hybrid monolithic columns. Experimental conditions: effective length, 300 mm × 100 μm i.d.; mobile phase, A, 0.1% TFA in water, B, 0.1% TFA in ACN; gradient, 20% B to 80% B in 30 min for 26

(a, b), 20% B to 60% B in 30 min for (c); flow rate, 358 nL min-1 (after split); detection wavelength, 214 nm. Analytes: 1, ribonuclease A; 2, ovalbumin; 3, horseradish peroxidase; 4, α-transferrin; 5, BSA; 6, myoglobin. Fig. 9. Separation chromatograms of purified TARG1 protein (a) and the expressed

IP T

TARG1 protein sample (b) on the prepared POSS-VBI-Cys hybrid monolithic

SC R

column. Experimental conditions: effective length, 300 mm × 100 μm i.d.; mobile

phase, A, 0.1% TFA in water, B, 0.1% TFA in ACN; gradient, 0% B to 75% B in 30 min and keep 75% B for additional 15 min; flow rate, 358 nL min-1 (after split);

ED

M

A

N

U

detection wavelength, 280 nm.

Table 1 Parameters for fabrication of the POSS-VBI-Cys hybrid monolithic columns POSS-MA

Methanol

Dodecanol

Temperature

(mg)

(mg)

(mg)

(µL)

(µL)

(°C)

PT

Cys

A

CC E

Column

VBIBr

Permeability (K) (× 10-14 m2)

A

9.2

4.3

27.2

150

55

60

0.79

B

9.2

4.3

27.2

145

60

60

2.60

C

9.2

4.3

27.2

140

65

60

9.32

D

9.2

4.3

27.2

135

70

60

19.87

E

9.2

4.3

27.2

145

60

55

42.68

F

9.2

4.3

27.2

145

60

65

0.40

27

A ED

PT

CC E

28

IP T

SC R

U

N

A

M Fig. 1

A ED

PT

CC E Fig. 2

29

IP T

SC R

U

N

A

M

Fig. 3

30

A ED

PT

CC E

IP T

SC R

U

N

A

M

31

A ED

PT

CC E

IP T

SC R

U

N

A

M

A ED

PT

CC E

Fig. 4

32

IP T

SC R

U

N

A

M

Fig. 5

33

A ED

PT

CC E

IP T

SC R

U

N

A

M

A ED

PT

CC E

Fig. 6

34

IP T

SC R

U

N

A

M

Fig. 7

35

A ED

PT

CC E

IP T

SC R

U

N

A

M

36

A ED

PT

CC E

IP T

SC R

U

N

A

M

A ED

PT

CC E

IP T

SC R

U

N

A

M Fig. 8

37

A ED

PT

CC E Fig. 9

38

IP T

SC R

U

N

A

M