Parameters Governing the Formation of Photopolymerized Silica Sol–Gel Monoliths in PDMS Microfluidic Chips

Although polydimethylsiloxane (PDMS) microfluidic chips provide an alternative to more expensive microfabricated glass chips, formation of monolithic stationary phases in PDMS is not a trivial task. Photopolymerized silica sol–gel monoliths were fabricated in PDMS-based microfluidic devices using 3-trimethoxysilylpropylmethacrylate and glycidyloxypropyltrimethoxysilane. The monolith formation was optimized by identifying a suitable porogen, controlling monomer concentration, functional additives, salts, porogen, wall attachment methods, and rinsing procedures. The resulting monoliths were evaluated using scanning electron microscopy, image analysis, differential scanning calorimetry, and separation performance. Monoliths functionalized with boronic acid ligands were used for the separation of cis-diol containing compounds both in batch mode and in the microfluidic chip.     

Parameters Governing the Formation of Photopolymerized Silica Sol–Gel Monoliths in PDMS Microfluidic Chips

Although polydimethylsiloxane (PDMS) microfluidic chips provide an alternative to more expensive microfabricated glass chips, formation of monolithic stationary phases in PDMS is not a trivial task. Photopolymerized silica sol–gel monoliths were fabricated in PDMS-based microfluidic devices using 3-trimethoxysilylpropylmethacrylate and glycidyloxypropyltrimethoxysilane. The monolith formation was optimized by identifying a suitable porogen, controlling monomer concentration, functional additives, salts, porogen, wall attachment methods, and rinsing procedures. The resulting monoliths were evaluated using scanning electron microscopy, image analysis, differential scanning calorimetry, and separation performance. Monoliths functionalized with boronic acid ligands were used for the separation of cis-diol containing compounds both in batch mode and in the microfluidic chip.

     

A facile and efficient strategy for one-step in situ preparation of hydrophobic organic monolithic stationary phases by click chemistry and its application on protein separation

A simple one-step in situ “click” modification strategy was developed for the preparation of hydrophobic organic monolithic columns for the first time. The column morphology and surface chemistry of the fabricated monolithic columns were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, respectively. The chromatographic performances of the C8/C18 “click” monoliths were evaluated through the separation of a mixture of five proteins such as ribonuclease A, soybean trypsin inhibitor, cytochrome c, bovine haemoglobin and bovine serum albumin. Compared with the blank column, the higher hydrophobicity stationary phases obtained from the “clicked” modification have longer retention times and higher resolution for the five proteins. The separation of five proteins mixture on click C18 monolith with gradient elution at different flow rates was also investigated, the baseline separation of five proteins could be achieved at three different flow rates.     

Monolithic poly[(trimethylsilyl-4-methylstyrene)-co- bis(4-vinylbenzyl)dimethylsilane] stationary phases for the fast separation of proteins and oligonucleotides

In this paper the synthesis, optimisation and application of a silane based monolithic copolymer for the rapid separation of proteins and oligonucleotides is described. The monolith was prepared by thermal initiated in situ copolymerisation of trimethylsilyl-4-methylstyrene (TMSiMS) and bis(4-vinylbenzyl)dimethylsilane (BVBDMSi) in a silanised 200 microm I.D. fused silica column. Different ratios of monomer and crosslinker, as well as different ratios of micro- (toluene) and macro-porogen (2-propanol) were used for optimising the physical properties of the stationary phase regarding separation efficiency. The prepared monolithic stationary phases were characterised by measurement of permeability with different solvents, determination of pore size distribution by mercury intrusion porosimetry (MIP). Morphology was studied by scanning electron microscopy (SEM). Applying optimised conditions, a mixture comprised of five standard proteins ribunuclease A, cytochrome c, alpha-lactalbumine, myoglobine and ovalbumine was separated within 1 min by ion-pair reversed-phase liquid chromatography (IP-RPLC) obtaining half-height peak widths between 1.8 and 2.4 s. Baseline separation of oligonucleotides d(pT)(12-18) was achieved within 1.8 min obtaining half-height peak widths between 3.6 and 5.4 s. The results demonstrate the high potential of this stationary phase for fast separation of high-molecular weight biomolecules such as oligonucleotides and proteins.     

Photografted methacrylate‐based monolithic columns coated with cellulose tris(3,5‐dimethylphenylcarbamate) for chiral separation in CEC

A chiral capillary monolithic column for enantiomer separation in capillary electrochromatography was prepared by coating cellulose tris(3,5‐dimethylphenylcarbamate) on porous glycidyl methacrylate‐co‐ethylene dimethacrylate monolith in capillary format grafted with chains of [2(methacryloyloxy)ethyl] trimethylammonium chloride. The surface modification of the monolith by the photografting of [2(methacryloyloxy)ethyl] trimethylammonium chloride monomer as well as the coating conditions of cellulose tris(3,5‐dimethylphenylcarbamate) onto the grafted monolithic scaffold were optimized to obtain a stable and reproducible chiral stationary phase for capillary electrochromatography. The effect of organic modifier (acetonitrile) in aqueous mobile phase for the enantiomer separation by capillary electrochromatography was also investigated. Several pairs of enantiomers including acidic, neutral, and basic analytes were tested and most of them were partially or completely resolved under aqueous mobile phases. The prepared monolithic chiral stationary phases exhibited a good stability, repeatability, and column‐to‐column reproducibility, with relative standard deviations below 11% in the studied electrochromatographic parameters.     

Controlling retention, selectivity and magnitude of EOF by segmented monolithic columns consisting of octadecyl and naphthyl monolithic segments--applications to RP-CEC of both neutral and charged solutes

Monolithic capillaries made of two adjoining segments each filled with a different monolith were introduced for the control and manipulation of the electroosmotic flow (EOF), retention and selectivity in reversed phase-capillary electrochromatography (RP-CEC). These columns were called segmented monolithic columns (SMCs) where one segment was filled with a naphthyl methacrylate monolith (NMM) to provide hydrophobic and π-interactions, while the other segment was filled with an octadecyl acrylate monolith (ODM) to provide solely hydrophobic interaction. The ODM segment not only provided hydrophobic interactions but also functioned as the EOF accelerator segment. The average EOF of the SMC increased linearly with increasing the fractional length of the ODM segment. The neutral SMC provided a convenient way for tuning EOF, selectivity and retention in the absence of annoying electrostatic interactions and irreversible solute adsorption. The SMCs allowed the separation of a wide range of neutral solutes including polycyclic aromatic hydrocarbons (PAHs) that are difficult to separate using conventional alkyl-bonded stationary phases. In all cases, the k' of a given solute was a linear function of the fractional length of the ODM or NMM segment in the SMCs, thus facilitating the tailoring of a given SMC to solve a given separation problem. At some ODM fractional length, the fabricated SMC allowed the separation of charged solutes such as peptides and proteins that could not otherwise be achieved on a monolithic column made from NMM as an isotropic stationary phase due to the lower EOF exhibited by this monolith.     

胃蛋白酶亲和有机聚合物毛细管整体柱的制备及性能考察

以热引发原位聚合方法制备了聚（甲基丙烯酸缩水甘油酯（glycidyl methacrylate,GMA）-乙二醇二甲基丙烯酸酯（ethyleneglycol dimethacrylate,EDMA））毛细管整体柱,对整体柱的性能进行了表征。结果表明,柱内部结构均匀、渗透性好;整体柱能够实现苯等中性小分子化合物的分离,具有反相色谱特征,重现性和稳定性良好。利用整体柱环氧基团的活性,采用间接法,以戊二醛为连接臂制备胃蛋白酶亲和手性整体柱。在毛细管电色谱模式下进行了柱分离性能研究,并对缓冲液pH值和运行电压等分离条件进行了考察。结果表明,亲和整体柱对4种碱性手性药物（奈福泮、氨氯地平、西酞普兰、扑尔敏）有拆分效果,奈福泮、氨氯地平、西酞普兰能达到基线分离。本文为蛋白质亲和毛细管电色谱整体柱的制备和应用提供了新的思路和方法。     

Graphene oxide-octadecylsilane incorporated monolithic nano-columns with 50 μm id and 100 μm id for small molecule and protein separation by nano-liquid chromatography

In this study, graphene oxide-octadecylsilane incorporated monolithic nano-columns were developed for protein analysis by nano liquid chromatography (nano LC). The monolithic column with 100 μm id was first prepared by an in situ polymerization using ethylene dimethacrylate (EDMA), 3-chloro-2-hydroxypropylmethacrylate (HPMA-Cl), and methacryloyl graphene oxide nanoparticles (MGONPs). MGONPs were synthesized by the treatment of 3-(trimethoxysilyl)propylmethacrylate (TMSPM) and GO. Tetrahydrofuran (THF) and dodecanol were used as the porogenic solvent. The resulting column was functionalized by dimethyloctadecylch lorosilane (DODCS) for the enhancement of hydrophobicity. The functionalization greatly improved the baseline separation of hydrophobic compounds such as polyaromatic hydrocarbons (PAHs). The optimized monolith with respect to total polymerization mixture was characterized by using Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) X-ray diffraction (XRD) and chromatographic analyses. The blank monoliths without functionalization exhibited poor separation while a good separation performance of MGONPs functionalized monoliths was achieved. The monolith with 100 μm id was evaluated in protein separation in nano LC using RNase A, Cytochrome C, Lysozyme, Trypsin, and Ca isozyme II as the test proteins. It was shown that protein separation mechanism was based on large π-system of GO and hydrophobicity of the monolithic structure. Theoretical plates number up to 57 600 plates were achieved. The nano-column with 50 μm id was also prepared using the same polymerization mixture under the same chemical conditions. These nano-columns were employed for protein separation by nano LC, and the dependence of both nano-column performance on the internal diameter was also discussed.     

Coupled affinity-hydrophobic monolithic column for on-line removal of immunoglobulin G, preconcentration of low abundance proteins and separation by capillary zone electrophoresis

A butyl methacrylate-co-ethylene dimethacrylate (BuMA-co-EDMA) monolith was synthesized by UV initiated polymerization at the inlet end of a 75 microm I.D. fused silica capillary that had been previously coated with a protein compatible polymer, poly(vinyl)alcohol. The monolith was used for on-line preconcentration of proteins followed by capillary electrophoresis (CE) separation. For the analysis of standard proteins (cytochrome c, lysozyme and trypsinogen A) this system proved reproducible. The run-to-run %RSD values for migration time and corrected peak area were less than 5%, which is typical of CE. As measured by frontal analysis using lysozyme as solute, saturation of a 1cm monolith was reached after loading 48 ng of protein. Finally, the BuMA-co-EDMA monolithic preconcentrator was coupled to a protein G monolithic column via a zero dead volume union. The coupled system was used for on-line removal of IgG, preconcentration of standard proteins and CE separation. This system could be a valuable sample preparation tool for the analysis of low abundance proteins in complex samples such as human serum, in which high abundance proteins, e.g., human serum albumin (HSA) and immunoglobulin G (IgG), hinder identification and quantification of low abundance proteins.     

Capillary electrochromatography with monolithic stationary phases. 4. Preparation of neutral stearyl-acrylate monoliths and their evaluation in capillary electrochromatography of neutral and charged small species as well as peptides and proteins

A neutral, nonpolar monolithic capillary column having a relatively strong electroosmotic flow (EOF) yet free of electrostatic interactions with charged solutes was developed for the reversed-phase capillary electrochromatography (RP-CEC) of neutral and charged species including peptides and proteins. The neutral nonpolar monolith is based on the in situ polymerization of pentaerythritol diacrylate monostearate (PEDAS) in a ternary porogenic solvent composed of cyclohexanol, ethylene glycol, and water. PEDAS plays the role of both the cross-linker and the ligand provider, generating a macroporous nonpolar monolith having C17 chains as the chromatographic ligands. Despite the fact that the neutral PEDAS monolith is devoid of fixed charges, the monolithic capillary columns exhibited a relatively strong EOF due to the ability of PEDAS to adsorb sufficient amounts of electrolyte ions from the mobile phase. The adsorbed ions imparted the neutral PEDAS monolith the zeta potential necessary to support the EOF required for mass transport across the monolithic column. The absence of fixed charges on the surface of the neutral PEDAS monolith and in turn the adsorption sites for electrostatic attraction of charged solutes allowed the rapid and efficient separations of proteins and peptides at pH 7.0, with an average plate number of 255,000 and 121,000 plates/m, respectively. To the best of our knowledge, this constitutes the first report on the separation of proteins at neutral pH by RP-CEC using a neutral monolithic column.     

Review of recent advances in the preparation of organic polymer monoliths for liquid chromatography of large molecules

In recent years the use of monolithic polymers in separation science has greatly increased due to the advantages these materials present over particle-based stationary phases, such as their relative ease of preparation and good permeability. For these reasons, these materials present high potential as stationary phases for the separation and purification of large molecules such as proteins, peptides, nucleic acids and cells. An example of this is the wide range of commercial available polymer-based monolithic columns now present in the market.     

Fast preparation of polystyrene-based monolith using microwave irradiation for micro-column separation

The conventional analytical HPLC was successfully developed for micro-column separation by using a simple eluate splitting system, a self-preparation of monolithic column and an on-capillary column detector in our laboratory. A typical polystyrene-based monolith was quickly prepared inside the fused-silica capillary, which in situ polymerization was carried out in 10 min by microwave irradiation. The reactant solution consisted of styrene (ST) as a functional monomer, divinylbenzene (DVB) as a cross-linking agent, toluene and isooctane as porogenic solvents, and azobisisobutyronitrile (AIBN) as an initiator. The monolith was proved to form in the center of the capillary and adhered to the column inner wall by the scanning electron micrograph. Its chromatographic behaviors were evaluated in detail by varying the flow rate and percentage of mobile phases, and under the optimal condition, baseline separation of the model analytes including thiourea, benzene, toluene, ethylbenzene was obtained with a highest theoretical plate number near 11,290 N/m by the developed capillary HPLC. Furthermore, the stability and porosity of the prepared monolith were systematically investigated by a simple flow method. Figure A polystyrene-based monolith was rapidly prepared inside the fused-silica capillary, which in situ polymerization was carried out about 10 min by microwave irradiation.     

On-chip solid phase extraction and enzyme digestion using cationic PolyE-323 coatings and porous polymer monoliths coupled to electrospray mass spectrometry

We evaluate the compatibility and performance of polymer monolith solid phase extraction beds that incorporate cationic charge, with a polycationic surface coating, PolyE-323, fabricated within microfluidic glass chips. The PolyE-323 is used to reduce protein and peptide adsorption on capillary walls during electrophoresis, and to create anodal flow for electrokinetically driven nano-electrospray ionization mass spectrometry. A hydrophobic butyl methacrylate-based monolithic porous polymer was copolymerized with an ionizable monomer, [2-(methacryloyloxy)ethyl] trimethylammonium chloride to form a polymer monolith for solid phase extraction that also sustains anodal electroosmotic flow. Exposure of the PolyE-323 coating to the monolith forming mixture affected the performance of the chip by a minor amount; electrokinetic migration times increased by ～5%, and plate numbers were reduced by an average of 5% for proteins and peptides. 1-mm long on-chip monolithic solid phase extraction columns showed reproducible, linear calibration curves (R(2)=0.9978) between 0.1 and 5 nM BODIPY at fixed preconcentration times, with a capacity of 2.4 pmol or 0.92 mmol/L of monolithic column for cytochrome c. Solution phase on-bed trypsin digestion was conducted by capturing model protein samples onto the monolithic polymer bed. Complete digestion of the proteins was recorded for a 30 min stop flow digestion, with high sequence coverage (88% for cytochrome c and 56% for BSA) and minimal trypsin autodigestion product. The polycationic coating and the polymer monolith materials proved to be compatible with each other, providing a high quality solid phase extraction bed and a robust coating to reduce protein adsorption and generate anodal flow, which is advantageous for electrospray.     

Preparation of a long‐alkyl‐chain‐based hybrid monolithic column with mixed‐mode interactions using a “one‐pot” process for pressurized capillary electrochromatography

A simple “one‐pot” approach for the preparation of a new vinyl‐functionalized organic–inorganic hybrid monolithic column is described. In this improved method, the hydrolyzed alkoxysilanes of tetramethoxysilane and triethoxyvinylsilane were used as precursors for the synthesis of a silica‐based monolith, while 1‐hexadecene and sodium ethylenesulfonate were used as vinyl functional monomers along with azobisisobutyronitrile as an initiator. The effects of reaction temperature, urea content, and composition of organic monomers on the column properties (e.g. morphology, mechanical stability, and chromatographic performance) were investigated. The monolithic column was used for the separation of neutral solutes by reversed‐phase pressurized capillary. Furthermore, the monolith can separate various aromatic amines, which indicated its excellent cation‐exchange capability and hydrophobic interactions. The baseline separation of the aromatic amines was obtained with a column efficiency of up to 78 000 plates/m.     

Novel Porous Monolithic Column Using Poly(high internal phase emulsion) Methacrylate as Materials for Immunoglobulin Separation Performance on HPLC

A novel porous polymeric monolithic column based on poly(high internal phase emulsion) methacrylate monolith was prepared and applied to fast separation of proteins. The block copolymer chemistry of high internal phase emulsions was used in the experiment. These unique properties, together with high porosity, good mechanical property, chemical modification of the surface make themselves superior in monolithic medium applications. Morphology of the monolithic material was studied by scanning electron microscopy. The stability of the emulsion and the load of hydroxyl groups–the active group of the monolithic column were investigated. Additionally, the capabilities of separation of this column in conjunction with high performance liquid chromatography (HPLC) were investigated. Immunoglobulin was separated from human plasma and chicken egg yolk with high resolution on the hydrophobic interaction chromatographic column in a short time. The effects of pH and concentration of mobile phase (buffer) on the elution of immunoglobulin were investigated. Moreover, fast separation of a two mode protein mixture (α‐amylase and lysozyme) on the monolith was achieved within 1.5 min at a velocity of 1445 cm·h^?1. As a result, good separation was achieved, and stable low back pressure drop was ensured at high throughput elution with an even longer column.     

Cation exchange/hydrophobic interaction monolithic chromatography of small molecules and proteins by nano liquid chromatography†

In this study, vinyl phenyl boronic acid modified lauryl methacrylate‐based monolithic column was successfully prepared for cation exchange/hydrophobic interaction monolithic chromatography of small molecules and proteins in nano LC. The polymeric mixture consisted of lauryl methacrylate, vinyl phenyl boronic acid as cation exchanger, ethylene dimethacrylate as cross‐linker, polyethylene glycol and methanol as binary porogenic solvent, and azobisisobutyronitrile as initiator. The resulting monolith showed good permeability and mechanical stability. Different ratios of monomer and porogens were used for optimizing the properties of the column. The monolithic column performance with respect to hydrophobic and cation exchange interactions was assessed by the separation a series of alkyl benzenes and anilines, respectively. cis‐Diol‐containing compounds such as phenols were also utilized to evaluate the retention behaviors of the vinyl phenyl boronic acid modified monolithic column. The monolithic column showed cation exchange interactions in the separation of aniline compounds. Theoretical plate number up to 52 000 plates/m was successfully achieved. The prepared monolith was further applied to the proteins with different acetonitrile content.     

Convenient enantioseparation by monolithic imprinted capillary clamped in a chip with electrochemical detection

A microchip integrated with a monolithic imprinted capillary has been manufactured for performing the chip-based capillary electrochromatographic enantioseparation. The microporous monolith anchored on the inner wall of the microchannel was prepared by in situ chemical copolymerization, and characterized with scanning electron microscopy, IR spectroscopy, and solid-state UV-vis spectroscopy. The monolithic network with high porosity gave a large surface area, good permeability, low mass-transfer resistance, and thus high separation efficiency. A portable microchip was conveniently constructed by integrating an imprinted capillary with 5-cm length as the separation channel and a carbon fiber microdisk working electrode for amperometric detection. Using L-tyrosine (L-Tyr) as the template molecule, Tyr enantiomers could be baseline separated within 55 s under the optimized preparation and separation conditions. The linear ranges for online amperometric detection of both Tyr enantiomers were from 20 to 2400 μM. The microporous monolithic chip strategy exhibited excellent separation efficiency and promising analytical application in enantioseparation. It opens an avenue for high-throughput screening of chiral compounds.     

Development of capillary electrochromatography with poly(styrene-divinylbenzene-vinylbenzenesulfonic acid) monolith as the stationary phase

A new polystyrene-based monolithic stationary phase, which was prepared by single step in situ copolymerization of styrene, divinylbenzene and vinylbenzenesulfonic acid (VBSA), was developed as a separation column for capillary electrochromatography, in which VBSA was employed as the charge-bearing monomer. Polymerization time of the polystyrene-based monolith had slightly influenced the separation time of the tested analytes, but it effectively altered their separation resolutions. Furthermore, baseline separation for a wider range of acetonitrile levels of mobile phase was achieved when a monolithic column prepared by a longer polymerization time was used. This novel polystyrene-based monolithic column provided an adequate electroosmotic flow either in basic or acidic mobile phase when VBSA level was maintained at 2.6% (w/w). Finally, this proposed polystyrene-based column allowed seven tested analytes to achieve a reproducible baseline separation within 2.2 min with theoretical plate numbers higher than 164 000 plates/m.     

Macrocyclic polyamine-modified poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith for capillary electrochromatography

1,4,10,13,16-Pentaazatricycloheneicosane-9,17-dione (macrocyclic polyamine)-modified polymer-based monolithic column for CEC was prepared by ring opening reaction of epoxide groups from poly(glycidyl methacrylate-co-ethylene dimethacrylate) (GMA-co-EDMA) monolith with macrocyclic polyamine. Conditions such as reaction time and concentration of macrocyclic polyamine for the modification reaction were optimized to generate substantial EOF and enough chromatographic interactions. Anodic EOF was observed in the pH range of 2.0-8.0 studied due to the protonation of macrcyclic polyamine at the surface of the monolith. Morphology of the monolithic column was examined by SEM and the incorporation of macrocyclic polyamine to the poly(GMA-co-EDMA) monolith was characterized by infrared (IR) spectra. Successful separation of inorganic anions, isomeric benzenediols, and benzoic acid derivatives on the monolithic column was achieved for CEC. In addition to hydrophobic interaction, hydrogen bonding and electrostatic interaction played a significant role in the separation process.     

Amylose-3,5-dimethylphenylcarbamate immobilized on monolithic silica stationary phases for chiral separations in capillary electrochromatography

Chiral monolithic silica capillary columns were prepared by immobilization of amylose-3,5-dimethylphenylcarbamate (ADMPC) bearing a small fraction of 3-(triethoxysilyl)propyl residues through intermolecular polycondensation of the triethoxysilyl groups. The obtained columns were used for chiral separations in capillary electrochromatography (CEC). The effects of the silica monolith nature and the used chiral selector concentration on the resulting enantiomeric separations were investigated. Fifteen chiral compounds, including acidic, neutral, and basic substances were evaluated and twelve showed partial or baseline separation at some of the different conditions tested. These results demonstrated the promising applicability of ADMPC-immobilized monolithic silica columns in CEC enantioseparations, but also revealed the need for further improvements on the level of baseline separations and efficiencies.