Comparative study on the separation behavior of monolithic columns prepared via ring-opening metathesis polymerization and via electron beam irradiation triggered free radical polymerization for proteins

Monolithic columns have been prepared via ring-opening metathesis polymerization using different monomers and crosslinkers, i.e. norborn-2-ene, 1,4,4a,5,8,8a-hexahydro-1,4,5,8-exo,endo-dimethanonaphthalene, cyclooctene and tris(cyclooct-4-en-1-yloxy)methylsilane. 2-Propanol and toluene were used as macro- and microporogens. Alternatively, monolithic supports were realized via electron beam triggered free radical polymerization using trimethylolpropane triacrylate and ethylmethacrylate. Here, 2-propanol, 1-dodecanol and toluene were used as porogens. The three monolithic supports were structurally characterized by inverse size exclusion chromatography and investigated for their separation capabilities for a series of proteins. Separation efficiencies are discussed within the context of the different structural features of the monolithic supports and are compared to the separation data obtained on a commercial silica-based Chromolith RP-18e column.     

Monolithic poly(ethylhexyl methacrylate-co-ethylene dimethacrylate) column with restricted access layers prepared via reversible addition-fragmentation chain transfer polymerization

The "living"/controlled radical polymerization has provided an opportunity in making a more homogeneous polymer, which is favorable for polymer-based monolithic column fabrication. To study its application in the preparation of separation material, a capillary poly(ethylhexyl methacrylate-co-ethylene dimethacrylate) monolithic column has been synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. The correlation between the synthetic conditions and the polymer structures and separation performance was studied. The result indicated RAFT-mediated reaction provides condition for creating polymers with narrower pore size distribution and higher column efficiency compared with traditional polymerization. The "living" property of the RAFT polymerization was further utilized to graft hydrophilic polymer on the surface of poly(ethylhexyl methacrylate-co-ethylene dimethacrylate). The hydrophilic chain modified monolithic column has both abilities of protein exclusion and small hydrophobic compound retention. The result indicated that RAFT polymerization can be used for making multifunctional material. The restricted access monolithic material synthesized by this method can be used in biological sample analysis with HPLC direct injection.     

Characterization of polymer monolithic stationary phases for capillary HPLC

Monolithic capillary columns have been prepared in fused‐silica capillaries by radical co‐polymerization of ethylene dimethacrylate and butyl methacrylate in the presence of porogen solvent mixtures containing various concentration ratios of 1‐propanol, 1,4‐butanediol, and water with azobisisobutyronitrile as the initiator of the polymerization reaction. The through pores in organic polymer monolithic columns can be characterized by “equivalent permeability particle size”, and the mesopores with stagnant mobile phase by “equivalent dispersion particle size”. Increasing the concentration of propanol in the polymerization mixture diminishes the pore volume and size in the monolithic media and improves the column efficiency, at a cost of decreasing permeability. Organic polymer monolithic capillary columns show similar retention behaviour to packed alkyl silica columns for compounds with different polarities characterized by interaction indices, I_x, but have different methylene selectivities. Higher concentrations of propanol in the polymerization mixture increase the lipophilic character of the monolithic stationary phases. Best efficiencies and separation selectivities were found for monolithic columns prepared using 62–64% propanol in the porogen solvent mixture. To allow accurate characterization of the properties of capillary monolithic columns, the experimental data should be corrected for extra‐column contributions.     

Photopolymerized organo‐silica hybrid monolithic columns: Characterization of their performance in capillary liquid chromatography

Porous hybrid organo‐silica monoliths have been prepared inside pretreated 100 μm id UV transparent fused‐silica capillaries using simultaneous sol‐gel transition and polymerization of 3‐(methacryloyloxy)propyl trimethoxysilane in the presence of toluene as a porogen. The sol‐gel reaction was catalyzed by hydrochloric acid while various photoinitiators including azobisisobutyronitrile, 2,2‐dimethoxy‐2‐phenylacetophenone, and Irgacure 819 were used for the photopolymerization carried out under irradiation with UV light at a wavelength of 254 or 365 nm. The chromatographic performance of photopolymerized monolithic columns in RP liquid chromatographic mode was assessed with respect to the following metrics: column efficiency, methylene and steric selectivity, effect of silanol groups, van Deemter plot, permeability, and pore size distribution. Columns with an efficiency of up to 77 000 plates/m for benzene has been achieved at a flow velocity of 0.47 mm/s. The performance of photopolymerized hybrid monolithic column was compared to the performance of columns prepared via thermally initiated polymerization.     

Preparation of high efficiency and highly retentive monolithic silica capillary columns for reversed-phase chromatography by chemical modification by polymerization of octadecyl methacrylate

High efficiency and highly retentive monolithic silica capillary columns were obtained by polymerization of octadecyl methacrylate using alpha,alpha'-azobis-isobutyronitrile (AIBN) as a free radical initiator. Hybrid type monolithic silica columns (25 cm total length x 200 microm I.D.) prepared from a mixture of tetramethoxysilane and methyltrimethoxysilane were used as a support. The effects of the monomer and the radical initiator concentrations in the reaction mixture were examined. The performance of the columns was tested in terms of column efficiency and retention behavior by using alkylbenzenes and a few other compounds as solutes and compared with that of hybrid monolithic silica columns modified with octadecylsilyl-(N,N-diethylamino)silane (ODS-DEA). Highly retentive monolithic silica columns were obtained by polymerization at high monomer concentrations. Although a decrease in column efficiency was observed with the increase in the monomer concentration in a feed mixture, an improvement in efficiency was achieved (a plate height value lower than 10 microm) by increasing an initiator concentration without significant variations in column retention properties. Results obtained by polymerization using other monomers are also presented to demonstrate the applicability of the preparation method.     

Enrofloxacin-imprinted monolithic columns synthesized using reversible addition-fragmentation chain transfer polymerization

Molecularly imprinted monolithic columns for selective separation of enrofloxacin were prepared by Reversible Addition-Fragmentation Chain Transfer (RAFT)-mediated radical polymerization. Different ratios of initiation system were used in the synthesis. The structures of the monoliths were characterized to study the relationship between the synthetic conditions and morphology of the monolithic material. The separation performance of the monoliths was evaluated by liquid chromatography. Under optimized synthetic conditions, a monolithic molecularly imprinted polymer (MIP) with high selectivity and improved column efficiency was obtained. The research has shown that RAFT polymerization provides more adjustable conditions for making monolithic materials with different morphologies. The results also demonstrated that homogeneous macro-pore size distribution and large specific surface area are the key factors providing good separation ability and column efficiency for MIP monolithic structures.     

CEC with monolithic poly(styrene-divinylbenzene-vinylsulfonic acid) as the stationary phase

In this study, poly(styrene-divinylbenzene-vinylsulfonic acid) monolithic columns, prepared by singlestep in situ polymerization, were first used as separation columns for CEC in which vinylsulfonic acid was employed as the charge-bearing monomer. The effects of the polymerization conditions (compositions of monomer and porogenic solvent, and polymerization time) on the chromatographic property of the polystyrene-based monolithic column were examined. Furthermore, an increase in water content of the porogenic solvent markedly altered the retention behaviors of these analytes, where the monolithic column was able to be produced in a higher ratio of vinylsulfonic acid. Finally, the study demonstrated that acidic analytes could be successfully separated by the polystyrene-based monolithic column under an acidic mobile phase.     

Molecularly imprinted monolithic stationary phases for liquid chromatographic separation of enantiomers and diastereomers

The method for preparation of molecularly imprinted monolithic stationary phase has been improved to achieve liquid chromatographic separation of enantiomers and diastereomers. By adopting low polar porogenic solvents of toluene and dodecanol and optimal polymerization conditions, the molecularly imprinted monolithic stationary phases with good flow-through properties and high resolution were prepared. Enantiomers of amino acid derivatives and diastereomers of cinchona alkaloids were completely resolved using the monolithic stationary phases. The influence of porogenic composition, monomer-template ratio and polymerization conditions on the chromatographic performance was investigated. Some chromatographic conditions such as the composition of the mobile phase and the temperature were characterized. Scanning electron microscopy showed that the molecularly imprinted monolithic stationary phase has a large through-pore structure to allow the mobile phase to flow through the column at very low backpressure. Accelerated separations of enantiomers and diastereomers were therefore achieved at elevated flow rates. Finally, the chiral recognition performance of the prepared stationary phase in aqueous media was investigated. Hydrophobic interaction, and ionic and/or hydrogen bonding interactions were proposed to be responsible for the recognition mechanism.     

Visible light initiated polymerization of styrenic monolithic stationary phases using 470 nm light emitting diode arrays

Poly(styrene‐co‐divinylbenzene) monolithic stationary phases have been synthesized for the first time by photoinitiated polymerization. An initiator composed of (+)‐(S)‐camphorquinone/ethyl‐4‐dimethylaminobenzoate/N‐methoxy‐4‐phenylpyridinium tetrafluoroborate was activated using a 470 nm light emitting diode array as the light source. Spatially controlled polymerization of styrenic monoliths has been achieved within specific sections of a 100 μm id polytetrafluoroethylene‐coated fused‐silica capillary using simple photo masking. The sharpness of the edges was confirmed by optical microscopy, while SEM was used to verify a typical porous, globular morphology. Flow resistance data were used to assess the permeability of the monoliths and they were found to have good flow through properties with a flow resistance of 0.725 MPa/cm at 1 μL/min (water, 20°C). Conductivity profiling along the length of the capillary was used to assess their lateral homogeneity. Monoliths which were axially rotated during polymerization were found to be homogeneous along the whole length of the capillary. The monolithic stationary phases were applied to the RP gradient separation of a mixture of proteins. Column fabrication showed excellent reproducibility with the retention factor (k) having a RSD value of 2.6% for the batch and less than 1.73% on individual columns.     

Organic polymer monoliths as stationary phases for capillary HPLC

Modern rigid porous polymer monoliths were conceived as a new class of stationary phases in classical columns in the early 1990s and later extended to the capillary format. These monolithic materials are typically prepared using a simple molding process carried out within the confines of the capillary. Polymerization of a mixture comprising monomers, initiator, and porogenic solvent affords macroporous materials with large through-pores that enable applications in a rapid flow-through mode. Since all the mobile phase must flow through the monolith, convection considerably accelerates mass transport within the monolithic separation medium and improves the separations. As a result, monolithic columns perform well even at very high flow rates. Various mechanisms including thermally and UV initiated free radical polymerization as well as ring opening metathesis copolymerizations were demonstrated for the preparation of monolithic capillary columns. The versatility of these preparation techniques was demonstrated by their use with hydrophobic (styrene, divinylbenzene, butyl methacrylate, ethylene dimethacrylate), hydrophilic (2-hydroxyethyl methacrylate, methacrylamide, methylenebisacrylamide), ionizable (vinylsulfonic acid, 2-acrylamido-2-methyl-propanesulfonic acid), and tailor-made (norborn-2-ene, 1,4,4a,5,8,8a-hexahydro-1,4,5,8-exo,endo-dimethanonaphthalene) monomers. Variation of polymerization conditions enables control of the porous properties of the monolith over a broad range and mediates the hydrodynamic properties of the monolithic columns. The applications of polymer-based monolithic capillary columns are demonstrated for numerous separations in the microHPLC mode.     

Thermoresponsive polymer brush on monolithic-silica-rod for the high-speed separation of bioactive compounds

Poly(N-isopropylacrylamide), one of the most utilized thermoresponsive polymers, brush-grafted monolithic-silica columns were prepared through surface-initiated atom transfer radical polymerization (ATRP) for effective thermoresponsive-chromatography matrices. ATRP initiator was grafted on monolithic silica-rod surfaces by flowing a toluene solution containing ATRP initiator into monolithic silica-rod columns. N-Isopropylacrylamide (IPAAm) monomer and CuCl/CuCl(2)/Me(6)TREN, an ATRP catalytic system, were dissolved in 2-propanol, and the reaction solution was pumped into the preprepared initiator-modified columns at 25 °C for 16 h. The constructed PIPAAm-brush structure on the monolithic silica-rod surface was confirmed by XPS, elemental analysis, SEM observation, and GPC measurement of grafted PIPAAm. The prepared monolithic silica-rod columns were also characterized by chromatographic analysis. PIPAAm-brush-modified monolithic silica-rod columns were able to separate hydrophobic steroids with a short analysis time (10 min), compared to PIPAAm-brush-modified silica-beads-packed columns, because of the horizontally limited diffusion path length of monolithic supporting materials. Additionally, diluted PIPAAm-brush monolithic silica-rod column gave a further shorting analysis time (5 min). These results indicated (1) surface-initiated ATRP constructed PIPAAm-brush structures on monolithic silica-rod surfaces and (2) PIPAAm-brush grafted monolithic silica-rod column prepared by ATRP was a promising tool for analyzing hydrophobic-bioactive compounds with a short analysis time.     

Evaluation of ring-opening metathesis polymerization (ROMP)-derived monolithic capillary high performance liquid chromatography columns

Novel monolithic capillary HPLC columns were prepared via ring opening metathesis polymerization (ROMP) within the confines of fused silica columns with 200 microm i.d. using norborn-2-ene (NBE), 1,4,4a,5,8,8a-hexahydro-1,4,5,8, exo, endo-dimethanonaphthalene (DMN-H6) as monomers, 2-propanol and toluene as porogens and RuCl2(PCy3)2(CHPh) as initiator. Using the monolithic capillary HPLC columns, different sets of analytes (i.e. standard systems) were used for the evaluation of the monolithic columns: (i) a protein standard consisting of six proteins in the range of 5000-66 000 g/mol, (ii) an insulin-albumin standard, and (iii) a peptide standard obtained from a tryptic digest of cytochrome C. With these three different standard systems the reproducibility of synthesis in terms of separation performance proved to be 1-2% relative standard deviation in tR. Variation of polymerization parameters had a significant influence on the monolithic morphology and therefore separation efficiency and back pressure. The maximum analytical loading capacity of ROMP-derived monolithic capillary columns for albumin was found to be 30-125 ng, depending on the monomer content. Long-term stability studies showed no alteration in separation performance.     

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A model of flow-through pore formation in methacrylate ester-based monolithic columns

The main factors affecting the porosity of methacrylate-ester based monolithic columns were investigated. We prepared 23 monolithic capillary columns with porosity controlled by varying the proportions of butyl methacrylate and ethylene dimethacrylate monomers and of 1,4-butanediol and 1-propanol as the porogen solvent in the polymerization mixtures by thermally initiated in-situ polymerization in fused-silica capillaries. Using mixture design software, we systematically varied the composition of the polymerisation mixtures to find significant factors affecting flow-through pore formation. Multivariate analysis of the experimental data obtained for the fabricated columns yielded a model for prediction of the flow-through porosity in monolithic beds as a function of the composition of the polymerization mixture used to prepare polymethacrylate monolithic capillary columns. The mean error of prediction was lower than 8% for eight columns prepared independently of the original set of 15 columns used to derive the flow-through model. The flow-through porosity increases with increasing concentration of the binary porogen solvent mixture, the concentration of 1,4-butanediol being the main factor enhancing flow-through pore formation. On the other hand, increasing concentrations of the hydrophobic monomer butyl methacrylate and increasing concentrations of 1-propanol have a negative effect on flow-through pore formation. The capillary columns prepared with a high proportion of flow-through pores and a minimum amount of mesopores can be used for fast gradient separations of both low-molecular weight compounds and biopolymers.     

Polymetacrylate and hybrid interparticle monolithic columns for fast separations of proteins by capillary liquid chromatography

Preparation of organic polymer monolithic columns in fused silica capillaries was aimed at fast gradient separation of proteins. For this purpose, polymerization in situ procedure was optimized, using ethylene dimetacrylate and butyl metacrylate monomers with azobisisobutyronitrile as initiator of the polymerization reaction in presence of non-aqueous porogen solvent mixtures composed of 1-propanol and 1,4-butanediol. The separation of proteins in totally monolithic capillary columns was compared with the chromatography on a new type of "hybrid interparticle monolithic" capillary columns, prepared by in situ polymerization in capillary packed with superficially porous spherical beds, 37-50 microm. The "hybrid" columns showed excellent stability and improved hydrodynamic flow properties with respect to the "totally" monolithic capillary columns. The separation selectivity is similar in the two types of columns. The nature of the superficially porous layer (bare silica or bonded C18 ligands) affects the separation selectivity less significantly than the porosity (density) of the monolithic moiety in the interparticle space, controlled by the composition of the polymerization mixture. The retention behaviour of proteins on all prepared columns is consistent with the reversed-phase gradient elution theory.     

Optimization of binary porogen solvent composition for preparation of butyl methacrylate monoliths in capillary liquid chromatography

Butyl methacrylate monolithic columns in 320 microm i.d. fused silica capillaries for reversed-phase capillary liquid chromatography were prepared by radical polymerization initiated thermally with azobisisobutyronitrile (AIBN). Polymerization mixture contained butyl methacrylate (BMA) as the function monomer and ethylene dimethacrylate (EDMA) as the crosslinking agent with 1,4-butanediol and 1-propanol as a binary porogen solvent. Ratio of 1,4-butanediol to 1-propanol in the porogen solvent was optimized regarding the monolithic column efficiency and performance. Total porosity, column permeability, separation impedance, Walters hydrophobicity index, retention factors, peak asymmetry factors, height equivalents to a theoretical plate and peak resolutions were used for characterization of the prepared monolithic columns. The polymerization mixture consisting of 17.8% of BMA, 21.8% of EDMA, 18.0% of 1,4-butanediol, 42.0% of 1-propanol and 0.4% AIBN generated monolithic columns of the best performance having a sufficient permeability and the lowest separation impedance. It was also demonstrated that monolithic columns of this composition exhibited good preparation reproducibility and an excellent pressure resistance when applied in capillary liquid chromatography.     

Peroxodisulfate as a chemical initiator for methacrylate-ester monolithic columns for capillary electrochromatography

Organic monolithic stationary phases for CEC were synthesized in situ in fused-silica capillaries. Polymerization mixtures were composed of butyl methacrylate, ethylene dimethacrylate, and [2-(methacryloyloxy)ethyl]trimethyl ammonium chloride in the presence of a porogenic solvent, using ammonium peroxodisulfate as chemical initiator, and N,N,N',N'-tetramethylethylenediamine to activate the reaction. The influence of the amount of initiator, temperature, and composition of porogenic solvent on the physical and chromatographic properties of monolithic stationary phases has been investigated. A minimum plate height of 14.5 microm was obtained at 18 wt% of 1,4-butanediol in the polymerization mixture. The produced monolithic stationary phases exhibited a good repeatability and batch-to-batch and mixture-to-mixture reproducibility, with RSD values below 5.6% in the electrochromatographic parameters studied. A comparison with columns prepared by thermal initiation with alpha,alpha'-azobisisobutyronitrile (AIBN) was also performed. The most efficient column initiated with peroxodisulfate showed better efficiencies and selectivities than that prepared with AIBN at the same composition mixture.     

Monolithic column with double mixed-modes of hydrophilic interaction/cation-exchange and reverse-phase/cation-exchange stationary phase for pressurized capillary electrochromatography

A monolithic capillary column with double mixed-modes of hydrophilic interaction/cation-exchange and RP/cation-exchange stationary phase was prepared by in situ thermal polymerization and then hydrolyzed with hydrochloric acid. The polymerization solution containing glycidyl methacrylate (GMA), 3-sulfopropyl methacrylate potassium salt (SPMA), and ethylene dimethacrylate (EDMA) in a binary porogenic solvent consisting of dimethylformamide (DMF) and 1,4-butanediol was polymerized in a fused-silica capillary pretreated with 3-(trimetoxysilyl) propyl methacrylate. The epoxy groups on the surface were hydrolyzed to diol groups with hydrochloric acid to enhance the polarity of the stationary phase. By simply altering the ACN content in the mobile phase, two mixed-mode mechanisms could be achieved on the same monolithic column in different mobile phase condition. Hydrophilic interaction (or hydrophilic interaction/cation-exchange) was observed at high ACN content, as well as RP (or RP/cation-exchange) was observed at low ACN content. The monolithic column provided good selectivity and high efficiency for separation of neutral polar analytes and basic compounds. Phenols, anilines, alkaloids, nucleic acid bases, and narcotic pharmaceuticals have been successfully separated. Effects of salt concentration and ACN content on the separation have also been investigated. High column efficiencies of up to 352 000 plates/meter were obtained by the separation of narcotic pharmaceuticals.     

Monolithic column plastic microfluidic device for peptide analysis using electrospray from a channel opening on the edge of the device

Electrospray from a channel exit at the edge of a fluorocarbon coated cycloolefin copolymer microfluidic device has been investigated. The fluorocarbon coating facilitated generation of a stable electrospray, thereby enhancing the detectability of electrospray ionization (ESI) mass spectrometry (MS). A microfluidic device of integrated ESI emitters and monolithic liquid chromatography columns has been fabricated on a cycloolefin copolymer chip. The monolithic columns were polymerized in situ using UV irradiation with a photomask to confine the porous polymer monolith to the desired regions of the channels. The monolithic stationary phase was homogenous and well bonded to the channel surfaces, which had been functionalized by graft polymerization. The ESI potential was applied within the channel via a carbon ink line. The performance of this microfluidic device was demonstrated by analysis of a tryptic digest of bovine serum albumin on an ion trap MS instrument.     

Capillary electrochromatography in anion-exchange and normal-phase mode using monolithic stationary phases

Hydrophilic macroporous weak and strong anion-exchange stationary phases have been prepared in a monolithic format within untreated fused-silica capillaries by the simple thermally or UV-initiated polymerization of 2-dimethylaminoethyl methacrylate, 2-hydroxyethyl methacrylate and ethylene dimethacrylate in the presence of a binary porogenic mixture of dodecanol and cyclohexanol. The tertiary amino functionalities were then alkylated in situ to afford strong anion-exchangers. These new monolithic stationary phases with optimized porous properties were used for the CEC separation of various organic anions. Thus, a mixture of 2-substituted propionic acid drugs (profens) was separated in 13 min and high column efficiencies of up to 231,000 plates/m were achieved. The separation of substituted benzoic acids indicates that the selectivity results primarily from the anion-exchange interactions, while electrophoretic migration contributes only slightly. In addition, these hydrophilic anion-exchangers are also able to separate weakly acidic, neutral and basic compounds such as phenols, xanthines and aromatic amines in normal-phase electrochromatographic mode.