Preparation of polymeric monoliths by copolymerization of acrylate monomers with amine functionalities for anion-exchange capillary liquid chromatography of proteins

Two novel polymeric monoliths for anion-exchange capillary liquid chromatography of proteins were prepared in a single step by a simple photoinitiated copolymerization of 2-(diethylamino)ethyl methacrylate and polyethylene glycol diacrylate (PEGDA), or copolymerization of 2-(acryloyloxy)ethyl trimethylammonium chloride and PEGDA, in the presence of selected porogens. The resulting monoliths contained functionalities of diethylaminoethyl (DEAE) as a weak anion-exchanger and quaternary amine as a strong anion-exchanger, respectively. An alternative weak anion-exchange monolith with DEAE functionalities was also synthesized by chemical modification after photoinitiated copolymerization of glycidyl methacrylate (GMA) and PEGDA. Important physical and chromatographic properties of the synthesized monoliths were characterized. The dynamic binding capacities of the three monoliths (24 mg/mL, 56 mg/mL and 32 mg/mL of column volume, respectively) were comparable or superior to values that have been reported for various other monoliths. Chromatographic performance was also similar to that provided by a modified poly(GMA-ethylene glycol dimethacrylate) monolith. Separation of standard proteins was achieved under gradient elution conditions using these monolithic columns. Peak capacities of 34, 58 and 36 proteins were obtained with analysis times of 20–30 min. This work represents a successful attempt to prepare functionalized monoliths via direct copolymerization of monomers with desired functionalities. Compared to earlier publications, additional surface modifications were avoided and the PEGDA crosslinker helped to improve the biocompatibility of the monolithic backbone.     

Effect of sequence structure on wetting behaviors of fluorinated methacrylate polymers based on perfluorohexylethyl methacrylate and stearyl acrylate

Due to the non-crystalline properties of short chain perfluoroalkyl groups,using short chain perfluoroalkyl to stabilize low surface free energy polymers has been a challenging task.In this study,we prepare a series of random copolymers poly(perfluorohexylethyl methacrylate)-co-poly(stearyl acrylate) (P13FMA-co-PSA) and block copolymers poly(perfluorohexylethyl methacrylate)-b-poly(stearyl acrylate) (P13FMA-b-PSA),and systematically investigate the effects of the sequence structure and the content of 13FMA of the fluorinated copolymers on surface free energy and surface reorganization.Static/dynamic contact angle goniometry and water/oil repellency analyses demonstrate that the random polymer P13FMA-co-PSA could not achieve low surface free energy and low surface reorganization at the same time.In contrast,for the block copolymer P13FMA-b-PSA,both low surface free energy and low surface reorganization are acquired simultaneously.The results of X-ray photoelectron spectroscopy (XPS),dynamic contact angle goniometry and differential scanning calorimetry (DSC) reveal the above-mentioned properties.The consecutive 13FMA segments improve the surface fluorine density,while the consecutive SA chains enhance the crystallinity of the SA segments,and further hinder the surface reorganization of the perfluoroalkyl groups.Therefore,P13FMA-b-PSA exhibits a higher utilization efficiency of fluorine atoms and a better structural stability than P13FMA-co-PSA.     

Fast separation of low molecular weight analytes on structurally optimized polymeric capillary monoliths

The optimization of recently introduced tetrakis(4-vinylbenzyl)silane (TVBS)-derived capillary monoliths to suit the fast separation of small molecules was accomplished by simultaneous changes in both the stoichiometry of the polymerization mixture and in the polymerization temperature. Four types of monoliths were prepared by varying the polymerization temperature within 60 and 65 °C as well as by changing the monomer content between 15.0 and 20.0 wt.%. Changes in the porosity and morphology of the capillary columns were studied applying inverse size exclusion chromatography (ISEC), nitrogen sorption (BET), and scanning electron microscopy (SEM). All monoliths were used for the reversed-phase separation of various sets of low molecular weight analytes such as alkyl benzenes, aryl amines, carboxylic acids, and a mixture of phenols and carbonyl compounds. Comparable efficiency and performance towards the different sets of analytes were observed. With optimized monolith structures, the fast separation of six-alkyl benzenes was accomplished within less than 2 min. This was made possible by a high linear solvent flow (10 mm/s) at low column pressure drops (<15 MPa).     

Monodisperse porous polymer particles with polyionic ligands for ion exchange separation of proteins

A new “grafting to” strategy was proposed for the preparation of polymer based ion exchange supports carrying polymeric ligands in the form of weak or strong ion exchangers. Monodisperse porous poly(glycidyl methacrylate-co-ethylene dimethacrylate), poly(GMA-co-EDM) particles 5.9 μm in size were synthesized by “modified seeded polymerization”. Poly(2,3-dihydroxypropyl methacrylate-co-ethylene dimethacrylate), poly(DHPM-co-EDM) particles were then obtained by the acidic hydrolysis of poly(GMA-co-EDM) particles. The hydroxyl functionalized beads were treated with 3-(trimethoxysilyl)propyl methacrylate to have covalently linked methacrylate groups on the particle surface. The selected monomers carrying weak or strong ionizable groups (2-acrylamido-2-methyl-1-propane sulfonic acid, AMPS; 2-dimethylaminoethylmethacrylate, DMAEM and N-[3-(dimethylamino)propyl] methacrylamide, DMAPM) were subsequently grafted onto the particles via immobilized methacrylate groups. The final polymer based materials with polyionic ligands were tried as chromatographic packing in the separation of proteins by ion exchange chromatography. The proteins were successfully separated both in the anion and cation exchange mode with higher column yields with respect to the previously proposed materials. The plate heights obtained for poly(AMPS) and poly(DMAEM) grafted poly(DHPM-co-EDM) particles by using proteins as the analytes were 80 and 200 μm, respectively. Additionally, the plate height exhibited no significant increase with the increasing linear flow rate in the range of 1–20 cm/min. The most important property of the proposed strategy is to be applicable for the synthesis of any type of ion exchanger both in the strong and weak form.     

Inertness and stability of a maltose-silica for the separation of proteins by HPLC

Summary A stationary phase consisting of maltose on a silica support, designed for the separation of proteins, is described. When maltose is coupled to aminopropylsilica an effective shielding of the silica surface can be obtained. A size exclusion experiment is used to investigate pore structure and the behaviour of proteins. The stability of the surface layer is studied, as influenced by silica pretreatments and cross-linking reactions. Inertness is studied via the denaturation of trypsin in columns of different length at a number of temperatures. An example of a derivatization of the maltose layer is given which demonstrates its use as an interactive separation medium.     

Preparation and characterization of methacrylate-based semi-micro monoliths for high-throughput bioanalysis

Hexyl methacrylate (HMA)-based monolithic semi-micro columns were prepared by in situ polymerization within the confines of 1.02-mm-i.d. silicosteel tubing for reversed-phase and/or precipitation–redissolution liquid chromatography. Practically useful monolithic columns with adequate separation efficiency, high permeability, and good mechanical strength were successfully obtained using a polymerization mixture comprising 24% hexyl methacrylate (HMA), 6% ethylene dimethacrylate (EDMA), 44.5% 1-propanol, and 25.5% 1,4-butanediol. The column performance was evaluated through the separations of a series of alkylbenzenes. At a normal flow rate of 50 μL min⁻¹, the produced HMA-based monolithic columns typically exhibited 3,000 theoretical plates for a 20-cm-long column, and the pressure drop was generally less than 1 MPa per 20 cm. The monolithic columns were resistant to at least 15 MPa, and could be properly operated at 15–20 times higher flow rate than normal, reducing the separation time to 1/15–1/20. The HMA-based monolithic columns were applied to rapid and efficient separations of proteins such as ribonuclease A, cytochrome c, transferrin, and ovalbumin in the precipitation–redissolution mode. Using a CH₃CN gradient elution at a flow rate of 1,000 μL min⁻¹, four proteins were baseline separated within 20 s.     

Synthesis and self-assembly of comb-like amphiphilic copolymer poly(maleic anhydride-<Emphasis Type="Italic">alt</Emphasis>-stearyl methacrylate)-<Emphasis Type="Italic">b</Emphasis>-poly(stearyl methacrylate)

Comb-like amphiphilic block copolymers of maleic anhydride (MA) and stearyl methacrylate (SMA) were prepared through the reversible-addition-fragmentation-transfer polymerization. The resultant copolymers were characterized by gel permeation chromatograph and ¹H NMR. The aggregation behaviors of P(MA-alt-SMA)-b-PSMA were investigated in tetrahydrofuran/water. It is of great interest that the aggregates with different morphologies and dimensions could be obtained by adjusting the polymer concentration, water content, and pH. The dimension and structure of these aggregates were investigated by transmission electron microscopy and dynamic light scattering. The effect of the copolymer–solvent interaction on these aggregations was discussed.     

Robust isocratic liquid chromatographic separation of functional poly(methyl methacrylate)

The separation of telechelic poly(methyl methacrylate) (PMMA) prepolymers based on the number of end-groups under critical liquid chromatography (LC) conditions has been studied using a bare-silica column, which can interact with polar functional groups. The critical solvent compositions for non-functional, mono-functional and bi-functional PMMAs were determined in normal-phase LC using mixtures of acetonitrile and dichloromethane (DCM) of varying composition as the mobile phase. The telechelic prepolymers were successfully separated according to hydroxyl (OH) functionality (with zero, one, or two OH groups, respectively) under the critical conditions, in which fast (5 min), base-line separations were obtained independent of molecular weight. Changing the column temperature, flow rate, and mobile-phase composition within a certain range did not affect the functionality separation. Therefore this isocratic LC separation method is quite robust. Evaporative light-scattering detector (ELSD) calibration curves were used for the quantitative analysis of functional PMMA prepolymers.     

Non-particulate (continuous bed or monolithic) restricted-access reversed-phase media for sample clean-up and separation by capillary-format liquid chromatography

Restricted-access reversed-phase non-particulate (continuous bed or monolithic) stationary phases of different hydrophobicity synthesized in 100 m i.d. fused silica capillaries have been evaluated. A specific property of restricted-access media (RAM) is that they interact with small analytes and exclude big molecules, e.g. proteins, from access to the active sites and adsorption on the surface. This dual property facilitates direct injection of biological fluids for drug or drug-metabolite analysis. Different RAM and RAM-precursor capillary columns were tested to assess the influence of chromatographic bed morphology on loadability. Inverse size-exclusion chromatography was used for investigation of pore structural properties of the capillary-format continuous beds. The data obtained were used to discuss the mechanism of separation of the biological samples using capillary columns and to propose a model for the topochemical architecture of the RAM investigated. Different morphology of the non-particulate reversed-phase precursors resulted in two types of RAM material shielded with hydrophilic polymer, classified as homogeneous or heterogeneous topochemistry stationary phases. Capillary columns were applied for chromatography of biological fluids. High resolution was obtained, without the need for column switching, when capillary columns operated in gradient conditions. Extensive evaluation of the chromatographic properties (hydrophobicity, efficiency, separation impedance, and loadability) of the non-particulate reversed-phase materials was performed before and after shielding with hydrophilic polymer to generate restricted-access properties. Minor changes of hydrophobicity, efficiency, or separation impedance were observed after the shielding.     

Restricted-access media development for direct analysis of drugs in biofluids using capillary liquid chromatography

In analytical sciences the design of novel materials and stationary phases for the sample preparation and separation of analytes from biological fluids is needed. In this work we present different strategies for modification of stationary phases to produce tailored solutions for the analytical problem. In this context a novel shielded polymeric reversed-phase monolithic material was prepared in the presence of different numbers of reactive groups and concentrations of the coating polymer. Chromatographic experiments were performed using benzoic acid propyl ester in order to characterize the hydrophobicity and efficiency of the different restricted-access continuous beds prepared. Inverse size-exclusion chromatography was used for investigation of the pore structure properties of the beds. Capillary columns were applied for nanochromatography of biological fluids containing a mixture of nitrazepamum and medazepamum. Presented at the 11th International Conference on Chemistry and the Environment, 9–12 September 2007, Torun, Poland     

Application of monoliths for plasmid DNA purification development and transfer to production

The demand of high-purity plasmid DNA (pDNA) for gene-therapy and genetic vaccination is still increasing. For the large scale production of pharmaceutical grade plasmids generic and economic purification processes are needed. Most of the current processes for pDNA production use at least one chromatography step, which always constitutes as the key-step in the purification sequence. Monolithic chromatographic supports are an alternative to conventional supports due to their excellent mass transfer properties and their high binding capacity for pDNA. Anion-exchange chromatography is the most popular chromatography method for plasmid separation, since polynucleotides are negatively charged independent of the buffer conditions. For the implementation of a monolith-based anion exchange step into a pDNA purification process detailed screening experiments were performed. These studies included supports, ligand-types and ligand-densities and optimization of resolution and productivity. For this purpose model plasmids with a size of 4.3 and 6.9 kilo base pairs (kbp) were used. It could be shown, that up-scaling to the production scale using 800 ml CIM Convective Interaction Media radial flow monoliths is possible under low pressure conditions. CIM DEAE was successfully implemented as intermediate step of the cGMP pDNA manufacturing process. Starting from 2001 fermentation aliquots pilot scale purification runs were performed in order to prove scale-up and to predict further up-scaling to 8 1 tube monolithic columns. The analytical results obtained from these runs confirmed suitability for pharmaceutical applications.     

Chromatographic selectivity of poly(alkyl methacrylate-co-divinylbenzene) monolithic columns for polar aromatic compounds by pressure-driven capillary liquid chromatography

In this study, divinylbenzene (DVB) was used as the cross-linker to prepare alkyl methacrylate (AlMA) monoliths for incorporating π-π interactions between the aromatic analytes and AlMA-DVB monolithic stationary phases in capillary LC analysis. Various AlMA/DVB ratios were investigated to prepare a series of 30% AlMA-DVB monolithic stationary phases in fused-silica capillaries (250-μm i.d.). The physical properties (such as porosity, permeability, and column efficiency) of the synthesized AlMA-DVB monolithic columns were investigated for characterization. Isocratic elution of phenol derivatives was first employed to evaluate the suitability of the prepared AlMA-DVB columns for small molecule separation. The run-to-run (0.16–1.20%, RSD; n = 3) and column-to-column (0.26–2.95%, RSD; n = 3) repeatabilities on retention times were also examined using the selected AlMA-DVB monolithic columns. The π-π interactions between the aromatic ring and the DVB-based stationary phase offered better recognition on polar analytes with aromatic moieties, which resulted in better separation resolution of aromatic analytes on the AlMA-DVB monolithic columns. In order to demonstrate the capability of potential environmental and/or food safety applications, eight phenylurea herbicides with single benzene ring and seven sulfonamide antibiotics with polyaromatic moieties were analyzed using the selected AlMA-DVB monolithic columns.     

Properties and performance of novel high-resolution/high-permeability ion-exchange media for protein chromatography

There is continued interest in the development of stationary phases for protein chromatography that can provide high resolution at elevated flow rates of the mobile phase. When using porous particles, resolution and dynamic binding capacity decline rapidly as the flow rate is increased. Monolithic columns have been developed to overcome these limitations. However, there are difficulties in manufacturing homogeneous larger scale monoliths. In this paper we investigate the morphology and performance characteristics of columns based on new ion exchangers obtained by mechanically disrupting continuous beds of acrylamido-based polymeric media. Near colloidal suspensions of loose particles obtained with this procedure can be flow-packed in ordinary chromatography columns resulting in beds of unexpectedly high hydraulic permeability. Columns up to 2.2 cm in diameter were studied with both Q and S functionalized media. The hydraulic permeability and interparticle porosity of these columns were rather high. The permeabilities of the S and Q media were 1.5 x 10(-13) and 2.4 x 10(-13) m2, respectively, while the corresponding porosities were 60 and 70%. These porosity values are similar to those of monoliths, suggesting that these particles assemble under flow to give high-porosity bridged structures. The structure of these packed beds was further characterized by embedding small packed columns in resins and obtaining sections for microscopic observation. The sections reveal the presence of small aggregates of non-porous 1-3 microm particles, surrounded by flow channels several micrometers in size. The height equivalent to a theoretical plate under isocratic and gradient elution conditions and the dynamic binding capacity were determined for several proteins and were found to be virtually independent of flow.     

Ionic liquid-modified materials for solid-phase extraction and separation: A review

In recent years, materials science has propelled to the research forefront. Ionic liquids with unique and fascinating properties have also left their footprints to the developments of materials science during the last years. In this review we highlight some of their recent advances and provide an overview at the current status of ionic liquid-modified materials applied in solid-phase extraction, liquid and gas chromatography and capillary electrochromatography with reference to recent applications. In addition, the potential of ionic liquids in the modification of capillary inner wall in capillary electrophoresis is demonstrated. The main target material modified with ionic liquids is silica, but polymers and monoliths have recently joined the studies. Although imidazolium is still clearly the most commonly used ionic liquid for the covalently modification of materials, the exploitation of pyridinium and phosphonium will most probably increase in the future.     

Incorporation of metal‐organic framework amino‐modified MIL‐101 into glycidyl methacrylate monoliths for nano LC separation

Metal‐organic frameworks consisting of amino‐modified MIL‐101(M: Cr, Al, and Fe) crystals have been synthesized and subsequently incorporated to glycidyl methacrylate monoliths to develop novel stationary phases for nano‐liquid chromatography. Two incorporation approaches of these materials in monoliths were explored. The metal‐organic framework materials were firstly attached to the pore surface through reaction of epoxy groups present in the parent glycidyl methacrylate‐based monolith. Alternatively, NH₂‐MIL‐101(M) were admixed in the polymerization mixture. Using short time UV‐initiated polymerization, monolithic beds with homogenously dispersed metal‐organic frameworks were obtained. The chromatographic performance of embedded UV‐initiated composites was demonstrated with separations of polycyclic aromatic hydrocarbons and non‐steroidal anti‐inflammatory drugs as test solutes. In particular, the incorporation of the NH₂‐MIL‐101(Al) into the organic polymer monoliths led to an increase in the retention of all the analytes compared to the parent monolith. The hybrid monolithic columns also exhibited satisfactory run‐to‐run and column‐to‐column reproducibility.     

Improvement of the liquid-chromatographic analysis of protein tryptic digests by the use of long-capillary monolithic columns with UV and MS detection

Optimisation of peak capacity is an important strategy in gradient liquid chromatography (LC). This can be achieved by using either long columns or columns packed with small particles. Monolithic columns allow the use of long columns at relatively low back-pressure. The gain in peak capacity using long columns was evaluated by the separation of a tryptic bovine serum albumin digest with an LC–UV–mass spectrometry (MS) system and monolithic columns of different length (150 and 750 mm). Peak capacities were determined from UV chromatograms and MS/MS data were used for Mascot database searching. Analyses with a similar gradient slope for the two columns produced ratios of the peak capacities that were close to the expected value of the square root of the column length ratio. Peak capacities of the short column were 12.6 and 25.0 with 3 and 15 min gradients, respectively, and 29.7 and 41.0 for the long column with 15 and 75 min gradients, respectively. Protein identification scores were also higher for the long column, 641 and 750 for the 3- and 15-min gradients with the short column and 1,376 and 993 for the 15- and 75-min gradients with the long column. Thus, the use of long monolithic columns provides improved peptide separation and increased reliability of protein identification.     

Selectivity of monolithic supports under overloading conditions and their use for separation of human plasma and isolation of low abundance proteins

Human serum albumin (HSA) and immunoglobulin G (IgG) represent over 75% of all proteins present in human plasma. These two proteins frequently interfere with detection, determination and purification of low abundance proteins that can be potential biomarkers and biomarker candidates for various diseases. Some low abundance plasma proteins such as clotting factors and inhibitors are also important therapeutic agents. In this paper, the characterization of ion-exchange monolithic supports under overloading conditions was performed by use of sample displacement chromatography (SDC). If these supports were used for separation of human plasma, the composition of bound and eluted proteins in both anion- and cation-exchange mode is dependent on column loading. Under overloading conditions, the weakly bound proteins such as HSA in anion-exchange and IgG in cation-exchange mode are displaced by stronger binding proteins, and this phenomenon was not dependent on column size. Consequently, small monolithic columns with a column volume of 100 and 200 μL are ideal supports for high-throughput screening in order to develop new methods for separation of complex mixtures, and for sample preparation in proteomic technology.     

High temperature liquid chromatography of intact proteins using organic polymer monoliths and alternative solvent systems

Alternative approaches to conventional acetonitrile gradient methods for reversed-phase liquid chromatographic analysis of intact proteins have been investigated using commercial poly(styrene-co-divinylbenzene) monolithic columns (Dionex ProSwift™ RP-2H and RP-4H). Alternative solvents to acetonitrile (2-propanol and methanol) coupled with elevated temperatures demonstrated complementary approaches to adjusting separation selectivity and reducing organic solvent consumption. Measurements of peak area at increasing isothermal temperature intervals indicated that only minor (<5%) decreases in detectable protein recovery occurred between 40 and 100 °C on the timescale of separation (2–5 min). The reduced viscosity of a 2-propanol/water eluent at elevated temperatures permitted coupling of three columns to increase peak production (peaks/min) by 16.5%. Finally, narrow-bore (1 mm i.d.) columns were found to provide a more suitable avenue to fast, high temperature (up to 140 °C) separations.     

Photopolymerized sol–gel monoliths for separations of glycosylated proteins and peptides in microfluidic chips

Photopolymerized silica sol–gel monoliths, functionalized with boronic acid ligands, have been developed for protein and peptide separations in polydimethylsiloxane microfluidic devices. Pore size characterization of the monoliths was carried out with SEM, image analysis, and differential scanning calorimetry to evaluate both the micron‐sized macropores and the nanometer‐sized mesopores. Monoliths were functionalized with boronic acid using three different immobilization techniques. Batch experiments were conducted to determine the capacity of the monoliths and selectivity toward cis‐diol‐containing compounds. Conalbumin was used as a model glycoprotein, and a tryptic digest of the glycoprotein horseradish peroxidase was used as a peptide mixture to demonstrate proof‐of‐concept extraction of glycoproteins and glycopeptides by the monoliths formulated in polydimethylsiloxane microfluidic chips. For proteins, fluorescence detection was used, whereas the peptide separations employed off‐line analysis using MALDI‐MS.