Capillary electrochromatography with zwitterionic stationary phase on the lysine-bonded poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolithic capillary column

A polymer-based neutral monolithic capillary column was prepared by radical polymerization of glycidyl methacrylate and ethylene dimethacrylate in a 100 mum id fused-silica capillary, and the prepared monolithic column was subsequently modified based on a ring opening reaction of epoxide groups with 1 M lysine in solution (pH 8.0) at 75 degrees C for 10 h to produce a lysine chemically bonded stationary phases in capillary column. The ring opening reaction conditions were optimized so that the column could generate substantial EOF. Due to the zwitterionic functional groups of the lysine covalently bonded on the polymer monolithic rod, the prepared column can generate cathodic and anodic EOF by varying the pH values of running buffer during CEC separation. EOF reached the maximum of -2.0 x 10(-8) m2v(-1)s(-1) and 2.6 x 10(-8) m2v(-1)s(-1) with pH of the running buffer of 2.25 and 10, respectively. As a consequence, neutral compounds, ionic solutes such as phenols, aromatic acids, anilines, and basic pharmaceuticals were all successfully separated on the column by CEC. Hydrophobic interaction is responsible for separation of neutral analytes. In addition, the electrostatic and hydrophobic interaction and the electrophoretic migration play a significant role in separation of the ionic or ionizable analytes.     

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.     

Performance of R-N(R')-R' functionalised poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) monolithic sorbent for plasmid DNA adsorption

High-throughput plasmid DNA (pDNA) manufacture is obstructed predominantly by the performance of conventional stationary phases. For this reason, the search for new materials for fast chromatographic separation of pDNA is ongoing. A poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) (GMA-EGDMA) monolithic material was synthesised via a thermal-free radical reaction, functionalised with different amino groups from urea, 2-chloro-N,N-diethylethylamine hydrochloride (DEAE-Cl) and ammonia in order to investigate their plasmid adsorption capacities. Physical characterisation of the monolithic polymer showed a macroporous polymer having a unimodal pore size distribution pivoted at 600 nm. Chromatographic characterisation of the functionalised polymers using pUC19 plasmid isolated from E. coli DH5alpha-pUC19 showed a maximum plasmid adsorption capacity of 18.73 mg pDNA/mL with a dissociation constant (KD) of 0.11 mg/mL for GMA-EGDMA/DEAE-Cl polymer. Studies on ligand leaching and degradation demonstrated the stability of GMA-EGDMA/DEAE-Cl after the functionalised polymers were contacted with 1.0 M NaOH, which is a model reagent for most 'cleaning in place' (CIP) systems. However, it is the economic advantage of an adsorbent material that makes it so attractive for commercial purification purposes. Economic evaluation of the performance of the functionalised polymers on the grounds of polymer cost (PC)/mg pDNA retained endorsed the suitability of GMA-EGDMA/DEAE-Cl polymer.     

Preparation and evaluation of hydroxylated poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolithic capillary for in-tube solid-phase microextraction coupled to high-performance liquid chromatography

A hydroxylated poly(glycidyl methacrylate-co-ethylene dimethacrylate) (GMA-co-EDMA) monolithic capillary was prepared and investigated for in-tube solid-phase microextraction (SPME). The polymer monolith was synthesized by in-situ polymerization of GMA and EDMA in the presence of dodecanol and toluene as the mixed porogenic solvents. After polymerization, glycidyl groups were hydrolyzed with sulfuric acid to produce diol groups at the surface of the porous monolith. To investigate the extraction mechanism, several groups of model analytes (including neutral, acidic and basic) were selected to perform extractions. The resulting monolith showed high extraction selectivity towards polar compounds, which resulted from the enhancement of dipole-dipole and hydrogen bonding interactions relative to hydrophobic interactions. The equilibrium extraction time profiles were also monitored for those model compounds to assess the extraction capacity of the monolithic capillary. Moreover, the hydroxylated poly(GMA-co-EDMA) monolithic capillary exhibited satisfactory reproducibility and stability. Finally, the in-tube SPME-HPLC method, based on the developed monolithic capillary as the extraction media, was successfully applied to the determination of five polar organic contaminants in lake water.     

Enantiopure poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate): a new material for supported catalytic asymmetric hydrogen transfer reduction

A novel copolymer containing chiral epoxy residues was prepared. Free radical initiated suspension copolymerization of (R)- or (S)-glycidyl methacrylate with ethylene glycol dimethacrylate afforded crosslinked copolymer 1 in high yield. Optically active polymers containing amino alcohol functionalities were then formed from 1 through epoxide ring opening with a number of achiral and homochiral amines. It was shown that ruthenium complexes based on these new polymeric amino alcohol ligands were effective catalysts for the asymmetric hydrogen transfer reduction of acetophenone.     

Extraction of clenbuterol from urine using hydroxylated poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith microextraction followed by high-performance liquid chromatography determination

A hydroxylated poly(glycidyl methacrylate-co-ethylene dimethacrylate) (GMA-co-EDMA) monolithic capillary was prepared for polymer monolith microextraction (PMME). Coupled to HPLC with UV detection, this extraction medium was successfully applied to establish a simple and fast method for the analysis of clenbuterol (CLB) in urine. To obtain optimum extraction performance, the effects of pH value and ionic strength of the sample matrix on extraction efficiency were investigated. The linearity of the method was evaluated over a concentration range of 10-2000 ng/mL and the correlation coefficient (R2 value) was 0.9985. The detection limit and quantification limit were 2.3 and 7.7 ng/mL, respectively. Good reproducibility of the method was obtained, yielding the intra- and interday RSDs less than 5.1 and 9.1%, respectively. Moreover, the hydroxylated poly(GMA-co-EDMA) monolithic capillary exhibited good preparation reproducibility and long-term extraction life. When applied to the determination of CLB in urine samples, an effective removal of interfering compounds was achieved and recoveries were in the range of 87.6-106%. The determination of CLB from one real sample including pretreatment, extraction, and analysis could be finished within 30 min.     

Preparation and characterization of polyethyleneimine modified ion-exchanger based on poly(methacrylate-co-ethylene dimethacrylate) monolith

A polyethyleneimine (PEI) modified ion-exchanger was prepared based on poly(methacrylate-co-ethylene dimethacrylate) monolith cast in 100 mm x 4.6 mm I.D. stainless steel tube with heptane as the porogenic solvent at 65 degrees C for 12 h. The pores larger than 500 nm presented 85% of total pore volume of PEI monolith and provided the better permeability for separation. Bovine serum albumin (BSA) binding capacity on the column was enhanced with increasing the molecular weight of PEI, indicated that the brush ligand emanated from the surface and captured more protein by multiple binding sites. Titration experiment as well as BSA retention versus the pH of mobile phase showed that the monolith exhibited weak ion-exchange property, and recovered BSA on the monolith reached 97% when NaCl content in mobile phase was higher than 0.5 M. Frontal analysis and gradient elution of BSA indicated that PEI monolith provided the rapid mass transfer in chromatographic procedure, which made the dynamic binding capacities as well as column efficiency keep as constants at high operating flow rate. Fast separation of three mode proteins mixture (lysozyme, hemoglobin and BSA) on the monolith was achieved within 3 min at velocity of 1445 cm/h. This demonstrated the potential of PEI monolith for the rapid analysis and separation of proteins.     

Stability and repeatability of capillary columns based on porous monoliths of poly(butyl methacrylate-co-ethylene dimethacrylate)

Monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) capillary columns have been prepared via either thermally or photochemically initiated polymerization of the corresponding monomers and the repeatability of their preparation has been explored. Three separate batches of 5 columns each were prepared using thermal and photochemical initiation for a total of 30 columns. All 30 capillary columns were tested in liquid chromatography-electrospray ionisation mass spectrometry mode for the separation of a model mixture of three proteins--ribonuclease A, cytochrome c and myoglobin. Excellent repeatability of retention times was observed for the proteins as evidenced by relative standard deviation (RSD) values of less than 1.5%. Somewhat broader variations with RSD values of up to 10% were observed for the pressure drop in the columns. The stability of retention times was also monitored using a single monolithic column and no significant shifts in either retention times or back pressure was observed in a series of almost 2200 consecutive protein separations.     

固载蛋白A交联聚甲基丙烯酸缩水甘油酯连续床亲合色谱柱的制备及性能考察

采用沉淀聚法“原位”（in-situ)聚合合成了交联聚甲基丙烯酸缩水甘油酯连续床色谱柱,对其进行化学改性后,分别得到含有11个碳原子间隔臂以及不含间隔臂键合了蛋白A的高效亲合色谱柱。考察了这两种色谱介质的性能,结果表明含有间隔臂的介质有一定的疏水性;对传统制备蛋白配基的亲合色谱介质的合成路线进行了改进;采用不含间隔臂的亲合柱测定了人血浆中人免疫球蛋白G（HIgG)的含量,所测得的定量标准曲线线性相关系数达到0．999;考察了流速对合成的连续床色谱柱柱压的影响,当流速高达9．0mL/min时,柱压也仅为6．5MPa;采用pH梯度对HIgG的不同亚基进行了分离。     

Effect of capillary cross-section geometry and size on the separation of proteins in gradient mode using monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) columns

Porous polymer monoliths have been prepared in capillaries with circular or square cross-sections and lateral dimensions of 50, 75, 100 μm as well as in a rectangular 38 μm × 95 μm capillary. These capillaries have been used to determine the effect of the size and shape of their cross-section on the porous and hydrodynamic properties of poly(butyl methacrylate-co-ethylene dimethacrylate) monoliths. The capillaries were studied by scanning electron microscopy and evaluated for their permeability to flow and their performance in the liquid chromatographic separation of a protein mixture comprising ribonuclease A, cytochrome c, myoglobin, and ovalbumin using a linear gradient of acetonitrile in the mobile phase. No differences resulting from channel geometry were found for the various capillary columns. These results demonstrate that standard capillaries with circular geometry are a good and affordable alternative conduit for modeling the processes carried out in microfluidic chips with a variety of geometries.     

Fabrication of a poly(styrene-octadecene-divinylbenzene) monolithic column and its comparison with a poly(styrene-divinylbenzene) monolithic column for the separation of proteins

In this paper, a poly(styrene-octadecene-divinylbenzene) (PS-OD-DVB) monolithic column was prepared in one step by introducing a C18 carbon chain as monomer. N,N-Dimethylformamide and decanol served as porogens to make a homogeneous polymerization mixture in a fused silica capillary (320 microm inner diameter). Its physical and chromatographic properties were compared with those of poly(styrene-divinylbenzene) (PS-DVB) monolithic column, which was also fabricated by in-situ polymerization in a fused silica capillary with the same inner diameter. Six standard proteins were used to evaluate the columns and their potential application for the separation of human hemoglobin was also discussed. It was shown that the PS-OD-DVB and PS-DVB monoliths appeared to have similar efficiency for rapid separation of six proteins within 3.5 min. The PS-OD-DVB monolith was found to have higher loading capacity and higher resolution for the separation of alpha and beta chains of hemoglobin because of the introduction of C18 carbon chains, and shows great potential for the separation of bio-macromolecules.     

正交试验法优化制备用于小分子物质分离的聚(甲基丙烯酸缩水甘油酯-二乙烯基苯)型整体柱

以甲基丙烯酸缩水甘油酯(GMA)为单体,二乙烯基苯(DVB)为交联剂,以环己醇和正十二醇为致孔剂,过氧化苯甲酰(BPO)为引发剂,直接以50 mm×4.6 mm色谱柱为模具,通过原位聚合制备聚(GMA-DVB)型整体柱。以GMA和DVB的体积比、环己醇和十二醇的体积比以及BPO占聚合物的质量分数为三因素,以分离苯和乙苯等小分子物质时的半峰宽分离度(R1/2)为考察指标,进行三因素三水平的正交试验,通过测定整体柱的比表面积、孔径和孔容分布对其进行表征。结果表明,制备整体柱的最优配方为V(GMA): V(DVB): V(环己醇): V(正十二醇)=0.825:0.825:1.32:2.03, BPO的质量分数为0.7%。应用所制备的整体柱分离苯和乙苯等小分子物质,理论塔板数达到37000塔板/m, R1/2值达到7.14,完全达到基线分离,分离时间小于10 min。该方法制备整体柱的重复性好,柱效较高,基本满足商品化要求。     

Rapid reversed-phase separation of proteins and peptides using optimized 'moulded' monolithic poly(styrene-co-divinylbenzene) columns

Monolithic macroporous poly(styrene-co-divinylbenzene) stationary phases have been prepared by free radical polymerization within the confines of 4.6-mm I.D. chromatographic columns. The optimized porous properties allow the mobile phase to flow through these columns at flow-rates of up to 10 ml/min. As opposed to the simultaneously tested columns packed with either silica or synthetic polymer beads, the monoliths exhibit only modest back pressure. The monolithic columns were able to separate mixtures of peptides and proteins in a very short time. Under the optimized conditions, the separation of five proteins can be easily achieved in less than 20 s.     

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.     

Synthesis of macroporous poly(glycidyl methacrylate) microspheres by surfactant reverse micelles swelling method

Macroporous poly(glycidyl methacrylate-ethylene dimethacrylate) [P(GMA-EDMA)] particles with pore size around 140-200 nm and poly(glycidyl methacrylate-divinylbenzene) [P(GMA-DVB)] particles with pore size of 450 nm were prepared by the surfactant reverse micelles swelling method. This method was similar with the conventional suspension polymerization, and the difference was that higher concentration of surfactant was added in the oil phase. When the oil phase containing surfactant was dispersed in aqueous phase, the surfactant reverse micelles in the oil droplets absorbed water from continuous phase. After polymerization, the large pores were formed by the absorbed water. The effects of the amount and type of surfactants, the cooperation of surfactant and diluents, and the crosslinking agent on the morphology of microspheres were investigated. This study provided a new and simple method to prepare microspheres with the pores of several hundred nanometers, which overcame the disadvantages found in the conventional preparation methods of macroporous microspheres.     

Effects of ethylene glycol dimethacrylate as cross-linker in ionic liquid gel polymer electrolyte based on poly(glycidyl methacrylate)

In this study, modified poly(glycidyl methacrylate)-based films for gel electrolyte were prepared by an in situ UV photopolymerization technique. The effects of adding ethylene glycol dimethacrylate (EGDMA) to the polymer host were studied through X-ray diffraction analysis and differential scanning calorimetry. The results from Fourier transform-infrared spectra indicate complete polymerization among the monomers. The addition of EGDMA to the formulation of gel polymer electrolyte increased the loading of 1-butyl-3-methylimidazolium bis(fluoromethylsulfonyl)imide up to 200 wt.% with the highest value of 8.2 × 10^?4 S cm^?1. All the gel polymer electrolyte membranes obeyed the Arrhenius law.     

Chromatographic evaluation of some stationary phases based on poly(methyloctylsiloxane) immobilized onto silica

Several stationary phases were prepared by thermal immobilization of poly(methyloctylsiloxane) onto a silica surface using different amounts of poly(methyloctylsiloaxane), and different times and temperatures of immobilization to provide different carbon contents for the phases. The chromatographic properties were evaluated using the Tanaka test. Comparison of the results obtained with literature data using hierarchical cluster analysis showed dissimilarity with most of the commercial phases. Some basic pharmaceuticals, including six benzodiazepines were separated on one of the better PMOS phases.     

Novel monolithic poly(p-methylstyrene-co-bis(p-vinylbenzyl)dimethylsilane) capillary columns for biopolymer separation

Hydrophobic organo-silane based monolithic capillary columns were prepared by thermally initiated free radical polymerisation within the confines of 200 microm i.d. fused silica capillaries. A novel crosslinker, namely bis(p-vinylbenzyl)dimethylsilane (BVBDMS), was copolymerised with p-methylstyrene (MS) in the presence of 2-propanol and toluene, using alpha,alpha'-azoisobutyronitrile (AIBN) as initiator. Monolithic capillary columns, differing in the total monomer, microporogen content and microporogen nature were fabricated and the chromatographic efficiency of each monolith, regarding the separation of proteins, peptides and oligonucleotides, was evaluated and compared. Changes in monolith morphology were monitored by scanning electron microscopy (SEM). Porosity and specific surface areas of the supports were studied by means of mercury intrusion porosimetry and BET measurements, respectively. Pressure drop vs. flow rate measurements proved the prepared poly(p-methylstyrene-co-bis(p-vinylbenzyl)dimethylsilane) (MS/BVBDMS) monoliths to be mechanically stable and swelling propensity (SP) factors of 0.78-1.10 indicate high crosslinking homogeneity.     

Development of erbium phosphate doped poly(glycidyl methacrylate/ethylene dimethacrylate) spin columns for selective enrichment of phosphopeptides

In this study, a novel method for the highly selective enrichment of phosphopeptides using erbium phosphate doped poly(glycidyl methacrylate/ethylene dimethacrylate) spin columns is presented. Erbium phosphate was synthesized by precipitation from boiling phosphoric acid and incubated overnight in erbium chloride solutions. The resulting powder was embedded in a monolithic poly(glycidyl methacrylate/ethylene dimethacrylate) polymer. The monolith was synthesized in a spin column by radical polymerization. Erbium phosphate demonstrated a high affinity and selectivity for phosphopeptides due to the strong interaction of trivalent erbium ions with the phosphate groups of phosphopeptides. The high selectivity and performance of the designed spin columns were demonstrated by successfully enriching phosphopeptides from tryptically digested protein mixtures containing the model phosphoproteins α‐ and β‐casein, bovine milk, and human saliva. By the implementation of several washing steps, unspecific components were removed and the enriched phosphopeptides were effectively eluted from the spin columns under alkaline conditions. The selective performance of the presented method was further demonstrated by the enrichment of two synthetic phosphopeptides, which were spiked in tryptically digested and dephosphorylated HeLa cell lysates at low ratios. Finally, the presented approach was compared to conventional phosphopeptide enrichment by titanium oxide and revealed higher recoveries for the erbium phosphate doped monoliths.     

Ensuring repeatability and robustness of poly(glycidyl methacrylate‐co‐ethylene dimethacrylate) HPLC monolithic columns of 3 mm id through covalent bonding to the column wall

Two different methods to reinforce the poly(glycidyl methacrylate‐co‐ethylene dimethacrylate) HPLC monolithic columns of 3 mm id in a glass column reservoir were studied: composite columns with polymeric particles in the monolith and surface treatment of the reservoir wall. Of the two methods used to counter the mechanical instability and formation of flow channels (composite columns and column wall surface treatment), we demonstrated that proper column wall surface treatment was sufficient to solve both problems. Our study also indicated that no surface treatment is efficient, and of the methods studied silanization in acidified ethanol solution and constant renewal of the reaction mixture (dynamic mode) proved to be the most effective. As a result of this study, we have been able to prepare repeatable and durable methacrylate HPLC columns with good efficiencies.