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.     

阴离子交换整体柱对蛋白质的分离与纯化

分别用乙二胺、二乙胺、三乙胺将自制的以甲基丙烯酸缩水甘油酯(GMA)为单体、乙二醇二甲基丙烯酸酯(EDMA)为交联剂的整体柱修饰为弱、强阴离子交换整体柱。考察了该整体柱的性能,选择出分离蛋白质(牛血清白蛋白、溶菌酶和谷胱甘肽)的最佳实验条件,并在最佳分离条件下考察了这些蛋白质在整体柱上的色谱行为和该整体柱对纤维素降解酶的分离纯化情况。实验结果表明,该整体柱性能良好,可以实现对纤维素降解酶的快速分离与纯化。同时,实验也证明采用梯度洗脱可以实现对某些蛋白质的分离纯化。     

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.     

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.     

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.     

Amino-Functionalized Monolithic Poly(glycidyl methacrylate-<Emphasis Type="Italic">co</Emphasis>-divinylbenzene) Ion-Exchange Stationary Phases for the Separation of Oligonucleotides

Monolithic capillary columns were prepared by thermal initiated copolymerization of glycidyl methacrylate (GMA) and divinylbenzene (DVB) inside silanized 200 µm i.d. fused silica capillaries. Polymerization mixtures containing different amounts of porogen (1-decanol and tetrahydrofuran (THF)) and different ratios of monomer and crosslinker were used for synthesis. For characterization the pore size distribution profiles of the resulting monoliths were determined by mercury intrusion porosimetry. The morphology of the copolymer was investigated by scanning electron micrographs (SEM). A high linear dependence between flow rate and pressure drop was achieved which indicates that the polymer is pressure-stable even at high flow rates. After characterization the produced GMA-DVB monoliths, which contain reactive epoxide groups, were modified by reaction with diethylamine to obtain a poly(3-diethylamino-2-hydroxypropyl methacrylate-co-divinylbenzene) ion-exchange monolithic stationary phase. The synthesized monoliths contain ionizable amino groups that are useful for anion-exchange chromatography (AEC). Poly(3-diethylamino-2-hydroxypropyl methacrylate-co-divinylbenzene) monolithic columns allowed a fast and highly efficient separation of a homologous series of phosphorylated oligothymidylic acids [d(pT)_12-18]. Since durability is an important parameter of chromatographic column characterization, the separation performance for d(pT)_12-18 in a freshly produced capillary column and on the same column after 100 chromatographic runs was compared.     

Alkylated poly(styrene-divinylbenzene) monolithic columns for mu-HPLC and CEC separation of phenolic acids

Macroporous poly(styrene-divinylbenzene) monolithic columns were prepared in fused silica capillaries of 100 microm id by in-situ copolymerization of styrene with divinylbenzene in the presence of propan-1-ol and formamide as the porogen system. The monoliths were subsequently alkylated with linear alkyl C-18 groups via Friedel-Crafts reaction to improve the retention and chromatographic resolution of strongly polar phenolic acids. A new thermally initiated grafting procedure was developed in order to shorten the time of the alkylation process. The grafting procedure was optimized with respect to the reaction temperature, time, the grafting reactant concentration, and the solvent used. The type of solvent and the grafting temperature are the most significant factors affecting the hydrodynamic properties, porosity, and efficiency of the columns. While the equivalent particle diameter of the grafted column increased, the capillary-like flow-through pore diameter decreased in comparison to non-alkylated monoliths. The hydrodynamic permeability of the monolith decreased, but the monolithic column still permitted fast micro-HPLC separations.     

Enantioseparation of 2-aryloxypropionic acids on chiral porous monolithic columns by capillary electrochromatography. Evaluation of column performance and enantioselectivity

The enantioseparation of 2-aryloxypropionic acids by capillary electrochromatography was tested on columns with a monolithic stationary phase prepared from silanized fused-silica capillaries (100 microm I.D.) by in situ copolymerization of glycidyl methacrylate, ethylene glycol dimethacrylate and methyl methacrylate in the presence of formamide and 1-propanol as the porogen solvents. The porous chiral monolithic stationary phases were prepared by reaction of the epoxy-groups at the surface of the monolith with (+)-1-(4-aminobutyl)-(5R,8S,10R)-terguride. To attain the minimum HETP values for the enantiodiscrimination of 2-phenoxypropionic acid, the influence of the composition of polymerization solution on column total porosity and efficiency was investigated. Optimum mobile phase conditions were found for all analytes tested using acetonitrile-methanol mixtures containing triethylamine and acetic acid as the buffer components. Furthermore, the chemical and mechanical stabilities of the columns were satisfactory, allowing hundreds of analyses.     

Reversed-phase and weak anion-exchange mixed-mode silica-based monolithic column for capillary electrochromatography

A silica-based CEC monolithic column with mixed modes of RP and weak anion-exchange (WAX) was successfully prepared by using the sol-gel technique at mild temperature. The synthesizing procedure was optimized by changing the ratios of tetraethoxysilane (TEOS), aminopropyltriethoxysilane (APTES), and octyltriethoxysilane (C(8)-TEOS) in the mixture. While serving as WAX group, the amino group dominated the charge on the surface of the capillary column and generated an EOF from cathode to anode at low pH. At pH above 7.5, a cathodic EOF was observed due to the full ionization of silanol group and the suppression in the ionization of amino group. The morphology of monolithic columns was examined by SEM, and the performance of column was evaluated in detail by separating different kinds of compounds. As expected, the monolithic column exhibited RP chromatographic behavior for neutral solutes. Fast and efficient separation of six aromatic acids was obtained using acidic mobile phase with column efficiency up to 160,000 plates/m. Symmetrical peaks can be obtained for aromatic amines because positively charged amino groups on the surface can effectively minimize the adsorption of positively charged analytes to the stationary phase.     

有机-无机杂化硅胶基质整体柱的制备及其电色谱性能

采用溶胶-凝胶法,以四乙氧基硅烷和苯基三乙氧基硅烷作为反应单体,通过酸碱两步催化在毛细管中进行原位缩聚反应,制备了新型有机-无机杂化硅胶基质毛细管整体柱,制备过程简单。整体柱基质中均匀分布的苯基基团可直接用于反相毛细管电色谱的分离,因而不需要对基质再进行衍生化。优化了整体柱的制备条件,采用扫描电镜和压汞法对整体柱的微观结构和孔径分布进行了表征。分别考察了溶胶-凝胶初始反应液中水的用量对柱床结构的影响和两种单体的配比对材料孔径分布的影响。研究了稠环芳烃类化合物在整体柱上的保留行为,用所制备的整体柱分离了7种苯酚类化合物,平均柱效达100000塔板/m。     

Comparison of monolithic and microparticulate columns for reversed-phase liquid chromatography of tryptic digests of industrial enzymes in cleaning products

Enzymes of several classes used in the formulations of cleaning products were characterized by trypsin digestion followed by HPLC with UV detection. A polymeric monolithic column (ProSwift) was used to optimize the separation of both the intact enzymes and their tryptic digests. This column was adequate for the quality control of raw industrial enzyme concentrates. Then, monolithic and microparticulate columns were compared for peptide analysis. Under optimized conditions, the analysis of tryptic digests of enzymes of different classes commonly used in the formulation of cleaning products was carried out. Number of peaks, peak capacity and global resolution were obtained in order to evaluate the chromatographic performance of each column. Particulate shell-core C18 columns (Kinetex, 2.6 μm) showed the best performance, followed by a silica monolithic column (Chromolith RP-18e) and the conventional C18 packings (Gemini, 5 μm or 3 μm). A polymeric monolithic column (ProSwift) gave the worst performances. The proposed method was satisfactorily applied to the characterization of the enzymes present in spiked detergent bases and commercial cleaners.     

Impact of pore structural parameters on column performance and resolution of reversed-phase monolithic silica columns for peptides and proteins

In this work, monolithic silica columns with the C4, C8, and C18 chemistry and having various macropore diameters and two different mesopore diameters are studied to access the differences in the column efficiency under isocratic elution conditions and the resolution of selected peptide pairs under reversed-phase gradient elution conditions for the separation of peptides and proteins. The columns with the pore structural characteristics that provided the most efficient separations are then employed to optimize the conditions of a gradient separation of a model mixture of peptides and proteins based on surface chemistry, gradient time, volumetric flow rate, and acetonitrile concentration. Both the mesopore and macropore diameters of the monolithic column are decisive for the column efficiency. As the diameter of the through-pores decreases, the column efficiency increases. The large set of mesopores studied with a nominal diameter of approximately 25 nm provided the most efficient column performance. The efficiency of the monolithic silica columns increase with decreasing n-alkyl chain length in the sequence of C18相似文献   

Reduced-bore monolithic silica column modified with C8-TEOS for reversed-phase electrochromatography

Monolithic silica columns of 2.7 mm ID were prepared and derivatized with C8-TEOS and TEOS by on-column sol-gel reaction. These C8 large diameter monolithic silica columns gave 21 000 theoretical plates for aromatic hydrocarbons in 60% acetonitrile and 40% Tris-HCI buffer. The surface areas as well as the separation reproducibility were improved on coating by the sol-gel approach. Joule heating was greatly reduced by using monolithic columns to which fine quartz sand had been added during column preparation. Since this is a preliminary investigation on a monolithic column with such a large inner diameter, the separation efficiency was not so high as that presently achieved in normal capillary electrochromatography (CEC). However, use of the columns improved sample loadability and concentration detectability of electrochromatography, and semi-preparative separations could be performed.     

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.     

Development of a γ‐alumina‐ nanoparticle‐functionalized porous polymer monolith for the enrichment of Sudan dyes in red wine samples

Monolithic materials were synthesized in capillaries by in situ polymerization with N‐isopropylacrylamide, glycidyl methacrylate, and ethylene dimethacrylate as the monomers, and methanol and PEG as the porogens. With γ‐alumina nanoparticles attached to the surface of the porous monolithic column via epoxide groups, a novel polymer monolith microextraction (PMME) material was prepared with a good mechanical stability and a high extraction capacity. SEM and X‐ray photoelectron spectroscopy were employed to characterize the modified monolithic column, demonstrating that γ‐alumina nanoparticles were effectively functionalized onto the monolithic column. In addition, a new method was developed for the analysis of Sudan I–IV dyes using PMME coupled with HPLC. In order to obtain the optimum extraction efficiency, the PMME conditions including desorption solvent type, sample pH, sample volume, sample flow rate, and eluent flow rate were investigated. Under the optimum conditions, we obtained acceptable linearities, low LODs, and good intra‐ and interday RDSs. When applied to the determination of Sudan I–IV dyes in red wine samples, satisfactory recoveries were obtained in the range of 84.0–115.9%.     

丁基胺丙基硅胶毛细管整体柱的制备及其电色谱性能研究

采用溶胶-凝胶技术制备了丁基胺丙基硅胶毛细管整体柱,此整体固定相表面同时含有能产生阳极的电渗流的仲胺官能团和产生疏水作用的正丁基和丙基官能团。对所制备的整体柱电色谱性能进行了详细的表征和分析。考察了流动相pH值对电渗流的影响;对烷基苯同系物、有机酸酸性化合物和苯胺类碱性化合物保留行为进行了研究,并对其可能的保留机理进行了探讨。实验结果表明,对于中性化合物的保留机理主要基于反相作用;而对于酸性化合物的保留行为则是基于混合模式作用机理,即除了电泳作用外,还包括阴离子交换和疏水作用。碱性化合物在丁基胺丙基硅胶毛细管整体柱上的峰形较好,没有明显的峰拖尾现象。     

Incorporation of carbon nanotubes in porous polymer monolithic capillary columns to enhance the chromatographic separation of small molecules

Multiwalled carbon nanotubes have been entrapped in monolithic poly(glycidyl methacrylate-co-ethylene dimethacrylate) capillary columns to afford stationary phases with enhanced liquid chromatographic performance for small molecules in the reversed phase. While the column with no nanotubes exhibited an efficiency of only 1800 plates/m, addition of a small amount of nanotubes to the polymerization mixture increased the efficiency to over 15,000 and 35,000 plates/m at flow rates of 1 and 0.15 μL/min, respectively. Alternatively, the native glycidyl methacrylate-based monolith was functionalized with ammonia and, then, shortened carbon nanotubes, bearing carboxyl functionalities, were attached to the pore surface through the aid of electrostatic interactions with the amine functionalities. Reducing the pore size of the monolith enhanced the column efficiency for the retained analyte, benzene, to 30,000 plates/m at a flow rate of 0.25 μL/min. Addition of tetrahydrofuran to the typical aqueous acetonitrile eluents improved the peak shape and increased the column efficiency to 44,000 plates/m calculated for the retained benzene peak.     

Monolithic silica columns with various skeleton sizes and through-pore sizes for capillary liquid chromatography

Reduction of through-pore size and skeleton size of a monolithic silica column was attempted to provide high separation efficiency in a short time. Monolithic silica columns were prepared to have various sizes of skeletons (approximately 1-2 microm) and through-pores (approximately 2-8 microm) in a fused-silica capillary (50-200 microm I.D.). The columns were evaluated in HPLC after derivatization to C18 phase. It was possible to prepare monolithic silica structures in capillaries of up to 200 microm I.D. from a mixture of tetramethoxysilane and methyltrimethoxysilane. As expected, a monolithic silica column with smaller domain size showed higher column efficiency and higher pressure drop. High external porosity (> 80%) and large through-pores resulted in high permeability (K = 8 x 10(-14) -1.3 x 10(-12) m2) that was 2-30 times higher than that of a column packed with 5-mirom silica particles. The monolithic silica columns prepared in capillaries produced a plate height of about 8-12 microm with an 80% aqueous acetonitrile mobile phase at a linear velocity of 1 mm/s. Separation impedance, E, was found to be as low as 100 under optimum conditions, a value about an order of magnitude lower than reported for conventional columns packed with 5-microm particles. Although a column with smaller domain size generally resulted in higher separation impedance and the lower total performance, the monolithic silica columns showed performance beyond the limit of conventional particle-packed columns under pressure-driven conditions.     

Ionic liquids-assisted fabrication of silica-based monolithic columns

The mesopores of a monolithic silica column are very important and useful for chromatographic separation since they can offer sufficiently large surface area. In this paper, a novel method with the assistance of an ionic liquid (1-butyl-3-methylimidazolium tetrafluoroborate ([bmin]BF₄)) was developed for the preparation of a C18-modified monolithic silica column for the first time, in which, the through pores and mesopores were formed simultaneously during the sol-gel reaction. The method is effective to simplify the preparation process of the silica-based monolithic columns. The factors influencing the sol-gel process, including the content of methanol and pH, were studied. The chromatographic performance of the prepared monolithic column was evaluated by the separation of alkylbenzenes.     

Characterization of some physical and chromatographic properties of monolithic poly(styrene-co-divinylbenzene) columns

Monolithic capillary columns were prepared by copolymerization of styrene and divinylbenzene inside a 200 microm i.d. fused silica capillary using a mixture of tetrahydrofuran and decanol as porogen. Important chromatographic features of the synthesized columns were characterized and critically compared to the properties of columns packed with micropellicular, octadecylated poly(styrene-co-divinylbenzene) (PS-DVB-C18) particles. The permeability of a 60 mm long monolithic column was slightly higher than that of an equally dimensioned column packed with PS-DVB-C18 beads and was invariant up to at least 250 bar column inlet pressure, indicating the high-pressure stability of the monolithic columns. Interestingly, monolithic columns showed a 3.6 times better separation efficiency for oligonucleotides than granular columns. To study differences of the molecular diffusion processes between granular and monolithic columns, Van Deemter plots were measured. Due to the favorable pore structure of monolithic columns all kind of diffusional band broadening was reduced two to five times. Using inverse size-exclusion chromatography a total porosity of 70% was determined, which consisted of internodule porosity (20%) and internal porosity (50%). The observed fast mass transfer and the resulting high separation efficiency suggested that the surface of the monolithic stationary phase is rather rough and does not feature real pores accessible to macromolecular analytes such as polypeptides or oligonucleotides. The maximum analytical loading capacity of monolithic columns for oligonucleotides was found to be in the region of 500 fmol, which compared well to the loading capacity of the granular columns. Batch-to-batch reproducibility proved to be better with granular stationary phases compared to monolithic stationary phase, in which each column bed is the result of a unique column preparation process.