Polymer-Modified Silica Adsorbent with Reversed-Phase and Weak Cation-Exchange Groups

Micro-spherical silica gel has been modified with a polymer containing two different functional groups (C₁₈ and COOH) by use of a copolymer of octadecyl methacrylate and butyl acrylate and introduction of maleic anhydride. The chromatographic properties of these stationary phases in reversed-phase high-performance liquid chromatography (HPLC) have been studied over a broad pH range. Introduction of maleic anhydride as the third component of the polymer layer on the surface of micro-spherical silica leads to repartitioning of C₁₈ groups, which affects the hydrophobicity of the materials obtained. The presence of maleic anhydride secures the availability of carboxyl groups on the surface, and thus the acidic properties of the material.     

包覆聚合物高效液相色谱柱填料的研究进展

评述了用于高效液相色谱的包覆聚合物填料的研究进展．该类填料以无机载体为基质，包覆以多种有机聚合物层．这些新型填料既保留了有机聚合物和无机基质的优点，又克服了它们各自的缺陷．包覆的聚合物类型包括聚烃、聚醚、聚酰胺、多糖、多肽、聚硅氧烷、聚胺及多核苷酸等，无机基质包括硅胶、铝胶、钛胶、钻胶及多孔石墨化碳等．包覆聚合物固定相可用于几乎所有色谱方式，如反相高效液相色谱、高效离子交换色谱、手性固定相、超临界流体色谱、体积－排阻色谱，以及亲合色谱．     

Open tubular capillary column of 50 μm internal diameter with a very high separation efficiency for the separation of peptides in CEC and LC

A specially designed long open tubular capillary column (50 μm internal diameter and 112 cm effective length) was prepared by fabrication of a thin three‐component co‐polymer layer on the inner surface of silica capillary. A pretreated silica capillary was reacted with 4‐(chloromethyl)phenyl isocyanate in the presence of dibutyltin dichloride as catalyst followed by sodium diethyl dithiocarbamate. Then a thin polymer layer was made on the inner surface of capillary by reversible addition‐fragmentation transfer polymerization of styrene, N‐phenylacrylamide, and methacrylic acid. A carefully adjusted formulation of reaction mixture and elaborated procedures were adopted to secure formation of the co‐polymer layer of enhanced separation performance. The co‐polymer immobilized open tubular capillary column was used for the separation of a synthetic mixture of five peptides and excellent separation efficiency (over 1.7 million per column) was obtained in the capillary electrochromatography mode. Such excellent separation efficiencies of ca. 1 m column have not been obtained in the isocratic elution mode so far. The column was also used for separation of the peptides in the liquid chromatography mode to show very good separation efficiency (average 286 700 per column).     

亲和膜色谱

亲和膜色谱又称亲和膜分离，其在蛋白质的分离纯化中作为一种综合性的技术出现在80年代末。亲和膜色谱主要优点是克服了颗粒状多孔载体扩散传质阻力大的缺点，代之以对流传质，这样就可以在较低的操作压和较高的流速下对目标蛋白进行快速的分离和纯化，从而缩短操作时间、提高纯化效率。本文将就近年来亲和膜色谱及其在蛋白质分离和纯化中的应用作一综述性介绍。     

The structure and chromatographic properties of poly(ethylene glycol) 20M layer on the surface of a diatomaceous earth type support

Summary The properties of poly(ethylene glycol) 20M (PEG 20M) coated on the hydroxylated surface of Chromaton N AW were investigated by inverse gas chromatography. It was found that the solid form of PEG 20M exhibits partition as the retention mechanism even below its melting range. The contribution of adsorption to the specific retention volume was also determined. It was demonstrated that inverse gas chromatography establishes the existence of a polymer surface layer but precise determination of its thickness is difficult. The limitation of the applicability of inverse gas chromatography results from the structure of the investigated surface layer which is thin and not coherent so that solute molecules can penetrate it.     

Stationary phase-based two-dimensional chromatography combining both covalent and noncovalent interactions on a single HPLC column

A new type of 2-D separation material was synthesized and studied. The material is suitable for 2-D chromatography utilizing both covalent and noncovalent interactions. The first dimension is boronate affinity chromatography, and the second dimension is RP chromatography (or vice versa). The polymeric media were prepared using p-vinylphenylboronic acid as the functional monomer. This monomer was selected due to the presence of the boronic acid group for the cis-diol/boronate interaction in boronate chromatography. Two crosslinkers were evaluated, namely ethylene glycol dimethacrylate and divinylbenzene. The crosslinker content was varied to maximize the polymer strength and the RP performance of the packed column. Several parameters were evaluated to define the optimum for polymer strength and column performance including crosslinker, porogen, initiator, and column-packing parameters. The polymer-based HPLC columns were successful in separating phenol, catechol, dimethylphthalate, and hydroquinone under RP conditions, and thus can be used as an RP HPLC column. The columns were also successful in separating catechol and adenosine under boronate chromatography conditions, and thus can be used as a boronate affinity column. Moreover, the two types of chromatography can be performed consecutively on the same column during one complete chromatographic run, making it a 2-D chromatography. Under these 2-D conditions, the catechol was separated from a mixture of phenol, catechol, dimethylphthalate, and hydroquinone; the adenosine ribonucleoside was separated from a mixture of adenosine ribonucleoside, adenosine deoxyribonucleoside, and uridine deoxyribonucleoside. This type of single-column 2-D HPLC eliminates the requirement of a complex and expensive multidimensional HPLC instrument and provides increased peak capacity for separation.     

Characterization by inverse gas chromatography of the surface properties of silicas modified by poly(ethylene glycols) and their models (oligomers,diols)

Summary Silicas are modified by esterification of the surface silanol groups either with poly(ethylene glycol), its oligomers or diols of the same chain length. A whole series of samples carrying grafts differing in chemical nature, molecular weight and number are prepared. These samples are examined by inverse gas chromatography. The London component of the surface energy, the acid/base interaction parameter and the enthalpy of adsorption of alkanes and polar probes are measured. It is apparent for PEG-grafted silicas that the most important variable is the surface coverage, i.e., the number of monomer units of the grafted PEG per unit surface area, and not its molecular weight. The fixation of PEG changes drastically the surface properties of the silicas. According to the surface coverage, silica may acquire base-like properties. The strong PEG-silica interactions cause the polymer to adopt a flat configuration by spreading on the solid surface. Grafted diols behave entirely differently: the conformation of the longer grafts is best described by a sandwich structure in which the outer surface layer is formed by associated alcohol groups, whereas the middle part is formed by the highly organized aliphatic parts of the chains. IR spectroscopy supports these conclusions.     

以桑色素为模板的分子烙印聚合物的制备、结合特性及其在薄层色谱固定相方面的应用

以桑色素为模板,选用不同的功能单体和致孔剂制备了一系列分子烙印聚合物,采用结合实验考察了它们对底物的吸附特性,发现以丙烯酰胺为功能单体,以乙酸乙酯为致孔剂制得的烙印聚合物对底物有很好的选择性,同时对一些结构相似的化合物具有一定的结合力.Scatchard方程研究结果表明,在研究的浓度范围内聚合物形成了一类等价的结合位点,其平衡离解常数为0.877mmol/L,对模板分子的最大表观结合量为59.18μmol/g.可认为桑色素分子中3,7,4'位的羟基与功能单体的氢键作用是分子烙印聚合物具有底物选择性的主要因素.以该分子烙印聚合物为薄层色谱固定相,可以把模板分子从结构相似的化合物中分离出来,最小分离度为1.19.     

Ion-Exchange Chromatographic Supports Obtained by Formation of Polyelectrolyte Multi-Layers for the Separation of Proteins

Summary High performance liquid chromatography (HPLC) was used to study the mechanism of formation of polyelectrolyte multilayers on porous silicas. The coatings were produced by alternating the adsorption of positively and negatively charged polymers. The stationary phases formed by adsorbing a single layer, double layers and triple layers were tested by studying the elution behavior of model proteins. The double polymer coating was achieved by adsorbing first a polycation such as hexadimethrine bromide (HB) on the HPLC silica support and then a polyanion such as dextran sulfate (DS) on the cationic layer formed. The retention properties of this support are mainly those of a cation exchanger as the negatively charged proteins were strongly retained while positively charged ones were weakly adsorbed. This work demonstrated the importance of the first underlying layer as the retention behavior of proteins was greatly affected by the properties of this coating. The triple polymer coating was achieved by adsorbing the polycation (HB) on the double layer coating (HB-DS). Its retention behavior was that of an anion exchange support. The HB-DS stationary phase displayed good chromatographic performances, with an adsorbed layer relatively stable. The polyelectrolyte multilayer coating procedure was useful to easily synthesize cation-exchange supports for the separation of basic proteins.     

Structures and chromatographic characteristics of capsule-type silica gels coated with hydrophobic polymers in reversed-phase liquid chromatography

Summary Silicone polymer-coated silica gels modified with octadecyl and octyl groups (S/S-C₁₈, S/S-C₈), or “capsule-type silica gels” were developed as packing materials for reversed-phase liquid chromatography. They were obtained by coating the surface of totally porous silica gel with a homogeneous silicone polymer film, and thereafter modifying the coating polymer with octadecyl or octyl groups. Retaining the advantages of silica-based packings, they show strong resistance of alkali-like organic porous polymeric materials.     

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.     

Initial evaluation of protein A modified capillary‐channeled polymer fibers for the capture and recovery of immunoglobulin G

A novel protein A affinity chromatography stationary phase has been developed from polypropylene capillary‐channeled polymer fibers modified with a recombinant protein A ligand for the capture and recovery of immunoglobulin G (IgG) with high specificity and yield. An SPE micropipette tip format was employed so that solvent, protein, and antibody consumption was minimized. The adsorption modification of the fiber surfaces with protein A was evaluated as a function of feed concentration and volume. Optimal modification of the fiber surface with protein A yielded a 5.7 mg/mL (bed volume) ligand capacity with the modified fibers showing stability across numerous solvent environments. Performance was evaluated through exposure to human IgG and myoglobin, individually and as a mixture. Myoglobin was used as a surrogate for host cell proteins common to growth media. The efficacy of the selective binding to the ligand is demonstrated by the 2.9:1 (IgG/protein A) binding stoichiometry. Elution with 0.1 M acetic acid yielded an 89% recovery of the captured IgG based on absorption measurements of the collected eluents. Regeneration was possible with 10 mM NaOH. Protein A modified polypropylene capillary‐channeled polymer fibers show promising initial results as an affinity phase for efficient capture and purification of IgG.     

稻壳纤维的柱层析性能研究

试验了稻壳纤维作为柱层析介质的性能,并制成亲和层析介质经脂肪酶。结果表明稻壳纤维层析过程为化学物理混合吸附过程,亲和层析性能良好,层析可将脂肪酶纯化２倍,回收率达８１．６％。适合较大规模层析分离。     

Methacrylate-based monolithic layers for planar chromatography of polymers

A series of macroporous monolithic methacrylate-based materials was synthesized by in situ free radical UV-initiated copolymerization of functional monomers, such as glycidyl methacrylate (GMA), butyl methacrylate (BuMA), 2-aminoethyl methacrylate (AEMA), 2-hydroxyethyl methacrylate (HEMA) and 2-cyanoethyl methacrylate (CEMA), with crosslinking agent, namely, ethylene glycol dimethacrylate (EDMA). The materials obtained were applied as the stationary phases in simple and robust technique - planar chromatography (PLC). The method of separation layer fabrication representing macroporous polymer monolith bound to the specially prepared glass surface was developed and optimized. The GMA-EDMA and BuMA-EDMA matrixes were successfully applied for the separation of low molecular weight compounds (the mixture of several dies), as well as poly(vinylpyrrolidone) and polystyrene homopolymers of different molecular weights using reversed-phase mechanism. The materials based on copolymers AEMA-HEMA-EDMA and CEMA-HEMA-EDMA were used for normal-phase PLC separation of 2,4-dinitrophenyl amino acids and polystyrene standards.     

Epoxy-functionalized large-pore SBA-15 and KIT-6 as affinity chromatography supports

Mesoporous silica supports are proposed as an alternative to polymeric stationary phases for fast affinity chromatography due to their better mechanical strength compared to polymers. Ideal supports should combine high surface area and large pore size to allow a high loading capacity of large molecules, such as proteins, and favor their accessibility. Increasing the pore size of large-surface area micelle-templated silicas (SBA-15, KIT-6) has been achieved by swelling the micelles by the addition of organic molecules and increasing synthesis time and temperature. The pore size of hexagonal silica mesostructured SBA-15 has been increased up to 35 nm. These materials could find therefore application as affinity chromatography for immunoextraction.     

Molecularly imprinted polymer layer-coated silica nanoparticles toward dispersive solid-phase extraction of trace sulfonylurea herbicides from soil and crop samples

This paper reports the preparation of metsulfuron-methyl (MSM) imprinted polymer layer-coated silica nanoparticles toward analysis of trace sulfonylurea herbicides in complicated matrices. To induce the selective occurrence of surface polymerization, the polymerizable double bonds were first grafted at the surface of silica nanoparticles by the silylation. Afterwards, the MSM templates were imprinted into the polymer-coating layer through the interaction with functional monomers. The programmed heating led to the formation of uniform MSM-imprinted polymer layer with controllable thickness, and further improved the reproducibility of rebinding capacity. After removal of templates, recognition sites of MSM were exposed in the polymer layers. As a result, the maximum rebinding capacity was achieved with the use of optimal grafting ratio. There was also evidence indicating that the MSM-imprinted polymer nanoparticles compared with nonimprinted polymer nanoparticles had a higher selectivity and affinity to four structure-like sulfonylurea herbicides. Moreover, using the imprinted particles as dispersive solid-phase extraction (DSPE) materials, the recoveries of four sulfonylurea herbicides determined by high-performance liquid chromatography (HPLC) were 80.2-99.5%, 83.8-102.4%, 77.8-93.3%, and 73.8-110.8% in the spiked soil, rice, soybean, and corn samples, respectively. These results show the possibility that the highly selective separation and enrichment of trace sulfonylurea herbicides from soil and crop samples can be achieved by the molecular imprinting modification at the surface of silica nanoparticles.     

Molecular imprinted polymers for separation science: A review of reviews

Molecular imprinted polymer is an artificial receptor made by imprinting molecules of a template in a polymer matrix followed by removing the template molecules via thorough washing to give the permanent template grooves. They show favored affinity to the template molecule compared to other molecules, and this property is the basic driving force for such diverse application of this techniques. Such techniques have been increasingly employed in a wide scope of applications such as chromatography, sample pretreatment, purification, catalysts, sensors, and drug delivery, etc., mostly in bioanalytical areas. A major part of them is related to development of new stationary phases and their application in chromatography and sample pretreatment. Embodiments of molecular imprinted polymer materials have been carried out in a variety of forms such as irregularly ground particles, regular spherical particles, nanoparticles, monoliths in a stainless steel or capillary column, open tubular layers in capillaries, surface attached thin layers, membranes, and composites, etc. There have been numerous review articles on molecular imprinted polymer issues. In this special review, the reviews in recent ca. 10 years will be categorized into several subgroups according to specified topics in separation science, and each review in each subgroup will be introduced in the order of date with brief summaries and comments on new developments and different scopes of prospects. Brief summaries of each categories and conclusive future perspectives are also given.     

Syntheses and Characterization of Conjugates of Nucleic Acid Probes and 6-Aminoglucose-based Polymers

Homopolymers based on 6-deoxy-6-methacryloylamido-d -glucopyranose (6-aminoglucose), obtained by radical-initiated polymerization, were used as reactive polymeric supports to tether oligodeoxyribonucleotides (ODNs) in order to make oligonucleotide–polymer conjugates of potential applications in diagnostics. The reducing ends of the homopolymers, used as self-protected groups during the radical polymerizations, served as reactive groups for the covalent attachment of 5′-amino-modified oligodeoxyribonucleotides via reductive amination, with sodium borohydride as a reducing agent. The best coupling conditions used dimethylformamide/20 mm sodium tetraborate as a 90/10 (v/v) mixture at 50°C. The main factors controlling the course of the reaction are electrostatic repulsive interactions and polymer chain complexation. A low ionic strength was essential for the reaction to occur in a pH range of 7.3 to 9.3. The polymer–ODN conjugates could be aggregated depending on the extent of polymer chain interactions. © 1997 John Wiley & Sons, Ltd.     

Influence of the thermal modification of controlled porosity glass on the affinity chromatography of fungal proteins

Summary The properties and the coverage density of chemically bonded phases depend among others on the properties of their supports. Controlled-porosity glasses (CPG) are materials used as a support of bio-active ligands. Their network mainly consist of SiO₂ as well as of a small amount of B₂O₃ and Na₂O. The characteristic feature of porous glasses is the possibility of a change in the surface boron concentration leading to the change of the properties of their surface.This paper deals with the results of the separation of fungal proteins on packings consisting of vanillin bonded to CPG with different boron concentration on its surface. It appears from the data obtained that the changes in the affinity of the fungal proteins to the packing are related to the extend of the thermal treatment of the CPG. The protein fractions separated by chromatography were confirmed to be homogeneous by poly-acryloamide gel disc electrophoresis.     

Bioaffinity chromatography on monolithic supports

Affinity chromatography on monolithic supports is a powerful analytical chemical platform because it allows for fast analyses, small sample volumes, strong enrichment of trace biomarkers and applications in microchips. In this review, the recent research using monolithic materials in the field of bioaffinity chromatography (including immunochromatography) is summarized and discussed. After giving an introduction into affinity chromatography, information on different biomolecules (antibodies, enzymes, lectins, aptamers) that can act as ligands in bioaffinity chromatography is presented. Subsequently, the history of monoliths, their advantages, preparation and formats (disks, capillaries and microchips) as well as ligand immobilization techniques are mentioned. Finally, analytical and preparative applications of bioaffinity chromatography on monoliths are presented. During the last four years 37 papers appeared. Protein A and G are still most often used as ligands for the enrichment of immunoglobulins. Antibodies and lectins remain popular for the analysis of mainly smaller molecules and saccharides, respectively. The highly porous cryogels modified with ligands are applied for the sorting of different cells or bacteria. New is the application of aptamers and phages as ligands on monoliths. Convective interaction media (epoxy CIM disks) are currently the most used format in monolithic bioaffinity chromatography.