Macroporous Particles Research Articles

Electron microscopic observation of uniform macroporous particles. II. Effect of DVB concentration

The electron microscopic observation of uniform and macroporous poly(styrene-co-divinylbenzene) particles prepared by a two-step seeded polymerization method was performed. In the synthesis of uniform macroporous particles, the uniform polystyrene latices produced by a dispersion polymerization method with two different sizes and average molecular weights were utilized as the seed particles. The seed particles were first swollen with dibutylphthalate and then with a monomer phase, including styrene and divinylbenzene. The macroporous structure of the final particles was achieved by using a porogen mixture consisting of dibutylphthalate and linear polystyrene. The linear polystyrene part of the porogen solution was directly obtained from the seed latex. The macroporous particles with different diameters and porosities were produced by changing the divinylbenzene concentration between 25 and 100% in the repolymerization step. The effect of divinylbenzene concentration on the size and the surface morphology of the final particles were investigated by scanning electron microscopy. The internal structure of the final particles was analyzed by transmission electron microscopy. The results indicated that the average size of the final particles increased with the increasing divinylbenzene concentration. The increase in the DVB concentration also led to an increase in the average pore size. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 71: 2291–2302, 1999

Cibacron blue F3G-A-attached uniform and macroporous poly(styrene-co-divinylbenzene) particles for specific albumin adsorption

Cibacron blue F3G-A-carrying uniform macroporous particles were proposed as an alternative sorbent for specific albumin adsorption. These particles were produced by a multistep polymerization procedure. In the first step of production, the uniform polystyrene seed particles were prepared by a dispersion polymerization method. Next, the polystyrene seed particles were first swollen by dibutylphthalate and then by styrene-divinylbenzene mixture in an aqueous emulsion medium. In the last step (i.e. repolymerization). styrene-divinylbenzene mixture was copolymerized within the swollen seed particles in the absence or presence of a stabilizer (e.g. poly(vinyl alcohol)). Although a considerable amount of non-specific BSA adsorption was observed on the surface of the particles produced in the absence of PVA, zero non-specific albumin adsorption could be achieved with the uniform macroporous particles produced in the presence of PVA. The stabilizer on the particle surface was also used as a ligand in the further derivatization of macroporous particles for specific albumin adsorption. Cibacron blue F3G-A was then covalently attached onto the surface of uniform macroporous particles. Specific albumin adsorption capacities up to 93 mgg-1 could be achieved with the cibacron blue F3G-A-carrying macroporous particles of 6.25 μm in size.

Chemiluminescent Imaging of Enzyme-Labeled Probes Using an Optical Microscope–Videocamera Luminograph

A chemiluminescent system has been developed for ultrasensitive, quantitative analysis as well as visualization of the spatial distribution of biomolecules such as antigens, enzymes, antibodies, DNA probes in tissue, or cells. The system consists of a low-light imaging Vidicon videocamera connected to an optical microscope, able to measure light at the single photon level and perform 3D image analysis of the subcellular distribution of the analyte. The concentration and the spatial distribution of enzymes, or enzyme-labeled biospecific reagents can be determined using appropriate chemiluminescent substrates. Analytes are also determined with coupled enzymatic reactions terminating in light emission. Oxirane acrylic beads (250-μm-diameter macroporous particles) with immobilized horseradish peroxidase have been used as a model system to optimize the experimental conditions in terms of signal intensity and spatial resolution as a function of different chemiluminescent substrates such as luminol/enhancer/H2O2and acridancarboxylate ester/H2O2. Localization of oxirane beads immobilized acetylcholinesterase has been also used to optimize a system in which the detection and localization of the primary enzyme involves two secondary enzymes in solution, choline oxidase and horseradish peroxidase, leading to a final light emission. Immunoenzymatic reactions for the detection of viral antigens andin situhybridization assays for the detection of viral DNAs (cytomegalovirus, herpes simplex virus) have been performed in cells using peroxidase-labeled antibodies or cDNA probes and the analytical performance of different chemiluminescent substrates for the enzyme has been evaluated. The results obtained showed the possibility to sharply image the bioprobes in single cells and peroxidase is a suitable label when luminol/H2O2system is used in conjunction with enhancer as in the ECL and SuperSignal Ultra reagents; other substrates such as Lumigen PS-3, despite adequate detectability, showed problems of localization of the signal as a result of the relatively long half-life of the excited emitting species and its diffusion in the chemiluminescent cocktail. The system has proven to be highly sensitive, able to perform quantitative analysis, and relatively simple.

Biochemical and biomedical application of monodisperse polymer particles

AbstractMacroporous particles with large pores are used for size exclusion chromatography of carbohydrates with very high molecular weights. Macroporous particles of different sizes are used for multiple immunofluorometric analysis by flow cytometry. Porous magnetic particles with protein A coupled to the surface of the pores combine a high capacity in binding of IgG with easy handling of the particles. Magnetic beads with boronic acid coupled to the surface are applied for selective isolation of subclasses of cells. The beads are detached from the cells by addition of sorbitol, leaving free, pure cell fractions in good yield. The use of magnetic particles in molecular biology is described.

Properties of poly(vinyl alcohol)/silica hybrid gel particles

We have been investigating the preparation of macroporous gel particles of poly(vinyl alcohol) [1-3]. These particles are obtained by the saponification of particles of poly(vinyl acetate) formed in the polymerization of vinyl acetate in a suspension process. The poly(vinyl alcohol) gel particles have been used as a column packing for aqueous separations. However, the gels have a disadvantage in pressure-resistant property because of poor mechanical strength. We expected that a gel having excellent mechanical stability might be obtained by reinforcing poly(vinyl alcohol) with an inorganic component. We report here that reinforcement using silica as an inorganic component is useful for the improvement of performance of poly(vinyl alcohol) gel particles used as a column packing for gel permeation chromatography in aqueous media. Tetraethoxy silane (TEOS) was used as the inorganic component of the hybrid gels throughout this study. The sol-gel method was applied to the preparation of hybrid gel [4-6]. The process is divided into two steps: hydrolysis of metal alkoxides to produce metal hydroxides, followed by polycondensation of hydroxyl groups. By carrying out this reaction in a poly(vinyl alcohol) gel matrix containing many hydroxyl groups, it is possible to copolymerize metal alcoxide with poly(vinyl alcohol). Thus a silica network may be incorporated into the poly (vinyl alcohol) gel matrix. The gel particles of poly(vinyl acetate) (DP = 1000) obtained by suspension polymerization were classified to give a particle size distribution with particle diameters in the range 350-400 #m. The saponification of poly(vinyl acetate) to poly(vinyl alcohol) was carried out at 30 ~ for 1 month by immersing the gel particles in a solution containing sodium hydroxide and methanol in an aqueous saturated sodium sulfate solution [7]. The gel particles of poty(vinyl alcohol) obtained above have sufficient mechanical stability in water below 55 ~ without crosslinking treatment. The gel particles (1 g) obtained above were immersed in 10 ml of ethanol for 24 h and then tetraethoxy silane and hydrochloric acid as catalyst were added to the particles. The reaction was carried out at 30 ~ For the purpose of removal of TEOS homopolymer the reaction product was then subjected to a Soxhlet extraction using tetrahydrofuran. Swelling behaviour and gel permeation chromatographic properties of the hybrid gel particles obtained above were investigated. The ratio of the volume of the particles in ethanol to that on swelling with water (swelling ratio) was evaluated from observations by optical microscopy. As shown in Fig. 1, swelling ratio decreases with increase of reaction time and levels off at about 2 h. This is due to the formation of the silica network in the poly(vinyl alcohol) gel matrix. The swelling behaviour is mainly controlled by the amount of TEOS added. The slurry of hybrid gel particles was packed into a 150 x 4 mm stainless column at a pressure of about 5 MPa. High-performance gel permeation chromatography (HP-GPC) separations were performed with a Shimazu LC-6AD, employing distilled water as eluent. Solutions (40 #1) of individual solutes were injected with an off-column syringe-septum arrangement. Detection of solutes was performed with a Shimazu Refracto Monitor Model RID-6A (cell volume = 10 ~tl, aqueous reference). The samples of poly(ethylene glycol) are designated PEG with a number which is the molecular weight provided by the suppliers (Kanto Chemicals, Tokyo). The calibration curve was established at a flow rate of 0.4cm3/min with a PEG concentration of 2.0% (w/v). The calibration curve of the hybrid gel particles established with PEG samples is shown in Fig. 2, in comparison with the poly(vinyl alcohol) gel particles. The value of the excluded molecular weight is almost the same for the hybrid gel particles and the poly(vinyl alcohol) gel particles. However, the calibration curve for the column packing containing the hybrid gel particles shifts to higher elution volumes. Since separation power is inversely proportional to the slope of the plot of molecular weight versus elution volume, it is clear that the column containing the hybrid gel particles shows better resolution separations. The shape of the slope

Poly(ethylene dimethacrylate) particles with poly(glycidyl methacrylate) functionalities

Macroporous particles, prepared by the suspension polymerization of ethylene dimethacrylate (EDMA), possessing high specific surface areas (558 m 2g −1) and pore volumes (6.9 ml g −1), contain unreacted double bonds which are accessible for free radical polymerization. The amount of unreacted double bonds was determined by 13C CP/MAS n.m.r. These bonds were used for the functionalization of poly(EDMA) particles by the solution grafting copolymerization of glycidyl methacrylate (GMA) into the pore structure of the particles. The poly(GMA) formed in this way is permanently attached to the particles. By this method composite particles, containing up to 77 wt% of oxirane groups (determined by i.r. spectroscopy), were obtained. However, the presence of grafted polymer in the pores leads to a decrease in both the pore volume and the specific surface area. The oxirane groups can be chemically modified, as evidenced by their reactions with hydrogen chloride and 2,2,6,6-tetramethyl-4-aminopiperidine.

Homogeneous immunofluorometric assays of alpha-fetoprotein with macroporous, monosized particles and flow cytometry

We evaluated two homogeneous immunofluorometric assays (IFMAs) of alpha-fetoprotein (AFP) based on new macroporous acrylate particles combined with flow cytometry. The standard IFMA, requiring 1 h of incubation, provided a working range from 1.8 to > 900 kIU/L (CV < 10%) and a detection limit of 0.6 kIU/L. Use of overnight incubation and a lower particle concentration extended the working range by 1 decade in the lower end. Analytical recoveries for the standard IFMA varied between 97% and 108%. The slope and y-intercept of the regression line correlating measurements by the standard IFMA and a routine immunoradiometric assay were not significantly different from 1 and 0, respectively (P > 0.5), and the correlation coefficient was 0.996. High precision and warning of spuriously high measurements were obtained by including in each sample separate particle types for detecting instrument instability and measuring nonspecific binding only.

Application of gel-integrated macroporous hydroxyapatite in the treatment of marginal periodontitis

Marginal periodontitis is a moderate to severe chronic inflammation of the gingiva and deep connective tissue around teeth with progressive resorption of bone and formed connective tissue caused by microbial plaque accumulation on the teeth and gingiva. Marginal adult periodontitis causes loss of attachment of tooth with the formed connective tissue at a different degree. The development of angular bone defect is a typical sign of this chronic inflammation of the periodontium. These angular alveolar bone defects are different in size and morphology following different plaque accumulation on the root surfaces, and the radius of influence of plaque toxins into the connective tissues (circumference of 2.5 mm). Other factors influencing the development of angular alveolar bone defects are the macromorphology of the jaw as well as the individual cyclic progression of marginal periodontitis. Angular bone defects are bordered with one, two or three walls of residual bone or of inflammed connective tissues only. The treatment of marginal periodontitis aims to repair and, if possible, to regenerate the bone. The surgical treatment measures are designed to improve accessibility for root scaling and root planing, the excision of inflamed connective tissue, and the modulation of alveolar bone to improve postoperative morphology. The additional implantation of granular macroporous hydroxyapatite (HAP) results in an improvement of the postoperative clinical judgment [1-3]. HAP aims to stabilise alveolar bone defects [4,5] and to create the possibility of repair and sometimes of regeneration. The HAP particles will be integrated into newly formed alveolar bone during a kind of foreign body reaction [6]. The application of macroporous HAP particles into periodontal bone defects is not guaranteed because single particles are too light. Therefore, aids for application are recommended. The fixation of HAP particles in an inert basic substance, which serves as an integration material for the HAP particles, could result in improving the application during the surgical procedures. The aim of this clinical study was to test a gel-integrated macroporous hydroxyapatite (Osprogel ®, Cerasiv) clinically and to compare this new material with non gel-integrated macroporous HAP (lnterpore 200®, Interpore Int.).

Monosized polymer particles in biochemical and biomedical separations

AbstractThe method of activated swelling allows production of highly monodisperse polymer particles in the range of 1 to 100μm, with identical bead‐to‐bead morphology. Monosized particles with a wide variety in content of crosslinker, pore size and pore size distribution have been evaluated in different areas of biotechnology. Underivatized macroporous particles have been developed for reversed phase and size exclusion chromatography in organic solvents. Supports for aqueous phase ion exchange have been produced by covalent coupling of a hydrophilic layer to the particle surface. These media give excellent characteristics with regard to selectivity, capacity and recoveries of biomolecules. In this paper proteins and peptides are used to illustrate chromatographic performance. Monosized polymer particles have been magnetized by a process where magnetic iron oxides are formed inside preformed monodisperse particles. These particles are used extensively in selective isolation of cells and bacteria and in DNA technology. This paper reviews some new results in the field of cell separation.

Reversed-Phase Chromatographic Properties of Monodispersed Macroporous Particles of Poly(styrene-divinylbenzene) Prepared by a Multi-Step Swelling and Polymerization Method

Abstract Monodispersed macroporous poly(styrene-divinylbenzene) particles in a 5-6 μm size range were prepared by a multi-step swelling and polymerization method with toluene as the porogenic solvent, and their properties were examined. The particles were found to possess relatively broad pore size distributions. HPLC columns packed with the monodispersed particles show good efficiencies and low column pressure drops in a reversed-phase chromatographic mode.

Synthesis and properties of uniform beads based on macroporous copolymer glycidyl methacrylate–ethylene dimethacrylate: A way to improve separation media for HPLC

AbstractMonodisperse beads based on hydrolyzed macroporous poly(glycidyl methacrylate‐co‐ethylene dimethacrylate) for use as size‐exclusion HPLC packings were synthesized by the method of “activated” swelling of polystyrene seeds followed by a suspension polymerization of both methacrylates. Effects of the type and fraction of the swelling agent, inert porogenic solvent, and cross‐linking monomer on the uniformity of the particles, extent of the specific surface area, pore volume, pore size, and pore‐size distribution and chromatographic properties (size‐exclusion limit and column effciency) have been investigated. Trends leading to the synthesis of macroporous particles with predesigned properties for use in size‐exclusion high‐performance liquid chromatography in both aqueous and organic mobile phase were studied. © 1992 John Wiley &amp; Sons, Inc.

Preparation and properties of porous particles from trimethylolpropane trimethacrylate

AbstractSpherical macroporous particles, intended for use as carrier particles in various types of column operations, were prepared by suspension and microsuspension polymerization of trimethylolpropane trimethacrylate (TRIM), in the presence of various amounts of porogenic agents (toluene or mixtures of toluene and isooctane). The texture and properties of the particles varied markedly with the amount and composition of the porogenic agent, and were also affected by the choice of suspension stabilizer. Macroporous particles were prepared, ranging in total pore volume from 0.4 to 3.6 cm3/g, and in specific surface area from 7 to &gt;450 m2/g. All of the particles contained macropores, and except for those with very low specific surface area, contained a certain fraction of micropores (diameter &lt;60 Å). The presence of micropores could be eliminated completely by decreasing the monomer concentration and increasing the isooctane content of the porogenic agent to 50 wt.‐%. Increasing the isooctane content also resulted in an increase in the total pore volume and a decrease in the tendency of the polymer matrix to swell in toluene. The effect on properties of particles by heat treatment at 130°C and by grafting poly(methyl methacrylate) onto the particles was also studied.The amount of unreacted double bonds of the particles could be varied by varying the polymerization temperature. The presence of easily accessible double bonds is important as they can be utilized for functionalization of the rigid macroporous particles.

Polymer-bound chiral polymers for use in enantiomer separations

A new kind of chiral stationary phase was prepared by immobilization of the synthetic chiral polymer poly(N-acryloyl-S-phenylalanine ethyl ester) in macroporous polymer particles. Immobilization greatly improved the chromatographic performance of the chiral polymer almost without loss of selectivity. Rigid, macroporous particles were prepared by suspension polymerization of trimethylolpropane trimethacrylate. The chiral polymer was introduced into these particles by polymerizing the chiral monomer in their pore system. Immobilization was obtained by copolymerization with unreacted double bonds present in the matrix particles. Composite particles were prepared with up to 40% (w/w) of chiral polymers. The new materials, and materials prepared using alternative immobilization techniques, were evaluated for their use in chromatography with racemic compounds. The results suggest that enantioselectivity is dependent on the conformational freedom of flexibility of the chiral polymer.

Monosized stationary phases for chromatography

Polymer particles with a highly monodisperse particle size distribution were produced by a two-step microsuspension method. This process is based on the activation of monosized polymer seed particles by the introduction of a low-molecular-weight material, which leads to a large increase in the monomer swelling capacity of the seed particles. The versatility of the process allows the preparation of polymer monosized compact or macroporous particles of predetermined particle size in the range 1–100 μm and with application of a wide selection of polymeric materials. Underivatized, rigid, porous particles were developed for size-exclusion chromatography in organic solvents. The uniform packing that may be achieved with monosized particles has resulted in chromatographic columns with unusual efficiency and separation capacity. By coating the particle surface with a hydrophilic cross-linked polymer, supports for aqueous phase ion-exchange and size-exclusion chromatography may be produced.

Internal structures of wide-pore packing materials for high-performance liquid chromatography studied by transmission electron microscopy

Internal structures of high-performance liquid chromatographic column packing materials, including porous glass particles, silica gels and polymer gels, were studied by transmission electron microscopy. Clear micrographs of the internal structures were obtained for macroporous particles by using ultrathin sections, with staining in the case of polymer gels. Corpuscular structures were seen with silica gels with relatively small pores, while porous glass particles showed a typical spongy structure. Spherical silica particles with large pores in the range 50–500 nm from different sources possess common spongy structures with a difference in the presence of a shell at the outer surface. Several silica packing materials of 30–400 nm nominal pore size were found to be mixtures of two or more kinds of silica particles of different pore sizes, which accounted for the broad, in some instances bimodal, pore size distribution found in nitrogen adsorption measurements and in inverse size-exclusion chromatography. A bimodal pore size distribution was also seen for unmixed macroporous polymer gel particles, with one of the maxima in a small pore size range, presumably due to the presence of micropores formed by the network structures in microgels.

Hydrogels in endovascular embolization. I. Spherical particles of poly(2-hydroxyethyl methacrylate) and their medico-biological properties

Spherical macroporous particles based on poly(2-hydroxyethyl methacrylate) with defined porosity, swelling and morphology have been developed, and are suitable for endovascular occlusion of various organs. Unlike cylindrical particles, spherical particles are specifically suited for transcatheteral introduction. The method chosen for the preparation of such particles was suspension radical polymerization, where the monomers were dissolved in a mixture of higher-boiling alcohols, and the solution dispersed in water. Physicochemical and medico-biological properties of spherical particles were examined. The residual amounts of monomers and other low-molecular compounds were checked; haematological analyses showed that the value 10 −5 g/g of the polymer was not toxic and contributed to an irreversible aggregation of thrombocytes. The occlusion effect in the vascular lumen was stable. The histomorphological results fully demonstrated the perfect biocompatifaility of artificial spherical emboli. The latter met the requirements of application to clinical practice.

Novel Packing for High Pressure Liquid Chromatography. Partially Alkylated and Cross-Linked PMLG Spherical Particles

Abstract Novel spherical and macroporous particles for the high pressure liquid chromatography were prepared by our unique method. The packing was composed of poly-γ-methyl-L-glutamate, and were functionalyzed by the partial cross-linking and long-chain alkylation. The packings are very rigid and show satisfactorily chromatographic properties. They are attributable to the suitable polarity and hydrophobicity introduced by polypeptide-units and long-chain alkyl groups, respectively.

Preparation and application of monodisperse polymer particles

AbstractThe swelling capacity of aqueous dispersions of particles (or droplets) containing a relatively low molecular weight, highly water‐insoluble compound (Y) may be more than a thousand times higher than that of particles consisting of polymer only. The rate, and also the degree of swelling, may furthermore be increased by having the swelling substance, which usually is a low molecular weight, slightly water soluble substance, present in a finely dispersed form. A two‐step swelling process has been described. In a first step, relatively small monodisperse polymer particles are swollen with a component Y and then with monomer or monomer mixtures, followed by polymerization. The method allows preparation of highly monodisperse particles of more than 50 μm diameter with a standard deviation of less than 2%. The particles may be prepared as core and shell particles of different density and with various surface layers, or as macroporous particles. The new particles have found application as standards in immunoassays and, most important up to now, in liquid chromatography. An extension of the process allows the production of monodisperse magnetizable particles which have found application in cell separation of cancer cells from normal cells.

Isolation of immunoreactive components from experimental and human tumour tissues and serums by high-performance gel chromatography

A method for the rapid separation of proteinous fractions by high-performance gel chromatography was described. Homogenates from tumorous and healthy tissues and blood were eluted by saline on a column packed with rigid hydrophilic macroporous particles of O-glucosa—Spheron 300. Fractions were collected and subjected to further analyses. Their antigenic activity was determined by the leucocyte adherence inhibition test method. For the specific immunoactive fractions a dependence of leucocyte adherence inhibition test values on the clinical state of sample donators has been found.