Inverse Size Exclusion Chromatography Research Articles

Influence of diluent compositions on the porous structure of methacrylate derivatives of aromatic diols and divinylbenzene

Synthesis and properties of the new aromatic tetrafunctional methacrylate monomer 2,7-(2-hydroxy-3-methacryloyloxypropoxy)naphthalene are presented. This monomer was applied for the synthesis of porous microspheres. It was copolymerized with divinylbenzene in the presence of pore-forming diluents, decan-1-ol and toluene. Influence of diluents composition on their porous structures was studied. Porous structures of the obtained microspheres in dry (from nitrogen adsorption–desorption measurements) and swollen (the inverse size exclusion chromatography method) states were compared with those synthesized from isomeric 2,3-(2-hydroxy-3-methacryloyloxypropoxy)-naphthalene and divinylbenzene.

Characterisation of porous materials for bioseparation

A set of chromatographic materials for bioseparation were characterised by various methods. Both commercial materials and new supports presenting various levels of rigidity were analysed. The methods included size-exclusion and capillary phenomena based techniques. Both batch exclusion and inverse size-exclusion chromatography were used. Gas adsorption, mercury porosimetry and thermoporometry were applied as well as a new method based on water desorption starting from the saturated state. When the rigidity of adsorbents is high enough, the agreement is reasonable between the values of the structural parameters that were determined (surface area, porosity, and pore size) by various methods. Nevertheless, a part of macroporosity may not be evidenced by inverse size-exclusion chromatography whereas it is visible by batch exclusion and the other methods. When the rigidity decreases, for example with soft swelling gels, where standard nitrogen adsorption or mercury porosimetry are no more reliable, two main situations are encountered: either the methods based on capillary phenomena (thermoporometry or water desorption) overestimate the pore size with an amplitude that depends on the method, or in some cases it is possible to distinguish water involved in the swelling of pore walls from that involved in pore filling by capillary condensation.

Effect of polymerization conditions on morphology and chromatographic characteristics of polyacrylamide‐based beds (monoliths) for capillary electrochromatography and capillary liquid chromatography

Pioneering research work on continuous or monolithic beds for LC was published in 1989. However, till date there is a lack of complex investigation of various factors affecting the morphology of the beds polymerized from water-soluble co-monomers. In this article the effect of buffer, used as polymerization medium, concentration and pH, and total monomer concentration on the morphology of the resultant continuous beds are discussed. Evaluation of the continuous beds was performed by means of capillary LC, CEC, and inverse size-exclusion chromatography.

Highly cross‐linked polymeric capillary monoliths for the separation of low, medium, and high molecular weight analytes

Highly rigid capillary monoliths with low swelling propensity were prepared within the confines of 200 microm ID fused silica capillaries via thermally induced free radical polymerization of tetrakis(4-vinylbenzyl)silane (TVBS) in the presence of 1-dodecanol and toluene. alpha,alpha'-Azobisisobutyronitrile (AIBN) was used as initiator. The resulting monoliths were optimized for the separation of low, medium, and high molecular weight analytes. The microstructure and porosity of the monoliths prepared were studied by SEM and inverse size-exclusion chromatography (ISEC). The porosity of the monolithic supports was tuned by varying the amount of initiator (i. e. AIBN) between 0.5 and 2 wt%. All monoliths were tested for a series of low molecular weight compounds including alkylbenzenes, amines, carboxylic acids, phenols, carbonyl compounds, and beta-blockers, as well as for the separation of medium molecular weight analytes such as peptides and high-molecular weight analytes such as proteins. Due to the microporous structure, the novel monoliths displayed high efficiency and performance particularly in the separation of low molecular weight analytes. Relevant chromatographic parameters including permeability, swelling propensity, and height equivalents to theoretical plates were determined.

Excess Adsorption of Commonly Used Organic Solvents from Water on Nonend-Capped C18-Bonded Phases in Reversed-Phase Liquid Chromatography

The excess adsorption isotherms of methanol, 2-propanol, acetonitrile, and tetrahydrofuran from water were measured on five silica-based packing materials by the minor disturbance method. The adsorbed amount of solvents was calculated per silica gel surface and per modeled surface of bonded phase which seem to give more correct data. Columns used in this study were nonend-capped with different coverage density of bonded C18 ligands. It allows investigating the influence of the density of residual silanols on the solvent adsorption and on adsorption properties of the surface. The stationary phases were characterized by elemental analysis and low-temperature nitrogen adsorption. Geometric parameters of the stationary phases in the column were measured by inverse size exclusion chromatography. The relative adsorption of the organic solvents from water informs on the heterogeneity of the stationary phases and on the accessibility of the unreacted silanol groups to the mobile phase. The differences in the organic solvent and water molecules for interaction with organic ligand and residual silanols are used for the estimation of the surface heterogeneity.

Migration of bacteria through a monolith

The separation of bacteria by electromigration techniques was a subject of several of our previous papers. This contribution presents the results of investigation of the porosity of the monolithic bed and migration of Staphylococcus aureus cells through it. The gigaporous monolith was thermally synthesized using glycidyl methacrylate, triethylene glycol dimethacrylate and trimethylolpropane trimethacrylate as the monomers in the presence of porogen solvent containing 1-decanol, polyethylene glycol and 2-methoxyethanol. The porous properties were evaluated by inverse size-exclusion chromatography (ISEC) using a wide range of polystyrene standards of different molecular weights. The results have shown, that large pores (ca. 300 nm) dominate in the monolithic bed structure, however much larger flow-through pores must also be present as ca. 1 μm sized S. aureus bacteria were able to migrate through the bed.

Preparation of Polymer Monoliths That Exhibit Size Exclusion Properties for Proteins and Peptides

Protein-resistant poly(ethylene glycol methyl ether acrylate-co-polyethylene glycol diacrylate) monoliths were prepared in 150 microm i.d. capillaries using novel binary porogenic solvents consisting of ethyl ether and poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) or PPO-PEO-PPO copolymer with molecular weights (MWs) from 2700 to 5800. The effects of the MWs and concentrations of these surfactants in the porogenic solvent mixture on the pore properties of the resultant monoliths were investigated. Several of the monoliths showed improvements in protein and peptide separations over an extended MW range compared to monoliths synthesized using non-surfactant porogens (i.e., low MW organic liquids). The pore size distributions were examined using inverse size-exclusion chromatography (ISEC) of a select series of proteins and peptides covering a wide MW range. It was found that the best monolith had relatively large fractions of micropores (<2 nm, 11.9%) and mesopores in the range from 2.8 to 15.7 nm (8.5%), which are important for size-exclusion separation of peptides and proteins, respectively. The new monoliths possessed high mechanical rigidity that enabled them to withstand pressures up to approximately 4000 psi.

Schrock Catalyst Triggered, Ring-Opening Metathesis Polymerization Based Synthesis of Functional Monolithic Materials

The synthesis of monolithic materials via the Schrock catalyst triggered ring-opening metathesis polymerization (ROMP) is described. Mo(N-2,6-(2-Pr)2-C6H3)(CHCMe2Ph)(OCMe3)2 (1) was used as an initiator. Using various ratios of norborn-2-ene (NBE) and 1,4,4a,5,8,8a-hexahydro-1,4,5,8-exo,endo-dimethanonaphthalene (DMN-H6) in different mixtures of micro- with macroporogens, i.e., 1,2-dichloroethane, toluene and THF with hexane or pentane, monolithic polymeric materials with continuous, interconnected pores in the micrometer range as well as with micro- and mesopores in the 1.5−300 nm range could be synthesized within the confines of 3 × 100 mm glass columns. The resulting monoliths were characterized via inverse size exclusion chromatography (ISEC) in terms of the total pore volume (Vp), the volume fraction of the pore porosity (ϵp), the volume fraction of the interstitial porosity (ϵz) and the pore size distribution. Finally, the novel monoliths were successfully used for the fast separation of proteins as well as of 9-fluorenylmethoxycarbonyl (FMOC) protected amino acids.

Synthesis and characterization of size-selective nanoporous polymeric adsorbents for blood purification

Crosslinked polystyrene co-divinylbenzene adsorbent microspheres with median diameters between 40 and 300 μm, with an enhanced proportion of mesopores in the range 4–10 nm, have been synthesized using a membrane emulsification technique. The aim was to develop an adsorbent for the selective removal of middle molecular weight uraemic toxins (size range 0.5–20 kDa), e.g. β 2-microglobulin (11.8 kDa), whilst size-excluding larger blood proteins, e.g. serum albumin (MW 69 kDa). Using inverse size exclusion chromatography, the role of size exclusion has been demonstrated using hen egg lysozyme (a surrogate for β 2-microglobulin) and human serum albumin as model proteins for batch adsorption studies. The adsorbent sample possessing a nanoporous structure with pores predominantly smaller than 10 nm successfully size-excludes serum albumin whilst displaying significant adsorption capacity for the middle molecular weight lysozyme (14.3 kDa).

Separation of Racemic Chiral Drugs Using Immobilized CHIRALPAK IA: Methodology for Preparative Scale Development

Immobilized stationary phases represent a new perspective for analytical as well as preparative scale separation of chiral species. In this study, the basic data necessary for further development of the production-scale processes using CHIRALPAK IA columns were acquired. The new immobilized chiral stationary phase CHIRALPAK IA has proven to be useful for the chiral separations of propranolol, metoprolol, guaifenesin, and α-Tetralol enantiomers. A broad range of standard and non-standard solvents used as components of mobile phases were tested and their influence on the separations was evaluated. A satisfactory separation considering further utilization of simulated moving bed chromatography was found for propranolol, guaifenesin, and α-tetralol using the following mobile phases n-Heptane/methanol/ethanolamine (05/95/0.1, v/v/v), n-Heptane/ethanol (85/15, v/v) and n-Heptane/DCM (85/15, v/v), respectively. Inverse size exclusion chromatography was implemented to measure the porosity of the column using the set of polystyrene standards. The multicomponent adsorption equilibrium was characterized by linear + Langmuir model for α-Tetralol and linear model for propranolol and guaifenesin due to their limited solubility in selected mobile phases. The isotherm model was successfully validated by comparison of pulse profiles obtained by solving the mathematical model and those obtained experimentally.

細孔を分析する手段としてのＳＥＣ，Ｉｎｖｅｒｓｅ‐ＳＥＣについて

Inverse size exclusion chromatography (Inverse - SEC) is used as a chromatographic method for determining the pore size of porous materials. Inverse SEC can be applied to swollen material particles where the conventional methods, gas adsorption and mercury porosimetry, cannot be used. This report reviews the principles and the methods of Inverse SEC.

Characterization of pore structure of a strong anion-exchange membrane adsorbent under different buffer and salt concentration conditions

The quantitative characterization of pore structure of Sartobind Q, a strongly basic membrane anion exchanger that is formed by cross-linked cellulose support and a hydrogel layer on its pore surface, was made combining the results obtained by several experimental techniques: liquid impregnation, batch size-exclusion, inverse size-exclusion chromatography, and permeability. Mercury intrusion and nitrogen sorption porosimetry were carried out for a dry cellulose support membrane in order to get additional information for building a model of the bimodal pore structure. The model incorporated the distribution of the total pore volume between transport and gel-layer pores and the partitioning of solutes of different molecular weights was expressed through the cylindrical pore model for the transport pores and random plane model for the gel layer. The effect of composition of liquid phase on the pore structure was investigated in redistilled water, phosphate and Tris–HCl buffers containing up to 1 M NaCl. Evident differences in the bimodal pore structure were observed here when both the specific volume and size of the hydrogel layer pores significantly decreased with the ionic strength of liquid phase.

Modeling electrostatic exclusion effects during ion exchange chromatography of monoclonal antibodies

Recent experimental studies have shown a reduction in dynamic- binding capacity for both monoclonal antibodies and antigen-binding fragments at very low conductivity, conditions that should generate the greatest electrostatic attraction. This behavior has been attributed to the steric and electrostatic exclusion of the charged protein from the entrance of the resin pores. This manuscript presents a quantitative mathematical description of this phenomenon. The protein partition coefficient was evaluated using models for the partitioning of a charged sphere into a charged cylindrical pore, with the pore size distribution evaluated by inverse size exclusion chromatography. The results were in very good agreement with experimental data for batch protein uptake and dynamic-binding capacity over a range of pH and conductivity. This theoretical framework provides important insights into the behavior of ion exchange chromatography for protein purification.

Rapid monoclonal antibody adsorption on dextran-grafted agarose media for ion-exchange chromatography

The binding capacity and adsorption kinetics of a monoclonal antibody (mAb) are measured for experimental cation exchangers obtained by grafting dextran polymers to agarose beads and compared with measurements for two commercial agarose-based cation exchangers with and without dextran grafts. Introduction of charged dextran polymers results in enhanced adsorption kinetics despite a dramatic reduction of the accessible pore size as determined by inverse size-exclusion chromatography. Incorporation of neutral dextran polymers in a charged agarose bead results instead in substantially lower binding capacities. The effective pore diffusivities obtained from batch uptake curves increase substantially as the protein concentration is reduced for the resins containing charged dextran grafts, but are much less dependent on protein concentration for the resins with no dextran or uncharged dextran grafts. The batch uptake results are corroborated by microscopic observations of transient adsorption in individual particles. In all cases studied, the adsorption kinetics is characterized by a sharp adsorption front consistent with a shell-progressive, diffusion limited mechanism. Greatly enhanced transport rates are obtained with an experimental resin containing charged dextran grafts with effective pore diffusivities that are 1–9 times larger than the free solution diffusivity and adsorption capacity approaching 300 mg/cm 3 of particle volume.

Characterization of pore structure of chromatographic adsorbents employed in separation of monoclonal antibodies using size-exclusion techniques

Structural properties of commercial chromatographic adsorbents designated for separation of monoclonal antibodies were investigated using size-exclusion techniques. A batch technique provided the specific pore volumes distributed among small, medium and large pores. Inverse size-exclusion chromatography yielded the partition coefficients of dextran solute probes in medium pores. These data were fitted with one- and two-parametric models corresponding to different pore-size distribution functions and the suitability of the individual models for the characterization of the examined materials was then assessed. The reliability of estimation of the parameters of log-normal distribution of cylindrical pores was investigated. Large uncertainties and a strong correlation of the mean pore diameter and width of distribution function were observed.

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

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

Textural characterization of native and n-alky-bonded silica monoliths by mercury intrusion/extrusion, inverse size exclusion chromatography and nitrogen adsorption

Native and n-alkyl-bonded ( n-octadecyl) monolithic silica rods with mesopores in the range between 10 and 25 nm and macropores in the range between 1.8 and 6.0 μm were examined by mercury intrusion/extrusion, inverse size exclusion chromatography (ISEC) and nitrogen sorption. Our results reveal very good agreement for the mesopore size distribution obtained from nitrogen adsorption (in combination with an advanced NLDFT analysis) and ISEC. Our studies highlight the importance of mercury porosimetry for the assessment of the macropore size distribution and show that mercury porosimetry is the only method which allows obtaining a combined and comprehensive structural characterization of macroporous/mesoporous silica monoliths. Our data clearly confirm that mercury porosimetry hysteresis and entrapment have different origin, and indicate the intrinsic nature of mercury porosimetry hysteresis in these silica monoliths. Within this context some silica monoliths show the remarkable result of no entrapment of mercury after extrusion from the mesopore system (i.e. for the first intrusion/extrusion cycle). The results of a systematic study of the mercury intrusion/extrusion behavior into native silica monoliths and monoliths with bonded n-alkyl groups reveals that the macro (through) pore structure, which controls the mass transfer to and from the mesopores, here mainly controls the entrapment behavior. Our data suggest that mercury intrusion/extrusion porosimetry does not only allow to obtain a comprehensive pore structure analysis, but can also serve as a tool to estimate the mass transport properties of silica monoliths to be employed in liquid-phase separation processes.

Characterization of polymer-based monolithic capillary columns by inverse size-exclusion chromatography and mercury-intrusion porosimetry

Organic-polymer monolithic capillary columns were prepared in fused-silica capillaries by a radical copolymerization reaction of butyl methacrylate and ethylene dimethacrylate monomers in the presence of 1,4-butanediol and 1-propanol as porogen solvents and azobisisobutyronitrile as the initiator. The porous properties could be influenced by changing the ratio of porogen solvents, while keeping the monomer content constant, or by changing the ratio of monomers to porogen in the polymerization mixture. The resulting chromatographic properties, such as porosity, permeability were determined under high-pressure liquid chromatography (HPLC) conditions. The mesopore size distributions of the monolithic materials determined with inverse size-exclusion chromatography (ISEC) were compared with those measured by mercury-intrusion porosimetry. Both techniques are complementary. While mercury-porosimetry measures the entire range of pore sizes and provides more physical information on the monoliths, ISEC is very suitable for determining the size of mesopores in the swollen monoliths.

Treatment in Swelling Solutions Modifying Cellulose Fiber Reactivity – Part 1: Accessibility and Sorption

AbstractOf prime importance in reactions involving insoluble cellulosic fibers is the sorption of reagents, which is governed by their degrees of accessibility in substrates. Swelling treatments of cellulosics in alkali solutions alter substrate accessibility leading to changes in their reactivity. In this paper, the first of a two‐part series, we collate and examine the results from various studies involving different techniques to characterize modifications in cellulosic fibers after swelling treatments in alkali solutions. Results from measurements of structure and accessibility in fibers with techniques such as water retention, inverse size exclusion chromatography (ISEC), iodine sorption, fiber diameters, and fiber‐splitting propensities indicate that the influence of swelling treatments on fiber structure/accessibility is differs with alkali type. The results show that a non‐uniform rather than uniform distribution of reagents within structures is a more accurate representation of reactions involving swollen cellulosic fibers. Hence, the observed changes in cellulose‐fiber reactivity are governed by the degrees of fiber swelling, and reagent sorption and accessibility during swelling treatments.

New chiral and restricted-access materials containing glycopeptides as selectors for the high-performance liquid chromatographic determination of chiral drugs in biological matrices

Two new chiral and restricted-access materials containing glycopeptide antibiotics as chiral selectors (chiro-Glyco-RAM) were designed, suitable for the direct HPLC injection of biological fluids containing chiral drugs without any sample pre-treatment or pre-columns coupling. The external surface of the porous silica support was covered with a bio-compatible hydrophilic polymeric network (polyvinyl alcohol, PVA) while the chiral phase based on either teicoplanin (TE) or teicoplanin aglycone (TAG) was exclusively confined to the internal region. The chiro-Glyco-RAM supports were synthesized by the following steps: (a) introduction of 3-aminopropyl groups on 100 Å pore size silica gel; (b) activation of the aminopropylated silica with 1,6-diisocyanatohexane; (c) functionalization of the external region of the porous silica with PVA; (d) covalent linking of TE/TAG to the internal surface. The average pore diameter of the chiro-Glyco-RAM supports, calculated by inverse size-exclusion chromatography (ISEC), was about 80 Å and able to exclude macromolecules heavier than about 20,000 Da (such as the most abundant serum proteins) from the pores. The recovery of bovine serum albumin (BSA) was almost quantitative. HPLC analyses of model chiral drugs were performed using hydro-organic mobile phases consisting of an organic solvent (acetonitrile or methanol) and aqueous solutions of ammonium acetate (0.020 M) or ammonium formate (0.0025–0.0050 M).