Supermacroporous cryogel matrix for integrated protein isolation. Immobilized metal affinity chromatographic purification of urokinase from cell culture broth of a human kidney cell line

A new type of supermacroporous, monolithic, cryogel affinity adsorbent was developed, allowing the specific capture of urokinase from conditioned media of human fibrosarcoma cell line HT1080. The affinity adsorbent was designed with the objective of using it as a capture column in an integrated perfusion/protein separation bioreactor setup. A comparative study between the utility of this novel cryogel based matrix and the conventional Sepharose based affinity matrix for the continuous capture of urokinase in an integrated bioreactor system was performed. Cu(II)-ion was coupled to epoxy activated polyacrylamide cryogel and Sepharose using iminodiacetic acid (IDA) as the chelating ligand. About 27-fold purification of urokinase from the conditioned culture media was achieved with Cu(II)-IDA-polyacrylamide cryogel column giving specific activity of about 814 Plough units (PU)/mg protein and enzyme yields of about 80%. High yields (95%) were obtained with Cu(II)-IDA-Sepharose column by virtue of its high binding capacity. However, the adsorbent showed lower selectivity as compared to cryogel matrix giving specific activity of 161 PU/mg protein and purification factor of 5.3. The high porosity, selectivity and reasonably good binding capacity of Cu(II)-IDA-polyacrylamide cryogel column make it a promising option for use as a protein capture column in integrated perfusion/separation processes. The urokinase peak pool from Cu(II)-IDA-polyacrylamide cryogel column could be further resolved into separate fractions for high and low molecular weight forms of urokinase by gel filtration chromatography on Sephacryl S-200. The selectivity of the cryogel based IMAC matrix for urokinase was found to be higher as compared to that of Cu(II)-IDA-Sepharose column.     

High throughput processing of particulate-containing samples using supermacroporous elastic monoliths in microtiter (multiwell) plate format

Two steps in parallel processing of multiple biosamples, namely, sample clarification and capture of the target protein, were integrated and combined with the direct assay of captured protein using a newly developed microtiter (96-well) plate system based on the monoliths of hydrophilic elastic supermacroporous material, cryogel. Cryogel monoliths have pore size large enough for microbial and mammalian cells to pass through unretained. Moreover, cryogel monoliths are elastic allowing them to be slightly compressed and easily introduced into the wells. When expanded, cryogel monoliths fill the well tightly with no risk of leakage in between the monolith and the walls of the well. The capillary forces keep the liquid inside the pores of the cryogel monolith making the monolith columns drainage protected. The application of a certain volume of liquid on top of a cryogel monolith column results in the displacement of exactly the same volume of liquid from the column. The concept of using supermacroporous gels in 96-well plate format offers new possibilities to the biotechnologist allowing separation of particulate matter, capturing of soluble material from particle containing media, and parallel assay of large number of non-clarified samples.     

Iminodiacetic acid functionalised organopolymer monoliths: application to the separation of metal cations by capillary high-performance chelation ion chromatography

Lauryl methacrylate-co-ethylene dimethacrylate monoliths were polymerised within fused silica capillaries and subsequently photo-grafted with varying amounts of glycidyl methacrylate (GMA). The grafted monoliths were then further modified with iminodiacetic acid (IDA), resulting in a range of chelating ion-exchange monoliths of increasing capacity. The IDA functional groups were attached via ring opening of the epoxy group on the poly(GMA) structure. Increasing the amount of attached poly(GMA), via photo-grafting with increasing concentrations of GMA, from 15 to 35 %, resulted in a proportional and controlled increase in the complexation capacity of the chelating monoliths. Scanning capacitively coupled contactless conductivity detection (sC⁴D) was used to characterise and verify homogenous distribution of the chelating ligand along the length of the capillaries non-invasively. Chelation ion chromatographic separations of selected transition and heavy metals were carried out, with retention factor data proportional to the concentration of grafted poly(GMA). Average peak efficiencies of close to 5,000 N/m were achieved, with the isocratic separation of Na, Mg(II), Mn(II), Co(II), Cd(II) and Zn(II) possible on a 250-mm-long monolith. Multiple monolithic columns produced to the same recipes gave RSD data for retention factors of <15 % (averaged for several metal ions). The monolithic chelating ion-exchanger was applied to the separation of alkaline earth and transition metal ions spiked in natural and potable waters.     

Preparation of composite monoliths of quaternized chitosan and diatom earth for protein separation

In this study, composite monoliths with porous structures were prepared using quaternized chitosan and diatom earth for protein separation. Quaternized chitosan (N-[(2-hydroxy-3-trimethylammonium)propyl] chitosan chloride) dissolved in water was mixed with diatom earth and crosslinked with glutaraldehyde under low-temperature conditions to form a cryogel. Interconnected porous monoliths were obtained after removing ice crystals from the cryogel. The monoliths adsorbed bovine serum albumin selectively from the solution mixture of bovine serum albumin and bovine ɤ-globulin, and bovine ɤ-globulin was recovered in the flow-through fraction. The adsorption selectivity was enhanced by changing the solution pH from 6.8 to 5.5. The adsorption of bovine serum albumin by the monolith was replicated at least five times following its washing with a buffer containing 400 mM NaCl and subsequent regeneration with a 10 mM acetate buffer. The composited monolith is a promising adsorbent for the removal of acidic proteins, such as serum albumin contamination in neutral proteins, for example, ɤ-globulins, in bioproduction processes.     

Novel separation medium spongy monolith for high throughput analyses

Sponge-like material was utilized as novel chromatographic media for high throughput analyses. The pore size of the sponge-like material was several dozen micrometer, and was named spongy monolith because it consists of continuous structured copolymers, which was made of poly(ethylene-co-vinyl acetate), such as monolithic materials including silica monoliths and organic polymer monoliths. The spongy monolith was packed into a stainless steel column (100 mm × 4.6 mm I.D.) and evaluated in liquid chromatography (LC) with an on-line column-switching LC concentration system. The results indicate that the packed column could be used with high flow rates and low back pressure (9.0 mL/min at 0.5 MPa). Furthermore, bisphenol A was quantitatively recovered by on-line column-switching LC concentration with the spongy monolithic column. Additionally, the adsorption capacity and physical strength of the media was enhanced via chemical modification of spongy monoliths using glycerol dimethacrylate. The results compared with original spongy monolith demonstrated that a higher adsorption capacity was achieved on a shorter column, and a stable low back pressure was obtained at high throughput elution even with a longer column.     

Column preconcentration of lead in aqueous solution with macroporous epoxy resin-based polymer monolithic matrix

The objective of this article was to investigate the feasibility of epoxy resin-based monoliths prepared by stepwise polymerization and column preconcentration of metal ions using large-scale monolithic matrix. A novel macroporous polymer monolith matrix was prepared from epoxy resin (EP) and ethylenediamine (EDA) and pore-forming reagent (polyethylene glycol, PEG-1000) by in situ step-addition polymerization. The morphology of the resulting polymer monolith was characterized by scanning electron microscopy (SEM). A solid-phase extraction (SPE) cartridge prepared from a simple glass-tube was used for the preconcentration and determination of Pb(II) combined with flame atomic absorption spectroscopy (FAAS). The characteristics of the monoliths for the extraction of Pb(II) in aqueous solution were investigated. The experimental results showed that trace Pb(II) ions could be quantitatively preconcentrated in the pH range of 4.0-9.0 with recoveries of >95%. The maximum static adsorption capacity of the monolith adsorbent was 106.8 mg g⁻¹. The column was eluted by 1.0 mol L⁻¹ HNO₃ and recovery of Pb(II) was more than 97%. Moreover, the polymer monolith adsorbent shows superior reusability and stability. The precision and the accuracy of the proposed procedure were satisfactory by analyzing a standard reference material and three natural water samples. It was shown that the EP-EDA monolith was suitable for the preconcentration of environmental Pb(II) as an ion-selective SPE adsorbent.     

Mass transfer characteristics of plasmids in monoliths

The hydrodynamic properties and pore-structure of monoliths based on functionalized poly(glycidyl methacrylate-ethylene dimethacrylate) were characterised by pulse response experiments using different probes representing a wide range of molecular mass. On a small scale, band spreading was found to be caused to the extent of more than 90% by extra-column effects. These monoliths have large channel diameters, providing a suitable chromatography adsorbent for processing of large molecules. Dynamic and static binding capacity for plasmid DNA was investigated. For our model plasmid, consisting of 4.9 kbp, a capacity of 7 mg/mL was observed in comparison to 0.3 mg/mL for a conventional medium designed for protein separation. When plasmids were loaded on the monolith a gradual increase in pressure drop was observed. The channels filled up and the cross-sectional area available for liquid flow decreased. Therefore, a higher pressure drop was observed during elution. This is caused by (i) shrinking of the channels as effect of the high salt concentration, (ii) high viscosity of the mobile phase due to high concentration of plasmids, and (iii) an increase of the hydrodynamic radius of the plasmid with salt concentration from 45 nm at 150 mM to 70 nm at 2 M NaCl, as measured by dynamic light scattering. These types of monoliths are considered to be the preferred adsorbents for plasmid separation.     

Poly-L-Histidine Attached Poly(glycidyl methacrylate) Cryogels for Heavy Metal Removal

Poly-L-histidine immobilized poly(glycidyl methacrylate) (PGMA) cryogel discs were used for the removal of heavy metal ions [Pb(II), Cd(II), Zn(II) and Cu(II)] from aqueous solutions. In the first step, PGMA cryogel discs were synthesized using glycidyl methacrylate (GMA) as a basic monomer and methylene bisacrylamide (MBAAm) as a cross linker in order to introduce active epoxy groups through the polymeric backbone. Then, the metal chelating groups are incorporated to cryogel discs by immobilizing poly-L-histidine (mol wt ≥ 5000) having poly-imidazole ring. The swelling test, fourier transform infrared spectroscopy and scanning electron microscopy were performed to characterize both the PGMA and poly-L-histidine immobilized PGMA [P-His@PGMA] cryogel discs. The effects of the metal ion concentration and pH on the adsorption capacity were studied. These parameters were varied between 3.0–6.0 and 10–800 mg/L for pH and metal ion concentration, respectively. The maximum adsorption capacity of heavy metal ions of P-His@PGMA cryogel discs were 6.9 mg/g for Pb(II), 6.4 mg/g for Cd(II), 5.6 mg/g for Cu(II) and 4.3 mg/g for > Zn(II). Desorption of heavy metal ions was studied with 0.1 M HNO₃ solution. It was observed that cryogel discs could be recurrently used without important loss in the adsorption amount after five repetitive adsorption/desorption processes. Adsorption isotherms were fitted to Langmuir model and adsorption kinetics were suited to pseudo-second order model. Thermodynamic parameters (i.e. ΔH° ΔS°, ΔG°) were also calculated at different temperatures.     

Preparation and characterization of imprinted monolith with metal ion as pivot

This report provided the first example of using pivot concept to prepare monolithic molecularly imprinted polymers (MIPs) with ketoprofen (KET) imprints, in which metal ions were employed as mediator between the functional monomer and the template to achieve higher fidelity of imprint. To solve metal ions in pre-polymerization system, a new ternary porogen of dimethyl sulfoxide-toluene-isooctane was developed for preparation of MIP monoliths with high porosity and good permeability. The effect of polymerization parameters such as the nature of metal ions, the ratio of template to metal ion and the degree of crosslinking, on the permeability, morphology and affinity of the metal ion mediated MIP monolith were studied. The experiments demonstrated that Ni(2+), Co(2+) and Zn(2+) can be applied as pivot to prepare KET-imprinted monolith. Relative to monolithic MIP without metal ions, all the ion-mediated macropore MIP monoliths showed enhanced permeability, capacity factor and selectivity factor. High permeability (1.06×10(-7)mm(2)) was obtained on the Co(2+)-mediated MIP monolith and great selectivity factor (3.84) was achieved on the Ni(2+)-mediated one. The stoichiometric displacement model was constructed to investigate the recognition mechanism of metal-ion mediated MIP. The results indicate that metal ion as pivot not only improves the affinity but also allows the fine-tuning on the macroporous structure of MIP monolith.     

Efficient Removal of Pb(II) from Aqueous Medium Using Chemically Modified Silica Monolith

The adsorptive removal of lead (II) from aqueous medium was carried out by chemically modified silica monolith particles. Porous silica monolith particles were prepared by the sol-gel method and their surface modification was carried out using trimethoxy silyl propyl urea (TSPU) to prepare inorganic–organic hybrid adsorbent. The resultant adsorbent was evaluated for the removal of lead (Pb) from aqueous medium. The effect of pH, adsorbent dose, metal ion concentration and adsorption time was determined. It was found that the optimum conditions for adsorption of lead (Pb) were pH 5, adsorbent dose of 0.4 g/L, Pb(II) ions concentration of 500 mg/L and adsorption time of 1 h. The adsorbent chemically modified SM was characterized by scanning electron microscopy (SEM), BET/BJH and thermo gravimetric analysis (TGA). The percent adsorption of Pb(II) onto chemically modified silica monolith particles was 98%. An isotherm study showed that the adsorption data of Pb(II) onto chemically modified SM was fully fitted with the Freundlich and Langmuir isotherm models. It was found from kinetic study that the adsorption of Pb(II) followed a pseudo second-order model. Moreover, thermodynamic study suggests that the adsorption of Pb(II) is spontaneous and exothermic. The adsorption capacity of chemically modified SM for Pb(II) ions was 792 mg/g which is quite high as compared to the traditional adsorbents. The adsorbent chemically modified SM was regenerated, used again three times for the adsorption of Pb(II) ions and it was found that the adsorption capacity of the regenerated adsorbent was only dropped by 7%. Due to high adsorption capacity chemically modified silica monolith particles could be used as an effective adsorbent for the removal of heavy metals from wastewater.     

Characterisation of grafted weak anion-exchange methacrylate monoliths

A weak ion-exchange grafted methacrylate monolith was prepared by grafting a methacrylate monolith with glycidyl methacrylate and subsequently modifying the epoxy groups with diethylamine. The thickness of the grafted layer was determined by measuring permeability and found to be approximately 90nm. The effects of different buffer solutions on the pressure drop were examined and indicated the influence of pH on the permeability of the grafted monolith. Protein separation and binding capacity (BC) were found to be flow-unaffected up to a linear velocity of 280cm/h. A comparison of the BC for the non-grafted and grafted monolith was performed using beta-lactoglobulin, bovine serum albumin (BSA), thyroglobulin, and plasmid DNA (pDNA). It was found that the grafted monolith exhibited 2- to 3.5-fold higher capacities (as compared to non-grafted monoliths) in all cases reaching values of 105, 80, 71, and 17mg/ml, respectively. It was determined that the maximum pDNA capacity was reached using 0.1M NaCl in the loading buffer. Recovery was comparable and no degradation of the supercoiled pDNA form was detected. Protein z-factors were equal for the non-grafted and grafted monolith indicating that the same number of binding sites are available although elution from the grafted monolith occurred at higher ionic strengths. The grafted monolith exhibited lower efficiency than the non-grafted ones. However, the baseline separation of pDNA from RNA and other impurities was achieved from a real sample.     

Fabrication of high-permeability and high-capacity monolith for protein chromatography

A novel approach for the fabrication of macroporous poly(glycidyl methacrylate-ethylene glycol dimethacrylate) monolith is presented. The method involved the use of sodium sulfate granules and organic solvents as co-porogens. Compared with the conventional monoliths [ML-(1-3)] using organic solvents only as a porogen, the improved monoliths [MLS-(1-3)] showed not only higher column efficiency and dynamic binding capacity (DBC) for protein (bovine serum albumin, BSA), but also higher column permeability and lower back pressure. It is considered that the superpores introduced by the solid granules played an important role for the improvement of the monolith performance. Moreover, poly(glycidyl methacrylate-diethylamine) tentacles were grafted onto the pore surface of MLS-3 monolith. This has further increased the DBC of BSA to 74.7 mg/ml, about three times higher than that of the monoliths without the grafted tentacles. This grafting does not obviously decrease the column permeability, so a new monolith of high column permeability and binding capacity has been produced for high-performance preparative protein chromatography.     

Affinity cryogel monoliths for screening for optimal separation conditions and chromatographic separation of cells

Suitable conditions for separating cells using a chromatographic procedure were evaluated in parallel chromatography on minicolumns. A 96-hole minicolumn plate filled with cryogel monoliths (18.8 mm x 7.1 mm ?) with immobilized concanavalin A was used. Chromatographic columns (113 mm x 7.1 mm ?) were used for chromatographic resolution of a mixture of Saccharomyces cerevisiae and Escherichia coli cells. Separation of a cell mixture containing equal amounts of cells of both types performed in a column format under the determined optimal conditions, resulted in a quantitative capture of applied S. cerevisiae cells, while E. coli passed through the column. Bound S. cerevisiae cells were released by flow-induced detachment and by compression of the adsorbent in the presence of 0.3 M methyl alpha-D-manno-pyranoside. The flowthrough and the eluted fractions were analyzed by plate counting and by registering metabolic activity of S. cerevisiae cells in the eluted fractions after capturing on ConA-cryogel monoliths in a 96-minicolumn plate format. The flowthrough fraction contained E. coli cells with nearly 100% purity, whereas the fraction eluted by compression of the adsorbent contained viable S. cerevisiae cells with 95% purity. Thus, an efficient chromatographic separation of cells was achieved using affinity cryogel column.     

Boronate Affinity Monolith with a Gold Nanoparticle‐Modified Hydrophilic Polymer as a Matrix for the Highly Specific Capture of Glycoproteins

As low abundance is the great obstacle for glycoprotein analysis, the development of materials with high efficiency and selectivity for glycoprotein enrichment is a prerequisite in glycoproteome research. Herein, we report a new kind of hydrophilic boronate affinity monolith by attaching 4‐mercaptophenylboronic acid (MPBA) with 2‐mercaptoethylamine (MPA) on the gold nanoparticle‐modified poly(glycidyl methacrylate‐co‐poly(ethylene glycol) diacrylate)) monolith for glycoprotein enrichment. With poly(ethylene glycol) diacrylate as the cross‐linker and the further modification of gold nanoparticles, the matrix has advantages of good hydrophilicity and enhanced surface area, which are beneficial to improve the enrichment selectivity and efficiency for glycoproteins. The attachment of MPBA and MPA provide intramolecular B?N coordination, which could further enhance the specificity of glycoprotein capture. Such a boronate affinity monolith was applied to enrich horseradish peroxidase (HRP) from the mixture of HRP and bovine serum albumin (BSA), and high selectivity was obtained even at a mass ratio of 1:1000. In addition, the binding capacity of ovalbumin on such monolith reached 390 μg g^?1. Furthermore, the average recovery of HRP on the prepared affinity monoliths was (84.8±1.9) %, obtained in three times enrichment with the same column. Finally, the boronate affinity monolith was successfully applied for the human‐plasma glycoproteome analysis. As a result, 160 glycoproteins were credibly identified from 9 μg of human plasma, demonstrating the great potential of such a monolith for large‐scale glycoproteome research.     

Chromatographic separation of cytidine triphosphate from fermentation broth of yeast using anion-exchange cryogel

A novel separation method was developed to isolate directly cytidine triphosphate (CTP) from fermentation broth of yeast using anion-exchange supermacroporous cryogel. The anion-exchange cryogel with tertiary amine groups was prepared by graft polymerization. The breakthrough characteristics and elution performance of pure CTP in the cryogel bed were investigated experimentally and the CTP binding capacity was determined. Then the separation experiments of CTP from crude fermentation broth of yeast using the cryogel column were carried out using deionized water and 0.01 M HCl as washing buffer, respectively. The chromatographic behavior was monitored and analyzed. The purity and concentration of the obtained CTP in these processes were determined quantitatively by HPLC. The maximal purity of CTP obtained at the condition of 0.01 M HCl as washing buffer and 0.5 M NaCl in 0.01 M HCl as elution buffer reached 93%.     

A New Metal-Chelated Cryogel for Reversible Immobilization of Urease

Poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) [poly(HEMA-GMA)] cryogel was synthesized by cryopolymerization technique at frozen temperature. Iminodiacetic acid (IDA) was then attached covalently to the cryogel as a chelating agent. Then, poly(HEMA-GMA)-IDA cryogel was chelated with Ni(II) ions and this novel metal affinity support was used for adsorption of urease from its aqueous solution. Urease adsorption experiments were carried out in a continuous system by using a peristaltic pump. Maximum urease adsorption onto poly(HEMA-GMA)-IDA-Ni(II) cryogel was found to be 11.30 mg/g cryogel at pH 5.0 acetate buffer and in 25 °C medium temperature. Urease adsorption capacity decreased with increasing ionic strength and increasing chromatographic flow rate. Adsorption kinetics of urease onto poly(HEMA-GMA)-IDA-Ni(II) cryogel was also investigated and it was found that Langmuir adsorption model is applicable for this adsorption study. This novel immobilized metal affinity chromatography support was used 10 times without any decrease at their adsorption capacity. It was also observed that urease enzyme was repeatedly adsorbed and desorbed without significant lost in enzymatic activity.     

Post‐polymerization modification of a new reactive monolith for reversed phase and hydrophilic interaction capillary electrochromatography of neutral,polar, and biologically active compounds

A reactive monolith based on the polymerization of 3‐chloro‐2‐hydroxypropyl methacrylate, (HPMA‐Cl), with a crosslinking agent, ethylene glycol dimethacrylate (EDMA), was synthesized and post‐functionalized with a macromolecular ligand polyethyleneimine. Monolithic columns with controlled permeability and pore structure were prepared by free radical polymerization in the presence of a binary porogenic mixture of isopropanol and decanol. The presence of chloropropyl functionality in the pristine monolith allowed the synthesis of a post‐fuctionalized monolith carrying cationic groups that was used to control the magnitude of electroosmotic flow (EOF) in electrochromatographic separation. In the synthesis of pristine monoliths, the feed concentration of functional monomer (ie, HPMA‐Cl) was changed between 30 and 60 v/v % for obtaining cationic monoliths providing satisfactory electrochromatographic separation. The best electrochromatographic performance was obtained with the polyethyleneimine functionalized monolith prepared by using the pristine monolith obtained by 60% (v/v) monomer concentration. This monolith was used in reversed phase and hydrophilic interaction capillary electrochromatography modes for the separation of alkylbenzenes, polycyclic aromatic hydrocarbons, phenols, and nucleosides, using mobile phases with low acetonitrile (ACN) contents ranging between 20% and 35% (v/v). This ACN range was remarkably lower than the content of ACN used on the hydrophilic polymethacrylate‐based monoliths reported previously (ie, >90%). The plate heights up to 5.3 μm were obtained for the separation of nucleosides with the environmental friendly mobile phases whose ACN contents were also remarkably lower than that of similar polymethacrylate‐based monoliths.     

Preparation of monoliths from single crosslinking monomers for reversed-phase capillary chromatography of small molecules

Highly cross-linked networks resulting from single crosslinking monomers were found to enhance the concentrations of mesopores in, and the surface areas of, polymeric monoliths. Four crosslinking monomers, i.e., bisphenol A dimethacrylate (BADMA), bisphenol A ethoxylate diacrylate (BAEDA, EO/phenol=2 or 4) and pentaerythritol diacrylate monostearate (PDAM), were used to synthesize monolithic capillary columns for reversed phase liquid chromatography (RPLC) of small molecules. Tetrahydrofuran (THF) and decanol were chosen as good and poor porogenic solvents for BAEDA-2 and BAEDA-4 monoliths. For the formation of the BADMA monolith, THF was replaced with dimethylformamide (DMF) to improve the column reproducibility. Appropriate combinations of THF, isopropyl alcohol and an additional triblock poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) or PPO-PEO-PPO porogen were found to be effective in forming rigid PDAM monoliths with the desired porosities. Selection of porogens for the BADMA and PDAM monoliths was investigated in further detail to provide more insight into porogen selection. Isocratic elution of alkyl benzenes at a flow rate of 0.3 μL/min was conducted for BADMA and PDAM monoliths. The peaks showed little tailing on both monoliths without addition of acid to the mobile phase. The column efficiency measured for pentylbenzene using the BADMA monolithic column was 60,208 plates/m (k=7.9). Gradient elution of alkyl benzenes and alkyl parabens was achieved with high resolution. Optimized monoliths synthesized from all four crosslinking monomers showed high permeability, and demonstrated little swelling or shrinking in different polarity solvents. Column preparation was highly reproducible; relative standard deviation (RSD) values were less than 1.2% and 7.5% based on retention times and peak areas, respectively, of alkyl benzenes.     

Organized cryogel composites with 3D hierarchical porosity as an extraction adsorbent for nucleosides

Macroscopic monoliths are highly desirable in many fields of application. Herein, well organized organic–inorganic cryogel composite with a three‐dimensional hierarchical meso‐ and macroporous structure are presented, which were produced by in situ copolymerization of mesoporous multifunctional silica (size: 1–20 μm; pore: 2–20 nm mostly) and monomers (hydroxyethyl methacrylate and diallyldimethylammonium chloride) in water below the freezing point. This copolymerization method effectively adjusted the macropores of the basic cryogel, and the nanosilica was more homogeneously dispersed in the basic cryogel. The specific surface area of the cryogel composite was increased 17 times versus than that of the basic cryogel. The abundant meso‐ and macroporous pores on the cryogel composite provided sufficient reactive sites favorable for the efficient mass transport of target compounds. When the cryogel composite, as solid phase extraction adsorbent, was coupled with high‐performance liquid chromatography, an analytical tool, the nucleosides were quantified with good selectivity, lower detection limits (0.9–1.3 ng/mL) and satisfactory recoveries of greater than 80% from spiked human serum.     

Silica nanoparticle-templated methacrylic acid monoliths for in-line solid-phase extraction–capillary electrophoresis of basic analytes

Polymeric ion-exchange monoliths typically exhibit low capacities due to the limited surface area on the globules of the monoliths. The ion-exchange binding of protonated weakly basic analytes on deprotonated carboxylate sites on methacrylate polymer monoliths has been increased by templating the monoliths with silica nanoparticles. The templating method is achieved by adding the nanoparticles as a suspension to the polymerisation mixture. After polymerisation, the nanoparticles are removed by washing the monolith with strong base. Monolithic columns prepared using this procedure have exhibited a 33-fold increase in ion-exchange capacity when compared to untemplated monoliths prepared and treated under similar conditions. The templating procedure does not alter the macroporous properties of the polymer monolith, confirmed through scanning electron microscopy and BET surface area analysis, but provides increased capacity predominantly through the re-orientation of more carboxylic acid groups. The resulting increase in ion-exchange capacity has proven to be useful for the preconcentration and separation of neurotransmitters by in-line solid-phase extraction–capillary electrophoresis. The increased capacity of the templated monolith allowed the injection time to be increased 10 times over that of an untemplated monolith, allowing 10 times more sample to be injected with the efficiencies and recoveries remaining unaffected. The enhancement in sensitivity for the test mixture of neurotransmitter (dopamine, norepinephrine and metanephrine) ranged 1500–1900 compared to a normal hydrodynamic injection in capillary electrophoresis. Efficiencies obtained for the neurotransmitters were 100 000–260 000 plates, typical of those obtained in capillary zone electrophoresis. The applicability of the increased capacity silica nano-templated polymer monolith was demonstrated by analysing trace levels of caffeine in biological, food and environmental samples.