Effect of hydrophilic/lipophilic balance on the porogenic properties of non-ionic surfactants for monolith preparation and chromatographic separation.

The effect of hydrophilic/lipophilic balance (HLB) of polyoxyethylene ethers of different chain lengths on the microporogenic properties of the Brij surfactants has been studied. The objective of this work is to help better understand the role of each porogen and to set criteria for selecting the proper non-ionic surfactant, based on the HLB value. Seven recipes of different porogen compositions were first prepared and the highest efficiency was achieved using decane/decanol/dodecanol mixture with Brij® 30. Then, four other Brij surfactants covering the entire HLB scale were tested, and the prepared monoliths were characterized by SEM, BET, FT-IR and chromatography. The results showed that increasing the HLB from 9.72 to 18.84 was accompanied by an increase in monolith density and surface areas. The optimum HLB range was found to be 10 to 15. Surfactants of lower HLB formed either nonporous or less efficient columns, while those of higher HLB formed non-permeable columns. Adjusting the HLB was possible by mixing surfactants of different HLB. The prepared monoliths could be used in the isocratic mode with a mobile phase consisting of a mixture of ACN and water (20:80, v/v) at a flow rate of 1.5 µL min-1 to separate five sulfa drugs. The separation results showed that the elution order of the compounds correlated with their lipophilicity, with sulfamerazine (logp = 0.52) being the first to elute, and sulfaquinoxoline (logp=1.70) being the most retained. The asymmetry factors of the separated compounds ranged between 1.18 and 1.25, and the resolution was found to be in the range 2.92-7.80. The prepared monoliths could be also successfully separate a mixture of four different nonsteroidal anti-inflammatory drugs and a mixture of four benzoic acid derivatives. This work assists in optimizing the surfactant-based porogenic mixture to meet the desired porosity, surface area, morphology and chromatographic separation requirements.

The development of microfluidic-based western blotting: Technical advances and future perspectives.

Over the past two decades significant technical advancement in the field of western blotting has been made possible through the utilization of microfluidic technologies. In this review we provide a critical overview of these advancements, highlighting the advantages and disadvantages of each approach. Particular attention is paid to the development of now commercially available systems, including those for single cell analysis. This review also discusses more recent developments, including algorithms for automation and/or improved quantitation, the utilization of different materials/chemistries, use of projection electrophoresis, and the development of triBlots. Finally, the review includes commentary on future advances in the field based on current developments, and the potential of these systems for use as point-of-care devices in healthcare.

Styrene-based polymerised high internal phase emulsions using monomers in the internal phase as co-surfactants for improved liquid chromatography.

Poly(styrene-co-divinylbenzene)-based monoliths were prepared from the polymerisation of water-in-monomer high internal phase emulsions, where the water-soluble monomers acrylamide (AAm) or poly(ethylene glycol) diacrylate (PEGDA) (M w 258) were also included in the 90 vol% internal phase. Both AAm and PEGDA were found to act as co-surfactants, resulting in the obtainment of monoliths with greater homogeneity in some cases. As a result these materials demonstrated significantly improved chromatographic performance for the separation of a standard mixture of proteins using reversed-phase liquid chromatography, in comparison to monoliths prepared with no internal phase monomer. In particular, the columns grafted with PEGDA were capable of separating a more complex mixture consisting of seven components. The inclusion of monomers in the internal phase also allowed for the functionalisation of the monolith's surface where the degree of polymerisation that occurred in the internal phase, which was governed by the monomer content in the internal phase and initiation location, determined whether polymeric chains or a hydrogel were grafted to the surface. A monolith grafted with AAm was also found to be capable of retaining polar analytes as a result of the increase in surface hydrophilicity.

Integrated Platform Addressing the Finger-Prick Blood Processing Challenges of Point-of-Care Electrical Biomarker Testing.

Continued advances in label-free electrical biosensors pave the way to simple, rapid, cost-effective, high-sensitivity, and quantitative biomarker testing at the point-of-care setting that would profoundly transform healthcare. However, implementation in routine diagnostics is faced with significant challenges associated with the inherent requirement for biofluid sample processing before and during testing. We present here a simple yet robust autonomous finger-prick blood sample processing platform integrated with nanoscale field-effect transistor biosensors and demonstrate the feasibility of measuring the SARS-CoV-2 nucleocapsid protein. The 3D-printed platform incorporates a high-yield blood-to-plasma separation module and a delay valve designed to terminate the assay at a specific time. The platform is driven by hydrostatic pressure to efficiently and automatically dispense plasma and washing/measurement buffer to the nanosensors. Our model study demonstrates the feasibility of detecting down to 1.4 pg/mL of the SARS-CoV-2 nucleocapsid protein within 25 min and with only minimal operator intervention.

Effect of ethoxylated sorbitan ester surfactants on the chromatographic efficiency of poly(ethylene glycol)-based monoliths.

The effect of adding ethoxylated sorbitan ester surfactants (Tweens®) to poly(ethylene glycol) diacrylate-based monolithic recipes was investigated. Five different Tweens® have been evaluated to investigate the exact role of non-ionic surfactants in poly(ethylene glycol) diacrylate-based monolith preparations. These monoliths were characterized by scanning electron microscopy, infrared spectroscopy, and nitrogen physisorption analysis. Different morphological features, and surface areas were observed when different types of Tween® were included in the recipe; Tween® 20 and 85 showed small globules, while Tween® 40, 60 and 80 exhibited larger globular structures with different sizes and degrees of coalescence. The different Tween®-based monoliths were investigated for the chromatographic separation of mixtures consisting of hydroxybenzoic acids and alkylbenzenes. These columns were mechanically stable, except for Tween® 80. The highest methylene selectivity and the best overall performance were achieved by Tween® 60. The efficiency was increased by increasing the concentration of the Tween® 60 and the amount of poly(ethylene glycol) diacrylate Mn 700 in the recipes up to 30 wt%, each. Further increases in either Tween® 60 or poly(ethylene glycol) diacrylate Mn 700 led to formation of non-permeable columns. The optimized column was successfully used for separation of mixtures of nonsteroidal anti-inflammatory and sulfa drugs, with a maximum efficiency of 60,000 plates/m.

Utilizing RAFT Polymerization for the Preparation of Well-Defined Bicontinuous Porous Polymeric Supports: Application to Liquid Chromatography Separation of Biomolecules.

Polymer-based monolithic high-performance liquid chromatography (HPLC) columns are normally obtained by conventional free-radical polymerization. Despite being straightforward, this approach has serious limitations with respect to controlling the structural homogeneity of the monolith. Herein, we explore a reversible addition-fragmentation chain transfer (RAFT) polymerization method for the fabrication of porous polymers with well-defined porous morphology and surface chemistry in a confined 200 µm internal diameter (ID) capillary format. This is achieved via the controlled polymerization-induced phase separation (controlled PIPS) synthesis of poly(styrene-co-divinylbenzene) in the presence of a RAFT agent dissolved in an organic solvent. The effects of the radical initiator/RAFT molar ratio as well as the nature and amount of the organic solvent were studied to target cross-linked porous polymers that were chemically bonded to the inner wall of a modified silica-fused capillary. The morphological and surface properties of the obtained polymers were thoroughly characterized by in situ nuclear magnetic resonance (NMR) experiments, nitrogen adsorption-desorption experiments, elemental analyses, field-emission scanning electron microscopy (FESEM), scanning electron microscopy-energy-dispersive X-ray (SEM-EDX) spectroscopy, and X-ray photoelectron spectroscopy (XPS) as well as time-of-flight secondary ion mass spectrometry (ToF-SIMS) revealing the physicochemical properties of these styrene-based materials. When compared with conventional synthetic methods, the controlled-PIPS approach affects the kinetics of polymerization by delaying the onset of phase separation, enabling the construction of materials with a smaller pore size. The results demonstrated the potential of the controlled-PIPS approach for the design of porous monolithic columns suitable for liquid separation of biomolecules such as peptides and proteins.

Polymeric stationary phases for size exclusion chromatography: A review.

Synthetic and natural macromolecules are commonly used in a variety of fields such as plastics, nanomedicine, biotherapeutics, drug delivery and tissue engineering. Characterising macromolecules in terms of their structural parameters (size, molar mass and distribution, architecture) is key to have a better understanding of their structure-property relationships. Size exclusion chromatography (SEC) is a commonly used technique for polymer characterization since it offers access to the determination of the size of a macromolecule, its molar mass and the molar mass distribution. Moreover, detectors that allow the determination of true molar masses, macromolecule's architecture and the composition of copolymers can be coupled to the chromatographic system. Like other chromatographic techniques, the stationary phase is of paramount importance for efficient SEC separations. This review presents the basic principles for the design of stationary phases for SEC as well as synthetic methods currently used in the field. Current status of fully-porous polymeric stationary phases used in SEC is reviewed and their advantages and limitations are also discussed. Finally, the potential of polymer monoliths in SEC is also covered, highlighting the limitations this column technology could address. However, further development in the polymer structure is needed to consider this column technology in the field of macromolecule separation.

Non-ionic Surface Active Agents as Additives toward a Universal Porogen System for Porous Polymer Monoliths.

The influence of the addition of various non-ionic surfactants to poly(ethylene glycol) diacrylate-based monolith formulations was studied. Eight non-ionic surfactants having different chemistries were chosen for this study. These surfactants were Brij L4, Span 80, IGEPAL CO-520, Tergitol 15S9, 2,4,7,9-tetramethyl-5-decyne-4,7-diol ethoxylate, Tween 40, Triton X-405, and Tetronic 701. The chemical structures of these surfactants have a variety of functional groups and cover a wide range of molecular weights (360-3600 g/mol), viscosities (60-1500 cP), and hydrophilic-lipophilic balances (1.0-17.6). The formed polymers were characterized by scanning electron microscopy, surface area measurement by the Brunauer-Emmet-Teller method, elemental analysis, and Fourier transform infrared. Four formulations, involving the use of surfactants, resulted in permeable materials when prepared in 150 µm ID silica capillaries. The chromatographic performance of the resulting columns in reversed-phase mode was evaluated and compared using a mixture of alkyl benzenes as test analytes. The highest efficiency and methylene selectivity were observed when Tween 40 was included in the formulation, using decane/decanol/dodecanol as coporogens. This porogenic mixture was successfully used for preparation of monolithic columns from a selection of methacrylate- and styrene-based monomers, including butylmethacrylate, hydroxyethymethacrylate, laurylmethacrylate, glycidyl methacrylate, bisphenol diacrylate, benzylmethacrylate, and N,N-dimethylacrylamide, as well as for divinylbenzene. These results show the applicability of this porogenic mixture for a variety of monolithic formulations, providing an approach for developing a universal porogen system.

Natural deep eutectic solvents and aqueous solutions as an alternative extraction media for propolis.

Ethanolic extracts of propolis are consumed for their health benefits even though direct consumption of alcoholic extracts is not always ideal. Natural Deep Eutectic Solvents (NADES) can potentially extract similar compounds as alcoholic extracts while being better for direct consumption. Therefore, in this work alternative solvents for the extraction of green propolis including its biomarker artepillin C were examined. Sixteen NADES made from low toxicity chemicals, including the essential amino acid l-lysine, were explored along with twelve individual NADES components and honey, which showed similar physical-chemical properties to NADES. At 50 °C NADES made from choline chloride-propylene glycol or lactic acid proved to be equal or better than the benchmark EtOH:Water 7:3 (v/v). Alternatively, aqueous l-lysine appeared as a potential solvent for the preparation of aqueous propolis extracts. From these findings NADES, honey and aqueous l-lysine solutions all demonstrated the potential to replace ethanol or water for extracting green propolis.

Using natural deep eutectic solvents for the extraction of metabolites in Byrsonima intermedia leaves.

Natural deep eutectic solvents have been used as an alternative to organic solvents for the extraction of plants metabolites, allowing for the extraction of compounds of different polarities, while being inexpensive, non-toxic, and easy to prepare. This work presents the comparison of the chromatographic profiles by high-performance liquid chromatography with diode-array detection obtained from Byrsonima intermedia (Malpighiaceae) using five choline chloride-based natural deep eutectic solvents, in addition to the most used traditional extraction solvents, methanol/water 7:3 and ethanol/water 7:3 v/v. A reference extract was used to tentatively identify compounds by high-performance liquid chromatography with tandem mass spectrometry. The water content appeared to be important for the extraction efficiency and the mixture choline chloride/glycerol was shown to be the best candidate for efficiently extracting this matrix when compared with the traditional extraction media in addition to being far greener as shown by the environmental analysis tool. Seven phenolic compounds (digalloyl quinic acid, proanthocyanidin dimer, galloylproanthocyanidin dimer, quercetin-O-hexoside, galloyl quercetin hexoside, quercetin-O-pentoside, and galloyl quercetin pentoside) were tentatively identified in all extracts. Moreover, the influence of these solvents on the antioxidant activity of the extracts was studied and the results for choline chloride/glycerol extracts were very similar to that of the traditional extraction solvents.

Effect of shearing stress on the radial heterogeneity and chromatographic performance of styrene-based polymerised high internal phase emulsions prepared in capillary format.

Poly(styrene-co-divinylbenzene) monoliths were prepared from the polymerisation of water-in-monomer high internal phase emulsions consisting of a 90 vol% internal phase and stabilised by the non-ionic surfactant Span 80®. The materials were prepared in capillary housings of various internal diameters ranging from 150 µm to 540 µm by simply passing the emulsion through the capillaries. When low shear (300 rpm) was used for emulsification, the droplet and resulting void size distributions were observed to shift towards lower values when the emulsions were forced through capillaries of internal diameter less than 540 µm and all columns exhibited significant radial heterogeneity. When high shear was employed (14 000 rpm) the resulting emulsions preserved their structure when forced through these capillaries and possessed narrower void size distributions with no obvious radial heterogeneity observed upon curing. This resulted in significantly improved chromatographic performance for the separation of a standard mixture of proteins when compared to the materials prepared under low shear.

Characterization of oligo(acrylic acid)s and their block co-oligomers.

Oligo(acrylic acid), oligoAA are important species currently used industrially in the stabilization of paints and also for the production of self-assembled polymer structures which have been shown to have useful applications in analytical separation methods and potentially in drug delivery systems. To properly tailor the synthesis of oligoAA, and its block co-oligomers synthesized by Reversible-Addition Fragmentation chain Transfer (RAFT) polymerization to applications, detailed knowledge about the chemical structure is needed. Commonly used techniques such as Size Exclusion Chromatography (SEC) and Electrospray Ionization-Mass Spectrometry (ESI-MS) suffer from poor resolution and non-quantitative distributions, respectively. In this work free solution Capillary Electrophoresis (CE) has been thoroughly investigated as an alternative, allowing for the separation of oligoAA by molar mass and the RAFT agent end group. The method was then extended to block co-oligomers of acrylic acid and styrene. Peak capacities up to 426 were observed for these 1D CE separations, 10 times greater than what has been achieved for Liquid Chromatography (LC) of oligostyrenes. To provide a comprehensive insight into the chemical structure of these materials 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy was used to provide an accurate average chain length and reveal the presence of branching. The chain length at which branching is detected was investigated with the results showing a degree of branching of 1% of the monomer units in oligoAA with an average chain length of 9 monomer units, which was the shortest chain length at which branching could be detected. This branching is suspected to be a result of both intermolecular and intramolecular transfer reactions. The combination of free solution CE and NMR spectroscopy is shown to provide a near complete elucidation of the chemical structure of oligoAA including the average chain length and branching as well as the chain length and RAFT agent end group distribution. Furthermore, the purity in terms of the dead chains and unreacted RAFT agent was quantified. The use of free solution CE and 1H NMR spectroscopy demonstrated in this work can be routinely applied to oligoelectrolytes and their block co-oligomers to provide an accurate characterization which allows for better design of the materials produced from these oligomers.

Natural deep eutectic solvents as the major mobile phase components in high-performance liquid chromatography-searching for alternatives to organic solvents.

Over the past six decades, acetonitrile (ACN) has been the most employed organic modifier in reversed-phase high-performance liquid chromatography (RP-HPLC), followed by methanol (MeOH). However, from the growing environmental awareness that leads to the emergence of "green analytical chemistry," new research has emerged that includes finding replacements to problematic ACN because of its low sustainability. Deep eutectic solvents (DES) can be produced from an almost infinite possible combinations of compounds, while being a "greener" alternative to organic solvents in HPLC, especially those prepared from natural compounds called natural DES (NADES). In this work, the use of three NADES as the main organic component in RP-HPLC, rather than simply an additive, was explored and compared to the common organic solvents ACN and MeOH but additionally to the greener ethanol for separating two different mixtures of compounds, one demonstrating the elution of compounds with increasing hydrophobicity and the other comparing molecules of different functionality and molar mass. To utilize NADES as an organic modifier and overcome their high viscosity monolithic columns, temperatures at 50 °C and 5% ethanol in the mobile phase were used. NADES are shown to give chromatographic performances in between those observed for ACN and MeOH when eluotropic strength, resolution, and peak capacity were taken into consideration, while being less environmentally impactful as shown by the HPLC-Environmental Assessment Tool (HPLC-EAT) metric. With the development of proper technologies, DES could open a new class of mobile phases increasing the possibilities of new separation selectivities while reducing the environmental impact of HPLC analyses. Graphical abstract Natural deep eutectic solvents versus traditional solvents in HPLC.

Review: Synthetic scaffolds to control the biochemical, mechanical, and geometrical environment of stem cell-derived brain organoids.

Stem cell-derived brain organoids provide a powerful platform for systematic studies of tissue functional architecture and the development of personalized therapies. Here, we review key advances at the interface of soft matter and stem cell biology on synthetic alternatives to extracellular matrices. We emphasize recent biomaterial-based strategies that have been proven advantageous towards optimizing organoid growth and controlling the geometrical, biomechanical, and biochemical properties of the organoid's three-dimensional environment. We highlight systems that have the potential to increase the translational value of region-specific brain organoid models suitable for different types of manipulations and high-throughput applications.

PEO-based brush-type amphiphilic macro-RAFT agents and their assembled polyHIPE monolithic structures for applications in separation science.

Polymerized High Internal Phase Emulsions (PolyHIPEs) were prepared using emulsion-templating, stabilized by an amphiphilic diblock copolymer prepared by reversible addition fragmentation chain transfer (RAFT) polymerization. The diblock copolymer consisted of a hydrophilic poly(ethylene glycol) methyl ether acrylate (PEO MA, average Mn 480) segment and a hydrophobic styrene segment, with a trithiocarbonate end-group. These diblock copolymers were the sole emulsifiers used in stabilizing "inverse" (oil-in-water) high internal phase emulsion templates, which upon polymerization resulted in a polyHIPE exhibiting a highly interconnected monolithic structure. The polyHIPEs were characterized by FTIR spectroscopy, BET surface area measurements, SEM, SEM-EDX, and TGA. These materials were subsequently investigated as stationary phase for high-performance liquid chromatography (HPLC) via in situ polymerization in a capillary format as a 'column housing'. Initial separation assessments in reversed-phase (RP) and hydrophilic interaction liquid chromatographic (HILIC) modes have shown that these polyHIPEs are decorated with different microenvironments amongst the voids or domains of the monolithic structure. Chromatographic results suggested the existence of RP/HILIC mixed mode with promising performance for the separation of small molecules.

Flow-dependent separation selectivity for organic molecules on metal-organic frameworks containing adsorbents.

A new flow-dependent separation selectivity (FDSS) effect was discovered in isocratic HPLC. Significant changes in chromatographic selectivity were achieved by simple variation of the mobile phase flow rate. The FDSS effect was observed for a core-shell stationary phase using silica particles as a core and a Zr-based metal-organic framework (UiO-66) as a shell.

Characterization of polymer monoliths containing embedded nanoparticles by scanning transmission X-ray microscopy (STXM).

The structural and chemical homogeneity of monolithic columns is a key parameter for high efficiency stationary phases in liquid chromatography. Improved characterization techniques are needed to better understand the polymer morphology and its optimization. Here the analysis of polymer monoliths by scanning transmission X-ray microscopy (STXM) is presented for the first time. Poly(butyl methacrylate-co-ethyleneglycoldimethacrylate) [poly(BuMA-co-EDMA)] monoliths containing encapsulated divinylbenzene (DVB) nanoparticles were characterized by STXM, which gives a comprehensive, quantitative chemical analysis of the monolith at a spatial resolution of 30 nm. The results are compared with other methods commonly used for the characterization of polymer monoliths [scanning electron microscopy (SEM), transmission electron microscopy (TEM), mercury porosimetry, and nitrogen adsorption]. The technique permitted chemical identification and mapping of the nanoparticles within the polymeric scaffold. Residual surfactant, which was used during the manufacture of the nanoparticles, was also detected. We show that STXM can give more in-depth chemical information for these types of materials and therefore lead to a better understanding of the link between polymer morphology and chromatographic performance.

Monolithic cryopolymers with embedded nanoparticles. II. Capillary liquid chromatography of proteins using charged embedded nanoparticles.

The preparation of composite monolithic cryopolymers is presented. These novel porous materials were prepared in capillary format at -70°C using poly(ethyleneglycol) diacrylate (PEGDA) Mw 258 as the single monomer and a mixture of dioxane and water as the porogen. Positively (NR4(+)) or negatively (SO3(-)) charged nanoparticles were incorporated within the polymeric structure by direct addition of their suspensions to the polymerisation mixture. In contrast to our previous report using neutral nanoparticles, the trapping of charged nanoparticles is mostly observed at the polymer surface. The incorporation of these nanostructures improved the chromatographic separations of standard proteins under a hydrophobic interaction chromatography (HIC) separation mode. Moreover, the presence of ionic groups on the polymer surface allowed the application of these columns under ion-exchange (IEX) conditions. The results obtained in this work show that the functionalisation of monolithic columns by direct addition of nanoparticles is a good alternative towards the modification of monolithic polymers without altering the polymeric scaffold.

Poly(ethylene glycol)-based monolithic capillary columns for hydrophobic interaction chromatography of immunoglobulin G subclasses and variants.

Polymer monoliths were prepared in 150 µm id capillaries by thermally initiated polymerization of PEG diacrylate for rapid hydrophobic interaction chromatography of immunoglobulin G (IgG) subclasses and related variants. Using only one monomer in the polymerization mixture allowed ease of optimization and synthesis of the monolith. The performance of the monolith was demonstrated by baseline resolution of IgG subclasses and variants, including mixtures of the κ variants of IgG1, IgG2, and IgG3 as well as the κ and λ variants associated with IgG1 and IgG2. The effect of eluent concentration and pH on the separation efficiency of studied proteins was also explored, allowing almost baseline resolution to be achieved for mixtures of the κ variants of IgG1, IgG2, IgG3, and IgG4 but also for the κ and λ variants of IgG1 and IgG2. The results showed significant improvement in the separations in terms of the tradeoff between analysis time and resolution, while maintaining a simple methodology, in comparison to previous reports. The synthesized monolith was also used for the separation of isoforms of a therapeutic monoclonal antibody.

Epoxy-based monoliths for capillary liquid chromatography of small and large molecules.

A versatile epoxy-based monolith was synthesised by polycondensation polymerisation of glycidyl ether 100 with ethylenediamine using a porogenic system consisting of polyethylene glycol, M(w) = 1000, and 1-decanol. Polymerisation was performed at 80 °C for 22 h. A simple acid hydrolysis of residual epoxides resulted in a mixed diol-amino chemistry. The modified column was used successfully for hydrophilic interaction liquid chromatography (HILIC) of small molecule probes such as nucleic acid bases and nucleosides, benzoic acid derivatives, as well as for peptides released from a tryptic digest of cytochrome c. The mixed-mode chemistry allowed both hydrophilic partitioning and ion-exchange (IEX) interactions to contribute to the separation, providing flexibility in selectivity control. Residual epoxide groups were also exploited for incorporating a mixed IEX chemistry. Alternatively, the surface chemistry of the monolith pore surface rendered hydrophobic via grafting of a co-polymerised hydrophobic hydrogel. The inherent hydrophilicity of the monolith scaffold also enabled high performance separation of proteins under IEX and hydrophobic interaction modes and in the absence of non-specific interactions.