3D-Printed MOF Monoliths: Fabrication Strategies and Environmental Applications.

Metal-organic frameworks (MOFs) have been extensively considered as one of the most promising types of porous and crystalline organic-inorganic materials, thanks to their large specific surface area, high porosity, tailorable structures and compositions, diverse functionalities, and well-controlled pore/size distribution. However, most developed MOFs are in powder forms, which still have some technical challenges, including abrasion, dustiness, low packing densities, clogging, mass/heat transfer limitation, environmental pollution, and mechanical instability during the packing process, that restrict their applicability in industrial applications. Therefore, in recent years, attention has focused on techniques to convert MOF powders into macroscopic materials like beads, membranes, monoliths, gel/sponges, and nanofibers to overcome these challenges.Three-dimensional (3D) printing technology has achieved much interest because it can produce many high-resolution macroscopic frameworks with complex shapes and geometries from digital models. Therefore, this review summarizes the combination of different 3D printing strategies with MOFs and MOF-based materials for fabricating 3D-printed MOF monoliths and their environmental applications, emphasizing water treatment and gas adsorption/separation applications. Herein, the various strategies for the fabrication of 3D-printed MOF monoliths, such as direct ink writing, seed-assisted in-situ growth, coordination replication from solid precursors, matrix incorporation, selective laser sintering, and digital light processing, are described with the relevant examples. Finally, future directions and challenges of 3D-printed MOF monoliths are also presented to better plan future trajectories in the shaping of MOF materials with improved control over the structure, composition, and textural properties of 3D-printed MOF monoliths.

N-doped nano-casted carbon monolith for Pb (II) removal and photocatalytic degradation of thiamethoxam from aqueous solution.

Single rock-like N-doped carbon monolith (ND-PFCM) was successfully constructed via nanocasting method. Phenol formaldehyde resin was taken as carbon source and nitrogen was incorporated in monoliths through NaNH2 activation. The synthesized monoliths were used for the removal of Pb (II) from aqueous solution. Various characterization techniques, namely Brunauer-Emmett-Teller (BET), Raman spectroscopy, UV-visible diffuse reflectance spectra (DRS) UV-DRS, zeta potential, scanning electron microscopy (SEM), TEM (transmission electron microscopy), TGA (thermogravimetric analysis), and XPS (X-ray photoelectron spectroscopy), were utilized to characterize synthesized monolithic samples. The different parameters such as pH, adsorbent dosage, and time were enquired on the removal efficiency of monoliths toward Pb(II). ND-PFCM exhibited the highest adsorption capacity of 330.03 mg g-1 in 180 min at pH 6. This is attributed to the fact that the better texture properties and presence of nitrogen functional groups enhance the uptake of Pb (II) ions on the monolith surface. In the kinetic studies, pseudo-second-order model fitted best with the experimental data. Furthermore, the removal of thiamethoxam (TM) from aqueous solution was done by using different weight ratios of ND-PFCM under the visible light. The maximum removal efficiency of 97.35% with rate constant of 0.02085 min-1 was obtained in 160 min. Moreover, monoliths exhibited good reusability for five consecutive cycles. The findings suggest that the synthesized monoliths exhibit characteristics suitable and eco-friendly for sustainable use in water treatment applications.

Chondroitin Sulfate and Hyaluronic Acid-Based PolyHIPE Scaffolds for Improved Osteogenesis and Chondrogenesis In Vitro.

Osteochondral damage, affecting the articular cartilage and the underlying subchondral bone, presents significant challenges in clinical treatment. Such defects, commonly seen in knee and ankle joints, vary from small localized lesions to larger defects. Current medical therapies encounter several challenges, such as donor shortages, drug side effects, high costs, and rejection problems, often resulting in only temporary relief. Highly porous emulsion-templated polymers (polyHIPEs) offer numerous potential benefits in the fabrication of scaffolds for tissue engineering and regenerative medicine. Polymeric scaffolds synthesized using a high internal phase emulsion (HIPE) technique, called PolyHIPEs, involve polymerizing a continuous phase surrounding a dispersed internal phase to form a solid, foam-like structure. A dense, porous design encourages cell ingrowth, nutrient delivery, and waste disposal from the scaffold, mimicking the cells' natural microenvironment. This study used hydroxyethyl methacrylate (HEMA) and acrylamide (AAM) polyHIPE scaffolds combined with extracellular matrix (ECM) components of the tissue, such as methacrylated hyaluronic acid (MHA) and methacrylated chondroitin sulfate (MCS), to prepare polyHIPE scaffolds. The mouse preosteoblast MC3T3-E1 cells and primary rat chondrocytes (harvested from male Wistar rats) were seeded on the scaffolds and cultured for 21 days to assess the osteogenesis and chondrogenesis in vitro. When compared to the AAM-MHA and AAM-MCS groups at day 21, scaffold groups HEMA-MHA and HEMA-MCS showed a significant rise in alkaline phosphatase (ALP) and calcium content. Chondrogenic markers such as glycosaminoglycan (GAG) and hydroxyproline were also assessed over a 21-day time point. On day 21, it was found that GAG and hydroxyproline production were considerably higher in the HEMA-MHA and HEMA-MCS scaffolds than in the AAM-MHA and AAM-MCS scaffolds. The overall studies showed that polyHIPE monolith scaffolds could favor cell adherence, survival ability, proliferation, differentiation, and ECM formation over 21 days. Thus, incorporating ECM components enhanced osteogenesis and chondrogenesis in vitro and can be further used as tissue repair models.

Influence of the Polymerization Parameters on the Porosity and Thermal Stability of Polymeric Monoliths.

Rigid porous polymeric monoliths are robust, highly efficient, versatile stationary phases. They offer simple preparation and convenient modification provided by a whole range of synthesis factors, e.g., starting monomers, cross-linkers, initiators, porogens, polymerization techniques, and temperature. The main aim of this study was to synthesize polymeric monoliths and determine the correlation between polymerization parameters and the porosity and thermal stability of the obtained materials. Polymeric monoliths were synthesized directly in HPLC columns using N-vinyl-2-pyrrolidone (NVP) and 4-vinylpiridine (4VP) as functional monomers, with trimethylolpropane trimethacrylate (TRIM) serving as the cross-linking monomer. During copolymerization a mixture of cyclohexanol/decane-1-ol was used as the pore-forming diluent. Polymerization was carried out at two different temperatures: 55 and 75 °C. As a result, monoliths with highly developed internal structure were synthesized. The value of their specific surface area was in the range of 92 m2/g to 598 m2/g, depending on the monomer composition and polymerization temperature. Thermal properties of the obtained materials were investigated by means of thermogravimetry (TG). Significant differences in thermal behavior were noticed between monoliths synthesized at 55 and 75 °C. Additionally, the poly(NVP-co-TRIM) monolith was successfully applied in GC analyses.

Immobilization of controlled Pu:Am ratio on actinide-specific affinity monolith support developed in capillary and coupled to inductively coupled plasma mass spectrometry.

The objective of this work was to develop an actinide-specific monolithic support in capillary designed to immobilize precise Pu:Am ratios and its coupling to inductively coupled plasma mass spectrometry (ICP-MS) for immobilized metal affinity chromatography applications. This format offers many advantages, such as reducing the sample amount and waste production, which are of prime importance when dealing with highly active radioelements. Four organic phosphorylated-based monoliths were synthesized in situ through UV photo-polymerization in capillary and characterized. The capillary coupling to ICP-MS was set up in conventional laboratory using Th and Sm as chemical analogues of Pu and Am. A dedicated method was developed to quantify online Th and Sm amounts immobilized on the monolithic capillaries, allowing to select the best monolith candidate poly(BMEP-co-EDMA)adp. By precisely adjusting the elemental composition in the loading solutions and applying the developed quantification method, the controlled immobilization of several Th:Sm molar ratios onto the monolith was successful. Finally, the capillary ICP-MS coupling was transposed in a glove box and by applying the strategy developed to design the monolithic support using Th and Sm, the immobilization of a 10.5 ± 0.2 (RSD = 2.3%, n = 3) Pu:Am molar ratio reflecting Pu ageing over 48 years was achieved in a controlled manner on poly(BMEP-co-EDMA)adp. Hence, the new affinity capillary monolithic support was validated, with only hundred nanograms or less of engaged radioelements and can be further exploited to precisely determine differential interactions of Pu and Am with targeted biomolecules in order to better anticipate the effect of Am on Pu biodistribution.

Cryogels and Monoliths: Promising Tools for Chromatographic Purification of Nucleic Acids.

The increasing demand for highly pure biopharmaceuticals has put significant pressure on the biotechnological industry to innovate in production and purification processes. Nucleic acid purification, crucial for gene therapy and vaccine production, presents challenges due to the unique physical and chemical properties of these molecules. Meeting regulatory standards necessitates large quantities of biotherapeutic agents of high purity. While conventional chromatography offers versatility and efficiency, it suffers from drawbacks like low flow rates and binding capacity, as well as high mass transfer resistance. Recent advancements in continuous beds, including monoliths and cryogel-based systems, have emerged as promising solutions to overcome these limitations. This review explores and evaluates the latest progress in chromatography utilizing monolithic and cryogenic supports for nucleic acid purification.

Sustainable and reusable probe-encapsulated porous poly(AMST-co-TRIM) monolithic sensor for the selective and ultra-sensitive detection of toxic cadmium(II) from industrial/environmental wastewater.

This study focuses on a new type of fast responsive solid-state visual colorimetric sensor, custom engineered with dual-entwined porous polymer imbued with chromoionophoric 4-(sec-butyl)- 2-((5-mercapto-1,3,4-thiadiazol-2-yl)diazenyl)phenol (SMDP) probe for selective and ultra-sensitive colorimetric sensing of Cd(II). The polymer monolith, i.e., poly(aminostyrene-co-trimethylolpropanetrimethacrylate) denoted as poly(AMST-co-TRIM), is designed through a stoichiometric blending of monomer, crosslinker, and porogens leading to superior surface area, pore and adsorption properties for the voluminous incorporation of SMDP probe for target specific ion sensing. The porosity, surface and structural characteristics of the poly(AMST-co-TRIM)monolith and poly(AMST-co-TRIM)SMDP sensor are investigated using p-XRD, XPS, TG-DTA, FT-IR, BET/BJH, FE-SEM, HR-TEM, EDAX, and SAED techniques. The poly(AMST-co-TRIM)SMDP sensor reveals a frozen geometrical orientation of SMDP molecules to bind selectively with Cd(II), forming stable charge-transfer complexes by exhibiting transitional visible color shifts from light yellow to dark green (λmax 608 nm). The sensor imposes a linear response from 0-200 ppb, with quantification and detection limits of 0.95 and 0.28 ppb. The fabricated sensor material is cost-effective and versatile in its solid-state naked-eye sensing, with excellent reusability. The sensor performance has been verified using various environmentally contaminated water and commercial cigarette samples, with a recovery of ≥ 99.12% and an RSD of ≤ 1.95%, thus reflecting exceptional data reproducibility/reliability.

New trends in modelling of breakthrough curves to remove pollutants using adsorption on advanced monoliths geometries.

This work proposes a rigorous mathematical model capable of reproducing the adsorption process in dynamic regime on advanced monoliths geometries. For this, four bed geometries with axisymmetric distribution of channels and similar solid mass were proposed. In each geometry a different distribution of channels was suggested, maintaining constant the bed dimensions of 15 cm high and 5 cm radius. The mathematical modeling includes mass and momentum transfer phenomena, and it was solved with the COMSOL Multiphysics software using mass transfer parameters published in the literature. The overall performance of the column was evaluated in terms of breakthrough (CA/CA0 = 0.1) and saturation times (CA/CA0 = 0.9). The mass and velocity distributions obtained from the proposed model show good physical consistency with what is expected in real systems. In addition, the model proved to be easy to solve given the short convergence times required (2-4 h). Modifications were made to the bed geometry to achieve a better use of the adsorbent material which reached up to 80%. The proposed bed geometries allow obtaining different mixing distributions, in such a way that inside the bed a thinning of the boundary layer is caused, thus reducing diffusive effects at the adsorbent solid-fluid interface, given dissipation rates of about 323 × 10-11 m2/s3. The bed geometry composed of intersecting rings deployed the best performance in terms of usage of the material adsorbent, and acceptable hydrodynamical behavior inside the channels (maximum fluid velocity = 35.4 × 10-5 m/s and drop pressure = 0.19 Pa). Based on these results, it was found that it is possible to reduce diffusional effects and delimit the mass transfer zone inside the monoliths, thus increasing the efficiency of adsorbent fixed beds.

Selective immunoglobulin aggregates removal in antivenoms by a simple chromatographic step based on a monolithic stationary phase.

Antivenom therapy is a critical intervention for treating the more than 5.000.000 envenomation accidents that occur each year around the world. These immunotherapeutic drugs are mostly produced following techniques developed more than fifty years ago with minor changes. Aggregate content has been described as one of the main causes of early adverse effects after intravenous administration of antivenoms. In this work we propose the introduction of a final polishing step to traditional antivenom manufacturing processes aimed at lowering the aggregate content in the final product. The refinement step proposed in this work is based on the selective capture of immunoglobulin aggregates by a cation exchange monolithic stationary phase. We show that this media can effectively remove aggregates in the final product under isotonic ion-strength and mildly acidic conditions following a negative chromatography strategy, thus making it a useful technique for producing higher quality products.

Direct Synthesis of Metal-Organic Framework Sols: Advances and Perspectives.

The intrinsic lack of processability in the conventional nano/microcrystalline powder form of metal-organic frameworks (MOFs) greatly limits their application in various fields. Synthesis of MOFs with certain flowability make them promising for multitudinous applications. The direct synthesis strategy represents one of the simplest and efficient method for synthesizing solution processable MOF sols/suspensions, compared with other approaches, for instance, the post-synthesis surface modification, the direct dispersion of MOFs in hindered ionic liquids, as well as the calcination method toward a few MOFs with melting behavior. This article reviews the recent direct synthesis strategies of solution processable MOF sols and their typical applications in different fields. The direct synthesis strategies of MOF sols can be classified into two categories: particle size reduction strategy, and selective coordination strategy. The synthesis mechanism of different strategies and the factors affecting the formation of sols are summarized. The application of solution processable MOF sols in different fields are introduced, showing great application potentials. Furthermore, the challenges faced by the direct synthesis of MOF sols and the main methods to deal with the challenges are emphasized, and the future development trend is prospected.

Characterization of hybrid organo-silica monoliths for possible application in the gradient elution of peptides.

We characterized thermally polymerized organo-silica hybrid monolithic capillaries to test their applicability in the gradient elution of peptides. We have used a single-pot approach utilizing 3-(methacryloyloxy)propyltrimethoxysilane (MPTMS), ethylene dimethacrylate (EDMA), and n-octadecyl methacrylate (ODM) as functional monomers. The organo-silica monolith containing MPTMS and EDMA was compared with the stationary phase prepared by adding ODM to the original polymerization mixture. Column prepared using a three-monomer system provided a lower accessible volume of flow-through pores, a higher proportion of mesopores, and higher efficiency. We utilized isocratic and gradient elution data to predict peak widths in gradient elution. Both protocols provided comparable results and can be used for peptide peak width prediction. However, applying gradient elution data for peak width prediction seems simpler. Finally, we tested the effect of gradient time on achievable peak capacity in the gradient elution of peptides with a column prepared with a three-monomer system providing a higher peak capacity. However, the performance of hybrid organo-silica monolithic stationary phases in gradient elution of peptides must be improved compared to other monolithic stationary phases. The limiting factor is column efficiency in highly aqueous mobile phases, which needs to be focused on.

CO2 adsorption on carbonaceous materials obtained from forestry and urban waste materials: a comparative study.

The increasing emissions of gaseous pollutants of anthropogenic origin, such as carbon dioxide (CO2), which causes global warming, have raised great interest in developing and improving processes that allow their mitigation. Among them, adsorption on porous materials has been proposed as a sustainable alternative. This work presents a study of CO2 equilibrium adsorption at low temperatures (0, 10, and 20 °C) over a wide range of low pressures, on activated carbon derived from Eucalyptus (ES) and Patula pine (PP) forest waste, and carbonaceous material derived from waste tires (WT). The precursors of these materials were previously prepared, and their physicochemical properties were characterized. ES and PP were thermochemically treated with phosphoric acid, and WT was oxidized with nitric acid. Additionally, these materials were used to obtain monoliths using uniaxial compaction techniques and different binding agents, with better results obtained with montmorillonite. A total of six adsorbent solids had their textural and chemical properties characterized and were tested for CO2 adsorption. The highest specific surface area (1405 m2 g-1), and micropore properties were found for activated carbon derived from Eucalyptus whose highest adsorption capacity ranged from 2.27 mmol g-1 (at 0 °C and 100 kPa) to 1.60 mmol g-1 (at 20 °C and 100 kPa). The activated carbon monoliths presented the lowest CO2 adsorption capacities; however, the studied materials showed high potential for CO2 capture and storage applications at high pressures. The isosteric heats of adsorption were also estimated for all the materials and ranged from 16 to 45 kJ mol-1 at very low coverage explained by the energetic heterogeneity and weak repulsive interactions among adsorbed CO2 molecules.

High Recovery Chromatographic Purification of mRNA at Room Temperature and Neutral pH.

Messenger RNA (mRNA) is becoming an increasingly important therapeutic modality due to its potential for fast development and platform production. New emerging RNA modalities, such as circular RNA, drive the need for the development of non-affinity purification approaches. Recently, the highly efficient chromatographic purification of mRNA was demonstrated with multimodal monolithic chromatography media (CIM® PrimaS), where efficient mRNA elution was achieved with an ascending pH gradient approach at pH 10.5. Here, we report that a newly developed chromatographic material enables the elution of mRNA at neutral pH and room temperature. This material demonstrates weak anion-exchanging properties and an isoelectric point of 5.3. It enables the baseline separation of mRNA (at least up to 10,000 nucleotides (nt) in size) from parental plasmid DNA (regardless of isoform composition) with both a NaCl gradient and ascending pH gradient approach, while mRNA elution is achieved in a pH range of 5-7. In addition, the basic structure of the novel material is a chromatographic monolith, enabling convection-assisted mass transfer of large RNA molecules to and from the active surface. This facilitates the elution of mRNA in 3-7 column volumes with more than 80% elution recovery and uncompromised integrity. This is demonstrated by the purification of a model mRNA (size 995 nt) from an in vitro transcription reaction mixture. The purified mRNA is stable for at least 34 days, stored in purified H2O at room temperature.

Sponge-nested polymer monoliths: Versatile materials for the solid-phase extraction of bisphenols.

Polymer monoliths are promising materials for sample preparation due to their high porosity, pH stability, and simple preparation. The use of melamine formaldehyde foams has been reported as an effective support to prepare highly robust silica and polymer monoliths. Herein, divinylbenzene monoliths based on a 50:50 (%, w/w) crosslinker/porogen ratio have been nested within a melamine-formaldehyde sponge, resulting in monoliths with a surface area higher than 400 m2 /g. The extraction performance of these monoliths was evaluated for the extraction of endocrine-disrupting bisphenols from aqueous solutions. We evaluated for the first time the versatility of sponge-nested polymer monoliths by comparing three different extraction modes (vortex mixing, magnetic stirring, and orbital shaking). Vortex mixing showed a comparable recovery of bisphenols (39%-81%) in a shorter extraction time (30 min, instead of 2 h). In addition, the robustness of the sponge-nested polymer monoliths was demonstrated for the first time by reshaping a larger monolithic cube (0.125 cm3 ) into four smaller pieces (4 × 0.03125 cm3 ) leading to a 16%-21% increase in extraction efficiency. This effect was attributed to an increase in the effective contact area with the sample, obtaining a higher analyte extraction capacity.

Construction of chiral capillary electrochromatography microsystems based on Aspergillus sp. CM96.

Novel chiral capillary electrochromatography (CEC) microsystems were constructed based on Aspergillus sp. CM96. As a newly discovered intrinsic characteristic of the cell, cell chirality occupies an essential position in life evolution. Aspergillus sp. CM96 spore (CM96s) was chosen as a proof of concept to develop chiral capillary columns. Interestingly, various types of amino acid (AA) enantiomers were baseline separated under the optimized conditions. Furthermore, the time-dependent chiral interactions between AAs and CM96s were explored in a wider space. Pectinases generated from Aspergillus sp. CM96 fermentation were immobilized onto graphene oxide-functionalized capillary silica monoliths for separating AA enantiomers. Molecular docking simulations were performed to explore chiral separation mechanisms of pectinase for AA enantiomers. These results indicated that Aspergillus sp. CM96-based CEC microsystems have a significant advantage for chiral separation.

Reducing pressure and increasing peak capacity by incorporating an adaptable flow stream splitting platform to high-performance liquid chromatography columns: A study on C18 silica-based monoliths.

In this study, an adaptable end-column platform was fitted to a commercially available monolith, which enabled the column to be fitted with a flow-splitting device. A variety of flow-splitting adapters could be incorporated into the platform, and in this study, a radial flow stream splitter was utilized. The advantage of the radial flow stream spitter was that it overcame issues relating bed density variations that could cause bands to distort in the radial cross-section of the column. Using propylbenzene as a test standard in isocratic elution mode, height equivalent to a theoretical plate curves were constructed across ten flow rates, and it was found that the column efficiency improved by as much as 73%. Furthermore, the dual outlet flow splitter enabled a very substantial reduction in column back pressure, with the decrease being consistently between 20 to 30% depending on the column length. Additionally, sensitivity increased by 45%, consistent with the observed increase in efficiency. The adaptable end-column platform could be retrofitted to almost any commercial column with the expectation of gaining efficiency, sensitivity, and reducing back pressure.

Segmented two-dimensional liquid chromatography. Proof of concept study.

The separation in liquid chromatography is defined either by the space domain where it proceeds until the least retained analyte reaches the outlet of the column or by the time when individual analytes elute out of the column. These two approaches lead to the four possible combinations of two-dimensional liquid chromatography with online space x time coupling being the least experimentally feasible. Here, we show the development of a novel two-dimensional liquid chromatography method combining separation defined by space and the conventional elution-based separation. First-dimension column consisted of four capillary segments coupled serially via two-position six-port valves allowing an online and comprehensive transfer of analytes from the first to the second dimension. After initial experiments using homemade monolithic capillary columns, we tested commercially available columns in both dimensions. We ended with the combination of packed capillary columns in the first dimension and monolithic capillary column in the second dimension. We used a reversed-phase retention mechanism in the first spatial dimension, while HILIC was in the second, time-based dimension. We also developed a theoretical model to describe the proposed two-dimensional separation that was further confirmed by utilizing both an isocratic and gradient elution in the second dimension. Finally, we applied our experimental setup to separate neurotransmitters contained in human urine.

Trends in monoliths: Packings, stationary phases and nanoparticles.

Monoliths media are gaining interest as excellent substitutes to conventional particle-packed columns. Monolithic columns show higher permeability and lower flow resistance than conventional liquid chromatography columns, providing high-throughput performance, resolution and separation in short run times. Monolithic columns with longer length, smaller inner diameter and specific selectivity to peptides or enantiomers have been played important role in hyphenated system. Monolithic stationary phases possess great efficiency, resolution, selectivity and sensitivity in the separation of complex biological samples, such as the complex mixtures of peptides for proteome analysis. The development of monolithic stationary phases has opened the new avenue in chromatographic separation science and is in turn playing much more important roles in the wide application area. Monolithic stationary phases have been widely used in fast and high efficiency one- and multi-dimensional separation systems, miniaturized devices, and hyphenated system coupled with mass spectrometers. The developing technology for preparation of monolithic stationary phases is revolutionizing the column technology for the separation of complex biological samples. These techniques using porous monoliths offer several advantages, including miniaturization and on-line coupling with analytical instruments. Additionally, monoliths are ideal support media for imprinting template-specific sites, resulting in the so-called molecularly-imprinted monoliths, with ultra-high selectivity. In this review, the origin of the concept, the differences between their characteristics and those of traditional packings, their advantages and drawbacks, theory of separations, the methods for the monoliths preparation of different forms, nanoparticle monoliths and metal-organic framework are discussed. Two application areas of monolithic metal-organic framework and nanoparticle monoliths are provided. The review article discusses the results reported in a total of 218 references. Other older references were included to illustrate the historical development of monoliths, both in preparation and types, as well as separation mechanism.

A Continuous-Flow Microreactor for Knoevenagel Condensation of Ethyl Cyanoacetate with Benzaldehyde: The Effect of Grafted Amino Groups on Catalytic Activity.

Ethyl α-cyanocinnamate was synthesized in the Knoevenagel condensation of benzaldehyde and ethyl cyanoacetate in flow monolithic microreactor of 0.63 cm3 volume. The catalytically active core was made of silica monolith modified with various amine group precursors. Structural properties of the support, surface density of NHx groups, and catalytic activity were investigated. It was found that the poly- or di-amine groups attached to the silica surface appeared to be more effective than the aminopropyl groups. Microreactors grafted with diamine functional groups, accompanied by hydrophobic methyl groups, showed the highest activity and stability. It was proved that the decisive role on the activity of catalysts was exerted by the presence of primary amines in diamine chain. The reaction conditions were optimized and it was found that almost full substrate conversion could be achieved in 6 min at 50 °C in the microreactor with low concentration of diamine groups equal to 0.33 mmol g-1 .

Influence of ion-pairing reagents on the separation of intact glycoproteins using hydrophilic-interaction liquid chromatography - high-resolution mass spectrometry.

Hydrophilic-interaction liquid chromatography (HILIC) of intact proteins offers high-resolution separations of glycoforms of glycoproteins differing in the number of (neutral) glycans. However, to obtain efficient separations it is essential that the positively charged sites of the proteins are shielded by acidic (negative) ion-pair reagents (IPRs), so as to enhance the contribution of the hydroxyl groups of the (neutral) sugars in the glycoprotein. Here, we studied the influence of various IPRs that differ in physico-chemical properties, such as hydrophobicity and acidity, on the capillary-scale HILIC separation of intact (glyco)proteins. We evaluated the use of fluoroacetic acid (MFA), difluoroacetic acid (DFA), trifluoroacetic acid (TFA), and heptafluorobutyric acid (HFBA) as diluents for sample preparation, as solvents for sample loading on a reversed-phase trap prior to the HILIC separation, and as mobile-phase components for HILIC and HILIC-MS. To reduce the contribution of ion-exchange interaction with the (silica-based) stationary phase, we used an acrylamide-based monolithic column. We studied the influence of the different IPRs on each step of the separation of a mixture of proteins of different size and hydrophilicity and on the separation of the five glycoforms of ribonuclease B. The content of IPR in the sample was shown not to affect the separation and the MS detection. However, a low content of TFA and DFA in the mobile phase is favourable, as it reduces adduct formation and leads to higher signal intensity. The optimized HILIC conditions successfully resolved nine major glycoforms groups of a ∼40 kDa glycoprotein horseradish peroxidase (HRP), as an example of a complex glycoprotein.