Introduction of Octadecyl-Bonded Porous Particles in 3D-Printed Transparent Housings with Multiple Outlets.

Microfluidic devices for comprehensive three-dimensional spatial liquid chromatography will ultimately require a body of stationary phase with multiple in- and outlets. In the present work, 3D printing with a transparent polymer resin was used to create a simplified device that can be seen as a unit cell for an eventual three-dimensional separation system. Complete packing of the device with 5-µm C18 particles was achieved, with reasonable permeability. The packing process could be elegantly monitored from the pressure profile, which implies that optical transparency may not be required for future devices. The effluent flow was different for each of the four outlets of the device, but all flows were highly repeatable, suggesting that correction for flow-rate variations is possible. The investigation into flow patterns through the device was supported by computational-fluid-dynamics simulations. A proof-of-principle separation of four standard peptides is described, with mass-spectrometric detection for each of the four channels separately. Supplementary Information: The online version contains supplementary material available at 10.1007/s10337-022-04156-w.

On the road toward highly efficient and large volume three-dimensional-printed liquid chromatography columns?

The current performance of commercially packed liquid chromatography columns is limited by the random structure of the packed bed and by the wall-to-center heterogeneity of its structure. The minimum reduced plate heights observed are not smaller than 1.4, whereas they could theoretically be as low as 0.1 for dense and perfectly ordered packings of spheres. To bridge this gap, a wide inner diameter column with an ordered macroporous structure is printed in three dimensions by stereolithography of poly(ethylene glycol diacrylate) resin. Feature sizes below 100 µm are achieved by combining conventional polymer stereolithography with photolithography using photomasks. A layer-by-layer polymerization is performed by alternating two distinct photomasks having horizontally and vertically oriented patterns. Despite the inevitable printing imperfections, minimum reduced plate heights around unity are measured for nonretained analytes. The next challenges for the successful printing of highly efficient and large volume liquid chromatography columns are threefold: reducing the feature size down to below 10 µm, keeping minimum the unevenness of the flow channel dimensions, and tackling additive manufacturing of silica aerogels at such small feature sizes for higher mechanical stability and broader range of retention/selectivity than those delivered by polymer materials.

Fabrication of monolithic frits and columns for chip-based multidimensional separation devices.

In this work, devices for two-dimensional separations are considered. The device contains a flow distributor, a first-dimension channel, and 17 second-dimension outlets. In the design, all connections between the first-dimension channel, the flow distributor, and the second-dimension outlets were tapered, with a minimal diameter of 20 µm. The use of photo-masking is explored for the fabrication of monolithic frits in all tapered connections. Monolithic frits with optimized permeability and length were successfully fabricated in all 33 tapered channels through light-induced polymerization, photo-masking, and selective exposure. The efficacy of the monolithic frits was demonstrated by creating a packed bed of 15-µm particles, confined within the first-dimension channel. The outlet of the first-dimension channel was successfully connected to a mass spectrometer. Effective flow confinement was demonstrated with a reversed-phase separation of a mixture of five standard peptides.

Theoretical framework for mixer design for noise reduction and gradient fidelity.

The mixing of two or more solvent streams to deliver a stable and accurate solvent composition is crucial to the performance, repeatability and reproducibility of a liquid chromatographic separation. We provide a theoretical treatment of axial mixing of a sequence of solvent packets with the framework of continuous stirred tank reactors (CSTRs) in series and investigate the tradeoffs presented between the primary goal of mixers (noise reduction) and it's necessary side-effects of gradient deformation and asymmetry. An experimental setup to mimic CSTR conditions was created using a stop-flow setup where the fluid flow was periodically paused and sonicated within pods of a certain volume. The effects of mixer volume relative to the volume of pump strokes and gradient volumes were investigated and discussed. A total mixer volume that is six-fold the pump stroke volume was found to be necessary to achieve sufficient (95%) noise reduction necessary for certain applications. A series of two or more CSTR elements was found to outperform a single CSTR element for larger mixer-to-pump stroke volume ratio in dampening baseline noise. For linear gradients, a gradient volume that is ten times larger than the mixer volume was found to sufficiently maintain gradient fidelity. For very small gradient volumes relative to the mixer volume, deformation of linear gradients was found to be significantly greater than predicted by the analytical solution. Furthermore, the nature of the solvent gradient deformation was asymmetric, with the latter half of the solvent gradient deforming significantly more than the first half. Combining analytically and numerically derived solutions for multiple CSTRs connected in series with experimental data, several suggestions can be made on mixer dimensions and design for a certain pump system and method transfer, given a pump stroke volume and gradient time.

Latest Trends on the Future of Three-Dimensional Separations in Chromatography.

Separation and characterization of complex mixtures are of crucial importance in many fields, where extremely high separation power is required. Three-dimensional separation techniques can offer a path toward achieving high peak capacities. In this Review, online three-dimensional separation systems are discussed, including three-dimensional gas chromatography, and hyphenated combinations of two-dimensional gas chromatography with liquid chromatography or supercritical-fluid chromatography. Online comprehensive two-dimensional liquid chromatography provides detailed information on complex samples and the need for higher peak capacities is pushing researchers toward online three-dimensional liquid chromatography. In this review, an overview of the various combinations are provided and we discuss and compare their potential performance, advantages, perspectives, and results obtained during the most recent 10-15 years. Finally, the Review will discuss a novel approach of spatial three-dimensional liquid separation to increase peak capacity.

Fabrication of polymer monoliths within the confines of non-transparent 3D-printed polymer housings.

In the last decade, 3D-printing has emerged as a promising enabling technology in the field of analytical chemistry. Fused-deposition modelling (FDM) is a popular, low-cost and widely accessible technique. In this study, RPLC separations are achieved by in-situ fabrication of porous polymer monoliths, directly within the 3D-printed channels. Thermal polymerization was employed for the fabrication of monolithic columns in optically non-transparent column housings, 3D-printed using two different polypropylene materials. Both acrylate-based and polystyrene-based monoliths were created. Two approaches were used for monolith fabrication, viz. (i) in standard polypropylene (PP) a two-step process was developed, with a radical initiated wall-modification step 2,2'-azobis(2-methylpropionitrile) (AIBN) as the initiator, followed by a polymerization step to generate the monolith; (ii) for glass-reinforced PP (GPP) a silanization step or wall modification preceded the polymerization reaction. The success of wall attachment and the morphology of the monoliths were studied using scanning electron microscopy (SEM), and the permeability of the columns was studied in flow experiments. In both types of housings polystyrene-divinylbenzene (PS-DVB) monoliths were successfully fabricated with good wall attachment. Within the glass-reinforced polypropylene (GPP) printed housing, SEM pictures showed a radially homogenous monolithic structure. The feasibility of performing liquid-chromatographic separations in 3D-printed channels was demonstrated.

Multichannel separation device with parallel electrochemical detection.

A device with four parallel channels was designed and manufactured by 3D printing in titanium. A simple experimental setup allowed splitting of the mobile phase in four parallel streams, such that a single sample could be analysed four times simultaneously. The four capillary channels were filled with a monolithic stationary phase, prepared using a zwitterionic functional monomer in combination with various dimethacrylate cross-linkers. The resulting stationary phases were applicable in both reversed-phase and hydrophilic-interaction retention mechanisms. The mobile-phase composition was optimized by means of a window diagram so as to obtain the highest possible resolution of dopamine precursors and metabolites on all columns. Miniaturized electrochemical detectors with carbon fibres as working electrodes and silver micro-wires as reference electrodes were integrated in the device at the end of each column. Experimental separations were successfully compared with those predicted by a three-parameter retention model. Finally, dopamine was determined in human urine to further confirm applicability of the developed device.

Confinement of Monolithic Stationary Phases in Targeted Regions of 3D-Printed Titanium Devices Using Thermal Polymerization.

In this study, we have prepared thermally initiated polymeric monolithic stationary phases within discrete regions of 3D-printed titanium devices. The devices were created with controllable hot and cold regions. The monolithic stationary phases were first locally created in capillaries inserted into the channels of the titanium devices. The homogeneity of the monolith structure and the interface length were studied by scanning a capacitively coupled conductivity contactless detector (C4D) along the length of the capillary. Homogeneous monolithic structures could be obtained within a titanium device equipped with a hot and cold jacket connected to two water baths. The confinement method was optimized in capillaries. The sharpest interfaces (between monolith and empty channel) were obtained with the hot region maintained at 70 °C and the cold region at 4 or 10 °C, with the latter temperature yielding better repeatability. The optimized conditions were used to create monoliths bound directly to the walls of the titanium channels. The fabricated monoliths were successfully used to separate a mixture of four intact proteins using reversed-phase liquid chromatography. Further chromatographic characterization showed a permeability (Kf) of ∼4 × 10-15 m2 and a total porosity of 60%.

Optimizing design and employing permeability differences to achieve flow confinement in devices for spatial multidimensional liquid chromatography.

In spatial multi-dimensional liquid chromatography (LC) devices the flow of each dimension has to remain in the corresponding region, otherwise the separation efficiency is undermined. Adequate flow-confinement measures are necessary. Here, the use of permeability differences across different compartments of spatial two-dimensional (2D) and three-dimensional (3D) LC devices as a method to guide fluid flow and reduce analyte loss during the first, second- and third-dimension development was investigated with computational fluid dynamics (CFD) simulations. In case of 2DLC devices, it was shown that porous barriers with a permeability on the order of 10-12 m2 suffice to keep the total sample spillage from an open 1D channel under 1%. In case of 3DLC devices, it was shown that flow confinement could be achieved using an open 1D channel in combination with a highly-permeable monolith (permeability on the order of 10-12 m2) in the second-dimension (2D) and a less permeable packing with a permeability on the order of 10-15 m2 (e.g. 1 µm particles) in the third-dimension (3D). Additionally, the impact of the 3D flow-distributor has been studied and a novel design, capable of limiting the spillage to the other dimensions to the absolute minimum, is proposed.

Experimental and numerical study of band-broadening effects associated with analyte transfer in microfluidic devices for spatial two-dimensional liquid chromatography created by additive manufacturing.

Conventional one-dimensional column-based liquid chromatographic (LC) systems do not offer sufficient separation power for the analysis of complex mixtures. Column-based comprehensive two-dimensional liquid chromatography offers a higher separation power, yet suffers from instrumental complexity and long analysis times. Spatial two-dimensional liquid chromatography can be considered as an alternative to column-based approaches. The peak capacity of the system is ideally the product of the peak capacities of the two dimensions, yet the analysis time remains relatively short due to parallel second-dimension separations. Aspects affecting the separation efficiency of this type of systems include flow distribution to homogeneously distribute the mobile phase for the second-dimension (2D) separation, flow confinement during the first-dimension (1D) separation, and band-broadening effects during analyte transfer from the 1D separation channel to the 2D separation area. In this study, the synergy between computational fluid dynamics (CFD) simulations and rapid prototyping was exploited to address band broadening during the 2D development and analyte transfer from 1D to 2D. Microfluidic devices for spatial two-dimensional liquid chromatography were designed, simulated, 3D-printed and tested. The effects of presence and thickness of spacers in the 2D separation area were addressed and leaving these out proved to be the most efficient solution regarding band broadening reduction. The presence of a stationary-phase material in the 1D channel had a great effect on the analyte transfer from the 1D to the 2D and the resulting band broadening. Finally, pressure limit of the fabricated devices and printability are discussed.

Experimental and computational analysis of a novel flow channel to assess the adhesion strength of sessile marine organisms.

Bioadhesives produced by marine macroalgae represent a potential source of inspiration for the development of water-resistant adhesives. Assessing their adhesion strength, however, remains difficult owing to low volumes of adhesive material produced, low solubility and rapid curing time. These difficulties can be circumvented by testing the adhesion strength of macroalgae propagules attached to a substrate. In this paper, we present a simple, novel flow channel used to test the adhesion strength of the germlings of the fucalean alga Hormosira banksii to four substrates of biomedical relevance (PMMA, agar, gelatin and gelatin + lipid). The adhesion strength of H. banksii germlings was found to increase in a time-dependent manner, with minimal adhesion success after a settlement period of 6 h and maximum adhesion strength achieved 24 h after initial settlement. Adhesion success increased most dramatically between 6 and 12 h settlement time, while no additional increase in adhesion strength was recorded for settlement times over 24 h. No significant difference in adhesion strength to the various substrates was observed. Computational fluid dynamics (CFD) was used to estimate the influence of fluid velocity and germling density on drag force acting on the settled organisms. CFD modelling showed that, on average, the drag force decreased with increasing germling number, suggesting that germlings would benefit from gregarious settlement behaviour. Collectively, our results contribute to a better understanding of the mechanisms allowing benthic marine organisms to thrive in hydrodynamically stressful environments and provide useful insights for further investigations.

3D printed porous media columns with fine control of column packing morphology.

In this paper we demonstrate, for the first time, the use of 3D printing (also known as additive manufacturing or rapid prototyping) to create porous media with precisely defined packing morphologies, directly from computer aided design (CAD) models. We used CAD to design perfectly ordered beds with octahedral beads (115 µm apothem) packed in a simple cubic configuration and monoliths with hexagonal channels (150 µm apothem) in parallel and herringbone arrangements. The models were then printed by UV curing of acrylonitrile-butadiene-styrene powder layers. Each porous bed was printed at 1.0, 1.5 and 2.0 mL volumes, within a complete column, including internal flow distributors and threaded 10-32 flow connectors. Close replication of CAD models was achieved. The resultant individual octahedral beads were highly uniform in size, with apothems of 113.6±1.9 µm, while the monolith hexagonal cross-section channels had apothems of 148.2±2.0 µm. Residence time distribution measurements show that the beds largely behaved as expected from their design void volumes. Radial and fractal flow distributor designs were also tested. The former displayed poor flow distribution in parallel and herringbone pore columns, while the fractal distributors provided uniform flow distribution over the entire cross section. The results show that 3D printing is a feasible method for producing precisely controlled porous media. We expect our approach to revolutionize not only fundamental studies of flow in porous media but methods of chromatography column production.