Immobilized-Enzyme Reactors Integrated into Analytical Platforms: Recent Advances and Challenges

Immobilized-enzyme reactors (IMERs) are flow-through devices containing enzymes that are physically confined or localized with retention of their catalytic activities. IMERs can be used repeatedly and continuously and have been applied for (bio)polymer degradation, proteomics, biomarker discovery, inhibitor screening, and detection. Online integration of IMERs with analytical instrumentation, such as high-performance liquid chromatography (HPLC) systems, reduces the time needed for multi-step workflows, reduces the need for sample handling, and enables automation. However, online integration can also be challenging, as reaching its full potential requires complex instrumental setups and experienced users. This review aims to provide an assessment of recent advances and challenges in online IMER-based (analytical) LC platforms, covering publications from 2014-2021. A critical discussion of challenges often encountered in IMER fabrication, sample preparation, integration into the analytical workflow, long-term usage, and of potential ways to overcome these is provided. Finally, the obstacles preventing the proliferation of IMERs as efficient tools for high-throughput pharmacological, industrial, and biological studies are discussed

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

Abstract 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%. Since their introduction in the chromatographic world, porous polymer monoliths have proven to be powerful separation media. These chromatographic supports have been widely applied for applications, such as microscale liquid chromatography (LC) of peptides and proteins, but have also been used in capillary electrochromatography (CEC),(1) gas chromatography (GC),(2) sample preparation,(3) and catalysis.(4) The ease of preparation of monoliths, diverse chemistry options, and high permeabilities have made them popular materials for analytical devices, such as microfluidic chips for LC. In the past decade, miniaturization has been realized by developing lab-on-a-chip solutions, where several analytical processes can be integrated within a few square centimeters. In such systems, due to the small channels and articulated geometries, the particle-packing procedure has proven to be challenging.(5) In contrast, monolithic beds are usually created in situ by free-radical polymerization of monomers in the presence of porogens and they are well-suited for chip-based separations. The proliferation of microfluidic devices has spurred new interest in polymer monoliths for applications such as enzymatic reactors(6,7) and microfluidic mixers.(8) This development has been boosted by the advent of additive manufacturing (or 3D-printing), which allows for rapid prototyping of complex structures, converting computer-aided-design (CAD) models into physical objects. Unfortunately, the use of 3D-printed analytical devices for chromatographic analysis is limited by the solvent compatibility of some materials (e.g., acrylate-based polymers) and in some cases by their transparency at the desired wavelength (e.g., UV or IR wavelengths). Several successful steps have been taken to locally photopolymerize monolithic stationary phases in discrete regions of microfluidic devices.(9−12) Heat is an alternative way to transfer energy to the monomer precursors for initiating the polymerization. However, accurate control of temperature in small confined spaces is more difficult to achieve, and so far only few steps have been taken in this direction.(13) In this work, two methods are explored to achieve confined thermal polymerization. The first approach involves direct contact (DC) between Peltier elements and the surface of a titanium device. In the second approach, recirculating jackets are used for localized heating and cooling (heating/cooling jackets, HCJ). The latter approach resembles a recirculation-based freeze–thaw valve.(14) In both approaches, defined hot (HR) and cold (CR) regions are created. We aim to fabricate poly(styrene-co-divinylbenzene) (PS-DVB) monolithic stationary phases within a 3D-printed titanium microfluidic device through polymerization at 70 °C, and to separate intact proteins using this device

Multichannel Capillaries and Photonic Crystal Fibres for Separation Sciences.

Capillaries of many dimensions are fabricated and modified, for use across a wide range of applications within analytical science. This extends from sample preparation and sample extraction, to separation sciences, and detection sciences. Photonic crystal fibres, emerged in the 1990\u27s, and were specifically designed for optical applications, predominantly as waveguides. Since then, a range of modifications have been reported, with the fibres applied to a wide variety of analytical applications, in liquid and gas phase analyses or detection. This review aims to provide an overview of photonic crystal fibres and related multi-channel capillaries, the chemical modifications that have been carried out, and the subsequent applications of the developed materials towards analytical separations, including sample preparation

Direct Production of Microstructured Surfaces for Planar Chromatography Using 3D Printing

Through optimization of the printing process and orientation, a suitably developed surface area has been realized upon a 3D printed polymer substrate to facilitate chromatographic separations in a planar configuration. Using an Objet Eden 260VS 3D printer, polymer thin layer chromatography platforms were directly fabricated without any additional surface functionalization and successfully applied to the separation of various dye and protein mixtures. The print material was characterized using gas chromatography coupled to mass spectrometry and spectroscopic techniques such as infrared and Raman. Preliminary studies included the separation of colored dyes, whereby the separation performance could be visualized optically. Subsequent separations were achieved using fluorescent dyes and fluorescently tagged proteins. The separation of proteins was affected by differences in the isoelectric point (p<i>I</i>) and the ion exchange properties of the printed substrate. The simple chromatographic separations are the first achieved using an unmodified 3D printed stationary phase

Front Cover: Miniaturized capillary ion chromatograph with UV light-emitting diode based indirect absorbance detection for anion analysis in potable and environmental waters

A miniaturized, flexible, and low-cost capillary ion chromatography system has been developed for anion analysis in water. The ion chromatography has an open platform, modular design, and allows for ease of modification. The assembled platform weighs ca. 0.6 kg and is 25 × 25 cm in size. Isocratic separation of common anions (F–, Cl–, NO2–, Br–, and NO3–) could be achieved in under 15 min using sodium benzoate eluent at a flow rate of 3 μL/min, a packed capillary column (0.150 × 150 mm) containing Waters IC-Pak 10 μm anion exchange resin, and light-emitting diode based indirect UV detection. Several low UV light-emitting diodes were assessed in terms of sensitivity, including a new 235 nm light-emitting diode, however, the highest sensitivity was demonstrated using a 255 nm light-emitting diode. Linear calibration ranges applicable to typical natural water analysis were obtained. For retention time and peak area repeatability, relative standard deviation values ranged from 0.60–0.95 and 1.95–3.53%, respectively. Several water samples were analysed and accuracy (recovery) was demonstrated through analysis of a prepared mixed anion standard. Relative errors of –0.36, –1.25, –0.80, and –0.76% were obtained for fluoride, chloride, nitrite, and nitrate, respectively

Miniaturized Capillary ion Chromatograph with UV Light-Emitting Diode Based Indirect Absorbance Detection for Anion Analysis in Potable and Environmental Waters

A miniaturized, flexible, and low-cost capillary ion chromatography system has been developed for anion analysis in water. The ion chromatography has an open platform, modular design, and allows for ease of modification. The assembled platform weighs ca. 0.6 kg and is 25 × 25 cm in size. Isocratic separation of common anions (F–, Cl–, NO2–, Br–, and NO3–) could be achieved in under 15 min using sodium benzoate eluent at a flow rate of 3 μL/min, a packed capillary column (0.150 × 150 mm) containing Waters IC-Pak 10 μm anion exchange resin, and light-emitting diode based indirect UV detection. Several low UV light-emitting diodes were assessed in terms of sensitivity, including a new 235 nm light-emitting diode, however, the highest sensitivity was demonstrated using a 255 nm light-emitting diode. Linear calibration ranges applicable to typical natural water analysis were obtained. For retention time and peak area repeatability, relative standard deviation values ranged from 0.60–0.95 and 1.95–3.53%, respectively. Several water samples were analysed and accuracy (recovery) was demonstrated through analysis of a prepared mixed anion standard. Relative errors of –0.36, –1.25, –0.80, and –0.76% were obtained for fluoride, chloride, nitrite, and nitrate, respectively

What\u27s in this Drink? Classification and Adulterant Detection in Irish Whiskey Samples Using Near Infrared Spectroscopy Combined with Chemometrics

BACKGROUND: Near-infrared (NIR) spectroscopy coupled with principal component analysis (PCA) and partial least squares (PLS) regression was used to analyse a series of different Irish Whiskey samples in order to dene their spectral prole and to assess the capability of the NIR method to identify samples based on their origin and storage (e.g. distiller, method of mat- uration). The ability of NIR spectroscopy to quantify the level of potential chemical adulterants was also investigated. Samples were spiked with 0.1%, 0.5%, 1.0%, 1.5% and 2.0% v/v of each adulterant (e.g. methanol, ethyl acetate, etc.) prior to NIR analysis. RESULTS: The results of this study demonstrated the capability of NIR spectroscopy combined with PLS regression to classify the whiskey samples and to determine the level of adulteration. Moreover, the potential of NIR coupled with chemometric anal- ysis as a rapid, portable, and non-destructive screening tool for quality control, traceability, and food/beverage adulteration for customs and other regulatory agencies, to mitigate beverage fraud was illustrated. CONCLUSION: Given the non-specicity of the NIR technique, these positive preliminary results indicated that this method of analysis has the potential to be applied to identify the level of adulteration in distilled spirits. The rapid nature of the technique and lack of consumables or sample preparation required allows for a far more time and cost-effective analysis per sample