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

β decay of 129Cd and excited states in 129In

J. Taprogge et al.; 11 pags.; 8 figs.; 2 tabs.; PACS number(s): 23.20.Lv, 23.40.−s, 21.60.Cs, 27.60.+j©2015 American Physical Society. The β decay of 129Cd, produced in the relativistic fission of a 238U beam, was experimentally studied at the RIBF facility at the RIKEN Nishina Center. From the γ radiation emitted after the β decays, a level scheme of 129In was established comprising 31 excited states and 69 γ -ray transitions. The experimentally determined level energies are compared to state-of-the-art shell-model calculations. The half-lives of the two β-decaying states in 129Cd were deduced and the β feeding to excited states in 129In were analyzed. It is found that, as in most cases in the Z < 50, N 82 region, both decays are dominated by the ν0g7/2 → π0g9/2 Gamow–Teller transition, although the contribution of first-forbidden transitions cannot be neglected.This work was supported by the Spanish Ministerio de Ciencia e Innovacion under contracts FPA2009-13377-C02 and FPA2011-29854- C04, the Generalitat Valenciana (Spain) under grant PROMETEO/2010/101, the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. NRF-2012R1A1A1041763), the Priority Centers Research Program in Korea (2009-0093817), OTKA contract number K-100835, JSPS KAKENHI (Grant No. 25247045), the European Commission through the Marie Curie Actions call FP7-PEOPLE-2011-IEF under Contract No. 300096, the US Department of Energy, Office of Nuclear Physics, under Contract No. DE-AC02-06CH11357, the “RIKEN foreign research program,” and the German BMBF (No. 05P12RDCIA and 05P12RDNUP) and HIC for FAIR.Peer Reviewe

Phosphopeptidomimetic substance libraries from multicomponent reaction: Enantioseparation on quinidine carbamate stationary phase

In the present contribution we report a HPLC enantioseparation method for a library of amido-aminophosphonate structures generated by a novel Ugi-multicomponent reaction. The enantioseparation of these novel potentially bioactive molecules was achieved by means of HPLC on cinchona-carbamate based chiral anion-exchangers under polar organic elution conditions. The compounds were synthesized with the aim of offering a wide degree of heterogeneity in terms of structural elements to evaluate the influence of various structural motifs on enantioselectivity. The resultant compound library allows to derive structure-enantioselectivity relationships useful for understanding the chiral recognition process. As part of the study mobile phase characteristics such as concentration and type of counterion, mobile phase composition, apparent pH and temperature were investigated with regards to their effect on the separation performances. Employing a single mass spectrometry-compatible mobile phase, 23 compounds out of a pool of 42 racemic amido-aminophosphonate structures reached full baseline separation (Rs>1.5) and 5 were almost baseline separated (

Not (Only) reversed-phase lc–ms:Alternative lc–ms approaches

Electrospray ionization (ESI) and other ambient ionization techniques have allowed a successful interface between liquid chromatography (LC) and mass spectrometry (MS). The coupling of these two high-resolution techniques has fostered the use of analytical science in various fields, including, but not limited to, the clinical, pharmaceutical, and forensic fields, enabling the analysis, identification, and characterization of thousands of molecular components in a large diversity of complex mixtures. For many years, reversed-phase LC has remained the most commonly adopted chromatographic mode, due to its rather straightforward applicability to the analysis of a wide variety of compounds (from small to large molecules), as well as its direct compatibility with ESI-MS. However, reversed-phase LC–MS has shown relevant limitations in a number of analytical applications. This encouraged the development of alternative MS-compatible chromatographic techniques, including hydrophilic interaction chromatography (HILIC), supercritical fluid chromatography (SFC), size-exclusion chromatography (SEC), ion-exchange chromatography (IEC), and hydrophobic interaction chromatography (HIC), which provide analyte separation in the liquid phase based on different retention mechanisms compared with reversed-phase LC. Here, we present these alternative chromatographic approaches, highlighting the recent relevant applications in various fields, and discussing their potential in future of analytical science investigations

Not (Only) reversed-phase lc–ms: Alternative lc–ms approaches

Electrospray ionization (ESI) and other ambient ionization techniques have allowed a successful interface between liquid chromatography (LC) and mass spectrometry (MS). The coupling of these two high-resolution techniques has fostered the use of analytical science in various fields, including, but not limited to, the clinical, pharmaceutical, and forensic fields, enabling the analysis, identification, and characterization of thousands of molecular components in a large diversity of complex mixtures. For many years, reversed-phase LC has remained the most commonly adopted chromatographic mode, due to its rather straightforward applicability to the analysis of a wide variety of compounds (from small to large molecules), as well as its direct compatibility with ESI-MS. However, reversed-phase LC–MS has shown relevant limitations in a number of analytical applications. This encouraged the development of alternative MS-compatible chromatographic techniques, including hydrophilic interaction chromatography (HILIC), supercritical fluid chromatography (SFC), size-exclusion chromatography (SEC), ion-exchange chromatography (IEC), and hydrophobic interaction chromatography (HIC), which provide analyte separation in the liquid phase based on different retention mechanisms compared with reversed-phase LC. Here, we present these alternative chromatographic approaches, highlighting the recent relevant applications in various fields, and discussing their potential in future of analytical science investigations

Hydrophilic interaction liquid chromatography-mass spectrometry for the characterization of glycoproteins at the glycan, peptide, subunit, and intact level

This chapter presents a methodical overview of recent research literature dealing with the characterization of glycoproteins by hydrophilic interaction chromatography separations coupled to mass spectrometry (HILIC-MS). This includes the analysis of intact glycosylated proteins as well as released glycans, glycopeptides, and protein subunits obtained after enzymatic digestion. The chapter provides basic background information on HILIC and glycoproteins, and the molecular levels at which glycosylation can be characterized. Next, essential HILIC parameters such as (the choice of) stationary phase type, mobile phase conditions, injection volume, and column temperature are reviewed. Herein focus is on the effect that these parameters have on both HILIC separation and MS detection. This is discussed in the context of the respective levels (glycan/peptide/subunit/intact) of glycoprotein analysis and current trends are highlighted. In the final part of this chapter, these developments are put in perspective by treating several applications of HILIC-MS for glycoprotein analysis in more detail, particularly in biopharmaceutical and clinical fields

Computer-aided gradient optimization of hydrophilic interaction liquid chromatographic separations of intact proteins and protein glycoforms

Protein glycosylation is one of the most common and critical post-translational modification, which results from covalent attachment of carbohydrates to protein backbones. Glycosylation affects the physicochemical properties of proteins and potentially their function. Therefore it is important to establish analytical methods which can resolve glycoforms of glycoproteins. Recently, hydrophilic-interaction liquid chromatography (HILIC)-mass spectrometry has demonstrated to be a useful tool for the efficient separation and characterization of intact protein glycoforms. In particular, amide-based stationary phases in combination with acetonitrile-water gradients containing ion-pairing agents, have been used for the characterization of glycoproteins. However, finding the optimum gradient conditions for glycoform resolution can be quite tedious as shallow gradients (small decrease of acetonitrile percentage in the elution solvent over a long time)are required. In the present study, the retention mechanism and peak capacity of HILIC for non-glycosylated and glycosylated proteins were investigated and compared to reversed-phase liquid chromatography (RPLC). For both LC modes, ln k vs. φ plots of a series of test proteins were calculated using linear solvent strength (LSS)analysis. For RPLC, the plots were spread over a wider φ range than for HILIC, suggesting that HILIC methods require shallower gradients to resolve intact proteins. Next, the usefulness of computer-aided method development for the optimization of the separation of intact glycoform by HILIC was examined. Five retention models including LSS, adsorption, and mixed-mode, were tested to describe and predict glycoprotein retention under gradient conditions. The adsorption model appeared most suited and was applied to the gradient prediction for the separation of the glycoforms of six glycoproteins (Ides-digested trastuzumab, alpha-acid glycoprotein, ovalbumin, fetuin and thyroglobulin)employing the program PIOTR. Based on the results of three scouting gradients, conditions for high-efficiency separations of protein glycoforms varying in the degree and complexity of glycosylation was achieved, thereby significantly reducing the time needed for method optimization

Enhancing detectability of anabolic-steroid residues in bovine urine by actively modulated online comprehensive two-dimensional liquid chromatography - high-resolution mass spectrometry

In this study we describe an approach to enhance the sensitivity of an online comprehensive two-dimensional liquid chromatography (LC × LC) high-resolution mass spectrometry method for the separation and detection of trace levels of anabolic-steroid residues in complex urine matrices.Compared to one-dimensional liquid chromatography (1D-LC), LC × LC methods offer higher separation power, thanks to the combined effect of two different selectivities and a higher peak capacity. However, when using state-of-the-art LC × LC instrumentation, the price paid for the increase in separation power is a decrease in sensitivity and detectability of trace-level analytes. This can be ascribed to the sample dilution that takes place during each of the two chromatographic steps. The way in which fractions are collected and transferred from the first to the second column is also of paramount importance, especially the volume and the solvent composition of the fractions injected in the second column.To overcome the detection limitation, we present an active-modulation strategy, based on concentrating the fractions of the first-dimension effluent using a modulation interface that employs trap columns. We obtained a signal enhancement for anabolic-steroid compounds in a bovine-urine sample by a factor of 2.4-7.6 and an increase in the signal-to-noise ratio up to a factor of 7 in comparison with a standard loop-based modulation interface. In addition, thanks to the increased sensitivity of our method, a substantially larger number of peaks were detected (76 vs. 36). Moreover, we could reduce the solvent consumption by a factor of three (160 mL vs. 500 mL per run)

Characterization of complex polyether polyols using comprehensive two-dimensional liquid chromatography hyphenated to high-resolution mass spectrometry

Polyether polyols are often used in formulated systems, but their complete characterization is challenging, because of simultaneous heterogeneities in chemical composition, molecular weight and functionality. One-dimensional liquid chromatography–mass spectrometry is commonly used to characterize polyether polyols. However, the separation power of this technique is not sufficient to resolve the complexity of such samples entirely. In this study, comprehensive two-dimensional liquid chromatography hyphenated with high-resolution mass spectrometry (LC × LC-HRMS) was used for the characterization of (i) castor oil ethoxylates (COEs) reacted with different mole equivalents of ethylene oxide and (ii) a blended formulation consisting of glycerol ethoxylate, glycerol propoxylate and glycerol ethoxylate-random-propoxylate copolymers. Retention in the first (hydrophilic-interaction-chromatography) dimension was mainly governed by degree of ethoxylation, while the second reversed-phase dimension resolved the samples based on degree of propoxylation (blended formulation) or alkyl chain length (COEs). For different COE samples, we observed the separation of isomer distributions of various di-, tri- and tetra-esters, and such positional isomers were studied by tandem mass spectrometry (LC–MS/MS). This revealed characteristic fragmentation patterns, which allowed discrimination of the isomers based on terminal or internal positioning of the fatty-acid moieties and provided insight in the LC × LC retention behavior of such species

Micro-flow size-exclusion chromatography for enhanced native mass spectrometry of proteins and protein complexes

Size-exclusion chromatography (SEC) employing aqueous mobile phases with volatile salts at neutral pH combined with native mass spectrometry (nMS) is a valuable tool to characterize proteins and protein aggregates in their native state. However, the liquid-phase conditions (high salt concentrations) frequently used in SEC-nMS hinder the analysis of labile protein complexes in the gas phase, necessitating higher desolvation-gas flow and source temperature, leading to protein fragmentation/dissociation. To overcome this issue, we investigated narrow SEC columns (1.0 mm internal diameter, I.D.) operated at 15-μL/min flow rates and their coupling to nMS for the characterization of proteins, protein complexes and higher-order structures (HOS). The reduced flow rate resulted in a significant increase in the protein-ionization efficiency, facilitating the detection of low-abundant impurities and HOS up to 230 kDa (i.e., the upper limit of the Orbitrap-MS instrument used). More-efficient solvent evaporation and lower desolvation energies allowed for softer ionization conditions (e.g., lower gas temperatures), ensuring little or no structural alterations of proteins and their HOS during transfer into the gas phase. Furthermore, ionization suppression by eluent salts was decreased, permitting the use of volatile-salt concentrations up to 400 mM. Band broadening and loss of resolution resulting from the introduction of injection volumes exceeding 3% of the column volume could be circumvented by incorporating an online trap-column containing a mixed-bed ion-exchange (IEX) material. The online IEX-based solid-phase extraction (SPE) or “trap-and-elute” set-up provided on-column focusing (sample preconcentration). This allowed the injection of large sample volumes on the 1-mm I.D. SEC column without compromising the separation. The enhanced sensitivity attained by the micro-flow SEC-MS, along with the on-column focusing achieved by the IEX precolumn, provided picogram detection limits for proteins