Sam Wouters

Advances in organic polymer-based monolithic column technology for high-resolution liquid chromatography-mass spectrometry profiling of antibodies, intact proteins, oligonucleotides, and peptides.

This review focuses on the preparation of organic polymer-based monolithic stationary phases and their application in the separation of biomolecules, including antibodies, intact proteins and protein isoforms, oligonucleotides, and protein digests. Column and material properties, and the optimization of the macropore structure towards kinetic performance are also discussed. State-of-the-art liquid chromatography-mass spectrometry biomolecule separations are reviewed and practical aspects such as ion-pairing agent selection and carryover are presented. Finally, advances in comprehensive two-dimensional LC separations using monolithic columns, in particular ion-exchange×reversed-phase and reversed-phase×reversed-phase LC separations conducted at high and low pH, are shown.

Design and performance evaluation of a microfluidic ion-suppression module for anion-exchange chromatography

A microfluidic membrane suppressor has been constructed to suppress ions of alkaline mobile-phases via an acid-base reaction across a sulfonated poly(tetrafluoroethylene)-based membrane and was evaluated for anion-exchange separations using conductivity detection. The membrane was clamped between two chip substrates, accommodating rectangular microchannels for the eluent and regenerant flow, respectively. Additionally, a clamp-on chip holder has been constructed which allows the alignment and stacking of different chip modules. The response and efficacy of the microfluidic chip suppressor was assessed for a wide range of eluent (KOH) concentrations, using 127 and 183μm thick membranes, while optimizing the flow rate and concentration of the regenerant solution (H2SO4). The optimal operating eluent flow rate was determined at 5μL/min, corresponding to the optimal van-Deemter flow velocity of commercially-available column technology, i.e. a 0.4mm i.d.×250mm long column packed with 7.5μm anion-exchange particles. When equilibrated at 10mM KOH, a 99% decrease in conductivity signal could be obtained within 5min when applying 10mM H2SO4 regenerant at 75μL/min. A background signal as low as 1.2μS/cm was obtained, which equals the performance of a commercially-available electrolytic hollow-fiber suppressor. When increasing the temperature of the membrane suppressor from 15 to 20°C, ion suppression was significantly improved allowing the application of 75mM KOH. The applicability of the chip suppressor has been demonstrated with an isocratic baseline separation of a mixture of seven inorganic ions, yielding plate numbers between 5300 and 10,600 and with a gradient separation of a complex ion mixture.

Monitoring the morphology development of polymer-monolithic stationary phases by thermal analysis

Thermal analysis and SEM were employed to gain insights in the different stages of morphology development and the thermal properties of polymer-monolithic stationary phases. The studied system was a thermally initiated free-radical copolymerization reaction at 70°C of styrene and divinylbenzene in the presence of tetrahydrofuran and 1-decanol. The key events in the early stages of morphology development are initiation, chain growth, branching, and cyclization, leading to microgel particles. Interparticle reactions through pendant vinyl groups lead to the formation of microgel clusters. The rapid increase in molecular weight and cross-link density of the microgel clusters causes a reaction-induced phase separation, and the formation of a macroscopic network of interconnected globules was observed (macrogelation) at around 45 min. After 3 h or 65% conversion, a space-filling macroporous monolithic network was observed. Afterwards, mainly growth of existing globules takes place, reducing the macropore size. The porogen ratio affects the timing of the reaction-induced phase separation, strongly influencing the morphology of the polymer material. The use of a mixture of divinylbenzene isomers yielded a monolithic material that is less cross-linked at the surface compared to the central part of the polymer backbone due to copolymerization-composition drift. The less cross-linked outer layer starts devitrifying at 100°C.

A cyclic-olefin-copolymer microfluidic immobilized-enzyme reactor for rapid digestion of proteins from dried blood spots

A critical step in the bottom-up characterization of proteomes is the conversion of proteins to peptides, by means of endoprotease digestion. Nowadays this method typically uses overnight digestion and as such represents a considerable bottleneck for high-throughput analysis. This report describes protein digestion using an immobilized-enzyme reactor (IMER), which enables accelerated digestion times that are completed within seconds to minutes. For rapid digestion to occur, a cyclic-olefin-copolymer microfluidic reactor was constructed containing trypsin immobilized on a polymer monolithic material through a 2-vinyl-4,4-dimethylazlactone linker. The IMER was applied for the rapid offline digestion of both singular protein standards and a complex protein mixture prior to liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) analysis. The effects of protein concentration and residence time in the IMER were assessed for protein standards of varying molecular weight between 11 and 240kDa. Compared to traditional in-solution digestion, IMER-facilitated protein digestion at room temperature for 5min yielded similar results in terms of sequence coverage and number of identified peptides. Good repeatability was demonstrated with a relative standard deviation of 6% for protein-sequence coverage. The potential of the IMER was also demonstrated for a complex protein mixture in the analysis of dried blood spots. Compared to a traditional workflow a similar number of proteins could be identified, while reducing the total analysis time from 22.5h to 4h and importantly omitting the sample-pre-treatment steps (denaturation, reduction, and alkylation). The identified proteins from two workflows showed similar distributions in terms of molecular weight and hydrophobic character.

Design and evaluation of microfluidic devices for two-dimensional spatial separations

Various designs of chips for comprehensive two-dimensional spatial liquid chromatography were investigated. The performance of these chips was initially evaluated using computational fluid dynamics (CFD). A bifurcating distributor with an angle of 140° between branches was implemented in order to achieve a homogeneous velocity field. The cross-sectional area of the channels of the flow distributor was fixed at 0.5 × 0.5 mm, which allows a robust micromilling technique to be used for chip manufacturing. Experiments were performed with chips featuring purposely introduced imperfections in the structure of the bifurcating flow distributor to study its capacity of overcoming potential local clogging. Split peaks were observed when 75% of one of the flow channels was obstructed, in line with the CFD predictions. The main bottlenecks for the performance of the spatial two-dimensional chips were identified, viz. sample injected in the first dimension diverging into the flow distributor and channel discretization (i.e., remixing of first-dimension separation peaks because of finite number of second-dimension channels). Solutions to the former problem were studied by applying a flow resistance in the vertical segments that formed the outlets of the flow distributor and by simulating the presence of constrictions. It was found that a flow resistance of 1.0×10(11) m(-2) reduced the amount of sample diverging into the flow distributor by a factor of 10. The presence of a constriction of 90% of the segment area and 50% of the segment length decreased the diverging flow by a factor of 5. The influence of the linear velocity was significant. Solutions to the channel discretization problem were sought by investigating different designs of spatial two-dimensional chips.

Using contemporary liquid chromatography theory and technology to improve capillary gradient ion-exchange separations.

The gradient-performance limits of capillary ion chromatography have been assessed at maximum system pressure (34.5 MPa) using capillary columns packed with 4.1 μm macroporous anion-exchange particles coated with 65 nm positively-charged nanobeads. In analogy to the van-Deemter curve, the gradient performance was assessed applying different flow rates, while decreasing the gradient time inversely proportional to the increase in flow rate in order to maintain the same retention properties. The gradient kinetic-performance limits were determined at maximum system pressure, applying tG/t0=5, 10, and 20. In addition, the effect of retention on peak width was assessed in gradient mode for mono-, di-, and trivalent inorganic anions. The peak width of late-eluting ions can be significantly reduced by using concave gradient, resulting in better detection sensitivity. A signal enhancement factor of 8 was measured for a late-eluting ion when applying a concave instead of a linear gradient. For the analysis of a complex anion mixture, a coupled column with a total length of 1.05 m was operated at the kinetic-performance limit applying a linear 250 min gradient (tG/t0=10). The peak capacity varied between 200 and 380 depending on analyte retention, and hence on charge and size of the ion.

Comprehensive study of the macropore and mesopore size distributions in polymer monoliths using complementary physical characterization techniques and liquid chromatography.

Poly(styrene-co-divinylbenzene) monolithic stationary phases with two different domain sizes were synthesized by a thermally initiated free-radical copolymerization in capillary columns. The morphology was investigated at the meso- and macroscopic level using complementary physical characterization techniques aiming at better understanding the effect of column structure on separation performance. Varying the porogenic solvent ratio yielded materials with a mode pore size of 200 nm and 1.5 μm, respectively. Subsequently, nano-liquid chromatography experiments were performed on 200 μm id × 200 mm columns using unretained markers, linking structure inhomogeneity to eddy dispersion. Although small-domain-size monoliths feature a relatively narrow macropore-size distribution, their homogeneity is compromised by the presence of a small number of large macropores, which induces a significant eddy-dispersion contribution to band broadening. The small-domain size monolith also has a relatively steep mass-transfer term, compared to a monolith containing larger globules and macropores. Structural inhomogeneity was also studied at the mesoscopic level using gas-adsorption techniques combined with the non-local-density-function-theory. This model allows to accurately determine the mesopore properties in the dry state. The styrene-based monolith with small domain size has a distinctive trimodal mesopore distribution with pores of 5, 15, and 25 nm, whereas the monolith with larger feature sizes only contains mesopores around 5 nm in size.

Prototyping of thermoplastic microfluidic chips and their application in high-performance liquid chromatography separations of small molecules

The present paper discusses practical aspects of prototyping of microfluidic chips using cyclic olefin copolymer as substrate and the application in high-performance liquid chromatography. The developed chips feature a 60mm long straight separation channel with circular cross section (500μm i.d.) that was created using a micromilling robot. To irreversibly seal the top and bottom chip substrates, a solvent-vapor-assisted bonding approach was optimized, allowing to approximate the ideal circular channel geometry. Four different approaches to establish the micro-to-macro interface were pursued. The average burst pressure of the microfluidic chips in combination with an encasing holder was established at 38MPa and the maximum burst pressure was 47MPa, which is believed to be the highest ever report for these polymer-based microfluidic chips. Porous polymer monolithic frits were synthesized in-situ via UV-initiated polymerization and their locations were spatially controlled by the application of a photomask. Next, high-pressure slurry packing was performed to introduce 3μm silica reversed-phase particles as the stationary phase in the separation channel. Finally, the application of the chip technology is demonstrated for the separation of alkyl phenones in gradient mode yielding baseline peak widths of 6s by applying a steep gradient of 1.8min at a flow rate of 10μL/min.

System Design and Emerging Hardware Technology for Ion Chromatography

This review provides an overview of advances in system design for ion chromatography (IC), focusing on the suppressed conductivity detection mode. In particular, advances in automated mobile-phase generation and suppressor technology based on different electrolytic concepts are addressed and novel detection approaches are discussed. Finally, advances in multi-dimensional IC and aspects of miniaturization, including capillary IC instrumentation and chip-based IC, are discussed.

Microfluidic membrane suppressor module design and evaluation for capillary ion chromatography

A microfluidic ion-suppression module for use in ion-exchange chromatography has been developed and evaluated. The device consists of an ion-exchange membrane clamped between two polymer chips featuring a 200×100μm (width×depth) eluent channel (l=60mm), and a 300×150μm regenerant channel (60mm), respectively. The suppression efficacy using a Nafion membrane was compared with that of a styrene-sulfonate grafted fluorinated ethylene propylene (FEP) membrane. The latter was found to outperform Nafion in terms of lowest attainable background signal (suppression efficacy) and dynamic suppression range. Increasing the suppressor temperature or the sulfuric acid regenerant concentration led to an extension of the operational suppression range, this however at the cost of an increased background signal due to enhanced diffusion, inducing sulfate bleed. Under optimized operating conditions, the microfluidic suppressor provided a dynamic capacity of 0.35μEq./min, being compatible with gradient separations applying up to 70mM KOH in combination with 400μm i.d. capillary columns operated at the optimal flow velocity. The applicability of the miniaturized suppressor is demonstrated for both isocratic and gradient separations of mixtures of inorganic anions. Band-broadening characteristics of the suppressor were optimized with respect to a commercial capillary hollow-fiber suppressor, yielding comparable overall system efficiency, e.g., 8500 plates for nitrate recorded on a 150mm long capillary column. A second chip device was also constructed, featuring suppression at both sides of the eluent flow path. This double-sided suppressor allowed to increase sample throughput and operate at eluent flow rates of 10μL/min, while maintaining efficient suppression characteristics.