Microfluidic Pressure Driven Liquid Chromatography of Biologically Relevant Samples

An overview of the literature regarding the most recent and innovative developments in microfluidic devices for pressure-driven chromatographic separations is given, with a focus on proteomics and metabolomics applications. The applications can be considered as the main driving force for the developments in this research field, since they put high demands on the analytical technology such as for throughput, efficiency, and sensitivity and for the possibilities to interface with mass spectrometry. The developments are evaluated based on the feasibility for use in work flows for the analysis of biologically relevant samples. The literature up to the first half of 2011 is covered. Electrophoretic separations are not within the scope of this review. Several strategies have been described to obtain a retentive phase in microfluidic channels. Open channels with the stationary phase bound to the walls appear to be relatively easy to make. However, the retention in such channels is generally very low for separations of relevant samples. Microfabrication of perfectly ordered topographic structures is the most innovative of the methods discussed for the creation of stationary phases in narrow channels. Several groups work on the improvement of the surface-to-volume ratio in such channels, using different methods, and the developments towards real applications are promising. Channels packed with spherical particles and in situ polymerized monoliths for pressure-driven separations are the most frequently applied. Microfluidic devices with an integrated injection system, a (packed) separation column and a spray tip for coupling to a mass spectrometer are already commercially available, and used in practice in proteomics and metabolomics. Finally, the inherent advantages of microfluidic devices for multidimensional separations have been shown in practice in a number of studies. In these studies, pressure-driven chromatography is coupled (in series or multiplexed) to an electrophoretic separation method. The high peak capacity of such 2-dimensional separations has been shown.

     

"Thiol-ene" click chemistry: a facile and versatile route for the functionalization of porous polymer monoliths

The preparation of porous polymer monoliths with dodecyl and zwitterionic functionalities via the "thiol-ene" click chemistry of thiol-containing monoliths with both hydrophobic and polar methacrylate "ene" monomers has been demonstrated. Selected separations confirmed the excellent potential of these monoliths in chromatography.     

Optimization of the single-step synthesis of hybrid C(8) silica monoliths dedicated to nano-liquid chromatography and capillary electrochromatography

Hybrid silica monoliths functionalized with octyl groups and dedicated to chromatographic separations in the reversed-phase mode were directly synthesized within capillaries according to the protocol described by Yan et al. [L.J. Yan, Q.H. Zhang, Y.Q. Feng, W.B. Zhang, T. Li, L.H. Zhang, Y.K. Zhang, J. Chromatogr. A 1121 (2006) 92]. Although these monoliths allowed reaching high efficiencies in capillary electrochromatography (CEC), serious limitations prohibited their application in nano-liquid chromatography (nano-LC). Such limitations observed as poor performances in the nano-LC mode and the lack of reproducibility of the synthesis were related to the longitudinal morphological inhomogeneities of the hybrid material along the capillary. Thus, several modifications were conducted in the synthesis protocol in order to improve the resulting morphology of the monolith making it suitable for nano-LC separations. The influence of several critical parameters (such as the addition temperature of the basic catalyst and the hydrolysis duration) on the textural and chromatographic properties had been extensively studied. It was found that a decrease (i.e. 0 degrees C) of the temperature addition of the basic catalyst associated with a shorter hydrolysis duration (1h instead of 6h) allowed (i) delaying the gelation time and consequently facilitating the capillary filling step, (ii) increasing the structural homogeneity of the hybrid monoliths, i.e. their chromatographic performances in nano-liquid chromatography also (iii) greatly improving the reproducibility of the synthesis within the capillary without impairing the material's carbon load, i.e. the incorporation of the less hydrolysable C(8) precursor. The resulting hybrid monoliths afforded retention factors comparable to that previously obtained for C(18) grafted silica monoliths and efficiencies that are the best ever recorded in nano-LC with hybrid monoliths and that are close to the ones achieved with grafted silica monoliths. In fact, this modified protocol allowed a significant improvement of the performances in nano-LC which could be observed by the decrease of the mean value of H(min) going from 123 microm (Yan's protocol) to 24 microm (modified protocol) for a same length of capillary (l = 8.5 cm). In addition, the reproducibility of the synthesis was greatly improved through a factor six of reduction on the calculated standard deviation of these efficiencies. The high permeability and longitudinal homogeneity of the synthesized monolith allowed increasing the capillary length (for example, a 75-cm capillary was conveniently filled with hybrid silica monolith) and the column could be eluted at a very low backpressure leading to chromatographic performances up to 40,000 plates. Finally, the good efficiencies in the nano-LC mode combined with the excellent performances already present in the CEC mode led to fast (less than 1 min) and high efficient separations in the pressurized capillary electrochromatography (p-CEC) mode.     

Proteomics based on high-efficiency capillary separations

Identifying and quantifying in a high throughput manner the proteins expressed by cells, tissues or an organism provides the basis for understanding the functions of its constituents at a "systems" level. As a result, proteome analysis has increasingly become the focus of significant interest and research over the past decade. This is especially true following the recent stunning achievements in genomics analyses. However, unlike the static genome, the complexities and dynamism of the proteome present significant analytical challenges and demand highly efficient separations and detection technologies. A number of recent technological advancements have been in direct response to these challenges. Currently, strategically mated combinations of sophisticated separations techniques and advanced mass spectrometric detection represent the best approach to addressing the intricacies of the proteome. Liquid-phase separations, often within capillaries, are increasingly recognized as the best separations technique for this approach. In combination on-line with mass spectrometry, liquid-phase separations provide the improved analytical sensitivity, sample throughput, and quantitation capabilities necessitated by the multifaceted problems within proteomics analyses. This review focuses primarily on current high-efficiency capillary separations techniques, including both capillary liquid chromatography and capillary electrophoresis, applied to the analysis of complex proteomic samples. We emphasize developments at our laboratory and illustrate technical advances that attempt to review the role of separations within the broader context of a state-of-the-art integrated proteomics effort.     

Photopolymerized sol–gel monoliths for separations of glycosylated proteins and peptides in microfluidic chips

Photopolymerized silica sol–gel monoliths, functionalized with boronic acid ligands, have been developed for protein and peptide separations in polydimethylsiloxane microfluidic devices. Pore size characterization of the monoliths was carried out with SEM, image analysis, and differential scanning calorimetry to evaluate both the micron‐sized macropores and the nanometer‐sized mesopores. Monoliths were functionalized with boronic acid using three different immobilization techniques. Batch experiments were conducted to determine the capacity of the monoliths and selectivity toward cis‐diol‐containing compounds. Conalbumin was used as a model glycoprotein, and a tryptic digest of the glycoprotein horseradish peroxidase was used as a peptide mixture to demonstrate proof‐of‐concept extraction of glycoproteins and glycopeptides by the monoliths formulated in polydimethylsiloxane microfluidic chips. For proteins, fluorescence detection was used, whereas the peptide separations employed off‐line analysis using MALDI‐MS.     

Recent strategies to enhance the performance of polymer monoliths for analytical separations

This review summarizes recent developments made in the incorporation of functional materials into organic polymer monoliths, together with new monolithic forms and formats, which enhance their application as supports and stationary phase materials for sample preparation and chromatographic separations. While polymer monoliths are well‐known supports for the separation of large molecules, recent developments have been made to improve their features for the separation of small molecules. The selectivity and performance of organic polymer monoliths has been improved by the incorporation of different materials, such as metal‐organic frameworks, covalent organic frameworks, or other types of nanostructured materials (carbon nanohorns, nanodiamonds, polyoxometalates, layered double hydroxides, or attapulgite). The surface area of polymer monoliths has been significantly increased by polymer hypercrosslinking, resulting in increased efficiency when applied to the separation of small molecules. In addition, recent exploration of less conventional supports for casting polymer monoliths, including photonic fibres and 3D printed materials, has opened new avenues for the applications of polymer monoliths in the field of separation science. Recent developments made in these topics are covered, focusing on the strategies followed by the authors to prepare the polymer monoliths and the effect of these modifications on the developed analytical applications.     

Chiral silica-based monoliths in chromatography and capillary electrochromatography

Chiral-modified silica-based monoliths have become well-established stationary phases for both high performance liquid chromatography (HPLC) and capillary electrochromatography (CEC). The silica-based monoliths were fabricated either in situ in the capillaries for nano-HPLC and CEC or in a mould for “conventional” HPLC. The present review summarizes the chiral modification of silica monoliths and the recent development in the field of enantioselective separations by nano-HPLC and CEC.     

Extending the array of crosslinkers suitable for the preparation of polymethacrylate-based monoliths

The in situ preparation of monolithic capillary columns comprising copolymers of butyl methacrylate with ethylene dimethacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, and pentaerythritol tetraacrylate using thermal polymerization within 250 microm ID capillaries and their application for micro-HPLC separations of proteins has been studied. For all crosslinkers, optimization of the porogenic mixture consisting of 1-propanol and 1,4-butanediol yielded monoliths with pore sizes above 1 microm suitable for rapid separations at low back pressure. Very good separations were achieved for a protein mixture consisting of ribonuclease A, cytochrome c, myoglobin, and ovalbumin with all tested columns.     

Polymethacrylate monoliths for preparative and industrial separation of biomolecular assemblies

Monoliths are considered as the fourth-generation chromatography material. Their use for preparative separation of biomolecules has been evolved over the past decade. Monolithic columns up to 8L in size are already commercially available for separation of large biomolecules such as proteins, protein aggregates, plasmid DNA, and viruses. These applications leverage monoliths' inherent properties, such as fast operation and high capacity for large biomolecules. The height equivalent to a theoretical plate (HETP) and dynamic binding capacity do not change with velocity. This is explained by the convective transport through the channels with a diameter of above 1000 nm and has been experimentally verified and also supported by theoretical analyses. Despite low absolute surface area, these large channels provide enough area for adsorption of these large biomolecules, which cannot penetrate into conventional chromatography media designed for protein separation. Monoliths for preparative separations are mainly cast as polymethacrylate or polyacrylamide blocks and have been functionalized as ion exchangers or hydrophobic interaction chromatography media. So-called cryogels have channels more than 30 microm wide, enabling efficient processing of suspensions or even cell-chromatography. This review discusses the pressure drop characteristics, mass transfer properties, scale-up, and applications of monoliths in the context of conventional chromatography media.     

Porous Monoliths: Stationary Phases of Choice for High Performance Liquid Chromatography in Various Formats

 Modern porous monoliths have been conceived as a new class of stationary phases for high performance liquid chromatography (HPLC) in classical columns in the early 1990s and later extended to the capillary format. These monolithic materials are prepared using simple processes carried out in an external mold (inorganic monoliths) or within the confines of the column (organic monoliths and all capillary columns). These methods afford macroporous materials with large through-pores that enable applications in a rapid flow-through mode. Since all the mobile phase must flow through the monolith, the convection considerably accelerates mass transport within the monolithic separation medium and improves the separations. As a result, the monolithic columns perform well even at very high flow rates. The applications of monolithic capillary columns are demonstrated on numerous separations in the HPLC mode.     

In situ sol-gel preparation of porous alumina monoliths for chromatographic separations of adenosine phosphates

A method enabling the in situ preparation of porous alumina monoliths within 100 μm i.d. fused silica capillaries has been developed. These monoliths were prepared using the sol-gel process from a mixture consisting of an inorganic aluminum salt, a porogen, an epoxide, and a solvent. We investigated the effects of varying the preparation conditions on the physical characteristics of the monoliths with respect to their potential application in chromatographic separations. The best columns were obtained from a mixture of aluminum chloride hexahydrate, N,N-dimethylformamide, water, ethanol and propylene oxide. Adenosine phosphates were then separated in the optimized column with retention increasing according to number of phosphate functionalities.     

Silica gel-based monoliths prepared by the sol-gel method: facts and figures

There is a great deal of interest in continuous beds as stationary phases for both HPLC and CEC. There are various ways to prepare monoliths, by polymerization of organic species or by polymerization of silicon alkoxides. The former method has recently been reviewed, while silica based monoliths are now commercially available. The purpose of this paper is to deal with the problems associated with silica based monoliths. The most important problem is obviously the cracking and the shrinkage of the bed during drying. The second problem is monolith cladding. Much literature has been published but no definitive solution is available and thus a wide research area remains open. Monoliths are a compromise between loadability, permeability and mass transfer kinetics. Due to the better mass transfer properties of a monolithic skeleton over distinct particles, high flow rates and high speed separations are possible.     

Synthesis of zirconia monoliths for chromatographic separations

The aim of this work is to join the advantages of two different kinds of stationary phases: monolithic columns and zirconia-based supports. On the one hand, silica monolithic columns allow a higher efficiency with a lower back-pressure than traditional packed columns. On the other hand, chromatographic stationary phases based on zirconia have a higher thermal and chemical stability and specific surface properties. Combining these advantages, a zirconia monolith with a macroporous framework could be a real improvement in separation sciences. Two main strategies can be used in order to obtain a zirconia surface on a monolithic skeleton: coating or direct synthesis. The coverage by a zirconia layer of the surface of a silica-based monolith can be performed using the chemical properties of the silanol surface groups. We realized this coverage using zirconium alkoxide and we further grafted n-dodecyl groups using phosphate derivatives. Any loss of efficiency was observed and fast separations have been achieved. The main advance reported in this paper is related to the preparation of zirconia monoliths by a sol-gel process starting from zirconium alkoxide. The synthesis parameters (hydrolysis ratio, porogen type, precursor concentration, drying step, etc.) were defined in order to produce a macroporous zirconia monoliths usable in separation techniques. We produced various homogeneous structures: zirconia rod 2 cm long with a diameter of 2.3 mm, and zirconia monolith inside fused silica capillaries with a 75 microm I.D. These monoliths have a skeleton size of 2 microm and have an average through pore size of 6 microm. Several separations have been reported.     

Less common applications of monoliths. III. Gas chromatography

Porous polymer monoliths emerged about two decades ago. Despite this short time, they are finding applications in a variety of fields. In addition to the most common and certainly best known use of this new category of porous media as stationary phases in liquid chromatography, monolithic materials also found their applications in other areas. This review article focuses on monoliths in capillaries designed for separations in gas chromatography.     

Incorporation of ionic liquid into porous polymer monoliths to enhance the separation of small molecules in reversed‐phase high‐performance liquid chromatography

An ionic liquid was incorporated into the porous polymer monoliths to afford stationary phases with enhanced chromatographic performance for small molecules in reversed‐phase high‐performance liquid chromatography. The effect of the ionic liquid in the polymerization mixture on the performance of the monoliths was studied in detail. While monoliths without ionic liquid exhibited poor resolution and low efficiency, the addition of ionic liquid to the polymerization mixture provides highly increased resolution and high efficiency. The chromatographic performances of the monoliths were demonstrated by the separations of various small molecules including aromatic hydrocarbons, isomers, and homologues using a binary polar mobile phase. The present column efficiency reached 27 000 plates/m, which showed that the ionic liquid monoliths are alternative stationary phases in the separation of small molecules by high‐performance liquid chromatography     

离子色谱整体固定相的最新进展

整体固定相是近年来新兴的一种多孔性固定相介质,它在离子态及极性化合物的分离中得到了越来越广泛的重视。本文就离子色谱领域整体固定相的发展以及最新的研究动向进行了综述,讨论了离子色谱整体固定相的优点、分类以及在分离分析离子态物质方面的应用等。     

Electrochemical interfaces for chemical and biomolecular separations

The design of molecularly selective interfaces can lead to efficient electrochemically-mediated separation processes. The fast growing development of electroactive materials has resulted in new electroresponsive adsorbents and membranes, with enhanced selectivity, higher uptake capacities, and improved energy performance. Here, we review progress on the interfacial design for electrochemical separations, with a focus on chemical and biological applications. We discuss the development of new electrode materials and the underlying mechanisms for selective molecular binding, highlighting areas of growing interest such as metal recovery, waste recycling, gas purification, and protein separations. Finally, we emphasize the need for integration between molecular level interface design and electrochemical engineering for the development of more efficient separation processes. We envision that electrochemical separations can play a key role towards the electrification of the chemical industry and contribute towards new approaches for process intensification.     

Comparing column dynamics in the liquid and vapor phase adsorption of biobutanol on an activated carbon monolith

Adsorption - Adsorbent monoliths are gaining increasing interest in gas phase separation processes, but have rarely been studied for liquid phase separations. In this work, we investigate an...     

Prospects for monolithic nano-LC columns in shotgun proteomics

We report a premier side-by-side comparison of two leading types of monolithic nano-LC column (silica-C(18), polystyrene) in shotgun proteomics experiments. Besides comparing the columns in terms of the number of peptides from a real-life sample (Arabidopsis thaliana chloroplast) that they identified, we compared the monoliths in terms of peak capacity and retention behavior for standard peptides. For proteomics applications where the mobile phase composition is constrained by electrospray ionization considerations (i.e., there is a restricted choice of ion-pairing modifiers), the polystyrene nano-LC column exhibited reduced identification power. The silica monolith column was superior in all measured values and compared very favorably with traditional packed columns. Finally, we investigated the performances of the monoliths at high flow rates in an attempt to demonstrate their advantages for high-throughput identification.     

Preparation and characterization of macroporous monoliths imprinted with erythromycin

The synthesis of macroporous molecularly imprinted monoliths was performed using the monomers system 2‐hydroxyethyl methacrylate‐ethylene glycol dimethacrylate and erythromycin as a template. The copolymerization was carried out in situ inside 50 mm × 4.6 mm i.d. stainless‐steel tubing. The morphology of the monoliths was examined with scanning electron microscopy. The porous characteristics were determined both from the data of hydrodynamic permeability of monoliths and by means of mercury intrusion porosimetry. The retention parameters of target substance (erythromycin), values of calculated imprinting factors and apparent dynamic dissociation constants were obtained for monoliths prepared with the application of different amount of template (4, 8 and 12 mol%). The separations of the mixtures azithromycin/erythromycin and ciprofloxacin/erythromycin were demonstrated. Additionally, the possibility of erythromycin quantification in human blood plasma was shown.