Simple capillary flow porometer for characterization of capillary columns containing packed and monolithic beds

A simple capillary flow porometer (CFP) was assembled for through-pore structure characterization of monolithic capillary liquid chromatography columns in their original chromatographic forms. Determination of differential pressures and flow rates through dry and wet short capillary segments provided necessary information to determine the mean diameters and size distributions of the through-pores. The mean through-pore diameters of three capillary columns packed with 3, 5, and 7 μm spherical silica particles were determined to be 0.5, 1.0 and 1.4 μm, with distributions ranging from 0.1 to 0.7, 0.3 to 1.1 and 0.4 to 2.6 μm, respectively. Similarly, the mean through-pore diameters and size distributions of silica monoliths fabricated via phase separation by polymerization of tetramethoxysilane (TMOS) in the presence of poly(ethylene glycol) (PEG) verified that a greater number of through-pores with small diameters were prepared in columns with higher PEG content in the prepolymer mixture. The CFP system was also used to study the effects of column inner diameter and length on through-pore properties of polymeric monolithic columns. Typical monoliths based on butyl methacrylate (BMA) and poly(ethylene glycol) diacrylate (PEGDA) in capillary columns with different inner diameters (i.e., 50–250 μm) and lengths (i.e., 1.5–3.0 cm) were characterized. The results indicate that varying the inner diameter and/or the length of the column had little effect on the through-pore properties. Therefore, the through-pores are highly interconnected and their determination by CFP is independent of capillary length.     

Improvements in the performance of etched glass capillary columns

Summary The surface of hydrogen chloride etched, glass capillary columns can be rendered less active by heating at 400°C for 3 hours in a stream of dry nitrogen prior to coating. The reduction of surface activity is probably caused by loss of water from surface silanol groups to produce siloxide bridges. In addition, such treatment almost halves the bleed rate of columns coated with OV 101 silicone oil stationary phase at 280°C and causes no deterioration in the wetting properties of the etched glass surface.     

Reproducibility of sample separation using liquid or forced air convection thermostatted high performance capillary electrophoresis systems

Run-to-run sample separation reproducibility has been compared on two commercial high performance capillary electrophoresis units which differ in the mode by which the capillary temperature is thermostatted. Three standard analytes, differing dramatically in molecular character and size, were used for the analysis: benzoic acid, a 14 amino acid peptide from human chorionic gonadotropin, and ribonuclease A represent, respectively, small stable organic molecules, small peptides with little or no secondary structure, and proteins with secondary structure. These standards were evaluated with regard to reproducibility of migration time, peak area, and peak height. The analyses, performed in buffers of optimum pH for the separations, demonstrated that the liquid and forced air convection thermostatted systems both performed extremely well. The reproducibility, as judged by the percent coefficient of variance (% CV) of replicate analyses, was generally found to be less than 1 % (migration time); the reproducibility decreased in the order migration time > peak height > peak area. Whereas the absolute % CV values for MT_rel (migration relative to a standard) observed with the liquid thermostatted system were 2- to 4-fold lower than those observed with the forced air convection thermostatted system, there was little statistically significant difference between the two. As expected, the data indicated a reduction in reproducibility as the complexity of the analyte increased, perhaps as the result of an increased potential for wall interactions. Comparing separations in which low (≈?1 watt/meter [W/m] of capillary) and high (>5 W/m) Joule heat was generated by altering the sodium chloride content of the buffer revealed few statistically significant differences in the reproducibility obtained from the two systems. With these particular standard analytes and their respective buffer systems, there appears to be little difference between forced air convection and liquid thermostatting of the capillary.     

Thermostating in capillary electrophoresis

Summary The use of high voltages across a electrophoresis capillary will increase the temperature of the buffer due to Joule heating. As a result temperature control in CE is rather important since variations in the buffer temperature will result in changes in the pH of the buffer, peak shape, migration time, reproducibility, efficiency, 3-D structure of macromolecular analytes, etc. Six different thermostating systems have been evaluated: (i) natural convection, (ii) fan, (iii) home-made and (iv and v) two commercially available high-speed air and a (vi) liquid thermostated device. In all cases the temperature of the buffer in the capillary is calculated according to the temperature-conductivity relationship. For this purpose two parameters are introduced describing temperature control: the temperature onset (δT) and the temperature rise factor (α). From these results, it can be concluded that high speed air thermostating can be as efficient as liquid thermostating.     

Splitless on-line coupling of capillary high-performance liquid chromatography,capillary electrochromatography and pressurized capillary electrochromatography with nuclear magnetic resonance spectroscopy

A mixture of unsaturated fatty acid methyl esters was separated with a new splitless capillary set-up. With the employed apparatus configuration different capillary separation techniques such as capillary high-performance liquid chromatography (cHPLC), capillary electrochromatography (CEC) and pressurized capillary electrochromatography (pCEC) could be applied. The detection and identification of the sample compounds were accomplished by hyphenating these capillary separation techniques with nuclear magnetic resonance (NMR) spectroscopy using a novel configuration of the detection capillary set-up. Using modified electrokinetically driven separation techniques, the electric field was applied solely across the separation column. With this improved interface for capillary liquid chromatography-NMR on-line coupling, the stereochemical assignment of the cis and trans configuration of unsaturated fatty acids could be easily accomplished. Finally, the results of cHPLC-NMR, CEC-NMR and pCEC-NMR coupling experiments were compared.Dedicated to Professor Günter Häfelinger on the occasion of his 65th birthday     

Capillary suspensions: Particle networks formed through the capillary force

The addition of small amounts of a secondary fluid to a suspension can, through the attractive capillary force, lead to particle bridging and network formation. The capillary bridging phenomenon can be used to stabilize particle suspensions and precisely tune their rheological properties. This effect can even occur when the secondary fluid wets the particles less well than the bulk fluid. These materials, so-called capillary suspensions, have been the subject of recent research studying the mechanism for network formation, the properties of these suspensions, and how the material properties can be modified. Recent work in colloidal clusters is summarized and the relationship to capillary suspensions is discussed. Capillary suspensions can also be used as a pathway for new material design and some of these applications are highlighted. Results obtained to date are summarized and central questions that remain to be answered are proposed in this review.     

Nanoparticle‐based capillary electroseparation of proteins in polymer capillaries under physiological conditions

Totally porous lipid‐based liquid crystalline nanoparticles were used as pseudostationary phase for capillary electroseparation with LIF detection of proteins at physiological conditions using unmodified cyclic olefin copolymer capillaries (Topas^®, 6.7 cm effective length). In the absence of nanoparticles, i.e. in CE mode, the protein samples adsorbed completely to the capillary walls and could not be recovered. In contrast, nanoparticle‐based capillary electroseparation resolved green fluorescent protein from several of its impurities within 1 min. Furthermore, a mixture of native green fluorescent protein and two of its single‐amino‐acid‐substituted variants was separated within 2.5 min with efficiencies of 400 000 plates/m. The nanoparticles prevent adsorption by introducing a large interacting surface and by obstructing the attachment of the protein to the capillary wall. A one‐step procedure based on self‐assembly of lipids was used to prepare the nanoparticles, which benefit from their biocompatibility and suspension stability at high concentrations. An aqueous tricine buffer at pH 7.5 containing lipid‐based nanoparticles (2% w/w) was used as electrolyte, enabling separation at protein friendly conditions. The developed capillary‐based method facilitates future electrochromatography of proteins on polymer‐based microchips under physiological conditions and enables the initial optimization of separation conditions in parallel to the chip development.     

Electrically assisted capillary liquid chromatography using a silica monolithic column

A silica monolithic capillary column was linked to an open capillary of the same internal diameter via a Teflon sleeve to form a duplex column to investigate the combination of chromatography and electrophoresis in the mode of electrically assisted capillary liquid chromatography (eCLC). Using a commercial CE instrument with an 8.5 cm long, 100 μm i.d. reversed phase silica monolithic section and a window 1.5 cm beyond the end of this in a 21.5 cm open section, a minimum plate height of 9 μm was obtained in capillary liquid chromatography (CLC) mode at a low driving pressure of 50 psi. In eCLC mode, high speed and high resolution separations of acidic and basic compounds were achieved with selectivity tuning based on the flexible combination of pressure (0–100 psi) and voltage. Taking advantage of the excellent permeability of silica monolithic columns, use of a step flow gradient enabled elution of compounds with different charge state.     

In-pore tensile stress by drying-induced capillary bridges inside porous materials

We present here some evidences that capillary liquid bridges are able to deform micrometric cylindrical pores by tensile stress. Brine-soaked filter membranes are submitted to drying conditions leading to NaCl precipitation inside the 5-10 μm pores. A close examination demonstrated that two forms of NaCl crystallites are successively generated. First, primary cubic crystals grow, driven by the permanent evaporation. When this angular primary solid gets near the pore wall, while the evaporation makes the pore volume to be partly invaded by air, capillary liquid can bridge the now-small gap between the halite angles and the pore wall. In a second step, these small capillary bridges are frozen by a secondary precipitation event of concave-shaped NaCl. The proposed interpretation is that the liquid capillary bridges deform the host matrix of the membrane, and the situation is fossilized by the growth of solid capillary bridges. A quantitative interpretation is proposed and the consequences towards the natural media outlined.     

Simultaneous separation of nitrate, nitrite and ammonium by capillary electrophoresis

Summary A capillary electrophoretic method for the simultaneous separation of nitrate, nitrite and ammonium has been developed. Direct (NO₃ ⁻, NO₂ ⁻) and indirect (NH₄ ⁺) UV detection at 214 nm in conjunction with electromigration sampling from both ends of the capillary was used. Two electrolyte systems based on imidazole-sulfate (pH 3.8) and copper(II)-ethylenediamine-chloride (pH 8.0) were investigated. Optimisation of the experimental parameters such as electrolyte concentration, pH, nature of the counter-ion, was studied. The method permits excellent separation of three nitrogen species in only 4 min. The analytical performance of both electrolyte systems is compared in terms of migration time and peak area repeatability and detectability. Alkaline electrolyte shows a better overall analytical performance.     

An amperometric detector with a tubular electrode deposited on the capillary for capillary liquid chromatography

A procedure is described for the preparation of a tubular electrode by chemical deposition of platinum at the end of a fused-silica capillary. The properties of the electrode were tested under liquid chromatographic conditions, demonstrating that both the static and the dynamic behaviour of the detection system satisfy the requirements of capillary chromatographic separations and compare well with a wall-jet amperometric system and with UV photometric detection. The detection system described is easy to prepare and does not require any time-consuming positioning of the electrode system as it is integrated into the separation part of the apparatus.     

Sample introduction in high speed capillary gas chromatography; input band width and detection limits

The speed of analysis in capillary gas chromatography can be substantially increased by reduction of the column inner diameter. However, special demands are then posed upon instrumental design. In particular, the sampling system is highly critical because it has to be capable of delivering extremely small injection band widths which must be compatible with the column inside diameter. This study focuses on the evaluation of two potentially suitable sample introduction systems with respect to input band width and detection limits and their compatibility with small bore (≦ 100 μm) columns in capillary gas chromatography. One of them allows liquid on-column injection, based on liquid splitting, of only a few nl onto small bore (≦ 100 ?m) fused silica columns. For gases, input band widths as low as 1 ms are obtained with this system. The other one is part of a miniaturized gas chromatograph with extremely low dead volume interfaces and detector volumes. It allows input band widths for gases of a few ms. Without any preconcentration ppm concentrations are measured in gaseous samples with a 80 ?m thick film capillary column. It will be shown that a further reduction of the minimum detectable amount and analysis time is possible with this equipment.     

Spontaneous formation of stable capillary bridges for firming compact colloidal microstructures in phase separating liquids: a computational study

Computer modeling and simulations are performed to investigate capillary bridges spontaneously formed between closely packed colloidal particles in phase separating liquids. The simulations reveal a self-stabilization mechanism that operates through diffusive equilibrium of two-phase liquid morphologies. Such mechanism renders desired microstructural stability and uniformity to the capillary bridges that are spontaneously formed during liquid solution phase separation. This self-stabilization behavior is in contrast to conventional coarsening processes during phase separation. The volume fraction limit of the separated liquid phases as well as the adhesion strength and thermodynamic stability of the capillary bridges are discussed. Capillary bridge formations in various compact colloid assemblies are considered. The study sheds light on a promising route to in situ (in-liquid) firming of fragile colloidal crystals and other compact colloidal microstructures via capillary bridges.     

On-line wall-free cell for laser-induced fluorescence detection in capillary electrophoresis

A wall-free detection method based on liquid junction in a capillary gap was proposed for laser-induced fluorescence (LIF) of capillary electrophoresis (CE). The capillary gap of the wall-free cell was fabricated by etching a 10-mm × 50-μm I.D. fused-silica capillary to obtain a polyimide coating sleeve, decoating about 6 mm at one end of both 50 μm I.D. separation and liquid junction capillary, inserting the treated capillary ends into the coating sleeve oppositely, fixing the capillaries with a gap distance of 140 μm by epoxy glue and removing the coating sleeve by burning. The theoretical model, experimental results and wall-free cell images indicated that the gap distance and applied voltage were main influence factors on the wall-free detection. Since the wall-free cell increased the absorption light path and avoided the stray light from the capillary wall, it improved the ratio of signal to noise and limit of detection (LOD) of CE-LIF. Three flavin compounds of riboflavin (RF), flavin mononucleotide sodium (FMN) and flavin adenine dinucleotide disodium (FAD) were used to evaluate the wall-free detection method. Compared with on-column cell, the LODs of the wall-free cell were improved 15-, 6- and 9-fold for RF, FMN and FAD, respectively. The linear calibration concentrations of the flavins ranged from 0.005 to 5.0 μmol/L. The column efficiency was in the range from 1.0 × 10⁵ to 2.5 × 10⁵ plates. The wall-free detection of CE-LIF was applied to the analysis of the flavins in spinach and lettuce leaves.     

A technique for capillary joining in capillary electrophoresis

Summary Aspects of cracking and joining capillaries have been investigated. Capillary coupling was achieved using various methods. The most successful used hydrofluoric acid-etched capillaries to form male and female ends which were then joined together. This type of joint was used to connect sections of capillary of similar and different internal diameters with minimal loss in resolution, peak width and number of theoretical plates. (Uridine and hypoxanthine was used as a test mixture). For hypoxanthine on a 50 m/50 m etched joined capillary 10 cm from the detector window the number of theoretical plates was 96.6% of that for hypoxanthine on an unbroken capillary. Following the relative success of capillary joining, a coupled capillary flowcell (50 m/200 m) was produced and evaluated.     

A new approach to the studies of submicron particle suspensions based on the effect of pressure in capillary zone electrophoresis

A new approach based on the effect of pressure in CZE is suggested for acceleration of particle migration in electrophoretic runs resulting in reduction of the analysis time. It provides conditions for studying fast processes in suspensions. The effect of pressure on the migration of silica spheres with average diameters of 100, 150, and 390 nm was studied by CZE at an applied voltage of 25 kV. The particle hydrodynamic behavior was also investigated under the same capillary dimensions and BGE composition. The total particle mobility (excluding the average flow rate) was found to increase with increasing the pressure applied and particle size. The particle migration mechanism explaining the effect of pressure on particle velocity was shown to be almost the same as in wide‐bore hydrodynamic chromatography. It is based on changing radial distribution of particle concentration along the capillary cross section depending on particle diffusivity. On the basis of this mechanism appearance of a zone of negatively charged particles in electropherograms ahead of the marker peak can be explained.     

Fluorescence detection system for capillary separations utilizing a liquid core waveguide with an optical fibre-coupled compact spectrometer

A fluorescence detection system for capillary liquid separation methods is described. The system is based on a silica capillary coated with a low refractive index fluoropolymer Teflon AF that serves both as a separation channel and as a liquid core waveguide (LCW). A fibre-coupled laser excites separated analytes in a detection point and arising fluorescence is collected at one end of the LCW capillary into the other optical fibre which brings it to a compact charge-coupled device (CCD) array spectrometer installed in a desktop computer. No additional components such as focusing optics or filters are necessary. This system was used for detecting isoelectrically focused fluorescent low-molecular-mass pI (isoelectric point) markers and fluorescein isothiocyanate (FITC) labelled proteins. The ability of the system to acquire fluorescent spectra is also demonstrated.     

Carbon nanotubes as separation carrier in capillary electrophoresis

The utility and versatility of carboxylic single-walled carbon nanotubes (c-SWNT) in capillary electrophoresis (CE) is demonstrated, using as model solutes homologues and structural isomers. In the case of homologues of caffeine and theobromine, distinct changes in the electrophoretic parameters occur at a critical concentration of c-SWNT in the run buffer. It is suggested that the c-SWNT of a definite concentration could form a network in the run buffer as a pseudostationary phase on the basis of the unique tubule structure, providing a different separation from sodium dodecyl sulfate (SDS) micelles. In the case of structural isomers of catechol and hydroquinone, differing from the homologues, it is mainly attributable to the functional groups on the c-SWNT that have an effect on the electrophoretic behaviors by forming intermolecular hydrogen bonding with analytes. Furthermore, aggregated c-SWNT serve as anticonvective media and minimize solute diffusion contributing to zone broadening. The presence of charged c-SWNT suppressed the electrodiffusion and decreased the adsorption between capillary wall and solutes, which led to better peak shapes of isomers.     

Electroosmotic pump‐supported molecularly imprinted monolithic column for capillary chromatographic separation of nitrophenol isomers

In this work, a novel molecularly imprinted polymer (MIP) monolithic column with integrated in‐column electroosmotic pump (EOP) was designed and successfully prepared to facilitate the capillary chromatography with MIP column. A silica‐based EOP was synthesized at the detection end of the MIP monolithic capillary column by so‐gel to provide the hydrodynamic driven force for the capillary chromatography. Because of large surface area and low fluidic resistance of the silica monolith,a strong and steady EOF was generated by silica‐based EOP, indicating that the EOP was quite compatible with MIP capillary column. With the sufficient EOF provided by EOP, the electro‐driven based capillary chromatographic separation of nitrophenol isomers was achieved in 4‐vinylpyridine‐based MIP monolithic capillary, which was originally proved infeasible because of the EOF shortage. No significant influence upon the specific recognition of the MIP was found due to the setting of EOP after the detection window of the column. The influence of experimental parameters on the EOF such as voltage and pH value of running buffer was investigated. The column was also evaluated by capillary liquid chromatographic mode to compare with EOP‐driven capillary chromatography. Higher column efficiency was obtained by EOP‐driven separation with improved peak shape. The results suggested that EOP‐supported technique would be a good way to solve the problem of weak EOF generation in electro‐driven capillary chromatography.     

Recent advances in capillary electrophoresis‐mass spectrometry: Instrumentation,methodology and applications

Capillary electrophoresis (CE) offers fast and high‐resolution separation of charged analytes from small injection volumes. Coupled to mass spectrometry (MS), it represents a powerful analytical technique providing (exact) mass information and enables molecular characterization based on fragmentation. Although hyphenation of CE and MS is not straightforward, much emphasis has been placed on enabling efficient ionization and user‐friendly coupling. Though several interfaces are now commercially available, research on more efficient and robust interfacing with nano‐electrospray ionization (ESI), matrix‐assisted laser desorption/ionization (MALDI) and inductively coupled plasma mass spectrometry (ICP) continues with considerable results. At the same time, CE‐MS has been used in many fields, predominantly for the analysis of proteins, peptides and metabolites. This review belongs to a series of regularly published articles, summarizing 248 articles covering the time between June 2016 and May 2018. Latest developments on hyphenation of CE with MS as well as instrumental developments such as two‐dimensional separation systems with MS detection are mentioned. Furthermore, applications of various CE‐modes including capillary zone electrophoresis (CZE), nonaqueous capillary electrophoresis (NACE), capillary gel electrophoresis (CGE) and capillary isoelectric focusing (CIEF) coupled to MS in biological, pharmaceutical and environmental research are summarized.