High-speed Separation Research Articles

Carrier ampholytes as potential buffers in electrophoresis: physico-chemical study

Joule heating is a limiting factor when separating proteins in capillary zone electrophoresis (CZE). Low conductivity buffers, are required for high-speed separations. We investigated the use of carrier ampholytes (CA) as background electrolytes (BGE) in CZE. We prepared 25 “narrow pH cuts” of wide pH range (3–10) CA mixture in order to know if these fractions were suitable to be used as BGE in CZE. Each fraction was characterised by CZE analysis, giving an idea of its heterogeneity (number and relative abundance of molecular ampholytes). Conductivities and buffering capacities of each fraction have been also measured. Our conclusion is that “narrow pH cuts” of CA might be well suited buffers for electrophoretic separations.

The contributions of Csaba Horváth to liquid chromatography

This article is not a biographic description of the life of Csaba Horváth, but an attempt to describe his most important contributions to liquid chromatography, as viewed by an author who himself started with HPLC in 1973. With the introduction of columns packed with rigid pellicular particles, which could withstand high pressures, the combination of fast separations with high efficiency was designated high pressure liquid chromatography about 35 years ago. The contribution of Horváth to the birth of modern LC was to imagine the potential for high-speed separations in LC by transferring ideas and technology from gas chromatography. From this time, his strong participation in understanding and describing the processes of separation science made him a leading scientist in fundamental studies of separation principles until his passing away in April 2004. In addition to being one of the forefathers of modern liquid chromatography, Csaba Horváth described both the ionic interactions and the hydrophobic interactions on ion exchangers as well as on reversed phase materials and developed a theoretical basis for understanding these processes. If one person should be mentioned as the inventor of ion-pair chromatography of organic compounds, of hydrophobic interaction chromatography, and of the theoretical basis for reversed phase chromatography, this person is Csaba Horváth. Throughout the last 30 years he also had a strong interest in studying proteins in separation processes, and in the last decade he made valuable contributions to capillary electrochromatography, particularly for separation of proteins on monolithic columns. For this author is difficult to find another person who has had a greater impact on liquid chromatography than Csaba Horváth.

Development and evaluation of gold-centered monolayer protected nanoparticle stationary phases for gas chromatography

The current status for the development of novel open-tubular gas chromatography (GC) stationary phases consisting of thin films of gold-centered monolayer protected nanoparticles (MPNs) is reported. Dodecanethiol MPNs, in which the monolayer is dodecanethiol linked to the gold nanoparticle, have shown great promise as a GC stationary phase with efficient columns having been produced in a variety of capillary i.d.'s with stationary phase film depths ranging from 10 to 60 nm, ±2 nm at a given film depth. Stationary phase operational parameters are discussed including maximum operating temperature, sample capacity, and stationary phase lifetime and robustness. An overview of the general method employed for column production is also included. The sample capacity was determined for a 2.5 m, 250 μm i.d. column with a stationary phase film thickness of 40 nm, at 50 °C using anisole ( k′ = 1.86) as the probe analyte. The sample capacity was experimentally found to be 2.3 ng under these conditions, similar to values reported for thicker, polymer stationary phases. The efficiency of the dodecanethiol MPN stationary phase was determined with a 100 μm i.d. capillary and found to have a reduced plate height h min value of 0.95 for octane ( k′ = 0.68). Areas of application illustrated and discussed utilizing the dodecanethiol MPN stationary phase include complementary separations such as two-dimensional GC (GC × GC), potential utilization within a model system for a micro-fabricated GC (μGC), as well as efficient single dimension high-speed separations. Initial development of polar stationary phases utilizing 4-chlorobenzenethiol MPNs and 4-(trifluoromethyl)benzenethiol MPNs is discussed. Included is a selectivity comparison of the retention behavior of the 4-chlorobenzenethiol MPN stationary phase and the dodecanethiol MPN stationary phase.

Capillary electrochromatography of peptides and proteins.

This paper reviews recent progress in bioanalysis using capillary electrochromatography (CEC), especially in the field of separation of proteins and peptides. Fundamentals of CEC are briefly discussed. Since most of the recent developments on CEC have focused on column technology, i.e., design of new stationary phases and development of new column configurations, we describe here a variety of column architectures along with their advantages and disadvantages. Newly emerged column technologies in CEC for high speed and high efficiency separation are also discussed. Different analytical platforms of CEC such as pressure-assisted CEC or voltage-assisted micro- high-performance liquid chromatography (HPLC), CEC with different detection techniques, CEC on microchip platforms and multidimensional electrochromatography with their applications in peptide and protein analysis are presented.

Monolayer-protected gold nanoparticles as an efficient stationary phase for open tubular gas chromatography using a square capillary: Model for chip-based gas chromatography in square cornered microfabricated channels

The application of a dodecanethiol monolayer-protected gold nanoparticle (MPN) stationary phase within a microchannel environment was explored using a square capillary column as a model for high-speed, microfabricated gas chromatography (μGC). Successful deposition and evaluation of a dodecanethiol MPN phase within a 1.3 m long, 100 μm×100 μm square capillary is reported. The thickness of the MPN phase was evaluated using SEM analysis. An average thickness of 15 nm along the capillary walls was determined. While the film depth along the walls was very uniform, the corner depths were greater with the largest observed depth being 430 nm. Overall, an efficient chromatographic system was obtained with a minimum reduced plate height, h min, of 1.2 for octane ( k=0.22). Characterization of the MPN column was completed using four compound classes (alkanes, alcohols, ketones, and aromatics) that were used to form a seven-component mixture with a 2-s separation. A mixture consisting of a nerve agent simulant in a sample containing analytes that may commonly interfere with detection was also separated in only 2 s, much faster than a similar separation previously reported using a μGC system requiring 50 s. A comparison of the MPN stationary phase to phases employed in previously reported μGC systems is also made. Application of the square capillary MPN column for a high-speed separation as the second column of a comprehensive 2-D gas chromatography system (GC×GC) was also explored.

High-Speed Micro-Thermal Focusing Field-Flow Fractionation of Micron-Size Particles

Micron-size polystyrene-based latex particles were separated by using new micro-thermal field-flow fractionation (micro-TFFF). The order of retention from the largest to the smallest particles that appears at high field strength and high flow rate corresponds to the focusing mechanism which itself is a consequence of the lift forces acting on the particles. The mechanism of steric exclusion can only be effective at low flow rates of the carrier liquid. Whenever high-speed separation was performed, the focusing effect clearly dominated the FFF mechanism. This application of micro-TFFF in focusing mode to the separation of the particles is the first one published. As a result, micro-TFFF thus became a very universal technique for the separation of synthetic and natural macromolecules and of particles of various origin and size up to large (micron-size) diameter.

Dodecylsulfate-coated monolithic octadecyl-bonded silica stationary phase for high-speed separation of hydrogen, magnesium and calcium in rainwater

The high-speed determination of hydrogen, magnesium and calcium ions by ion chromatography (IC) is demonstrated on a monolithic octadecyl-boned silica (ODS) column coated with lithium dodecylsulfate (Li-DS). This stationary phase, when used in conjunction with a 2 mM ethylenediamine and 0.1 mM Li-DS solution as eluent at pH 6.0, was found to be suitable for the rapid and efficient separation of hydrogen and magnesium and calcium in the order H +<Mg 2+<Ca 2+ within 4 min at a flow rate of 4.0 ml/min. Under the conditions, linear calibration plots of conductivity versus concentration were obtained for the cations over about three orders of magnitude, and the detection limits were 1 μM for H +, 2 μM for Mg 2+ and Ca 2+. Rainwater was analyzed directly using this IC system with satisfactory results.

Micro-thermal focusing field-flow fractionation

Focusing mechanism was effectively exploited to separate large (micrometer-size) particles by using new micro-thermal field-flow fractionation (micro-TFFF). It has been shown that the retention order of micrometer-size particles at high field strength can be explained by the mechanism of steric exclusion only at lowest flow rates of the carrier liquid. A simplistic, purely mechanical model of steric exclusion is not accurate to describe the retention at higher flow rates where the focusing phenomenon appears. Despite the fact that the thickness of the channel for micro-FFF cannot be reduced without taking into account a possible deterioration of the separation due to the contribution of “steric exclusion” mechanism, this paper demonstrates, in agreement with our previous results, that if the operational conditions were conveniently chosen, namely a low flow rate, a reasonable fit of the experimental retention data with the theory of steric exclusion mechanism in FFF was found and the separation of micron-size particles can be accomplished. However, high selectivity and resolution and high-speed separation were achieved if the focusing effect has clearly dominated the FFF mechanism. As a result, it seems that the micro-TFFF is the most universal technique which can be applied for the separation of the synthetic and natural macromolecules within an extended range of molar masses up to ultra-high molar masses and for the particles of various chemical nature and origin in a nano-size range as well as for large (micrometers) particles. Until nowadays, only sedimentation and flow field-flow fractionation techniques in so called “steric” modes were applied for the separations of large size particles. This application of micro-TFFF in focusing mode for the separation of large size particles is the first one described in the literature.

Development of a novel running buffer for the simultaneous determination of nitrate and nitrite in human serum by capillary zone electrophoresis

In order to improve NO 2 − peak height and obtain a convenient buffer system for the assay of nitrogen monooxide metabolites, we developed a novel running buffer for the simultaneous determination of nitrite and nitrate in human serum by capillary electrophoresis. The addition of cetyltrimethylammonium chloride to the running buffer resulted in high-speed separation using reverse electroosmotic flow. Highly sensitive determination was also achieved using stacking with 10-fold diluted sample solutions. The samples were injected hydrodynamically for 100 s into a 50 cm×75 μm I.D. capillary. The separation voltage was 10 kV (negative polarity). UV detection was performed at 214 nm. We obtained complete separation of nitrite and nitrate in deproteinized human serum within 6 min with optimum analytical conditions. Linear calibration curves for nitrite and nitrate for both peak height and peak area were obtained with standard addition method. The limits of detection obtained at a signal-to-noise ratio of 3 for nitrite and nitrate were 4.1 and 2.0 μ M, while the values of relative standard deviation of peak height were 2.4 and 2.6%, respectively.

An automated orthogonal two-dimensional liquid chromatograph.

A simple approach to two-dimensional liquid chromatography has been developed by coupling columns of different selectivity using a 12-port, dual-position valve and a standard HPLC system. The valve at the junction of the two columns enables continuous, periodic sampling (injection) of the primary column eluent onto the secondary column. The separation in the primary dimension is comparable to conventional HPLC, whereas the secondary column separation is fast, lasting several seconds. The high-speed separation in the secondary dimension enables the primary column eluent to be sampled with fidelity onto the secondary column throughout the chromatographic run. One might expect a coupled column liquid chromatography system operating in reverse-phase mode to be strongly correlated and, hence, inefficient. However, by applying a solvent gradient in the primary dimension and by progressively incrementing the solvent strength in the secondary dimension (tuning), the inefficiency or cross correlation between the two dimensions is minimized. In a tuned two-dimensional system, the influence of primary column retention (usually hydrophobicity) is minimal on secondary column retention. This enables subtle differences in component interaction with the two stationary phases to dominate the secondary column retention. The peaks are randomly dispersed over a retention plane rather than along a diagonal, resulting in an orthogonal separation. The peak capacity is multiplicative, and each component has a unique pair of retention times, enabling positive identification. In addition, the location of the component provides two independent measures of molecular properties. The 2D-LC system was evaluated by analyzing a test mixture made of some aromatic amines and non-amines on different secondary columns (ODS-AQ/ODS monolith, ODS/amino, ODS/cyano). The relative location of sample components in the two-dimensional plane varied significantly with change in secondary column. Among the secondary columns, the amino and cyano columns offered the most complementary separation, with the retention order of several components reversed in the secondary dimension. The theoretical peak capacity of the 2D-LC system was around 450 for a separation lasting 30 min. A 2D-LC system involving amino and cyano columns resulted in a high-speed separation of the test mixture, with most of the chemical components resolved within a few minutes.

High-speed simultaneous ion-exclusion/cation-exchange chromatography of anions and cations on a weakly acidic cation-exchange resin column

The simultaneous ion-exclusion/cation-exchange separation column packed with a polymethacrylate-based weakly acidic cation-exchange resin of 3 μm particle size was used to achieve the simultaneous high-speed separation of anions and cations (Cl −, NO 3 −, SO 4 2−, Na +, K +, NH 4 +, Ca 2+ and Mg 2+) commonly found in environmental samples. The high-speed simultaneous separation is based on a combination of the ion-exclusion mechanism for the anions and the cation-exchange mechanism for cations. The complete separation of the anions and cations was achieved in 5 min by elution with 15 m M tartaric acid–2.5 m M 18-crown-6 at a flow-rate of 1.5 ml/min. Detection limits at S/ N=3 ranged from 0.36 to 0.68 μ M for anions and 0.63–0.99 μ M for cations. This method has been applied to the simultaneous determination of anions and cations in several environmental waters with satisfactory results.

Hadamard Transform Time‐of‐Flight Mass Spectrometry: More Signal, More of the Time

AbstractFor Abstract see ChemInform Abstract in Full Text.

Monolithic octadecylsilyl-silica gel column for the high-speed ion chromatographic determination of acidity

A monolithic ODS-silica gel column modified by saturating it with lithium dodecylsulfate (Li-DS) was used to demonstrate the high-speed separation of H + from other mono- and divalent cations, such as Na +, NH 4 +, K +, Mg 2+ and Ca 2+ using ion chromatography (IC). Using a 5 m M EDTA-2K solution containing 0.10 m M Li-DS (pH 4.80) as eluent, H + was eluted with a sharp and symmetrical peak within 1.0 min before other cations at a flow-rate of 1.5 ml min −1. The rapid elution of H + and its conductimetric detection could be attributed to the presence of EDTA (HY 2−), which can convert H + ions as anions, i.e. H ++H 2Y 2−→H 3Y −. The acidity of rainwater and deionized water samples was determined using this IC system with satisfactory results.

Ultrafast analysis of oligosaccharides on microchip with light-emitting diode confocal fluorescence detection.

We have developed a new method for the high-speed separation and high-sensitivity detection of complex oligosaccharides based on microchip electrophoresis (nu-CE) with light-emitting diode (LED) confocal fluorescence detection. Oligosaccharides labeled with 8-aminopyrene-1,3,6-trisulfonate (APTS) were found to strongly adsorb to the surface of polymethylmethacrylate (PMMA) microchips. Accordingly, three classes of major dynamic coating additives were systematically investigated, and cellulose derivatives were found to specifically suppress such adsorption and allow high-performance separation on PMMA chips. Additive concentration, buffer pH and applied field strength were found to be key factors in the high-performance separation& of APTS-labeled oligosaccharides on PMMA chips. Under optimal conditions, 15 oligosaccharides in dextrin hydrolysate can be separated within 45 s with an electrophoretic separation efficiency of over 400 000 theoretical plates per meter. The relative standard deviation (RSD) values of migration times of fourteen oligosaccharides were less than 0.50% between six different channels, and the detection limit for APTS-labeled glucose was about 1.98 x 10(-8) mol/L or 8.61 amol with a signal-to-noise ratio (S/N) of 3. The high speed, high efficiency and high sensitivity of this micro-CE-based method indicate that it can be widely applied to analysis of complex oligosaccharides.

Hadamard Transform Time‐of‐Flight Mass Spectrometry: More Signal, More of the Time

Hadamard transform time-of-flight mass spectrometry (HT-TOF MS) is a type of mass analysis that was developed to couple continuous ion sources to the inherently pulsed nature of time-of-flight measurements. Unlike conventional TOF MS, the Hadamard transform method offers a duty cycle of 50%, with the possibility of extending it to 100%. Because it is a multiplexing technique, the attainable signal-to-noise ratio (SNR) is also significantly higher than that of conventional TOF MS. This review covers the basic principles behind HT-TOF MS. We illustrate, through examples, the source of the high-duty cycle and the increase in SNR. These features translate to a mass spectral storage rate that is the fastest among similar instruments, which enables its use as a detector for high-speed separations.

Bacteria sorting by field-flow fractionation. Application to whole-cell Escherichia coil vaccine strains.

Sorting and quantification of deactivated bacteria is an important way of quality control for whole-cell bacterial vaccines. In general, surface features of deactivated bacteria used for whole-cell bacterial vaccines affect the immunoresponse to bacteria-associated antigens. Enumeration of bacteria is also an important process development parameter for these vaccines. Field-flow fractionation (FFF) was previously applied to the separation of bacteria. For the first time, FFF is used for sorting bacteria strains of the same species on the basis of differences in bacterial membrane characteristics. Two FFF techniques, gravitational FFF (GrFFF) and asymmetrical flow FFF (AsFIFFF), are shown to be able to fractionate, distinguish, and quantify different deactivated Escherichia coli strains used for vaccines. E. coli can differ in the presence of fimbriae on the bacterial membrane. Fimbriae affect E. coli pathology and thus the use of E. coli for vaccines. GrFFF and AsFIFFF are able to fractionate fimbriated/ nonfimbriated cells in mixtures of different strains. While GrFFF is characterized by low cost and simplicity, As-FIFFF shows a higher performance in size fractionation with a high-speed separation. Coupled, on-line UV/visible turbidimetry yields the relative numbers of fractionated cells and sample recovery. Scanning electron microscopy and quasi-elastic light scattering are employed as uncorrelated techniques for size and morphology analysis of the E. coli strains.

High-speed separations by electrokinetic chromatography using polymeric pseudostationary phases

High-speed separations by electrokinetic chromatography using polymeric pseudostationary phases

High‐speed separations by electrokinetic chromatography using polymeric pseudostationary phases

High-speed separations of hydrophobic solutes by capillary-based electrokinetic chromatography (EKC) are reported in this short communication with analysis speed comparable to that of high-speed capillary electrochromatography (CEC). Polymeric pseudostationary phases can provide highly efficient separations of hydrophobic solutes in the presence of high concentration of organic solvents, and some of them have low electrical conductivity compared with sodium dodecyl sulfate micelles, so that a high electric field can be used without a significant loss of efficiency or resolution. Using poly(sodium 2-acrylamido-2-methylpropane sulfonate-co-dihydrocholesteryl acrylate) as the pseudostationary phase on a capillary column of 31 cm total length (23 cm effective length) and under an electric field strength of 962 V/cm, a separation of 12 out of 15 polycyclic aromatic hydrocarbons (PAHs) was achieved within 138 seconds. The RSD of retention times were less than 1.5% (n = 25).

Multiplex high-throughput solid-phase minisequencing by capillary electrophoresis and liquid core waveguide fluorescence detection

Minisequencing, solid-phase single-nucleotide primer extension reaction, is a robust method for performing multiplex single-nucleotide polymorphism (SNP) analysis. We have combined this technology with capillary gel electrophoresis in a multicapillary format, using liquid core waveguide (LCW) fluorescence detection. Polymerase chain reaction (PCR) amplification of multiple DNA targets is performed with one primer for each target biotinylated. Separation of the complementary strands, minisequencing and washing steps are carried out using streptavidin-coated magnetic beads. Dideoxynucleotides analogues labelled with different fluorophores are used for the extension of the minisequencing primers. The extended oligonucleotides, the length of which defines the position on the target and the color the identity of the polymorphism, are then separated in a gel-filled array of capillaries, coated on the outside with a layer of a fluoropolymer to provide the liquid core waveguide characteristics. The technology has a potential for extremely high throughputs when a combination of multiplex PCR-minisequencing is used together with a large array of capillaries, four-color detection and high-speed separation.

Electrophoretic Deposition (EPD) Coatings of Sol-Gel Solutions and Suspensions

question, ∼300 m 2 /g, is made available by the mesopores. Because the total porosity of the monolithic silica matrix is higher than 80%, the user is able to perform his chromatography using a much lower back pressure than with conventional particles that have a total porosity of only app. 65%. By optimising the ratio of throughpores to total porosity and the silica gel skeleton thickness, separations become possible at higher flow rates. This is due to accelerated adsorption and desorption of the substances to be separated by the silica gel surface. Due to better mass transfer properties of a monolithic skeleton over distinct particles, highspeed separation is possible without noticeable impact on resolution. For example, five β-blockers were separated at high α values (α = k‘Substance2/k‘Substance1) and with excellent peak symmetry. At a flow rate of 1 ml/min, the 5 substances were separated within 5 minutes. Under these conditions, the total system backpressure was 13 bar. At a flow rate of 9 ml/min., the 5 β-blockers can be baseline separated within