Moderation of the electroosmotic flow in capillary electrophoresis by chemical modification of the capillary surface with tentacle-like oligourethanes

The surface chemistry of the inner wall of fused-silica capillaries is one important means to control the magnitude as well as the direction of the electroosmotic flow and the adsorption activity. A method was developed to change the surface characteristics of fused-silica capillaries by binding tentacle-like oligourethane groups onto the inner surface. The electroosmotic flow at a buffer pH of 6-9 was reduced by 15 to 40% compared to that in a bare fused-silica tubing, dependent on the type of coating. Sample adsorption is diminished at the same time resulting in a separation of proteins with higher resolution and good migration time precision. At a pH below 4.5 the electroosmotic flow is reversed into the anodic direction, which offers further possibilities for the separation of positively charged analytes as demonstrated for the separation of aromatic and biogenic amines.     

Capillary electrochromatography of peptides on a neutral porous monolith with annular electroosmotic flow generation

A new kind of monolithic capillary column was prepared for capillary electrochromatography (CEC) with a positively charged polymer layer on the inner wall of a fused-silica capillary and a neutral monolithic packing as the bulk stationary phase. The fused-silica capillary was first silanized with 3-glycidoxypropyltrimethoxysilane (GPTMS). Polyethyleneimine (PEI) was then covalently bonded to the GPTMS coating to form an annular positively charged polymer layer for the generation of electroosmotic flow (EOF). A neutral bulk monolithic stationary phase was then prepared by in situ copolymerization of vinylbenzyl chloride (VBC) and ethylene glycol dimethacrylate in the presence of 1-propanol and formamide as porogens. Benzyl chloride functionalities on the monolith were subsequently hydrolyzed to benzyl alcohol groups. Effects of pH on the EOF mobility of the column were measured to monitor the completion of reactions. Using a column with this design, we expected general problems in CEC such as irreversible adsorption and electrostatic interaction between stationary phase and analytes to be reduced. A peptide mixture was successfully separated in counter-directional mode CEC. Comparison of peptide separations in isocratic monolithic CEC, gradient HPLC and capillary zone electrophoresis (CZE) indicated that the separation in CEC is governed by a dual mechanism that involves a complex interplay between selective chromatographic retention and differential electrophoretic migration.     

Multiplex Detection of Enzymatic Activity with Responsive Lanthanide‐Based Luminescent Probes

Multiplex analyte detection in complex dynamic systems is desirable for the investigation of cellular communication networks as well as in medical diagnostics. A family of lanthanide‐based responsive luminescent probes for multiplex detection is reported. The high modularity of the probe design enabled the rapid assembly of both green and red emitters for a large variety of analytes by the simple exchange of the lanthanide or an analyte‐cleavable caging group, respectively. The real‐time three‐color detection of up to three analytes was demonstrated, thus setting the stage for the non‐invasive investigation of interconnected biological processes.     

High-performance thin-layer chromatography plate blotting for liquid microjunction surface sampling probe mass spectrometric analysis of analytes separated on a wettable phase plate

A blotting method that transfers analytes separated on wettable high‐performance thin‐layer chromatography (HPTLC) plates to a hydrophobic reversed‐phase C8 HPLTC plate suitable for analysis with a liquid microjunction surface sampling probe electrospray ionization mass spectrometry system was described and demonstrated. The simple blotting procedure transfers the analyte from the wettable plate to the topmost surface of a rigidly backed, easy‐to‐mount hydrophobic substrate that already has been proven viable for analysis by this sampling probe/mass spectrometry system. The utility of the approach was demonstrated by the analysis of a four‐component peptide mixture originally separated on a ProteoChrom® HPTLC cellulose sheet and then blotted onto the reversed‐phase HPTLC plate. Published in 2011 by John Wiley & Sons, Ltd.     

Mathematical modelling of adsorption and transport processes in capillary electrochromatography: open-tubular geometry

A mathematical modelling approach for open-tubular capillary electrochromatography is presented. The spatially one-dimensional model takes into account (i) a coupling of (non)linear adsorption of positively or negatively charged analyte molecules (at a negatively charged capillary inner surface) with the equilibrium electrokinetics at this solid-liquid interface, (ii) mobile phase transport by electroosmosis and pressure-driven flow, as well as (iii) transport of species by electrophoresis and molecular diffusion. Under these conditions the local zeta-potential and electroosmotic mobility become a function of the concentration of the charged analyte. The resulting inhomogeneity of electroosmotic flow through the capillary produces a compensating pore pressure as requirement for incompressible mobile phase flow (i.e., for constant volumetric flow along the capillary). The results of the simulations are discussed in view of the surface-to-volume ratio of the capillary lumen, the analyte concentration (in combination with a Langmuir isotherm for the adsorption process), and buffer effects.     

Open-tubular capillary electrochromatography/electrospray ionization-mass spectrometry using polymeric surfactant as a stationary phase coating

We describe the use of the polymeric surfactant poly(sodium undecylenic sulfate) (poly-SUS) as a stationary phase coating in open-tubular capillary electrochromatography (OT-CEC) coupled with electrospray ionization-mass spectrometry (ESI-MS) for the analysis of beta-blocker and benzodiazepine analytes. The production of a polymeric surfactant coating on the capillary inner wall involves (i) adsorption of the cationic polymer poly(diallyldimethylammonium chloride) (PDADMAC) to the inner surface of capillary, and (ii) adsorption of the negatively charged poly-SUS onto the cationic polymer layer via strong physical interaction of the two polymer layers. As compared with micellar electrokinetic chromatography (MEKC) coupled with ESI-MS, the main advantage of this proposed method is minimization of introduction of the monomeric or polymeric surfactant into the mass spectrometer, thus avoiding the interference of the nonvolatile micelle in ESI-MS. The effects of buffer pH and applied voltage on the separation of the analytes are also discussed. Under optimum conditions, four of the five beta-blockers and four benzodiazepines are separated.     

Why robust background electrolytes containing multivalent ionic species can fail in capillary zone electrophoresis

Previous models for the retention behaviour of carboxylic acids in ion-exclusion chromatography are applicable only when the degree of ionisation of the analyte is constant over the entire chromatographic peak. When solutions of sulfuric acid are used as eluents, this condition applies only when the eluent concentration is considerably higher than that of the analyte. Since it is common for dilute solutions of sulfuric acid to be used as eluents, a retention model which accounts for unbuffered eluents has been developed. This model also considers the effects on retention of hydrophobic adsorption of the undissociated and dissociated forms of the analyte onto the stationary phase substrate, as well as the effects of organic solvents added to the eluent. The derivation of this model is presented and it has been evaluated using a comprehensive set of retention data obtained using three different sulfonated stationary phases over a range of eluent conditions. The adsorption coefficients calculated from the model are in accordance with expected trends and showed that both the undissociated and dissociated forms of the analyte acids were retained by hydrophobic adsorption effects, although this adsorption was much stronger for the undissociated analytes.     

Tungsten atomizer--theory of atomization mechanism of some volatile analytes

Several types of chemical reactions may participate in the evolution of free atoms in a tungsten furnace. Reactions may take place either in the homogeneous or heterogeneous phase. The assumed reactions may be classified into four types according to the phases in which they take place. Reactions occurring in the gaseous phase, i.e. in the inner volume of the furnace, are kinetically more significant. However, for atomization of easily volatile analytes heterogeneous reaction between gaseous compounds and between condensed salts of analytes and the solid surface of the furnace become significant. With regards to the reaction mechanisms during drying, pyrolysis and atomization of nitrates of volatile analytes, three basic types of chemical reactions may be assumed. Free atoms of analytes arise by evaporation of the elementary form of analytes at atomization temperature, where the particular analyte in its elementary form is produced by direct reduction of analyte nitrate by tungsten or by hydrogen at higher temperatures. Precursory reactions of atom formation are reduction reactions which occur between analyte nitrates and tungsten, between analyte nitrates and hydrogen, as well as reactions of thermal dissociation of relevant nitrates. The importance of particular types of precursory reactions for formation of metallic analytes or their oxides is documented by dependence of Gibbs energy values of particular reactions on the temperature.     

3-Mercaptopropionic acid modified ZnSe quantum dots as the matrix for direct surface-assisted laser desorption/ionization mass spectrometric analysis of peptides/proteins from sodium salt solution

This study describes a strategy of using zinc selenium quantum dots (ZnSe QDs) modified with 3-mercaptopropionic acid (3-MPA) as the matrix for direct analysis of peptides and proteins from sodium salt solution in surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS). The enhancement of detection sensitivity for these biomolecules was due to the adsorption of positively charged peptides or proteins onto the surfaces of negatively charged ZnSe-3MPA QDs via electrostatic interactions resulting in an increase in ionization efficiency for sodium adduct ions ([M+Na](+)). The applicability of the current approach was demonstrated for a variety of peptides, including leucine-enkephalin, methione-enkephalin, HW6, substance P and angiotensin II, and proteins (cytochrome c, myoglobin and lysozyme). Signal intensities of these peptides or proteins can be enhanced by 25-95 times compared with those obtained by LDI-MS in the absence of ZnSe-3MPA QDs. Applying ZnSe-3MPA QDs to serve as the matrix in SALDI-MS is a simple and effective approach for direct analysis of peptide and protein molecules from sodium salt solution without any pretreatment as the peptides and proteins can be successfully detected as sodium adduct ions ([M+Na](+)).     

Detection of fuel-oxidizer explosives utilizing portable capillary electrophoresis with wipe-based sampling

Portable analytical instrumentation that can provide an alarm indication for the presence of explosives and related components is critical for the identification of explosives-based hazards and threats. Many explosives incident reports involve an inorganic oxidizer-fuel mixture which can include pyrotechnics, fireworks, flash powders, black powders, black powder substitutes, and improvised or homemade explosives. A portable CE instrument with targeted analysis of common inorganic oxidizer ions, for example, chlorate, perchlorate, and nitrate, was used here as a rapid detection platform. Unlike frequently used gas-phase separation and detection instrumentation such as ion mobility spectrometry (IMS), an automated liquid extraction mechanism is required for CE separation using acetate paper sample collection wipes. Target inorganic oxidizers were inkjet-printed onto sample wipes to investigate instrument response relative to the collected analyte spatial distribution. Overall, analyte signal intensities increased with off-center sample deposition due to improved sample extraction from wipes and no change in response was observed for varied array distributions across wipes. The system demonstrated sub 200 ng detection limits for all target analytes, with further improvement when normalizing to an internal standard.     

Design and synthesis of labeling reagents (MS probes) for highly sensitive electrospray ionization mass spectrometry and their application to the detection of carbonyl, alcohol, carboxylic acid and primary amine samples.

Novel labeling reagents, called MS probes, which possess a positively charged quaternary amine moiety and can transform a neutral analyte into a charged compound by simply mixing with the analyte and allowing the mixture to stand from several minutes to 30 min at room temperature or while heating to 50 degrees C, were designed and synthesized for the highly sensitive detection of carbonyl, alcohol, carboxylic acid and primary amine samples by electrospray ionization mass spectrometry (ESI-MS). The positively charged products can be detected with high sensitivity in an ESI-MS system, which is the most popular liquid MS instrument. All of the labeled products showed a remarkably large increase in the molecular-ion peak abundance detection sensitivity of over 500-fold at picomolar concentration levels compared to that of unlabeled analytes in an ESI-MS system. These MS probes, used together with liquid MS detection, are widely applicable as a convenient method for the highly sensitive detection of less than picomolar levels of analytes, and therefore greatly enhance the power of ESI-MS analysis.     

Tungsten atomizer – theory of atomization mechanism of some volatile analytes

Several types of chemical reactions may participate in the evolution of free atoms in a tungsten furnace. Reactions may take place either in the homogeneous or heterogeneous phase. The assumed reactions may be classified into four types according to the phases in which they take place. Reactions occurring in the gaseous phase, i.e. in the inner volume of the furnace, are kinetically more significant. However, for atomization of easily volatile analytes heterogeneous reaction between gaseous compounds and between condensed salts of analytes and the solid surface of the furnace become significant. With regards to the reaction mechanisms during drying, pyrolysis and atomization of nitrates of volatile analytes, three basic types of chemical reactions may be assumed. Free atoms of analytes arise by evaporation of the elementary form of analytes at atomization temperature, where the particular analyte in its elementary form is produced by direct reduction of analyte nitrate by tungsten or by hydrogen at higher temperatures. Precursory reactions of atom formation are reduction reactions which occur between analyte nitrates and tungsten, between analyte nitrates and hydrogen, as well as reactions of thermal dissociation of relevant nitrates. The importance of particular types of precursory reactions for formation of metallic analytes or their oxides is documented by dependence of Gibbs energy values of particular reactions on the temperature.     

Magnetic force-based multiplexed immunoassay using superparamagnetic nanoparticles in microfluidic channel

This paper describes a novel microfluidic immunoassay utilizing binding of superparamagnetic nanoparticles to beads and deflection of these beads in a magnetic field as the signal for measuring the presence of analyte. The superparamagnetic 50 nm nanoparticles and fluorescent 1 microm polystyrene beads are immobilized with specific antibodies. When target analytes react with the polystyrene beads and superparamagnetic nanoparticles simultaneously, the superparamagnetic nanoparticles can be attached onto the microbeads by the antigen-antibody complex. In the poly(dimethylsiloxane)(PDMS) microfluidic channel, only the microbeads conjugated with superparamagnetic nanoparticles by analytes consequently move to the high gradient magnetic fields under the specific applied magnetic field. In this study, the magnetic force-based microfluidic immunoassay is successfully applied to detect the rabbit IgG and mouse IgG as model analytes. The lowest concentration of rabbit IgG and mouse IgG measured over the background is 244 pg mL(-1) and 15.6 ng mL(-1), respectively. The velocities of microbeads conjugated with superparamagnetic nanoparticles are demonstrated by magnetic field gradients in microfluidic channels and compared with the calculated magnetic field gradients. Moreover, dual analyte detection in a single reaction is also performed by the fluorescent encoded microbeads in the microfluidic device. Detection range and lower detection limit can be controlled by the microbeads concentration and the higher magnetic field gradient.     

Bare silica nanoparticles as concentrating and affinity probes for rapid analysis of aminothiols, lysozyme and peptide mixtures using atmospheric-pressure matrix-assisted laser desorption/ionization ion trap and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The analysis of peptide mixtures from urine and plasma samples using bare (uncapped) SiO2 nanoparticles (NPs) with atmospheric-pressure matrix-assisted laser desorption/ionization mass spectrometry (AP-MALDI-MS) has been reported. The method was based on the adsorption of positively charged peptides on the surface of negatively charged SiO2 NPs through the electrostatic force of attraction. The adsorption on the surface of SiO2 NPs caused enhancement of ionization efficiency of analytes and subsequently increased the signal intensity of peptides. Maximum signal intensity was obtained at optimized concentration of SiO2 NPs and pH of the aqueous solution. The limits of detection (LODs) obtained for different peptides in deionized water with and without using SiO2 NPs were in the range 4.7-360 nM and 0.1-18.0 microM, respectively. The sensitivity of the proposed method was 21-53-fold better than conventional use of AP-MALDI-MS. In addition, linearity in the range 9.5-95 nM was obtained for the peptide angiotensin-II in deionized water with a correlation of estimation of 0.992 using an internal standard. The proposed method provided a simple way to facilitate the ionization of peptides, reduce sample complexity and increase the tolerance to salts and surfactants in the analysis of biological samples. The applicability of the present method was also demonstrated in the real-world sample analysis of aminothiols and lysozyme using MALDI-time-of-flight (TOF)-MS.     

Minimizing analyte electrolysis in an electrospray emitter

The electrospray (ES) ion source is a controlled-current electrolytic flow cell. Electrolytic reactions in the ES emitter capillary are continually ongoing to sustain the production of charged droplets and ultimately gas-phase ions from this device. Under certain circumstances, the analytes under study may be directly involved in these electrolytic processes. It is demonstrated that a simple means to minimize analyte electrolysis is to exchange the normal metal emitter capillary of commercial ES sources with one made of fused silica. This change is shown to provide an ES mass spectrometric system of similar performance in terms of gas-phase ion signal generated for non-electroactive analytes and also assures minimal oxidation of electroactive analytes even at low (2.0 microl x min(-1)) solution flow-rates and high (millimolar) solution electrolyte concentrations.     

Separation of peptides and proteins by capillary electrophoresis using acidic buffers containing tetraalkylammonium cations and cyclodextrins

A method for improving separations of peptides and other positively charged species in capillary zone electrophoresis with untreated capillaries using acidic buffers containing tetraalkylammonium cations is described. Tetramethylammonium and tetrabutylammonium cations dynamically modify the capillary surface, leading to a reversal in the direction of the electroosmotic flow. As a result, the adsorption of positively charged peptides and proteins is minimized, and resolution and peak capacity are improved as the migration of cationic analytes is counterbalanced by the electroosmotic flow. The combining effect of reversing electroosmotic flow and cyclodextrin inclusion complexation on separations of closely related peptides and a protein mixture, as well as tryptic digest of hemoglobin is demonstrated.     

A glass capillary ultramicroelectrode with an electrokinetic sampling ability.

A glass capillary ultramicroelectrode (tip diameter approximately 1.2 microm) having an electrokinetic sampling ability is described. It is composed of a pulled glass capillary filled with an inner solution and three internal electrodes (Pt working and counter electrodes and an Ag/AgCl reference electrode). The voltammetric response of the capillary electrode is based on electrokinetic transport of analyte ions from the sample solution into the inner solution across the conical tip. It was found that the electrophoretic migration of analytes at the conical tip is faster than electroosmotic flow, enabling electrokinetic transport of analyte ions into the inner solution of the electrode. By using [Fe(CN)6]4- and (ferrocenylmethyl)trimethylammonium (FcTMA+) ions as model analytes, differential pulse voltammetric responses of the capillary electrode were investigated in terms of tip diameter of the capillary, sampling voltage, sampling time, detection limit and selectivity. The magnitude of the response depends on the size and charge of analyte ions. With a capillary electrode having a approximately 1.2-microm tip diameter, which minimizes non-selective diffusional entry of analytes, the response after 1 h sampling at +1.7 V is linearly related to [Fe(CN)6]4- concentration in the range of 0.50-5.0 mM with the detection limit of 30 microM. Application of a potential of the same sign as that of the analyte ion forces the analyte to move out from the electrode to the solution, enabling reuse of the same capillary electrode. The charge-selective detection of analytes with the capillary electrode is demonstrated for [Fe(CN)6]4- in the presence of FcTMA+.     

Silanized quantum dots as labels in lateral flow test strips for C-reactive protein

The paper describes the first use of silanized semiconductor core-shell quantum dots as fluorescent labels for macromolecule, C-reactive protein determination in blood plasma. The controlled synthesis of CdSe cores, with successive shells of CdS, CdZnS, ZnS and coating with transparent, stable, and inert silica shell, provides quantum dots with a narrow emission band, high quantum yield, and prolonged signal stability. Finally, the quantum dots were conjugated with specific antibodies via carboxylic groups on the silica surface. The method was further used for the immunochromatographic assay of C-reactive protein, a diagnostically important inflammatory biomarker. Assays with both the fluorescent QDs and a widely used colloidal gold label were developed in parallel and compared. The silanized quantum dots provide a more sensitive assay with a detection limit of 1?ng/mL for C-reactive protein in standard solutions, whereas the common assay has a detection limit of 10?ng/mL. The possibility of quantitative evaluation of analyte content by a portable device was demonstrated; the accuracy of the measurements was in the range of 5%–10%. The tests were used to determine C-reactive proteins in human plasma samples. The selected optimized protocol for these samples is based on a 4-fold dilution. The final working range of the assay, 4–1,200?ng/mL, covers practically all important interval of C-reactive protein values for the characterization of acute, chronic, and local inflammatory processes. Due to their high physical stability and inertness as well as intense, stable, and reproducible fluorescence, silanized quantum dots may be applied for high-sensitive assays for different analytes.     

Solid phase microextraction with matrix assisted laser desorption/ionization introduction to mass spectrometry and ion mobility spectrometry

Solid phase microextraction (SPME) with matrix assisted laser desorption/ionization (MALDI) introduction was coupled to mass spectrometry and ion mobility spectrometry. Nicotine and myoglobin in matrix 2,5-dihydroxybenzonic acid (DHB), enkephalin and substance P in alpha-cyano-4-hydroxy cinnaminic acid were investigated as the target compounds. The tip of an optical fiber was silanized for extraction of the analytes of interest from solution. The optical fiber thus served as the sample extraction surface, the support for the sample plus matrix, and the optical pipe to transfer the laser energy from the laser to the sample. The MALDI worked under atmospheric pressure, and both an ion mobility spectrometer and a quadrupole/time-of-flight mass spectrometer were used for the detection of the SPME/MALDI signal. The spectra obtained demonstrate the feasibility of the SPME with MALDI introduction to mass spectrometry instrumentation.     

The dipole approach in ion-interaction chromatography of zwitterions

Summary The chromatographic behavior of zwitterions in Ion-interaction chromatography (IIC) is, investigated theoretically for the first time. The modification of the stationary phase in the presence of Ion-interaction reagent (IIR), and adsorption competition between test analytes and IIR for inner layer sites are shown theoretically to change the partition coefficient for zwitterions. Experimental results from the literature concerning retention behavior of zwitterions in IIC, were used to test the new thermodynamic theory. Very reasonable estimates of (i) ΔG ^o values for the IIR adsorption onto the stationary phase (II) total ligand concentration, and (iii) dipolar moments validate the present thermodynamic model for the IIC of zwitterionic analytes. Retention equations are compared to those which can be obtained, if the net charge of the analyte is zero, from the most important retention models in IIC. None of them is able to explain, even in a qualitative way, the retention behavior of zwitterions in IIC whereas, the present model is quantitatively able to do this.