Advances in capillary electrochromatography and micro-high performance liquid chromatography monolithic columns for separation science

Over the last decade, monoliths or continuous beds have emerged as an alternative to traditional packed-bed columns for use in capillary electrochromatography (CEC) and micro-high performance liquid chromatography (micro-HPLC). Monolithic columns can be divided into two categories: silica-based monolithic columns and rigid organic polymer-based monolithic columns resulting from the polymerization of acrylamide, styrene, acrylate or methacrylate monomers. In this paper, the chemistry and most recent applications of these various types of monoliths in both CEC and micro-HPLC are presented.     

Sensitive determination of polycyclic aromatic hydrocarbons in water samples using monolithic capillary solid-phase extraction and on-line thermal desorption prior to gas chromatography-mass spectrometry

A methacrylate-based monolithic capillary column has been evaluated for the preconcentration of polycyclic aromatic hydrocarbons (PAHs) from environmental water samples. For this purpose, the monolyte was in situ synthesized in a 6cm×0.32mm id fused-silica capillary. The microextraction unit was fitted to a micro-HPLC pump to pass 10mL of sample. The isolated pollutants were eluted by means of 10μL of methanol, the organic phase being directly collected in a specific interface that can be fitted to the injection port of the gas chromatograph without modification. The interface allows the on-line thermal desorption of the PAHs, avoiding the dilution and providing enough sensitivity to reach the legal limits established for these pollutants in the matrices selected. The limits of detection achieved for 10mL of water ranged between 2.8ng/L (indeno(1,2,3-cd)pyrene) and 11.5ng/L (acenaphthene) with acceptable precision (between 4.5 and 18.2% RSD). The method was applied to the determination of the selected PAHs in tap, river waters and sewage, being fluoranthene and pyrene detected in all of them at concentrations lower than the legal limits established for these compounds in the matrices assayed.     

Enantioseparation of dipeptides by capillary electrochromatography on a teicoplanin aglycone chiral stationary phase

This work deals with investigations on the enantioseparation of glycyl-dipeptides by capillary electrochromatography (CEC) on a capillary packed with teicoplanin aglycone immobilized on 3.5 μm silica gel. The results were compared to those obtained with micro-HPLC using the same chiral stationary phase. Polar organic and reversed-phase mode were checked, whereby the latter showed better results. Out of 12 glycyldipetides investigated, all compounds showed baseline separation with R_s values up to 20. Plate numbers were in the range of 10 000–300 000/m. The choice of organic modifier was found to be crucial. While methanol increased retention time, acetonitrile reduced it. A ternary mixture of ethanol–acetonitrile–aqueous triethylamine acetate solution pH 4.1 was found to be a useful compromise, providing excellent resolution with retention times less than 25 min. Efficiency and resolution were generally found to be higher in CEC than with micro-HPLC.     

填充毛细管液相色谱-毛细管气相色谱在线联用分析八角茴香挥发油

首次将填充毛细管高效液相色谱-毛细管气相色谱在线联用技术(μ-HPLC-CGC)用于分离分析八角茴香果实的挥发油成分。液相色谱选用氰基分析柱(250 mm×0.32 mm i.d.),正己烷-乙腈-二氯甲烷(体积比为80∶8∶12)为流动相,对挥发油样品做族组分分离,得到的5个族组分被依次存放在多位储存接口内,然后不分流分别转入毛细管气相色谱仪做详细分析。气相色谱柱由10 m×0.53 mm i.d.保留间隔柱和30 m×0.53 mm i.d.×1.0 μm SE-54分析柱组成。采用了不分流柱内进样模     

On-line on-chip post-column derivatisation reactions for pre-ionisation of analytes and cluster analysis in gradient micro-liquid chromatography/electrospray mass spectrometry

A system is presented that demonstrates the principle of on-line and on-chip post-column derivatisation reactions in micro-high-performance liquid chromatography (micro-HPLC) hyphenated to electrospray time-of-flight mass spectrometry (ESI-TOFMS). In this micro-HPLC-chip-MS set-up, the analytes are separated using gradient micro-HPLC and subsequently derivatised on-chip and detected. One of the major limitations of MS detection is its dependency on the degree of ionisation, which is widely variable and compound-specific. Optimising and controlling the degree of ionisation in a simple manner would allow MS detection to be truly generic. One way of achieving this is by pre-ionisation of analytes using simple derivatisation procedures that are both rapid and quantitative. Performing this in situ on the system described here overcomes issues of sample handling and efficiency losses when time-consuming "bench chemistry" is necessary prior to analysis. The power of the system is demonstrated by the separation of primary and secondary amines, which are subsequently derivatised with a positively charged phosphonium complex and detected in an enhanced manner. Typically, molecular cations (M(+)) are detected showing that the ionisation process is dominated by the phosphonium species, leading to more constant ionisation for a variety of compounds. In addition, stable isotopically labelled ((12)C/(13)C)-phosphonium reagent is used for the reactions, allowing for inherent signal/noise (S/N) improvement and automated data processing using cluster analysis. A similar reaction scheme is used for the derivatisation of ketones and aldehydes, also demonstrating dramatic increases in sensitivity, especially with increasing temperature. Minimal loss in chromatographic fidelity in terms of retention times is observed by the introduction of the micromixer chip into the system. Optimal flow rates in micro-HPLC and ESI-MS are compatible with flow rates for the chip as well as a multitude of in-line optical detectors including UV and fluorescence. In addition, the micromixer chip can be positioned pre-column if preferred. The system is robust, easily fully automated and applicable to a wide variety of reactions. The system has a major advantage in its simple robust connection to the "normal scale" outside world.     

Chromatographic evaluation of perphenylcarbamoylated β-cyclodextrin bonded stationary phase for micro-high performance liquid chromatography and pressurized capillary electrochromatography

Application of mono (6^A-N-ethylenediamine-6^A-deoxy) perphenylcarbamoylated β-cyclodextrin (β-CD) bonded stationary phase (CSP) in micro-high performance liquid chromatography (micro-HPLC) and pressurized capillary electrochromatography (p-CEC) was firstly presented. A series of racemic α-amidophosphonates were resolved in reversed- and normal-phase modes on this CSP. The investigated chromatographic parameters include retention factor (k′), separation factor (α) and resolution (Rs) of solutes. In addition, the structural variation of the solutes and the experimental factors affecting chiral separations have been examined, including the percentage of alcohol modifier, the linear velocity (u) of the mobile phase, electrical field strength, etc. Baseline separation was achieved for most of the entities. Hydrophobic interaction, steric effect and π-π interaction contribute to the possible mechanism. Comparative results indicate that higher Rs value up to 3.1 was found in micro-HPLC, higher efficiency up to 29,970 in p-CEC.     

Capillary electroendoosmotic chromatography of peptides

This review focuses on the current state of peptide separation by capillary electroendoosmotic chromatography (CEC). When carried out under optimised conditions, peptide separation by CEC methods represents an orthogonal and complementary technique to micro-HPLC (micro-HPLC) and high-performance capillary zone electrophoresis (HPCZE). The origin of the selectivity differences that can be achieved with these three separation techniques (CEC, micro-HPLC and HPCZE), respectively are discussed, and the current limits of performance with CEC methods documented. Peptide separations by CEC methods with n-alkyl bonded silicas or mixed-mode phases are also illustrated. The development of different variants of CEC and pressurised CEC (also commonly referred to in the literature as electrically-assisted micro-HPLC) are examined. The potential of coupling CEC systems to mass spectrometers for real-time analyses of peptides or protein digests has been examined. Several future directions for the application of this technique in phenotype/proteomic and zeomic mapping of naturally occurring peptides and proteins are highlighted.     

Microcolumn high pressure liquid chromatography with a glass-frit nebulizer interface for plasma emission detection

Microcolumn high pressure liquid chromatography (micro-HPLC) is rapidly gaining recognition as a practical separation tool for organometallic compounds. The use of the inductively coupled plasma (ICP) as a detector for micro-HPLC is studied. Several miniaturized glass-frit nebulizers are investigated as interfaces between the output of the microbore column and the ICP torch. Their performance with aqueous and methanolic solutions is evaluated by direct nebulization and flow injection analysis. The most efficient of these nebulizers is used in the micro-HPLC/ICP study of some Cd, Pb, and Zn organometallic compounds. Detection limits of 1.92 ng of Pb for tetramethyllead and 5.01 ng of Pb for tetraethyllead are obtained and compared with regular HPLC/ICP of these same compounds. Approximately equivalent detection limits were obtained when using a microwave induced plasma as an alternate plasma source.     

Micro-HPLC 1: Linear “breakthrough-gradients” using the discontinuous interface between eluents in a packed gradient-generator column

A new approach is described for micro-HPLC for generating nearly linear gradients in the 40 to 550 μl volume range. These “linear” gradients have a straight line middle portion with a gentle curved onset of the gradient andd rapid attainment of the final 100% composition. Such “breakthrough-gradients” are produced by the interface between weak and strong eluent when a small (3 × 0.46 cm) packed “gradient-generator” column is switched abruptly from weak to strong eluent. The model system described here, without an analytical column, shows gradients from weak eluent (water) to strong eluent (modele with water plus acetone) using a 12-port high pressure valve to 1) vent the “flush” eluent, and simultaneously 2) begin the gradient and 3) make the sample injection. The volume of the gradient can be changed in two ways. If the breakthrough-generator is packed with very large (300 to 1600 μm nonporous particles, the gradient volume is nearly independent of the flush flow rate. The flush flow is used to quickly move the breakthrough-interface to the head of the micro-HPLC column. However, the gradient volume can be increased by using larger solid particles in the gradient-generator column because of increased eddy diffusion. If the gradient-generator is packed with porous particles (75–150 μm), the gradient volume can be changed over a broad range by simply changing the flush flow rate. This simple control over the gradient volume is due to the increased mass transfer of weak eluent out of the porous particles into the strong eluent at higher flush flows. The breakthrough-interface gradient-generator column approach provides the following advantages for micro-HPLC:

• – Gradient volumes can be readily varied over the micro-HPLC range 40–550 μl.
• – Gradient are linear with smooth and rapid onset of intial and final concentrations.
• – Gradient are produced inexpensively with a single high-pressure precision pump.
• – Gradient can be automated with a timer and single 12-port valve that provides both column regeneration and sample injection.

     

Electrochromatography and micro high-performance liquid chromatography with 320 μm I.D. packed columns

Electrochromatography is a chromatographic method in which the mobile phase (liquid or supercritical fluid) is “pumped” through a stationary phase in a microbore or capillary column by electroosmosis using an electric field. The technique permits separation of charged and uncharged compounds with higher resolution and superior efficiency when compared with micro-HPLC with an identical column. It is desirable to work with packed capillary columns with wide diameter in electrochromatography in order to improve detectability and column loadability. This study shows that we have moved a step forward towards this goal in spite of problems and difficulties, due to Joule heating, frit making and column packing in using wide-diameter columns. The paper demonstrates that the pressure pump of micro-HPLC with a commercially available 320 μm I.D. column can be replaced by the electroosmotic “pump” of capillary zone electrophoresis. Experiments were carried out in a chromatographic system under both electroosmosis and pressure-driven flow with 320 and 50 μm I.D. columns packed with 3- and 5-μm ODS. The advantage of electrochromatography over conventional micro-HPLC is shown.     

Stir membrane liquid-liquid microextraction

In this article, a novel liquid phase microextraction technique, called stir membrane liquid-liquid microextraction (SM-LLME), is presented. The new approach combines the advantages of liquid phase microextraction and stirring in the same unit allowing the isolation and preconcentration of the analytes in a simple and efficient way. In the construction of the unit, a polymeric membrane is employed to protect the small volume of the extractant phase. The extraction technique is characterized for the resolution of a model analytical problem: the determination of five selected chlorophenols in water. A two-phase extraction mode is used for the extraction of the analytes with an organic solvent in which an in situ derivatization reaction takes place. The analytes are finally analyzed by gas chromatography/mass spectrometry. All the variables involved in the extraction process have been clearly identified and optimized. The new extraction mode allows the determination of chlorophenols with limits of detection in the range from 14.8 ng/L (for 2,4,5-trichlorophenol) to 22.9 ng/L (for 3-chlorophenol) with a relative standard deviation lower than 8.7% (for 2,6-dichlorophenol).     

An improved interface for universal acoustic flame detection in modified supercritical fluid chromatography

A novel method of interfacing the acoustic flame detector (AFD) with modified supercritical fluid chromatography (SFC) is presented. By applying resistive heating directly to the burner region between the restrictor outlet and the acoustic flame, infrequent severe noise, baseline drifting, and peak deformations that can occasionally be observed with the AFD are eliminated. For example, by increasing the interface temperature only a few hundred degrees Celsius, such sporadic noise in the detector can be reduced nearly ten-fold resulting in smooth stable operation of the AFD. Further, for various levels of methanol modified supercritical carbon dioxide mobile phase examined, the interface was observed to reduce detector noise in each to a common minimal range near 10-25 Hz when an appropriate temperature was achieved. The method is simply assembled, inexpensive to construct, and robust in its daily operation. Overall, the heated interface developed and presented facilitates reliable AFD operation in modified SFC, and supports further exploration and implementation of this sensor as an alternative universal detector in separations requiring an organic cosolvent in the mobile phase.     

The study of organic semiconductor/ferromagnet interfaces in organic spintronics: A short review of recent progress

An overview is given on recent results in organic spintronic research. In particular, so‐called spinterfaces, spin‐injecting interfaces involving organic semiconductor (OSC) molecules and ferromagnetic metals, are discussed. The interfaces are classified in different categories depending on the type and strength of interface interaction and the relevant physics concerning energy level alignment and spin polarization of interface states are explained. Examples are given on characterization of both interface energetics and spin‐related properties obtained from a wide variety of experimental techniques, highlighting the different ways contacting can modify the electronic and magnetic properties of the OSC molecules and the ferromagnetic metals at the resulting spinterfaces. Finally, models for spin injection at spinterfaces are presented and discussed, followed by some speculations on consequences for device design and performance. © 2012 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2012     

Energy level alignment at metal/organic semiconductor interfaces: "pillow" effect, induced density of interface states, and charge neutrality level

A unified model, embodying the "pillow" effect and the induced density of interface states (IDIS) model, is presented for describing the level alignment at a metal/organic interface. The pillow effect, which originates from the orthogonalization of the metal and organic wave functions, is calculated using a many-body linear combination of atomic orbitals Hamiltonian, whereby electron long-range interactions are obtained using an expansion in the metal/organic wave function overlap, while the electronic charge of both materials remains unchanged. This approach yields the pillow dipole and represents the first effect induced by the metal/organic interaction, resulting in a reduction of the metal work function. In a second step, we consider how charge is transferred between the metal and the organic material by means of the IDIS model: Charge transfer is determined by the relative position of the metal work function (corrected by the pillow effect) and the organic charge neutrality level, as well as by an interface parameter S, which measures how this potential difference is screened. In our approach, we show that the combined IDIS-pillow effects can be described in terms of the original IDIS alignment corrected by a screened pillow dipole. For the organic materials considered in this paper, we see that the IDIS dipole already represents most of the realignment induced at the metal/organic interface. We therefore conclude that the pillow effect yields minor corrections to the IDIS model.     

Temperature effects on nano-LC column packing technology

We investigated the effect of temperature on the packing procedure of nano-LC columns (up to 50 cm) and on their performance. Several slurries of stationary phase were prepared using different solvent mixtures. Their stability was evaluated at several temperatures: 70°C, 50°C, and room temperature. At the higher temperature (70°C) the suspensions resulted to be stable for a longer time. For each slurry, we compared nano-LC columns packed with ultrasounds at 70°C and at room temperature. All the columns were tested with a standard mixture at 70°C, to reduce the solvent viscosity and the backpressure. Main chromatographic parameters such as the asymmetry factor, As, the reduced plate high, h, pattern in a Van Deemter plot, the total porosity, ε(t), and the permeability, k, were calculated and discussed. One of the nano-LC columns was used to separate a mixture of pesticides in a LC-MS system with an electron ionization LC-MS interface (Direct-EI). From our knowledge, this is the first study on the role of temperature in the efficiency of slurry-packing procedure.     

Kinetics of the reaction between 2-phenylpropionitrile and 2-chloro-5-nitrotrifluoromethylbenzene under phase-transfer catalysis

Phase-transfer catalysis (PTC) is a widely accepted methodology in organic synthesis. Although a great number of organic syntheses were reported in PTC conditions, systematic kinetic studies are scarce. In the present report, a detailed study of the kinetics of the reaction between 2-chloro-5-nitrotrifluoromethylbenzene (CNTFB) and 2-phenylpropionitrile anion (HPP-), under PTC, was performed under several conditions. The reaction was carried out either in toluene or chlorobenzene as the organic phase, in the presence of a concentrated aqueous solution of NaOH using tetraalkylammonium (Q+X-) salts as phase-transfer catalysts. The major product was 2-(4-nitro-2-trifluoromethylphenyl)-2-phenylpropionitrile, and its yield depends on the experimental conditions. Different aspects of the mechanism are discussed and quantified. Kinetic data were explained by means of an interfacial mechanism that involves the deprotonation of the adsorbed nucleophile precursor followed by its catalyst-mediated extraction to the organic phase. A multicomponent Langmuir-type interface was assumed. Although the extraction of OH- by catalyst to the organic phase is usually disregarded, the formation of the substrate hydrolysis product that leads to catalyst poisoning was also investigated. The influence of this side reaction on the yield of the main product was established. A discussion about the influence of this side process on the main reaction and the operating mechanism is presented.     

Mass transfer model of triethylamine across the n-decane/water interface derived from dynamic interfacial tension experiments

This publication presents a detailed experimental and theoretical study of mass transfer of triethylamine (TEA) across the n-decane/water interface. In preliminary investigations, the partition of TEA between n-decane and water is determined. Based on the experimental finding that the dissociation of TEA takes place in the aqueous and in the organic phase, we assume that the interfacial mass transfer is mainly affected by adsorption and desorption of ionized TEA molecules at the liquid/liquid interface. Due to the amphiphilic structure of the dissociated TEA molecules, a dynamic interfacial tension measurement technique can be used to experimentally determine the interfacial mass transport. A model-based approach, which accounts for diffusive mass transport in the finite liquid bulk phases and for adsorption and desorption of ionized TEA molecules at the interface, is employed to analyze the experimental data. In the equilibrium state, the interfacial tension of dissociated TEA at the n-decane/water interface can be adequately described by the Langmuir isotherm. The comparison between the theoretical and the experimental dynamic interfacial tension data reveals that an additional activation energy barrier for adsorption and desorption at the interface has to be regarded to accurately describe the mass transport of TEA from the n-decane phase into the aqueous phase. Corresponding adsorption rate constants can be obtained by fitting the theoretical predictions to the experimental data. Interfacial tension measurements of mass transfer from the aqueous into the organic phase are characterized by interfacial instabilities caused by Marangoni convection, which result in an enhancement of the transfer rate across the interface.     

311 - A new model of membrane transport: electrolysis at the interface of two immiscible electrolyte solutions

A simple membrane model is the interface between water and an organic liquid immiscible with water, with a strongly hydrophilic electrolyte dissolved in the aqueous phase and a strongly hydrophobic electrolyte in the organic phase. This interface can be electrochemically polarized in the same way as the interface electrode |electrolyte solution using various modes of voltammetry or the galvanostatic method. A four-electrode potentiostatic system is required for such studies. An electrolyte dropping electrode, analogous to Heyrovsky's DME, was also constructed. The voltammograms fully resemble those obtained with metallic electrodes.The faradaic proceses studied so far are mainly connected with the transfer of hydrophobic ions across the interface. These processes are quite rapid and the half-wave potential of a particular ion is related to its standard Gibbs transfer energy. Observed electron-transfer effects model redox processes at membranes.Macrocyclic ionophores facilitate transfer of alkali metal ions across this interface. Very fast ion transfer as well as complex formation was observed in the systems under investigation so that, generally, the diffusion of the ionophore toward the interface and of the complex into the organic phase is the rate-controlling step, no surface reaction retarding the overall process.Apart from the investigation of membrane processes, this approach can be used for elucidation of processes in ion-selective electrodes and in phase-transfer catalysis.     

Mixed-mode reversed-phase and ion-exchange monolithic columns for micro-HPLC

This paper describes the fabrication of RP/ion-exchange mixed-mode monolithic materials for capillary LC. Following deactivation of the capillary surface with 3-(trimethoxysilyl)propyl methacrylate (gamma-MAPS), monoliths were formed by copolymerisation of pentaerythritol diacrylate monostearate (PEDAS), 2-sulphoethyl methacrylate (SEMA) with/without ethylene glycol dimethacrylate (EDMA) within 100 microm id capillaries. In order to investigate the porous properties of the monoliths prepared in our laboratory, mercury intrusion porosimetry, SEM and micro-HPLC were used to measure the monolithic structures. The monolithic columns prepared without EDMA showed bad mechanical stability at high pressure, which is undesirable for micro-HPLC applications. However, it was observed that the small amount (5% w/w) of EDMA clearly improved the mechanical stability of the monoliths. In order to evaluate their application for micro-HPLC, a range of neutral, acidic and basic compounds was separated with these capillaries and satisfactory separations were obtained. In order to further investigate the separation mechanism of these monolithic columns, comparative studies were carried out on the poly(PEDAS-co-SEMA) monolithic column and two other monoliths, poly(PEDAS) and poly(PEDAS-co-2-(methacryloyloxy)ethyl-trimethylammonium methylsulphate (METAM)). As expected, different selectivities were observed for the separation of basic compounds on all three monolithic columns using the same separation conditions. The mobile phase pH also showed clear influence on the retention time of basic compounds. This could be explained by ion-exchange interaction between positively charged analytes and the negatively charged sulphate group.