Exploiting sequential injection analysis with lab-at-valve (LAV) approach for on-line liquid-liquid micro-extraction spectrophotometry

Sequential injection analysis (SIA) with lab-at-valve (LAV) approach for on-line liquid-liquid micro-extraction has been exploited. Sample, reagent and organic solvent were sequentially aspirated into a coil attached to a central port of a conventional multiposition selection valve, where the extraction process was performed. The aqueous and organic phases were separated in a conical separating chamber LAV unit attached at one port of the valve. The organic phase containing extracted product was then monitored spectrophotometrically. The system offers a novel alternative on-line automated extraction in a micro-scale and has been successfully demonstrated for the assays of diphenhydramine hydrochloride (DPHH) in pharmaceutical preparations and anionic surfactant in water samples.     

Capillary electrophoresis with contactless conductivity detection coupled to a sequential injection analysis manifold for extended automated monitoring applications

A capillary electrophoresis (CE) instrument with capacitively coupled contactless conductivity detection (C⁴D) based on a sequential injection analysis (SIA) manifold was refined. Hydrodynamic injection was implemented to avoid a sampling bias by using a split-injection device based on a needle valve for precise adjustment. For safety and reliability, the integrity of the high voltage compartment at the detection end was fully maintained by implementing flushing of the high voltage interface through the capillary. With this set-up, extended fully automated monitoring applications are possible. The system was successfully tested in the field for the determination of the concentration levels of major inorganic cations and anions in a creek over a period of 5 days.     

On-line introduction of high-pressure gas-liquid sample for capillary gas chromatographic analysis

An on-line sample introduction technique in capillary gas chromatograph (CGC) for the analysis of high-pressure gas-liquid mixtures has been designed and evaluated. A sample loop of 0.05 microL and a washing solvent loop of 0.5 microL are mounted on a 10-port switching valve, which serves as the injection valve. A capillary resistor was connected to the vent of sample loop in order to maintain the pressure of the sample. Both the sample and the washing solvent are transferred into the split-injection port through a narrow bore fused silica capillary inserted into the injection liner through a septum. The volume of the liner is used both as the pressure-release damper and evaporation chamber of the sample. On-line analysis of both reactants and resultants in ethylene olimer reaction mixture at 5 MPa was carried out, which demonstrated the applicability of the technique.     

Capillary electrophoretic analysis of carbohydrates derivatized by in-capillary condensation with 1-phenyl-3-methyl-5-pyrazolone

Our previous papers on capillary electrophoresis (CE) have shown that samples can be derivatized in a capillary and the derivatives can be analyzed immediately after derivatization, provided that the derivatization reaction is so rapid as to complete in seconds. The present paper presents extended application of in-capillary derivatization to a much slower reaction such as the condensation of reducing carbohydrates with 1-phenyl-3-methyl-5-pyrazolone (PMP) which requires 30 min at 70 degrees C in pre-column derivatization by manual operation. It was necessary to first drive the introduced plugs of sample and reagent solutions to put them together at the entrance of the heated portion of a capillary, then to allow the superimposed plugs to react for a relevant period. We showed how to determine the introduction times of the sample and the reagent solutions as well as intermediate running buffer, the voltages to be applied for plug driving and product analysis, and the duration of voltage application, all of which are important for effective in-capillary derivatization. An example of the analysis of maltooligosaccharides by this technique is presented. It was shown that maltooligosaccharides were quantitatively derivatized with PMP in 35 min at 57 degrees C, and the derivatives could be analyzed in ca. 15 min by CE immediately after derivatization. Separation was satisfactory in 200 mM borate buffer, pH 8.2 containing sodium dodecyl sulfate to a concentration of 200 mM. Although the theoretical plate number, and accordingly the resolution, were significantly lower than the corresponding values in pre-capillary derivatization, reasonable reproducibility was ensured for both migration time (RSD 3.5% on average) and peak area (RSD less than 3%) under the optimized conditions. It is notable that sample amount could be lowered to the 10 fmol level, in contrast to the 10 pmol level in pre-capillary derivatization. In addition, since the technique employed here (the modified at-inlet technique of in-capillary derivatization) is easily automated, the established system will be highly beneficial for routine analysis of carbohydrates. Analysis by this technique was also shown to be useful for kinetic study of the derivatization reaction.     

On-line coupling of SPE and CE-MS for peptide analysis

An on-line SPE-CE-MS system has been developed for the analysis of peptides. Analytes are preconcentrated using a C(18) microcolumn (5 x 0.5 mm id), and then introduced into the CE system via a valve interface. The CE system with a Polybrene-poly(vinylsulfonate) bilayer coated capillary is combined with an ion-trap mass spectrometer via ESI using a coaxial sheath-liquid sprayer. The on-line coupling of the SPE and CE step by the valve interface is advantageous because it allows an independent functioning of the system parts. Optimization of the SPE-CE system was performed using UV detection. Subsequently, the SPE-CE system has been coupled to the ion-trap mass spectrometer. Test solutions with enkephalin peptides (50 ng/mL) were used for evaluation of system performance. Repeatability of effective mobility and peak area ratio of the two enkephalins were within 1.2% and 9% RSD, respectively. The analysis of 1:1 v/v diluted cerebrospinal fluid samples spiked with enkephalin peptides showed detection limits (S/N = 3) in the range of 1.5-3 ng/mL (around 5 nM), which were similar to those obtained for enkephalin test solutions. Moreover, the potential of the on-line SPE-CE-MS system was demonstrated by the analysis of a cytochrome C digest. Some hydrophilic peptides did not show sufficient retention on the SPE column, and were lost during preconcentration. Nonetheless, positive identification of the protein was achieved, indicating the feasibility of the system for proteomics.     

Continuous on-line derivatization and selective separation of D-aspartic acid by a capillary electrophoresis system with a continuous sample introduction interface

A rapid and selective method is described for the separation of D-aspartic acid (D-Asp) using a continuous on-line derivatization system coupled to capillary electrophoresis (CE). D-Asp was derivatized using o-phthaldialdehyde/N-acetyl-L-cysteine (OPA/NAC). By on-line derivatization, amino acid enantiomers were automatically and reproducibly converted to the UV-absorbing diastereomer derivatives which were separated by capillary zone electrophoresis (CZE) in the presence of 10 mmol/L beta-cyclodextrin (beta-CD). Under the investigated separation conditions, D-Asp is resolved from L-aspartic acid (L-Asp) and other amino acids in a standard mixture of amino acids. The separation could be achieved within 4 min and the sample throughput rate can reach up to 16 h(-1). The repeatability (defined as relative standard deviation, RSD) was 3.21%, 3.58% with peak area evaluation and 3.72%, 4.03% with peak height evaluation for L-Asp and D-Asp.     

On-column derivatization and analysis of amino acids, peptides, and alkylamines by anhydrides using capillary electrophoresis

This work demonstrates the use of an in-capillary procedure for derivatization of amino acids, peptides, and alkylamines by anhydrides using capillary electrophoresis (CE). Migrating in an uncoated fused-silica capillary, plugs of substrate and anhydride are injected separately and electrophoresed. Differential transport velocities permit the separate zones to penetrate each other under an applied field, thereby facilitating reaction. In initial experiments the extent of reaction between tryptophan and acetic anhydride was examined and product amounts quantitated by CE. In separate experiments a series of amino acids and peptides were injected into the capillary and reacted with phthalic anhydride on-column to yield the phthalic derivatized species. Finally, on-column derivatization of alkylamines with phthalic anhydride was investigated and electrophoretic mobility related to molecular weight of the derivatized amines. These procedures illustrate the use of the capillary as a microreactor in the facile synthesis of derivatized molecules and ease of quantitation of reaction products under conditions of electrophoresis.     

Continuous flow derivatization system coupled to capillary electrophoresis for the determination of amino acids

A derivatization system coupled to capillary electrophoresis for the determination of amino acids using 1,2-naphthoquinone-4-sulfonate as a labeling agent is described. In this system, amino acids are derivatized on-line in a three-channel flow manifold for sample, reagent and buffer solutions. The reaction takes place in a PTFE coil heated at 80 degrees C. The resulting solution, which contains the amino acid derivatives, is introduced into the electrophoretic system by means of an appropriate interface. Subsequently, amino acid derivatives are separated at 25 kV using a 40 mM sodium tetraborate aqueous solution with 30% (v/v) isopropanol solution as a running buffer. The electropherograms are monitored spectrophotometrically at 230 nm. The method has been applied to the determination of amino acids in feed samples and pharmaceutical preparations. A good concordance of the predicted values with those given by a standard amino acid analyzer is shown.     

On-line hyphenation of flow injection, miniaturized capillary electrophoresis and atomic fluorescence spectrometry for high-throughput speciation analysis

A hyphenated technique was developed for high-throughput speciation analysis by on-line coupling of flow injection (FI), miniaturized capillary electrophoresis (CE) and atomic fluorescence spectrometry (AFS). Two interfaces were used to couple all three systems: the first to couple FI and CE and the second to couple miniaturized CE and AFS. The first interface was a modified flow through chamber, connected to the FI valve with a piece of PTFE tube (0.1mm i.d.x 20 cm long). The capillary outlet was coupled to the AFS by using the second concentric "tube-in-tube" interface. Split sampling was achieved in the electrokinetic mode. Inorganic mercury (Hg(II)) and methylmercury (MeHg(I)) were taken as model analytes to demonstrate the performance of the developed hyphenated technique. A volatile species generation (VSG) technique was employed to convert the analytes from the CE effluent into their respective volatile species. Baseline separation of Hg(II) and MeHg(I) was achieved by CE in a 50 microm i.d.x 8 cm long capillary at 3.0 kV within 60s. The precisions (RSD, n=12) were in the range of 0.7-0.9% for migration time, 3.8-4.2% for peak area, and 2.1-3.5% for peak height. The detection limits were 0.1 and 0.2 microgmL(-1) (as Hg) for Hg(II) and MeHg(I) with a sample throughput of 60 samples h(-1). The recoveries of both mercury species in the water samples studied were in the range of 93-106%.     

Trace level determination of substance P using capillary electrophoresis and laser-induced fluorescence

An analytical method was developed to determine the undecapetide substance P (SP) based on capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. SP was derivatized with the fluorogenic reagent 2,3-naphthalenedicarboxaldehyde (NDA) prior to injection into the CE-LIF system. The pre-column derivatization scheme combined with injection enhancement techniques extends the detectability of SP to the subnanomolar level. Limit of detection (LOD) of 100 pM was achieved without pre-concentrating the sample prior to injection. The reproducibility for six different preparations of a standard sample containing 5 nM of SP was 6.8% RSD and that of the CE migration time was 0.08% RSD. The method was used to determine SP in a saliva sample.     

Sequential injection analysis system for on-line monitoring of l-cysteine concentration in biological processes

A sequential injection analysis (SIA) system was developed to monitor the concentration of l-cysteine in biological processes on-line. It is based on the redox reaction of l-cysteine with iron(III) in the presence of 1,10-phenanthroline (phen) and the detection of the red-iron(II)-phen complex with a spectrophotometry. The system was fully automated using software written in the LabVIEW™ development environment. A number of system variables such as the flow rate of the carrier buffer solution, the volume ratio of the sample to the reagents, and the reaction coil length, etc., were evaluated to increase the sensitivity and performance of the SIA system. Under partially optimized operating conditions the performance of the SIA system was linear up to a concentration of l-cysteine of 1 mM (R² = 0.998) with a detection limit of 0.005 mM and a sample frequency of 15 hr⁻¹. The SIA system was employed to monitor the concentration of l-cysteine on-line in a continuously stirred reactor and a fermentation process of Saccharomyces cerevisiae. The on-line monitored data were in good agreement with the off-line data measured by a HPLC with a fluorescence detector (n = 15, R² = 09899).     

Single-step analysis of low abundance phosphoamino acids via on-line sample preconcentration with chemical derivatization by capillary electrophoresis

New strategies for rapid, sensitive and high-throughput analysis of low abundance metabolites in biological samples are required for future metabolomic research. In this report, a direct method for sub-micromolar analyses of phosphoamino acids was developed using on-line sample preconcentration with 9-fluorenylmethyloxycarbonyl chloride (FMOC) derivatization by capillary electrophoresis (CE) and UV detection. Analyte focusing by dynamic pH junction and FMOC labeling efficiency were influenced by several experimental factors including buffer pH, ionic strength, sample injection length and FMOC concentration. About a 200-fold enhancement in concentration sensitivity was achieved under optimal conditions relative to conventional off-line derivatization, as reflected by a detection limit (S/N approximately 3) of 0.1 microM. In-capillary sample preconcentration with chemical labeling by CE offers a unique single-step analytical platform for high-throughput screening of low abundance metabolites without intrinsic chromophores.     

On-line preparation of microsamples prior to CE

An experimental setup is presented here for the automated analysis of microsamples, based on the on-line coupling of a capillary SPE module and a CE unit using a two-position six-port valve, an open-closed valve to isolate electrically the sample preparation from the CE unit and a "T" interface. A C18 trapping microcolumn (dimensions 2.5 cm x 100 microm id x 360 microm od) was used for the SPE step. The utility of the proposed experimental setup was demonstrated by applying it to the determination of quinolone antibiotics in serum microsamples, which was efficiently carried out in less than 20 min (4 min for protein denaturation and 15 min for analytes preconcentration and CE-UV separation-determination). A complete optimization study was performed for preconcentration and cleanup of quinolones, the coupling of sample preparation module to the CE unit and electrophoretic separation of quinolones. A preconcentration factor of 10.4 was achieved. The volume injected with the proposed method was 125 nL versus 160 nL introduced by hydrodynamic injection. The volume required for the analysis was 2 microL, which makes the proposed experimental setup very useful for the analysis of microsamples in fields of current interest such as metabolomics or proteomics.     

An automated method of on-line extraction coupled with flow injection and capillary electrophoresis for phytochemical analysis

In this study, an automated system for phytochemical analysis was successfully fabricated for the first time in our laboratory. The system included on-line decocting, filtering, cooling, sample introducing, separation, and detection, which greatly simplified the sample preparation and shortened the analysis time. Samples from the decoction extract were drawn every 5 min through an on-line filter and a condenser pipe to the sample loop from which 20-μL samples were injected into the running buffer and transported into a split-flow interface coupling the flow injection and capillary electrophoresis systems. The separation of glycyrrhetinic acid (GTA) and glycyrrhizic acid (GA) took less than 5 min by using a 10 mM borate buffer (adjusted pH to 8.8) and +10 kV voltage. Calibration curves showed good linearity with correlation coefficients (R) more than 0.9991. The intra-day repeatabilities (n = 5, expressed as relative standard deviation) of the proposed system, obtained using GTA and GA standards, were 1.1% and 0.8% for migration time and 0.7% and 0.9% for peak area, respectively. The mean recoveries of GTA and GA in the off-line extract of Glycyrrhiza uralensis Fisch root were better than 99.0%. The limits of detection (signal-to-noise ratio = 3) of the proposed method were 6.2 μg/mL and 6.9 μg/mL for GTA and GA, respectively. The dynamic changes of GTA and GA on the decoction time were obtained during the on-line decoction process of Glycyrrhiza uralensis Fisch root.     

Application of capillary electrophoresis with field-amplified sample injection for the detection of new adrenoreceptor antagonist enantiomers in plasma in the low ng/mL concentration range

Throughout the separation of chiral basic drugs by capillary electrophoresis (CE) with neutral hydroxypropyl-beta-cyclodextrin (HP-beta-CD) as chiral selector, the sensitivity of detection has been improved by using field-amplified sample injection (FASI). In the present work, this on-line stacking method has been used to detect low ng/mL levels of cationic enantiomers of a new adrenoreceptor antagonist in plasma. A systematic study of the parameters affecting on-line concentration of these enantiomers (nature of the preinjection plug, composition of sample solvent, injection times of water and sample plugs) has been performed enabling the detection sensitivity of antagonist enantiomers to be improved by 180 times compared with usual hydrodynamic injection. The quantification of each adrenoreceptor antagonist enantiomer in plasma samples was then performed in the 2-100 ng/mL (or 8-400 nM) concentration range after a solid-phase extraction step. Using this FASI-CE-UV procedure, the limit of quantification (LOQ) for each enantiomer was in the low ng/mL concentration range (3 ng/mL or 10 nM).     

On-line conversion and determination of artemisinin using a flow-injection capillary electrophoresis system

A novel, rapid, and simple capillary electrophoresis (CE) method has been developed for the determination of antimalarial artemisinin by on-line treatment with alkaline. By on-line reaction, artemisinin was automatically and reproducibly converted to the strongly UV-absorbing compound, Q292, by treating it with 0.20 mol/L NaOH solution for 3 min at 40 degrees C. Analysis was carried out in less than 12 min after conversion of artemisinin in a flow injection (FI) system that was coupled to CE equipment via a split-flow interface cell, and a sampling frequency of 8 h(-1) is achievable. The on-line conversion method has been applied to the determination of artemisinin in the traditional Chinese herbal drug Artemisia annua L., and the results are satisfactory.     

A simplified multidimensional approach for analysis of complex biological samples: on-line LC-CE-MS

Information on protein expression, disease biomarkers or surrogate markers and genetic disorders can nowadays be achieved from analysis of complex biological samples by liquid separation coupled to mass spectrometric (MS) detection. This paper describes fast multidimensional separation by on-line liquid chromatography (LC) and capillary electrophoresis (CE), followed by electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) MS detection. This detector provides ultrahigh resolution of the detected ions, mass accuracy at the ppm-level and high sensitivity. Most of the challenge of this system lies in the development of a new interface for the on-line coupling of LC to CE. The interface developed in poly(dimethylsiloxane) provides a RSD for injection repeatability of <3.5% and surface control for unspecific binding by deactivation with a cationic polymer, PolyE-323. We have evaluated the interface, as well as the overall system, with respect to robustness and deconvolution ability. Sequence coverage for bovine serum albumin (BSA) of 93% showed a high recovery of sample in the different transfer steps through the system. The detection limit for identification is 277 ng mL(-1) (or 280 nM) on average for peptides. In the future, we expect LC-CE-MS to be a novel strategy for elucidating the chemistry of biological matrices.     

Determination of multiple drugs of abuse in human urine using capillary electrophoresis with fluorescence detection

Methods for separation and determination of multiple drugs of abuse in biological fluids using capillary electrophoresis (CE) with native fluorescence and laser-induced fluorescence (LIF) detection are described herein. Using native fluorescence, normorphine, morphine, 6-acetyl morphine (6-AM), and codeine were analyzed by CE without any derivatization procedure and detected at an excitation wavelength of 245 nm with a cut-off emission filter of 320 nm, providing a rapid and simple analysis. The detection limits were in the range of 200 ng/mL. For a highly sensitive analysis, LIF detection was also examined using a two-step precolumn derivatization procedure. In this case, drugs extracted from human urine were first subjected to an N-demethylation reaction involving the use of 1-chloroethyl chloroformate (ACE-Cl) and then derivatized using fluorescein isothiocyanate isomer I (FITC) and analyzed by CE coupled to a LIF detector. Variables affecting this derivatization: yield of demethylation reaction, FITC concentration, reaction time and temperature, were studied. The estimated instrumental detection limits of the FITC derivatives were in the range of 50-100 pg/mL, using LIF detection with excitation and emission wavelengths of 488 nm and 520 nm, respectively. The linearity, reproducibility and reliability of the methods were evaluated. In addition, a comparison of the characteristics for both native fluorescence and LIF detections was also discussed.     

Derivatization trends in capillary electrophoresis: an update

This survey as a sequel of two earlier reports gives an overview of recent developments, starting from 1999, in the use of derivatization protocols in capillary electrophoretic (CE) analysis. Derivatization is mainly used for enhancement of the detection sensitivity in CE, for which a combination of fluorescence labeling and laser-induced fluorescence detection is favorable. Moreover, especially in the field of saccharide assay, derivatization to introduce charge into the molecule, is common. Derivatization procedures are classified in tables, focused on precapillary, on-line, on-capillary and postcapillary arrangements and divided in sections concerning the functional group that is derivatized. The most frequently reported groups are amines and the reducing end of (oligo)saccharides, but thiols, carbonyl and carboxyl groups, steroids and inorganic ions have also been reported about. Other reasons for derivatization are to enhance chiral separation, introduction of a suitable charge into the molecule or to improve mass spectrometric detection. The use of derivatization techniques for special cases, such as the analysis of neurotransmitters, insulin antibodies and mitochondria has also been incorporated as well as a study on the adsorption of proteins onto capillary walls during CE in which derivatization plays a role.     

Constant pressure-assisted head-column field-amplified sample injection in combination with in-capillary derivatization for enhancing the sensitivity of capillary electrophoresis

In this work, a novel method combining constant pressure-assisted head-column field-amplified sample injection (PA-HC-FASI) with in-capillary derivatization was developed for enhancing the sensitivity of capillary electrophoresis. PA-HC-FASI uses an appropriate positive pressure to counterbalance the electroosmotic flow in the capillary column during electrokinetic injection, while taking advantage of the field amplification in the sample matrix and the water of the “head column”. Accordingly, the analytes were stacked at the stationary boundary between water and background electrolyte. After 600 s PA-HC-FASI, 4-fluoro-7-nitro-2,1,3-benzoxadiazole as derivatization reagent was injected, followed by an electrokinetic step (5 kV, 45 s) to enhance the mixing efficiency of analytes and reagent plugs. Standing a specified time of 10 min for derivatization reaction under 35 °C, then the capillary temperature was cooled to 25 °C and the derivatives were immediately separated and determined under 25 °C. By investigating the variables of the presented approach in detail, on-line preconcentration, derivatization and separation could be automatically operated in one run and required no modification of current CE commercial instrument. Moreover, the sensitivity enhancement factor of 520 and 800 together with the detection limits of 16.32 and 6.34 pg/mL was achieved for model compounds: glufosinate and aminomethylphosphonic acid, demonstrating the high detection sensitivity of the presented method.