Analyte focusing by micelle collapse for liquid extraction surface analysis coupled with capillary electrophoresis of neutral analytes on a solid surface

Liquid extraction surface analysis (LESA) has an advantage of directly sampling analytes on a surface, thus avoiding unnecessary dilution by homogenization of the bulk sample commonly practiced in solid sample analysis. By combining LESA with CE, the additional advantage of separating analytes before detection can be accomplished. For neutral molecules, MEKC needs to be used. Since the detection sensitivity of CE in general suffers from the small capillary dimension, analyte focusing by micelle collapse was employed for enhanced extraction in LESA and sample preconcentration for MEKC. In addition, using a commercial CE instrument, the LESA process was performed much faster and more reliably compared to our first demonstration of LESA‐CE using a homemade CE setup. Three neutral water‐insoluble pesticides sprayed on an apple skin were directly extracted, preconcentrated, and analyzed by the automated LESA‐analyte focusing by micelle collapse‐MEKC with high sensitivity in 10 min. The relative standard deviations of the migration times and peak heights were 0.8–2.1 and 1.2–3.0%, respectively when ametryn was used as an internal standard. The limits of detection obtained with UV absorbance at 200 nm were 1.8–6.4 ppb.     

In-line coupling of two-phase single drop microextraction and large volume stacking using an electroosmotic flow pump in nonaqueous capillary electrophoresis

In order to improve the concentration sensitivity of capillary electrophoresis (CE), two sample preconcentration techniques, single drop microextraction (SDME) and large volume stacking using an electroosmotic flow pump (LVSEP), were coupled in-line in a commercial CE instrument. By simple programming of liquid handling sequences, a pentanol drop was prepared at the tip of a fused silica capillary over which a Teflon tube had been sleeved to serve as a hydrophobic support. After extraction of the analytes from an aqueous donor solution into the drop, the entire capillary column was filled with enriched pentanol extract. LVSEP, in which the sample matrix is automatically removed by the EOF, was then carried out using a methanolic run buffer. The overall enrichment factors for the analytes pentachlorophenol (PCP), 3-bromobenzoic acid (3-BBA), and 4-iodobenzoic acid (4-IBA), from a combination of 30 min SDME and LVSEP on a 27 cm capillary, were about 7000, even without agitation of the donor solution. The resulting limits of detection for PCP, 3-BBA, and 4-IBA were 0.7, 0.3 and 0.7 nM, respectively. Since no modification of the existing CE instrument is necessary and a bare capillary is used for LVSEP, this scheme can be adapted quite easily for many CE applications that require high concentration sensitivity.     

Miniaturized capillary electrophoresis system with ultraviolet photometric detection combined with flow injection sample introduction using a modified falling-drop interface

A miniaturized capillary electrophoresis (CE) system with UV-Vis detection was coupled to a flow injection (FI) system for achieving high throughput continuous sample introduction. The cassette of a commercial CE instrument was modified to hold a 6.5 cm long silica capillary and a flow-through waste reservoir. The cassette was inserted into the flow-cell chamber of a commercial UV detector, with the light beam focused on the capillary and collected by two ball lenses on the cassette. The capillary inlet, left outside the cassette and detector, was positioned on the top of a vertical 3.5 mm diameter glass rod, in close contact with an electrode. Samples injected through the FI system dropped freely on top of the pillar, covering the capillary inlet and electrode. Continuous sample introduction was achieved for CE separations under non-interrupted separation voltage, which was isolated from the FI system through the discontinuity of droplets. The newly developed interface and UV detection system was used for fast separation of sulphamethoxazole (SMZ) and trimethoprim (TMP) in sulphatrim tablets, achieving a high throughput of over 48 h⁻¹, and a low carryover of 2%. Separation efficiencies of 8 μm plate height and detection limits of 1.0 mg l⁻¹ for SMZ and 0.5 mg l⁻¹ (3σ) for TMP were obtained.     

Simplified universal method for determining electrolyte temperatures in a capillary electrophoresis instrument with forced-air cooling

Temperature increase due to resistive electrical heating is an inherent limitation of capillary electrophoresis (CE). Active cooling systems are used to decrease the temperature of the capillary, but their capacity is limited; and in addition, they leave "hot spots" at the detection interface and at the capillary ends. Until recently, the matter was complicated by the lack of a fast and generic method for temperature determination in efficiently and inefficiently cooled regions of the capillary. Our group recently introduced such a method, termed "Universal Method for determining Electrolyte Temperatures" (UMET). UMET is a probe-less approach that requires only measuring current versus voltage for different voltages and processing the data using an iterative algorithm. Here, we apply UMET to develop a Simplified Universal Method of Temperature Determination (SUMET) for a CE instrument with a forced-air cooling system using an Agilent 7100 CE instrument (Agilent Technologies, Saint Laurent, Quebec, Canada) as an example. We collected a wide set of empirical voltage-current data for a variety of buffers and capillary diameters. We further constructed empirical equations for temperature calculation in efficiently and inefficiently cooled parts of the capillary that require only the data from a single 1-min voltage-current measurement. The equations are specific for the Agilent 7100 CE instrument (Agilent Technologies) but can be applied to all kinds of capillaries and buffers. Similar SUMET approaches can be developed for other CE instruments with forced-air cooling using our approach.     

On-line combination of single-drop liquid–liquid–liquid microextraction with capillary electrophoresis for sample cleanup and preconcentration: A simple and efficient approach to determining trace analyte in real matrices

In order to improve the sensitivity of capillary electrophoresis (CE) and overcome the deficiency of commercial CE instruments in handling complex matrices directly, we proposed a novel technique which combined single-drop liquid–liquid–liquid microextraction (SD-LLLME) with CE on-line. In this technique, SD-LLLME was realized using a commercial CE instrument and, to further concentrate the target analyte, large-volume sample stacking combined sweeping without polarity switching was utilized. Even though without agitating the donor phase in the extraction process, the model compound, adenine was enriched 550-fold in only 10 min. The enrichment factors were 760 and 1030 when the extraction time was extended to 30 and 60 min, respectively. The relative standard deviations (RSDs) of adenine were 5.24% and 2.29% for peak area and migration time, respectively, which indicated that this method was much more reproducible compared to the existing methods that combined sample-preparation strategies with CE. In addition, this approach was selective while cleaning up target analyte. These mentioned advantages allowed the developed method to be an attractive approach to determining trace target compounds in complex real samples.     

An automated instrument for human STR identification: design, characterization, and experimental validation

The microfluidic integration of an entire DNA analysis workflow on a fully integrated miniaturized instrument is reported using lab‐on‐a‐chip automation to perform DNA fingerprinting compatible with CODIS standard relevant to the forensic community. The instrument aims to improve the cost, duration, and ease of use to perform a “sample‐to‐profile” analysis with no need for human intervention. The present publication describes the operation of the three major components of the system: the electronic control components, the microfluidic cartridge and CE microchip, and the optical excitation/detection module. Experimental details are given to characterize the level of performance, stability, reliability, accuracy, and sensitivity of the prototype system. A typical temperature profile from a PCR amplification process and an electropherogram of a commercial size standard (GeneScan 500?, Applied Biosystems) separation are shown to assess the relevance of the instrument to forensic applications. Finally, we present a profile from an automated integrated run where lysed cells from a buccal swab were introduced in the system and no further human intervention was required to complete the analysis.     

On-line process monitoring of water-soluble ions in pulp and paper machine waters by capillary electrophoresis

In this study, capillary electrophoresis (CE) was used for separation of inorganic and organic ions from waters of paper and paperboard machines at mills. The instrument was constructed for on-line measurements by building a batch-type sample feeding unit. Chloride, thiosulphate, sulphate, oxalate, sulphite, hydrogen sulphide, formate, carbonate, phosphate and acetate in the process water samples were separated using an ion-specific separation system in CE with dicarboxylic acid buffer (pH 8.2), with pyridinium-2,3-dicarboxylic acid modified with commercial NICE-Pak OFM-OH solution (pH 12.0) or with a cetyltrimethylammonium bromide solution modified with chromate (pH 10.6). In addition, Analis CEofix Anions 8 electrolyte solution was tested in on-line studies at mills. It allowed 5 min separation time for the anions. Aluminium was determined at pH 3.6 in 10 min by using a laboratory-made imidazole buffer modified with 18-crown 6-ether. The developed CE systems were used to monitor the concentrations of sulphur species in dithionite degradation, to estimate corrosion degree in the water tanks, to monitor formaldehyde as the biocide chemical in wire washing and, in general, to observe process disturbances resulting from chemical feedings and their sites. The CE combination was on-line coupled to eight different process machines for continuous monitoring of concentrations for periods between two weeks and one month at paper and pulp mills in Finland.     

Photo-induced chemiluminescence from fibrous polymers and proteins

A commercial thermal chemiluminescence (TCL) instrument was modified to allow in situ sample irradiation at wavelengths in the range 320-700 nm under a controlled atmosphere at constant temperature. Weak photo-induced chemiluminescence (PICL) emission was observed from commercial poly(ethylene terephthalate), polyacrylonitrile, and polyamide 6 fabrics, cotton fabric and from the fibrous proteins wool and feather keratin, silk fibroin and bovine skin collagen (Type 1) after exposure to UVA (320-400 nm) or visible light in nitrogen, followed by a change of the atmosphere to oxygen. Collagen emits PICL of similar intensity to keratin, which demonstrates that tryptophan is not essential for PICL emission from proteins. In all cases the decay of PICL with time is complex and does not follow simple first- or second-order kinetics. The effects of experimental variables, including wavelength of radiation and exposure time, sample temperature and fabric pH on the PICL intensity and decay profile are reported for wool keratin.     

Integrated 2-D CE

A simple methodology for converting a commercial CE-MS instrument into an integrated 2-D CE system has been developed. The first-dimensional capillary operates as a typical CE instrument with UV/visible detection. Fractions leaving the first dimension are automatically collected and introduced into the second dimension, performed on a CE-MS apparatus, for analysis. The integrated system allows fractions in the second dimension to be analyzed using various electrophoretic modes. As an example, in this work we performed the separation of two families of antibiotics (nitroimidazoles and tetracyclines) in the first dimension and the subsequent resolution of the antibiotics in each family (nitroimidazoles were resolved by MEKC and tetracyclines by CZE) in the second dimension. The proposed system, which operates in an highly automatic manner, is flexible and allows various combination of electrophoretic modes to be implemented. In addition, the use of a mass spectrometer detector in the second dimension further increases the analytical potential of the system as a result of the high selectivity and wealth of structural information provided by the MS detector.     

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.     

Comparative study between a commercial and a homemade capillary electrophoresis instrument for the simultaneous determination of aminated compounds by induced fluorescence detection

The performance of two capillary electrophoresis (CE) instruments, one commercial and one homemade device, were compared for the determination of derivatised aminated compounds with fluorescein isothiocyanate (FITC). The commercial CE system first uses an argon ion laser as excitation source; the homemade CE device uses an inexpensive blue-light-emitting diode (LED) as the light source and a charge-coupled device (CCD) as the detection system. After fine optimisation of several separation parameters in both devices, a co-electroosmotic flow CE methodology was achieved in coated capillary tubing with 0.001% hexadimetrine bromide (HDB), and 50 mmol L⁻¹ sodium borate at pH 9.3 with 20% 2-propanol for the determination of several amines and aminoacids. Analytical performances, applicability in beer samples and other aspects such as cost or potential for miniaturization have been compared for both devices.     

Highly sensitive chiral analysis of amino acids by in-line single drop microextraction and capillary electrophoresis with laser-induced fluorescence detection

A highly sensitive method for chiral analysis of amino acids by in-line single drop microextraction (SDME) and chiral capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was developed. In SDME, a drop of a basic aqueous acceptor phase covered with a thin organic layer was formed at the tip of a capillary by simple combination of sample-handling sequences of a CE apparatus. Then fluorescein isothiocyanate (FITC)-derivatized amino acids in an acidic donor solution were enriched into the drop through the organic layer. The enriched enantiomers were then resolved using a dual chiral selector of β-cyclodextrin (β-CD) and sodium taurodeoxycholate (STC). Here, in addition to serving as a labeling reagent providing high fluorescence signal, hydrophobic FITC was primarily used as a modifier aiding the extraction of zwitterionic amino acids by blocking the amino groups and increasing the hydrophobicity, yielding 220 times increase in extraction efficiency. Several hundred-fold enrichments were achieved with 10 min SDME, yielding LODs of 30-60 pM and enabling direct analysis of d-AAs in a 99% enantiomeric excess mixture. In view of no additional modification of the existing commercial CE instrument, this method without stirring can be easily realized using known operations. When a microstirrer was customized to the CE instrument several thousand-fold enrichments could be obtained with LODs in the low picomolar range of 1-3 pM.     

A simple sample pretreatment device with supported liquid membrane for direct injection of untreated body fluids and in‐line coupling to a commercial CE instrument

A simple sample pretreatment device was developed employing extractions across supported liquid membranes (SLMs) and in‐line coupling to a commercial CE instrument. The device consisted of two polypropylene conical units interspaced with a polypropylene planar SLM, which were impregnated with 1‐ethyl‐2‐nitrobenzene. The two units and the SLM were pressed against each other, donor unit was filled with 40 μL of an untreated body fluid and acceptor unit with 40 μL of DI water. The device was then placed into conventional CE vial fitted with a soft spring, which was depressed during injection into CE capillary and ensured that the SLM was not ruptured. Position of separation capillary injection end and high‐voltage electrode in the CE instrument was optimized in order to ensure efficient injection of pretreated body fluids. The device can be easily assembled/disassembled and SLMs can be replaced after each extraction thus minimizing sample carry‐over, avoiding tedious SLM regeneration, and reducing total pretreatment time and costs. The pretreatment device was examined by direct injection of human urine and serum spiked with nortriptyline, haloperidol, and loperamide. The basic drugs were diffusionaly transported across the SLM within 10 min and were injected into the separation capillary directly from the SLM surface in the acceptor unit, whereas matrix components were retained by the SLM. The in‐line SLM‐CE method showed good repeatability of peak areas (3.8–11.0%) and migration times (below 1.4%), linear relationship (r² = 0.990–0.999), and low LODs (12–100 μg/L).     

Direct Determination of Glyphosate in Environmental Waters Using Capillary Electrophoresis with Electrospray Condensation Nucleation Light Scattering Detection

An electrospray condensation nucleation light scattering detector (ESI-CNLSD) was coupled with capillary electrophoresis (CE) for analysis of glyphosate, a chemical of agricultural interest, which is otherwise difficult to detect owing to its lack of chromophores or fluorophores. To reduce the absorption of glyphosate on the CE capillary and to reduce the separation time, an N-cetyltrimethylammonium bromide (CTAB) pre-rinsing CE method was developed here. The protocol consisted of 15 min pre-rinsing of the capillary before analysis with CTAB solution and 5 min with ammonium acetate buffer at pH 2.8. The capillary inner wall coating established by this treatment lasted up to 10 h without bleeding to interfere with CNLSD signal. Calibration data were linear over two orders of magnitude, the instrument detection limit was 0.06 µg/mL and the method detection limit was 0.2 µg/mL. The method was applied to the analysis of local (rural area) lake water and commercial herbicide samples.     

In-line preconcentration of oxidized and reduced glutathione in capillary zone electrophoresis using transient isotachophoresis under strong counter-electroosmotic flow

Transient isotachophoresis (tITP) can improve the sensitivity of capillary electrophoresis (CE). In general, it was carried out under the condition of suppressed electroosmotic flow (EOF). However, some special conditions, such as extreme low pH background electrolyte and coating were needed to achieve the requirements of suppressed EOF. In this work, an approach of tITP under the strong counter-EOF in open system (counter-EOF-tITP) is presented for the rapid and sensitive preconcentrating the reduced glutathione (GSH) and the oxidized glutathione (GSSG) without modifying the capillary and the commercial CE instrument. The parameters of the experimental system, such as the concentration of leading electrolyte, the injected amount of terminating electrolyte and the injected pressure of sample were investigated in detail to understand the mechanism of counter-EOF-tITP. The sensitivity enhancement factors were of 320 for GSH and 280 for GSSG. In addition, the detection limit of 23.4 and 18.0 μg L⁻¹ for GSH and GSSG was achieved, respectively. The method's applicability was demonstrated by determining GSH and GSSG in tomato and human serum.     

用于电渗控制的新型毛细管电泳装置

提出了一种可利用径向电场控制电渗的新型毛细管电泳装置，着重讨论了其工作原理，电渗控制方法及相关结果。该装置不仅能方便地实现电渗的电场控制，而且通过略加改进，还能与商品仪器兼容。     

Automated analysis of individual particles using a commercial capillary electrophoresis system

Capillary electrophoretic analysis of individual submicrometer size particles has been previously done using custom-built instruments. Despite that these instruments provide an excellent signal-to-noise ratio for individual particle detection, they are not capable of performing automated analyses of particles. Here we report the use of a commercial Beckman P/ACE MDQ capillary electrophoresis (CE) instrument with on-column laser-induced fluorescence (LIF) detection for the automated analysis of individual particles. The CE instrument was modified with an external I/O board that allowed for faster data acquisition rates (e.g. 100 Hz) than those available with the standard instrument settings (e.g. 4 Hz). A series of eight hydrodynamic injections expected to contain 32 +/- 6 particles, each followed by an electrophoretic separation at -300 V cm(-1) with data acquired at 100 Hz, showed 28 +/- 5 peaks corresponding to 31.9 particles as predicted by the statistical overlap theory. In contrast, a similar series of hydrodynamic injections followed by data acquisition at 4 Hz revealed only 8 +/- 3 peaks suggesting that the modified system is needed for individual particle analysis. Comparison of electropherograms obtained at both data acquisition rates also indicate: (i) similar migration time ranges; (ii) lower variation in the fluorescence intensity of individual peaks for 100 Hz; and (iii) a better signal-to-noise ratio for 4 Hz raw data. S/N improved for 100 Hz when data were smoothed with a binomial filter but did not reach the S/N values previously reported for post-column LIF detection. The proof-of-principle of automated analysis of individual particles using a commercially available CE system described here opens exciting possibilities for those interested in the study and analyses of organelles, liposomes, and nanoparticles.     

Interlaboratory method validation of capillary electrophoresis sodium dodecyl sulfate (CE-SDS) methodology for analysis of mAbs

Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) is an analytical method to assess the purity of proteins, commonly applied to monoclonal antibodies (mAbs) in the biopharmaceutical industry. To address the need to standardize the CE-SDS method in the pharmaceutical industry and to enhance the confidence in method transfer between laboratories operating different commercial capillary electrophoresis (CE) instrument platforms, an interlaboratory CE-SDS method validation was organized involving 13 laboratories in 13 companies on four different types of commercial capillary electrophoresis instruments. In the validation, a commercial mAb therapeutic was used as the sample. The validation process followed the analytical guidelines set by the ICH guidelines (International Conference for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use). The method's precision, accuracy, linearity and range, and limit of quantitation (LOQ) were validated in the study. Variations of all the parameters validated in the study passed the pre-set criteria defined at the beginning of the study. The definition was based on previously published works and the intended application purpose of the CE-SDS method for mAbs. The study proved that the CE-SDS method fits its intended application purpose as a size impurity assay and size heterogeneity characterization assay for mAb therapeutic products. This study is the first time a CE-SDS method is validated by multiple laboratories using different commercial CE instrument platforms and on a commercial mAb therapeutic. Its results will enhance the confidence of the biopharmaceutical industry to develop CE-SDS methods and transfer CE-SDS methods between different laboratories.     

Headspace‐single drop microextraction with a commercial capillary electrophoresis instrument

Automated coupling of headspace‐single drop microextraction (HS‐SDME) and CE has been demonstrated using a commercial CE instrument. When a drop hanging at the inlet tip of a capillary for CE is used as the acceptor phase, HS‐SDME becomes a simple but powerful sample pretreatment technique for CE before injection to facilitate sample cleanup and enrichment. By combining HS‐SDME with an on‐line sample preconcentration technique, large volume sample stacking using an electroosmotic flow pump, the sensitivity can be improved further. The overall enrichment factors for phenolic compounds were from 1900 to 3400. HS‐SDME large volume sample stacking using an electroosmotic flow pump was successfully applied to a red wine sample to obtain an LOD of 4 nM (0.8 ppb) for 2,4,6‐trichlorophenol which is a precursor for 2,4,6‐trichloroanisole causing the foul odor in wine called cork taint.     

Unmanned platform for long-range remote analysis of volatile compounds in air samples

This paper describes a long-range remotely controlled CE system built on an all-terrain vehicle. A four-stroke engine and a set of 12-V batteries were used to provide power to a series of subsystems that include drivers, communication, computers, and a capillary electrophoresis module. This dedicated instrument allows air sampling using a polypropylene porous tube, coupled to a flow system that transports the sample to the inlet of a fused-silica capillary. A hybrid approach was used for the construction of the analytical subsystem combining a conventional fused-silica capillary (used for separation) and a laser machined microfluidic block, made of PMMA. A solid-state cooling approach was also integrated in the CE module to enable controlling the temperature and therefore increasing the useful range of the robot. Although ultimately intended for detection of chemical warfare agents, the proposed system was used to analyze a series of volatile organic acids. As such, the system allowed the separation and detection of formic, acetic, and propionic acids with signal-to-noise ratios of 414, 150, and 115, respectively, after sampling by only 30 s and performing an electrokinetic injection during 2.0 s at 1.0 kV.