Chemometrical examination of active parameters and interactions in flow injection-capillary electrophoresis

The first detailed examination of flow injection-capillary electrophoresis (FI-CE) active parameters and their interactions via response surface methodology (RSM) is presented. Specifically, RSM in the form of a Box-Behnken design was implemented to effectively predict the significance of capillary length, voltage and injection volume on the optimization of an in-house built FI-CE analyzer. Initial studies were performed assessing peak height and peak shape of the model compound N,N-dimethylformamide. Optimum model conditions were then derived and used in the model separation of two small molecules, nicotinamide adenine dinucleotide, reduced form (NADH) and benzenesulfonamide. By implementing the RSM approach, detailed examination of active FI-CE parameters was possible, including the ability to reveal a significant interactive effect. This work is not only highly significant for advancing FI-CE developments, but instructive for investigators actively exploring other coupled analytical techniques and associated experimental parameters.     

A flow injection-capillary electrophoresis system with high-voltage contactless conductivity detection for automated dual opposite end injection

The system comprises two flow injection-capillary electrophoresis interfaces into which the opposite ends of the separation capillary are inserted. The electrolyte solution flows through both interfaces by use of hydrostatic pressure. The injection of the samples into the electrolyte flow is accomplished by a rotary-type chromatographic valve at the grounded side and by a pinch-valve injector at the high-voltage side that provides sufficient isolation from the high electric field. The system allows a fully automated dual-injection sequence of samples from both capillary ends and simultaneous electrophoretic separation of anions and cations in the samples. The analytes are detected by a high-voltage contactless conductometric detector positioned approximately in the middle of the separation capillary. The parameters of the system were evaluated. The repeatability of the flow injection-capillary electrophoresis system for the simultaneous determination of anions and cations was evaluated for ten consecutive injections and relative standard deviation (RSD) values for peak areas were better than 1.0%. The sample throughput for total ionic analysis was estimated to be 25 samples per hour. The system was used for automated simultaneous analysis of anions and cations in various real samples. Using a short separation capillary, rapid total ionic analysis in less then 1 min is demonstrated.     

Separation and determination of honokiol and magnolol in herbal medicines by flow injection-capillary electrophoresis

A simple, rapid, and accurate method for the separation and determination of honokiol and magnolol in Magnolia officinalis and related herbal medicines was developed by combination of flow injection (FI) and capillary zone electrophoresis (CZE). The analysis was carried out using an unmodified fused-silica capillary (50-μm I.D.; total length 7.5 cm; effective length 4.5 cm). A series of optimization steps afforded the following conditions: the sample solvent consisted of 150 mM NaOH and a running buffer composed of 10 mM sodium tetraborate/10 mM sodium dihydrogenphosphate (NaH₂PO₄) at pH 12 was applied for the separation of the analytes. The separation could be achieved within 5 min with a sample throughput rate of up to 28 h⁻¹. The repeatability (defined as the relative standard deviation, RSD) for honokiol and magnolol was 2.0% and 1.6% with peak area evaluation, 3.6% and 2.0% with peak height evaluation, and 2.0% and 1.4% with migration time evaluation, respectively. Regression equations revealed linear relationships (r = 0.9991–0.9998) between the peak area of each analyte and the concentration.     

Determination of sulfa antibiotic residues in river and particulate matter by field-amplified sample injection-capillary zone electrophoresis

In the present research, field-amplified sample injection–CZE (FASI–CZE) coupled with a diode array detector was established to determine trace level sulfa antibiotic. Sulfathiazole, sulfadiazine, sulfamethazine, sulfadimethoxine, sulfamethoxazole, and sulfisoxazole were selected as analytes for the experiments. The background electrolyte solution consisted of 70.0 mmol/L borax and 60.0 mmol/L boric acid (including 10% methanol, pH 9.1). The plug was 2.5 mmol/L borax, which was injected into the capillary at a pressure of 0.5 psi for 5 s. Then the sample was injected into the capillary at an injection voltage of –10 kV for 20 s. The electrophoretic separation was carried out under a voltage of +19 kV. The capillary temperature was maintained at 20˚C throughout the analysis, and six sulfonamides were completely separated within 35 min. Compared with pressure injection-CZE, the sensitivity of FASI-CZE was increased by 6.25–10.0 times, and the LODs were reduced from 0.2–0.5 to 0.02–0.05 μg/mL. The method was applied to the determination of sulfonamides in river water and particulate matter samples. The recoveries were 78.59–106.59%. The intraday and interday precisions were 2.89–7.35% and 2.77–7.09%, respectively. This provides a simpler and faster method for the analysis of sulfa antibiotic residues in environmental samples.     

Separation and determination of four active anthraquinones in Chinese herbal preparations by flow injection-capillary electrophoresis

A simple, rapid, and accurate method for the separation and determination of physcion, chrysophanol, aloe-emodin, and emodin in Rhubarb, Juemingzi, and Chinese herbal preparations was developed by combination of flow injection-capillary zone electrophoresis for the first time. The analysis was carried out using an unmodified fused-silica capillary (75 mm x 50 microm ID x 375 microm OD, effective separation length of 48 mm) and direct ultraviolet detection at 254 nm. By a series of optimization, the sample solvent consisted of NaOH (100 mmol/L) and ACN (1:1 v/v), and a running buffer composed of 15 mmol/L sodium borate - 12.5 mmol/L sodium dihydrogen phosphate - 42% v/v ACN (pH 10.1) was applied for the separation of the four anthraquinones. The separation was rapid and highly reproducible, with complete resolution of all four compounds within 6 min. The sample throughput rate could reach up to 12 per h. The repeatability (defined as relative standard deviation) was 4.45, 4.44, 4.34, 0.61% with peak height evaluation and 1.62, 0.89, 2.49, 2.19% with peak area evaluation for physcion, chrysophanol, aloe-emodin, and emodin, respectively.     

A capillary electrophoresis chip with hydrodynamic sample injection for measurements from a continuous sample flow

A microchip-based capillary electrophoresis device supported by a microfluidic network made of poly(dimethylsiloxane), used for measuring target analytes from a continuous sample flow, is presented. The microsystem was fabricated by means of replica molding in combination with standard microfabrication technologies, resulting in microfluidic components and an electrochemical detector. A new hydrodynamic sample injection procedure is introduced, and the maximum number of consecutive measurements that can be made with a poly(dimethylsiloxane) capillary electrophoresis chip with amperometric detection is investigated with respect to reproducibility. The device features a high degree of functional integration, so the benefits associated with miniaturized analysis systems apply to it.     

On-line simultaneous and rapid separation of anions and cations from a single sample using dual-capillary sequential injection-capillary electrophoresis

A novel capillary electrophoresis (CE) approach has been developed for the simultaneous rapid separation and identification of common environmental inorganic anions and cations from a single sample injection. The method utilised a sequential injection-capillary electrophoresis instrument (SI-CE) with capacitively-coupled contactless conductivity detection (C⁴D) constructed in-house from commercial-off-the-shelf components. Oppositely charged analytes from a single sample plug were simultaneously injected electrokinetically onto two separate capillaries for independent separation and detection. Injection was automated and may occur from a syringe or be directly coupled to an external source in a continuous manner. Software control enabled high sample throughput (17 runs per hour for the target analyte set) and the inclusion of an isolation valve allowed the separation capillaries to be flushed, increasing throughput by removing slow migrating species as well as improving repeatability. Various environmental and industrial samples (subjected only to filtering) were analysed in the laboratory with a 3 min analysis time which allowed the separation of 23 inorganic and small organic anions and cations. Finally, the system was applied to an extended automated analysis of Hobart Southern Water tap water for a period of 48 h. The overall repeatability of the migration times of a 14 analyte standard sample was less than 0.74% under laboratory conditions. LODs ranged from 5 to 61 μg L⁻¹. The combination of automation, high confidence of peak identification, and low limits of detection make this a useful system for the simultaneous identification of a range of common inorganic anions and cations for discrete or continuous monitoring applications.     

A microchip device to enhance free flow electrophoresis using controllable pinched sample injections

Micro free flow electrophoresis (µFFE) is a valuable technique capable of high throughput rapid microscale electrophoretic separation along with mild operating conditions. However, the stream flow separation nature of free flow electrophoresis affects its separation performance with additional stream broadening due to sample stream deflection. To reduce stream broadening and enhance separation performance of µFFE, we presented a simple microfluidic device that enables injection bandwidth control. A pinched injection was formed in the reported µFFE system using operating buffer at sample flow rate ratio (r) setting. Initial bandwidth at the entrance of separation chamber can be shrunk from 800 to 30 µm when r increased from 1 to 256. Stream broadening at the exit of separation chamber can be reduced by about 96% when r increased from 4 to 128, according to both theoretical and experimental results. Moreover, the separation resolution for a dye mixture was enhanced by a factor of 4 when r increased from 16 to 128, which corresponded to an 80% reduction in sample initial bandwidth. Furthermore, a similar enhancement on amino acids separation was obtained by using injection control in the reported µFFE device and readily integrated into online/offline sample preparation and/or downstream analysis procedures.     

Study of interaction between drug enantiomers and human serum albumin by flow injection-capillary electrophoresis frontal analysis

Flow injection (FI)-CE coupled with frontal analysis (FA) was applied to the study of stereoselectivity binding of amlodipine (AL) to HSA. Under protein-drug binding equilibrium, the unbound concentrations of drug enantiomers were measured by plateau height. The stereoselectivity of AL binding to HSA was proved by the different free fractions of two enantiomers. In physiological phosphate solution (pH 7.4, ionic strength 0.17) when 200 microM (+/-)AL was equilibrated with 300 microM HSA, the concentration of unbound R-AL was about 1.5 times higher than that of its antipode. The binding constants of two enantiomers, KR-AL and KS-AL, were 9910-11200 and 90200-104000 M(-1), respectively. The results obtained by the method were compared with those determined by conventional equilibrium dialysis (ED)-CE and fluorescence spectra. Hydroxypropyl-beta-CD (HP-beta-CD) (10 mM) was used as a chiral selector in pH 3.7 phosphate buffer. L-tryptophan (L-try) and ketoprofen (Ket) were used as displacement reagents to investigate the binding sites of AL to HSA. A binding synergism effect between hydrochlorothiazide (QL) and AL was observed and the results suggested that QL can destroy binding equilibrium of R-AL and S-AL toward HSA and they can occupy the same binding site of HSA (site I). The reproducibility was confirmed by RSD (RSD<1.5%) of the plateau height determined by FI-CE frontal analysis (FI-CE-FA). The FI-CE-FA was a good method to study protein-drug interaction.     

Efforts to improve detection sensitivity for capillary electrophoresis coupled to atmospheric pressure photoionization mass spectrometry

Electrospray ionization performs best with volatile buffers. However, generally the best separation performance for capillary electrophoresis (CE) is achieved with non‐volatile buffers. Hyphenation of CE with mass spectrometry (MS) utilizing atmospheric pressure photoionization (APPI) enables use of a wider range of separation buffers without compromising detection sensitivity. As APPI is considered to be mass flow sensitive, the use of a larger inner diameter separation capillary (75 µm) allows larger volumes to be injected, without decreased separation performance, thus providing improved sensitivity (approx. a factor of 10), compared to the use of a 25 µm capillary. However, nebulizing gas flow and position of capillary tip in the sprayer have to be carefully optimized to prevent excessive band broadening. Further improvement in sensitivity (approx. a factor of 2) was obtained by decreasing the distance between the sprayer and ionization region, indicating that a specially designed CE/APPI‐MS interface for low flow rates will be favourable. Copyright © 2010 John Wiley & Sons, Ltd.     

A novel approach to improve operation and performance in flow field-flow fractionation

A new system design and setup are proposed for the combined use of asymmetrical flow field-flow fractionation (AF4) and hollow-fiber flow field-flow fractionation (HF5) within the same instrumentation. To this purpose, three innovations are presented: (a) a new flow control scheme where focusing flow rates are measured in real time allowing to adjust the flow rate ratio as desired; (b) a new HF5 channel design consisting of two sets of ferrule, gasket and cap nut used to mount the fiber inside a tube. This design provides a mechanism for effective and straightforward sealing of the fiber; (c) a new AF4 channel design with only two fluid connections on the upper plate. Only one pump is needed to deliver the necessary flow rates. In the focusing/relaxation step the two parts of the focusing flow and a bypass flow flushing the detectors are created with two splits of the flow from the pump. In the elution mode the cross-flow is measured and controlled with a flow controller device. This leads to reduced pressure pulsations in the channel and improves signal to noise ratio in the detectors. Experimental results of the separation of bovine serum albumin (BSA) and of a mix of four proteins demonstrate a significant improvement in the HF5 separation performance, in terms of efficiency, resolution, and run-to-run reproducibility compared to what has been reported in the literature. Separation performance in HF5 mode is shown to be comparable to the performance in AF4 mode using a channel with two connections in the upper plate.     

On-line flow sample stacking in a flow injection analysis-capillary electrophoresis system: 2000-fold enhancement of detection sensitivity for priority phenol pollutants

A flow injection analysis-capillary electrophoresis system has been used for on-line flow stacking of 11 US Environmental Protection Agency priority phenol pollutants. Samples containing low concentrations of phenols dissolved in deionised water are continuously delivered to the capillary opening by means of a peristaltic pump. The sample components stack at the boundary between the highly conductive separation electrolyte and the introduced sample. By selecting an appropriate electrolyte and stacking conditions the movement of the electrolyte solution inside the capillary can be reduced, thereby improving the stacking efficiency. The electrolyte used here contained 20 mM phosphate, 8% 2-butanol, and 0.001% hexamethonium bromide at pH 11.95, and the stacking was carried out at 2 kV for 240 s. These conditions allowed up to 2000-fold preconcentration of the selected phenols. No matrix removal was necessary.     

A sheath flow gating interface for the on-line coupling of solid-phase extraction with capillary electrophoresis

A sheath flow gating interface (SFGI) is presented for the on-line coupling of solid-phase extraction (SPE) with capillary electrophoresis (CE). The design, construction and operation of the SFGI are described in detail. After operating conditions were investigated and selected, the SFGI was evaluated on a SPE–CE–UV setup using hydroxylated poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith as the absorbent and using three phenols as the test analytes. The preconcentration factors obtained with the SPE–CE–UV system and the SPE–UV part are 530 and 550, respectively. The plate numbers obtained using the SPE–CE–UV system are slightly better than or comparable to those with the CE–UV part. The precisions (RSDs) of 100 consecutive injections are 2.43%, 3.86%, and 4.25% for peak height, peak area and migration time, respectively. The measured recoveries for the river water samples spiked at three different levels are in the range of 93.6–102.8% with the interday RSD values ranging from 2.0 to 4.5% (n = 3). These data collectively demonstrate that the SFGI has the ability to exactly and reproducibly transfer nanoliters of fractions from SPE onto CE with no degradation of the efficiencies of SPE and CE, suggesting a great potential to be routinely used for the coupling of SPE, microcolumn LC or FIA with CE.     

Strategies to improve the sensitivity in capillary electrophoresis for the analysis of drugs in biological fluids

Capillary electrophoresis (CE) is a useful method to quantify drugs in biological fluids. However, especially for blood or plasma samples, the sensitivity is not sufficient to quantify drugs and their metabolites as they often need to be quantified in the lower microg/L range. To overcome this limitation and to increase the sensitivity, two strategies are applied: first, to increase the amount of analyte added to the capillary and, second, to increase the sensitivity on the detector site. To improve the sensitivity on the detector site, alternative detection techniques to UV detection, e.g., laser-induced fluorescence detection (LIF) or mass spectroscopy (MS), can be applied. However, LIF detection can only be used for fluorescent analytes and the current equipment for CE-MS coupling provides only small improvements in sensitivity compared to UV detection. The detection window for UV detection can be enhanced using capillaries with an extended light path (bubble cell) or Z-shaped capillaries. Sensitivity improvements up to a factor of 10 have been reported. Increasing the amount of analyte in the capillary can be done either by chromatographic or by electrokinetic methods. Chromatographic methods such as on-capillary membrane preconcentration have been used for several analytes. However, no validated application has been reported to date. In contrast, several validated examples can be found in which electrokinetic techniques like sample stacking have been applied to achieve limits of quantification in the lower microg/L range. In conclusion, to date, electrokinetic techniques such as field-amplified sample injection offer the most promising results in achieving a sufficient sensitivity to quantify drugs in biological fluids.     

Use of large-volume sample stacking in on-line solid-phase extraction-capillary electrophoresis for improved sensitivity

We present a new system for the sensitive analysis of cephalosporins by CE using both on-line SPE and large-volume sample stacking (LVSS). Sample volumes of 250 muL were loaded onto the SPE microcolumn which was then desorbed with 426 nL of ACN. The SPE elution plug was injected into the CE system via an in-line valve interface filling approximately 60% of the volume of the separation capillary. Subsequently, LVSS was performed by applying a voltage of -5 kV, which resulted in the simultaneous removal of the elution solvent and the preconcentration of the analytes in a narrow zone. This way the amount of analyte loaded into the capillary could be considerably increased without serious loss of CE separation efficiency. LODs for cefoperazone and ceftiofur were in the ng/L range which represents an improvement of a factor of 8450 and 11 450 when compared with direct CE injection. The cephalosporin test compounds presented a good linear response (corrected peak area) between 0.5 and 10 mug/L with correlation coefficients higher than 0.995. The final method is compared with previously reported LVSS-CE and SPE-CE systems for the analysis of cephalosporins.     

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.     

Sensitivity enhancing injection from a sample reservoir and channel interface in microchip electrophoresis

The stacking of a cationic analyte (i.e., rhodamine B) at the interface between a sample reservoir and channel in a microchip electrophoresis device is described for the first time. Stacking at negative polarity was by micelle to solvent stacking where the dye was prepared in a micellar solution (5 mM sodium dodecyl sulfate in 25 mM phosphoric acid, pH 2.5) and the channel was filled with high methanol content background solution (70% methanol in 50 mM phosphoric acid, pH 2.5). The injection of the stacked dye into the channel was by simple reversal of the voltage polarity with the sample solution and background solution at the anodic and cathodic reservoirs of the straight channel, respectively. The enrichment of rhodamine B at the interface and injection of the stacked dye into the channel was clearly visualized using an inverted fluorescence microscope. A notable sensitivity enhancement factor of up to 150 was achieved after 2 min at 1 kV of micelle to solvent stacking. The proposed technique will be useful as a concentration step for analyte mixtures in simple and classical cross‐channel microchip electrophoresis devices or for the controlled delivery of enriched reagents or analytes as narrow plugs in advanced microchip electrophoresis devices.     

Automated sample preparation and analysis using a sequential-injection-capillary electrophoresis (SI-CE) interface

A fully automated sequential-injection-capillary electrophoresis (SI-CE) system was developed using commercially available components as the syringe pump, the selection and injection valves and the high voltage power supply. The interface connecting the SI with the CE unit consisted of two T-pieces, where the capillary was inserted in one T-piece and a Pt electrode in the other (grounded) T-piece. By pressurising the whole system using a syringe pump, hydrodynamic injection was feasible. For characterisation, the system was applied to a mixture of adenosine and adenosine monophosphate at different concentrations. The calibration curve obtained gave a detection limit of 0.5 microg g(-1) (correlation coefficient of 0.997). The reproducibility of the injection was also assessed, resulting in a RSD value (5 injections) of 5.4%. The total time of analysis, from injection, conditioning and separation to cleaning the capillary again was 15 minutes. In another application, employing the full power of the automated SIA-CE system, myoglobin was mixed directly using the flow system with different concentrations of sodium dodecyl sulfate (SDS), a known denaturing agent. The different conformations obtained in this way were analysed with the CE system and a distinct shift in migration time and decreasing of the native peak of myoglobin (Mb) could be observed. The protein samples prepared were also analysed with off-line infrared spectroscopy (IR), confirming these results.     

A novel sheathless interface for capillary electrophoresis/electrospray ionization mass spectrometry using an in-capillary electrode

A simple and rugged sheathless interface for capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) was designed using common laboratory tools and chemicals. The interface uses a small platinum (Pt) wire that is inserted into the CE capillary through a small hole near the terminus. The position of the wire inside the CE capillary and within the buffer solution is analogous to standard CE separation operations where the terminus of the CE capillary is placed inside a buffer reservoir along with a grounded platinum electrode. By combining the use of the in-capillary electrode interface with sharpening of the fused silica tip of the CE capillary outlet, a stable electrospray current was maintained for an extended period of time. The design was successfully applied to CE/ESI-MS separations and analysis of mixtures of peptides and proteins. A detection limit of approximately 4 femtomole (S/N = 3) was achieved for detection of myoglobin utilizing a 75-µm-i.d. aminopropylsilane treated CE column and using a wide scan range of 550–1300 Da. The advantages of this new design include (1) a stable CE and ESI current, (2) durability, (3) a reduced risk of sparking between the capillary tip and the inlet of the mass spectrometer, (4) lack of any dead volume, and (5) facile fabrication with common tools and chemicals.     

Flow injection-capillary electrophoresis system with contactless conductivity detection and hydrostatic pressure generated flow. Application to the quantitative analysis of inorganic anions in water samples

A simple and inexpensive flow injection-capillary electrophoresis (FI-CE) system with contactless conductivity detection (CCD) for automated quantitative analysis of chloride, nitrate, and sulfate in various water samples is demonstrated. A glass bottle containing the background electrolyte that is raised above the FI-CE interface generates a pulse-free, highly reproducible flow of the electrolyte through the FI-CE interface. The system operates at a flow rate of 300 microLmin(-1) with an injection volume of only 4 microL. The repeatability of peak areas (n = 18) was better than 0.81% RSD and the sample throughput was 90 samples per hour using the background electrolyte containing 12 mM L-histidine adjusted to pH 4.00 with acetic acid. The limits of detection were better than 125 microgL(-1) and were comparable to those obtained by conventional CE systems with CCD. Various calibration methods for FI-CE system with electrokinetic injection were tested and their suitability for the analysis of anions in real samples was evaluated.