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.     

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 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.     

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.     

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.     

Improving sensitivity by large-volume sample stacking using the electroosmotic flow pump to analyze some nonsteroidal anti-inflammatory drugs by capillary electrophoresis in water samples

Large-volume sample stacking using the electroosmotic flow (EOF) pump (LVSEP) has been used to analyze some nonsteroidal anti-inflammatory drugs (NSAIDs) in water samples. With methanol as the run buffer solvent to suppress the EOF, sensitivity was enhanced by 80-100-fold. The sample for the analysis of real water sample was pretreated by solid-phase extraction (SPE). When the method was based on off-line SPE-LVSEP-CE, sensitivity improved by as much as 1000 times.     

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.     

Application of experimental design in optimization of the separation condition for determination of four active components in cold medicines by flow injection-capillary electrophoresis

Orthogonal design (OD) was employed to optimize the separation condition of flow injection-capillary electrophoresis (FI-CE). In order to compare the optimum condition, uniform design and univariate approach were also adopted. The influences of variables such as buffer pH, buffer concentration, acetonitrile (ACN) percentage, and separation voltage were discussed. The optimum separation condition was established. The limits of detection were 1.94 × 10(-2), 6.40 × 10(-3), 1.16 × 10(-2) and 1.94 × 10(-2) μg/mL for dextromethorphan hydrobromide (Dex), chlorphenamine hydrogen maleate (Chl), pseudoephedrine hydrochloride (Pse), and paracetamol (Par), respectively. The RSDs of peaks areas were less than 2.0%. The results showed the OD was an effective method among experimental designs for optimizing the separation conditions of CE. The optimum condition was used for separation and determination of Dex, Chl, Pse, and Par in cold medicines. The average recovery was between 96.68-101.25%.     

A novel interface for variable flow nanoscale LC/MS/MS for improved proteome coverage

A variable flow "peak trapping" liquid chromatography (LC) interface has been developed for the coupling of nanoscale LC to electrospray ionization mass spectrometry (ESI-MS). The presented peak trapping LC interface allows for the extended analysis time of co-eluting compounds and has been employed for the identification of proteins via tandem mass spectrometry (MS/MS). The variable flow process can be controlled either manually or in a completely automated manner where the mass spectrometer status determines the status of the variable flow interface. When the mass spectrometer operates in MS survey mode, the interface is operated in a so-called "high-flow" mode. Alternatively, the interface is operated in a "low-flow" mode during MS/MS analysis. In the "high-flow" mode of the variable flow process the column flow rate is typically around 200 nL/min, whereas in the "low-flow" mode the column effluent is introduced into the source of the mass spectrometer at 25 nL/min. In addition to the flow reduction during MS/MS analysis, the gradient is paused to preserve the peptide separation on the analytical nanoscale LC column. The performance of the variable flow nanoscale LC/MS/MS interface is demonstrated by the automated analysis of standard peptide mixtures and protein digests utilizing variable flow, data-dependent scanning MS/MS techniques, and automated database searching.     

A sheath-flow nanospray interface for capillary electrophoresis/mass spectrometry

We have developed a novel sheath-flow interface for low-flow electrospray ionization mass spectrometry (ESI-MS) and capillary electrophoresis/electrospray mass spectrometry (CE/ESI-MS). The interface is composed of two capillaries. One is a tapered fused-silica ESI emitter suitable for microliter and nanoliter flow rate electrospray and the other is a tail-end gold-coated CE separation column that is inserted into the emitter. A sheath liquid is supplied between the column and the emitter capillaries. The gold coating and the sheath liquid are used as the conducting media for ESI and the CE circuit. This novel design was initially evaluated by an infusion ESI-MS analysis of the most common antiretroviral dideoxynucleosides, followed by CE/MS coupling analysis of several antidepressant drugs. With infusion studies, the effects of the sheath liquid and the sample flow rates on detection sensitivity and signal stability were investigated. For an emitter with an internal diameter of 30 microm, the optimum flow rates for the sheath and the sample were 200 and 300 nL/min, respectively. The main improvement of this approach in comparison with conventional sheath liquid approaches using an ionspray interface is the gain in sensitivity. Sensitivities were three times better for dideoxynucleosides analyzed by infusion and 12 times higher for antidepressant drugs analyzed by CE/MS with this interface compared with ionspray. The emitter is durable, disposable, and simple to fabricate.     

A new sheathless electrospray interface for coupling of capillary electrophoresis to ion-trap mass spectrometry

A simple laboratory-made sheathless electrospray interface for coupling of capillary electrophoresis to ion-trap mass spectrometry (CE/MS) was developed. The interface was machined in-house and it was designed to be freely interchangeable with the commercially available ionization sources for the mass spectrometer. Sharpened fused-silica capillaries were coated with nickel by a simple electrodeless plating procedure and were used as all-in-one columns/emitters. The electrodeless plating produced a 2-5- micro m thick smooth nickel layer that lasted for more than 8 h of continuous electrospraying. The performance of the CE/MS interface was examined by using four cationic imipramine derivatives as test substances. Relative detection limits were calculated on the basis of the extracted ion electrophorograms and were in the range 6-130 nmol/L, corresponding to absolute detection limits in the range of 20-400 amol. The system was applied for analysis of impurities in an impure imipramine N-oxide preparation, and two of the impurities could be identified on the basis of online-MS(MS) spectra recorded in scan-dependent mode.