Application of capillary electrophoresis with capacitively coupled contactless conductivity detection (CE‐C4D): An update

Capacitively coupled contactless conductivity detection (C⁴D) has appeared as a powerful technique for the detection of compounds lacking chromogenic or fluorogenic group. Since our last review (Biomedical Chromatography 2014; 28 : 1502–1506) several new capillary electrophoresis (CE)‐C⁴D methods have been reported. This review provides an update of the most recent utilization of CE‐C⁴D in the field of pharmaceutical, biomedical and food analysis covering the period from February 2014 to October 2016. The use of CE with C⁴D in the pharmaceutical field has been shown in many papers. Examples illustrate the applicability of CE‐C⁴D in the fields of pharmaceutical, biomedical and food analysis. Finally, general conclusions and perspectives are provided.     

Advancements in capacitance-to-digital converter-based C4D technology for detection in capillary electrophoresis using amplified excitation voltages and comparison to classical and open-source C4Ds

This work introduces new hardware configurations for a capacitively coupled contactless conductivity detector (C⁴D) based on capacitance-to-digital conversion (CDC) technology for CE. The aim was to improve sensitivity, handling, price, and portability of CDC-based C⁴D detectors (CDCD) to reach LODs similar to classic C⁴Ds with more sophisticated electric circuits. To achieve this, a systematic study on the CDCDs was carried out including a direct comparison to already established C⁴D setups. Instrumental setups differing in electrode lengths, measurement modes, and amplification of excitation voltages were investigated to achieve LODs for alkali metal ions of 4 to 12 μM, similar to LODs obtained by classic C⁴D setups. Lowest LODs were achieved for a setup with two 10 mm electrodes at a distance of 0.2 mm and an excitation voltage of 24 V. The detection head was exceptionally lightweight with only 2.6 g and covered only 20 mm of the capillary on total. This allowed the use of multiple detectors along the separation path to enable spatial tracking of analytes during separation. The entirely battery-powered detector assembly weighs less than 200 g, and the data are transmitted wirelessly for possible portable applications. The freely accessible hardware and software were optimized for fully automated measurements with real time data plotting and allowed handling multidetector setups. The new developments were applied to quantify the potassium salt of glyphosate in its herbicide formulation.     

A capillary electrophoresis system with dual capacitively coupled contactless conductivity detection and electrospray ionization tandem mass spectrometry

A commercial system that is comprised of a CE coupled to an ESI triple quadrupole mass spectrometer was equipped with two capacitively coupled contactless conductivity detectors (C⁴Ds). The first C⁴D was positioned inside the original cartridge, and the second C⁴D was positioned as close as possible to the ESI probe entrance by using a 3D‐printed support. The C⁴Ds electropherograms were matched to the ESI‐MS electropherogram by correcting their timescales by the factor L_T/L_D, where L_T and L_D are the total capillary length and the length until the C⁴D, respectively. A general approach for method development supporting the simultaneous conductivity and MS detection is discussed, while application examples are introduced. These examples include the use of C⁴D as a simple device that dismiss the use of an EOF marker, a low‐selectivity detector that continuously provide information about unexpected features of the sample, and even a detector that can be more sensitive than ESI‐MS. The C⁴D used in this setup proved to have a smaller contribution to the peak broadening than ESI‐MS, which allowed that a C⁴D, positioned at 12 cm from the inlet of an 80‐cm‐long capillary, could be used to foresee position and shape of the peaks being formed 6.8 times slower at the ESI‐MS electropherogram.     

A review of the recent achievements in capacitively coupled contactless conductivity detection

Capacitively coupled contactless conductivity detection (C⁴D) in the axial electrode configuration was introduced in 1998 as a quantification method for capillary electrophoresis. Its universality allows the detection of small inorganic ions as well as organic and biochemical species. Due to its robustness, minimal maintenance demands and low cost the popularity of this detector has been steadily growing. Applications have recently also been extended to other analytical methods such as ion chromatography, high-performance liquid chromatography and flow-injection analysis. C⁴D has also found use for detection on electrophoresis based lab-on-chip devices. Theoretical aspects of C⁴D in both the capillary and microchip electrophoresis format have been comprehensively investigated. Commercial devices are now available and the method can be considered a mature detection technique. In this article, the achievements in C⁴D for the time period between September 2004 and August 2007 are reviewed.     

Application of a low impedance contactless conductometric detector for the determination of inorganic cations in capillary monolithic column chromatography

Poly(glycidyl methacrylate) cation exchange monolithic column was prepared in fused-silica capillaries of 320 μm i.d. by thermally initiated radical polymerization and utilized in capillary ion chromatography. With 15 mM methanesulfonic acid as the mobile phase, the separations of a mixture of inorganic cations (Li⁺, Na⁺, NH₄⁺, K⁺) was tested by using a capacitively coupled contactless conductivity detector (C⁴D) and a low impedance C⁴D (LIC⁴D). The LIC⁴D is the series combination of a C⁴D and a quartz crystal resonator. At the resonant frequency of the series combination, the capacitor impedance from capillary wall was offset by the inductance impedance from the quartz crystal resonator. A minimum impedance was obtained in the impedance-frequency curve of the combination. The responses of the C⁴D and LIC⁴D were analyzed based on an equivalent circuit model. It was shown that the sensitivity of the C⁴D to the change in analyte concentration is rather poor due to the high ratio of the impedance from the capillary wall capacitor to the solution impedance. The LIC⁴D has the similar sensitivity as a contact conductivity detector but a much smaller cell volume. The on-column detection model was realized by LiC⁴D without preparation of optical detection window in monolithic column.     

A Comparison of the Properties of Contactless Conductivity and Diode‐Array Photometric Detectors in Analyses of Low‐Molecular,Biologically Active Substances by Capillary Electrophoresis in Acetic Acid Solutions

The performance of the contactless conductivity (C⁴D) and diode array photometric (DAD) detectors has been compared for CE separations of creatinine, arginine and 3‐methylhistidine in acetic acid background electrolytes. The contactless conductivity detector response has also been modeled. It has been found that the two detectors provide similar responses and can readily be used for dual CE detection. Changes in the acetic acid concentration affect the C⁴D noise less than the DAD noise, but their effect on the C⁴D response to the analytes is greater than with DAD. In general, C⁴D provides better detection results at higher acetic acid concentrations, while DAD is more sensitive and reliable at very low ones. Capillaries with greater internal diameters are preferable for both detectors, provided that the separation efficiency is not adversely affected. Acetic acid is a suitable background electrolyte for CE separations of small, basic organic molecules.     

Simultaneous determination of atenolol and amiloride in pharmaceutical preparations by capillary zone electrophoresis with capacitively coupled contactless conductivity detection

Capillary zone electrophoresis coupled with a capacitively coupled contactless conductivity detector (CE‐C⁴D) has been employed for the determination of atenolol and amiloride in pharmaceutical formulations. Acetic acid (150 mm ) was used as background electrolyte. The influence of several factors (detector excitation voltage and frequency, buffer concentration, applied voltage, capillary temperature and injection time) was studied. Non‐UV‐absorbing L‐valine was used as internal standard; the analytes were all separated in less than 7 min. The separation was carried out in normal polarity mode at 28°C, 25 kV and using hydrodynamic injection (25 s). The separation was effected in an uncoated fused‐silica capillary (75 μm, i.d. × 52 cm). The CE‐C⁴D method was validated with respect to linearity, limit of detection and quantification, accuracy, precision and selectivity. Calibration curves were linear over the range 5–250 μg/mL for the studied analytes. The relative standard deviations of intra‐ and inter‐day migration times and corrected peak areas were less than 6.0%. The method showed good precision and accuracy and was successfully applied to the simultaneous determination of atenolol and amiloride in different pharmaceutical tablet formulations. Copyright © 2010 John Wiley & Sons, Ltd.     

Capillary electrophoresis procedure for the simultaneous analysis and stoichiometry determination of a drug and its counter-ion by using dual-opposite end injection and contactless conductivity detection: Application to labetalol hydrochloride

In this work, a capillary electrophoresis (CE) procedure was developed for the simultaneous determination of a pharmaceutical drug and its counter-ion, namely labetalol hydrochloride. For this purpose, an uncoated fused-silica capillary, a low conductivity background electrolyte (BGE) and a capacitively coupled contactless conductivity detector (C⁴D) were employed. This detection system is highly sensitive and enables detection of inorganic as well as organic ions unlike with direct UV detection. Moreover, to be able to simultaneously analyze the cationic drug (labetalol⁺) and its anionic counter-ion (Cl⁻) in the same electrophoretic run without the need of a coated capillary, a dual-opposite end injection was performed. In this technique, the sample is hydrodynamically injected into both ends of the capillary. This method is simple and easy to perform since the different injection steps are automated by the CE software.This novel CE-C⁴D procedure with dual-opposite end injection has been successfully validated and applied for the analysis of chloride content in an adrenergic antagonist (labetalol hydrochloride). Thus, the hereby developed method has been shown to enable fast (analysis time < 10 min), precise (repeatability of migration times < 0.7% and of corrected-peak areas < 3.3%; n = 6) and rugged analyses for the simultaneous determination of a pharmaceutical drug and its counter-ion.     

Determination of γ‐hydroxybutyric acid in saliva by capillary electrophoresis coupled with contactless conductivity and indirect UV absorbance detectors

The aim of the current study was to optimise and validate the methodology for determination of γ‐hydroxybutyric acid (GHB) in saliva by CE combined with a contactless conductivity detector (C⁴D) and indirect UV absorbance detection (λ_ABS = 210 nm). The optimized BGE, consisting of 8.5 mM maleic acid, 17 mM arginine, 255 μM cetyltrimethylammonium bromide (CTAB), and 15% acetonitrile, was evaluated for the separation of GHB in saliva within 6 min. The performance characteristics of the CE‐C⁴D‐indirect UV methodology was validated. The instrument detection and quantification limits were 0.49 and 1.6 mg/L for C⁴D, and 5.1 mg/L and 17.0 mg/L for indirect UV, respectively. The linearity was obtained over the range from 2.5 to 400 mg/L for C⁴D and from 12.5 to 400 mg/L for indirect UV. The interday precisions were within 2.3–5.7% and intraday precisions were within 1.6–9.0% for C⁴D as well as 2.1–9.3%, 5.6–10.1% for indirect UV in spiked saliva, respectively. The recoveries were within 87.2–104.4%. The matrix effects were +53.2% for small concentrations up to 25 mg/L for C⁴D and +23.6% for concentrations up to 75 for mg/L for indirect UV detection. No matrix effects were observed for higher concentration levels. In conclusion, CE‐C4D‐indirect UV can offer a rapid, accurate, sensitive, and definitive method for the determination of GHB abuse in saliva samples as a forensic screening tool.     

A Convenient Approach to Simultaneous Analysis of a Pharmaceutical Drug and Its Counter-Ion by CE Using Dual-Opposite End Injection and Contactless Conductivity Detection

Capillary electrophoresis (CE) coupled to a capacitively coupled contactless conductivity detector (C⁴D) was used for the determination in a single analysis of a pharmaceutical drug and its counter-ion. Dual-opposite end injection (DOI) was used to introduce hydrodynamically the analytes at each end of the capillary. No modification of the commercial apparatus is required. After applying the voltage, the cations and anions migrate from each end of the capillary in opposite directions toward the detector placed near the cathode outlet. The electrophoretic conditions were initially developed with three test drugs (chlorpheniramine maleate, metoprolol tartrate, clomiphene citrate) and then applied to two Vinca alkaloids (catharanthine sulfate, vinorelbine ditartrate). The 10 mM histidine–50 mM acetic acid buffer (pH 4.1)–methanol 90:10 (v/v) electrolyte was suitable for the analysis of these high or medium mobile anions by CE–C⁴D due to its low conductivity background and high buffer capacity. Finally, the CE procedure developed was successfully validated for catharanthine sulfate. The method developed herein is fast (<10 min) and accurate (repeatability on migration time < 0.6% and peak areas < 1.3%, n = 6).

     

Fast Determination of Ciprofloxacin by Batch Injection Analysis with Amperometric Detection and Capillary Electrophoresis with Capacitively Coupled Contactless Conductivity Detection

We report fast, precise, selective, and sensitive electroanalytical methods for the determination of ciprofloxacin in milk and pharmaceutical samples by batch‐injection analysis with amperometric detection (BIA‐AMP) and by capillary electrophoresis with capacitively‐coupled contactless conductivity detection (CE‐C⁴D). Both methods required simple sample preparation protocols before analysis (milk samples were just diluted and tablets powdered and dissolved in electrolyte/water). The analytical features of BIA‐AMP and CE‐C⁴D methods include, respectively, low relative standard deviation values for repetitive measurements (2.8 % and 1.7 %, n=10), low detection limits (0.3 and 5.0 µmol L^?1), elevated analytical frequency (80 and 120 h^?1) and satisfactory accuracy (based on comparative determinations by HPLC and recovery values for spiked samples).     

Monitoring of circulating amino acids in patients with pancreatic cancer and cancer cachexia using capillary electrophoresis and contactless conductivity detection

Branched chain amino acids (BCAAs), alanine and glutamine are determined in human plasma by capillary electrophoresis with contactless conductivity detection (CE/C⁴D). The baseline separation of five amino acids from other plasma components is achieved on the short capillary effective length of 18 cm in 3.2 mol/L acetic acid with addition of 13% v/v methanol as background electrolyte. Migration times range from 2.01 min for valine to 2.84 min for glutamine, and LODs for untreated plasma are in the interval 0.7–0.9 μmol/L. Sample treatment is based on the addition of acetonitrile to only 15 μL of plasma and supernatant is directly subjected to CE/C⁴D. Circulating amino acids are measured in patients with pancreatic cancer and cancer cachexia during oral glucose tolerance test. It is shown that patients with pancreatic cancer and cancer cachexia syndrome exhibit low basal circulating BCAAs and glutamine levels and loss of their insulin-dependent suppression.     

Fully electrochemical hyphenated flow system for preconcentration,cleanup, stripping,capillary electrophoresis with stacking and contactless conductivity detection of trace heavy metals

Successful coupling of electrochemical preconcentration (EPC) to capillary electrophoresis (CE) with contactless conductivity detection (C⁴D) is reported for the first time. The EPC–CE interface comprises a dual glassy carbon electrode (GCE) block, a spacer and an upper block with flow inlet and outlet, pseudo-reference electrode and a fitting for the CE silica column, consisting of an orifice perpendicular to the surface of a glassy carbon electrode with a bushing inside to ensure a tight press fit. The end of the capillary in contact with the GCE is slant polished, thus defining a reproducible distance from the electrode surface to the column bore. First results with EPC–CE–C⁴D are very promising, as revealed by enrichment factors of two orders of magnitude for Tl, Cu, Pb and Cd ion peak area signals. Detection limits for 10 min deposition time fall around 20 nmol L^?1 with linear calibration curves over a wide range. Besides preconcentration, easy matrix exchange between accumulation and stripping/injection favors procedures like sample cleanup and optimization of pH, ionic strength and complexing power. This was demonstrated for highly saline samples by using a low conductivity buffer for stripping/injection to improve separation and promote field-enhanced sample stacking during electromigration along the capillary.     

Dead volume–free flow splitting in capillary electrophoresis

In recent years, several dual detection concepts (DDCs) for CE were developed, which consisted of at least one nondestructive detector. For these DDCs, a linear detector arrangement could be used, which is not possible when both detectors are destructive. To overcome this problem, we developed a concept for the splitting of the CE stream utilizing commercially available flow splitters (FSs) that allow the parallel positioning of two destructive detectors. In this proof-of-concept study, T- and Y-shaped FSs were characterized regarding their suitability for DDCs. To keep it simple, a UV detector (UV) and a C⁴D were used for the characterization. The model system consisted of an acetonitrile-based background electrolyte and the two model substances, (ferrocenylmethyl)trimethylammonium iodide and caffeine. CE hyphenated to a UV detector (CE-UV) measurements revealed that the split ratio was about 50% for both FSs. CE-C⁴D was used to evaluate the peak shape in front of and behind the FSs. These measurements showed that there was no significant peak broadening introduced by the FSs. Additionally, there were no changes in the LODs in front of and behind the FSs. Furthermore, the flexibility of the new FS approach allowed the usage of capillaries with different ids (25–75 µm) for injection and detection.     

Non-aqueous electrolytes for isotachophoresis of weak bases and its application to the comprehensive preconcentration of the 20 proteinogenic amino acids in column-coupling ITP/CE–MS

Isotachophoresis (ITP) has long been used alone but also as a preconcentration technique for capillary electrophoresis (CE). Unfortunately, up to now, its application is restricted to relatively strong acids and bases as either the degree of (de)protonation is too low or the water dissociation is too high, evoking zone electrophoresis. With the comprehensive ITP analysis of all 20 proteinogenic amino acids as model analytes, we, here, show that non–aqueous ITP using dimethylsulfoxide as a solvent solves this ITP shortcoming. Dimethylsulfoxide changes the pH regime of analytes and electrolytes but, more importantly, strongly reduces the proton mobility by prohibiting hydrogen bonds and thus, the so-called Zundel–Eigen–Zundel electrical conduction mechanism of flipping hydrogen bonds. The effects are demonstrated in an electrolyte system with taurine or H⁺ as terminator, and imidazole as leader together with strong acids such as oxalic and even trifluoroacetic acid as counterions, both impossible to use in aqueous solution. Mass spectrometric as well as capacitively coupled contactless conductivity detection (C⁴D) are used to follow the ITP processes. To demonstrate the preconcentration capabilities of ITP in a two-dimensional set-up, we, here, also demonstrate that our non-aqueous ITP method can be combined with capillary electrophoresis–mass spectrometry in a column-coupling system using a hybrid approach of capillaries coupled to a microfluidic interface. For this, C⁴D was optimized for on-chip detection with the electrodes aligned on top of a thin glass lid of the microfluidic chip.     

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.     

Rapid screening of inorganic and organic anions in liquid by‐products from hydrothermal treatment of biomass by capillary electrophoresis

A simple and rapid capillary electrophoresis method with capacitively coupled contactless conductivity detection (CE‐C⁴D) for the simultaneous determination of inorganic and organic anions in liquid product obtained from the hydrothermal treatment of biomass residues is presented. Under the optimal analytical conditions, limits of detection ranged from 1.8 to 9.4 μM for most target solutes and 53 μM for citrate. Relative standard deviations were below 0.5% for migration times and within 0.6–6.5% for peak areas for all solutes. The proposed method was successfully applied for the rapid determination and screening of inorganic and organic anions in liquid product produced following differing hydrothermal treatment temperatures for banana and pineapple biomass, and the contribution of organic acid formation to acidity in the liquid was evaluated. CE‐C⁴D could be a suitable method for the optimization or tailoring of HTT conditions for desired liquid product composition, and additionally for determination of the best variety(s) of biomass to use in such processes.     

Mass Spectrometric and Contactless Conductivity Detection Approaches in the Determination of Muscle Relaxants by Capillary Electrophoresis

Novel and rapid capillary electrophoresis-coupled tandem mass spectrometry (CE-MS/MS) and capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C⁴D) methods have been developed for the separation and determination of three neuromuscular blocking agents: pancuronium, vecuronium, and rocuronium. In both cases, the separation was conducted in background electrolytes based on acidic acetate-ammonium buffers to avoid possible decomposition of the analytes that are known to be unstable in alkaline media. Baseline resolution of the analytes was achieved in the presence of modified γ-cyclodextrin by CE with C⁴D detection. The two detection techniques were compared with regard to analytical figures of merit including linear dynamic range, limit of detection, limit of quantification, precision, and accuracy. The calibration curves showed good linearity for both detection methods examined (characterized by r² ≥ 0.9908). The LODs of the CE-MS/MS and the CE-C⁴D methods differed at least by two orders of magnitude considering all analytes. The differences in precision and accuracy of these methods were evaluated and discussed. The assays of pancuronium, vecuronium, and rocuronium in commercial injection solutions by CE-MS/MS and CE-C⁴D were performed and the results compared.     

Capillary electrophoresis for the analysis of paralytic shellfish poisoning toxins in shellfish: Comparison of detection methods

Paralytic shellfish toxins (PSTs) are produced by marine and freshwater microalgae and accumulate in shellfish including mussels, oysters, and scallops, causing possible fatalities when inadvertently consumed. Monitoring of PST content of shellfish is therefore important for food safety, with currently approved methods based on HPLC, using pre‐ or postcolumn oxidation for fluorescence detection (HPLC‐FLD). CE is an attractive alternative for screening and detection of PSTs as it is compatible with miniaturization and could be implemented in portable instrumentation for on‐site monitoring. In this study, CE methods were developed for C⁴D, FLD, UV absorption detection, and MS—making this first report of C⁴D and FLD for PSTs detection. Because most oxidized toxins are neutral, MEKC was used in combination with FLD. The developed CZE‐UV and CZE‐C⁴D methods provide better resolution, selectivity, and separation efficiency compared to CZE‐MS and MEKC‐FLD. The sensitivity of the CZE‐C⁴D and MEKC‐FLD methods was superior to UV and MS, with LOD values ranging from 140 to 715 ng/mL for CZE‐C⁴D and 60.9 to 104 ng/mL for MEKC‐FLD. With the regulatory limit for shellfish samples of 800 ng/mL, the CZE‐C⁴D and MEKC‐FLD methods were evaluated for the screening and detection of PSTs in shellfish samples. While the CZE‐C⁴D method suffered from significant interferences from the shellfish matrix, MEKC‐FLD was successfully used for PST screening of a periodate‐oxidized mussel sample, with results confirmed by HPLC‐FLD. This confirms the potential of MEKC‐FLD for screening of PSTs in shellfish samples.     

Conductivity detection for conventional and miniaturised capillary electrophoresis systems

Since the introduction of capillary electrophoresis (CE), conductivity detection has been an attractive means of detection. No additional chemical properties are required for detection, and no loss in sensitivity is expected when miniaturising the detector to scale with narrow-bore capillaries or even to the microchip format. Integration of conductivity and CE, however, involves a challenging combination of engineering issues. In conductivity detection the resistance of the solution is most frequently measured in an alternating current (AC) circuit. The influence of capacitors both in series and in parallel with the solution resistance should be minimised during conductivity measurements. For contact conductivity measurements, the positioning and alignment of the detection electrodes is crucial. A contact conductivity detector for CE has been commercially available, but was withdrawn from the market. Microfabrication technology enables integration and precise alignment of electrodes, resulting in the popularity of conductivity detection in microfluidic devices. In contactless conductivity detection, the alignment of the electrodes with respect to the capillary is less crucial. Contactless conductivity detection (CCD) was introduced in capillary CE, and similar electronics have been applied for CCD using planar electrodes in microfluidic devices. A contactless conductivity detector for capillaries has been commercialised recently. In this review, different approaches towards conductivity detection in capillaries and chip-based CE are discussed. In contrast to previous reviews, the focus of the present review is on the technological developments and challenges in conductivity detection in CE.