Light-emitting diode-based indirect fluorescence detection for simultaneous determination of anions and cations in capillary electrophoresis

This report presents simultaneous analysis of cations and anions by capillary electrophoresis (CE) in conjunction with indirect fluorescence detection using a blue light-emitting diode (LED), based on the displacement of fluorescein with anionic EDTA-metal complexes and anions. A new focusing system combined with a plastic lens and a 40x objective was developed and used effectively to focus the diverging beam of the LED on the capillary. The optimum compositions for simultaneous analysis of metal ions and anions are the samples prepared in 5 mM borate, pH 9.2, containing 2 mM EDTA and the background electrolytes (BGEs) consisting of 5 mM borate buffer, 5 microM fluorescein, and 1 microM NaCl at pH 9.2. Using this pre-capillary complexation method, the analysis of a sample containing five metal ions and eight anions was accomplished in 8 min, with the relative standard deviation values for the migration times less than 2.0%. The peak heights against the concentrations of the metal ions and anions are linear in 10-1000 and 50-2000 microM, with correlation coefficients better than 0.998, and 0.982, respectively. The limits of detection at a signal-to-noise ratio 3 of up to 14.6 microM for formate and as low as 3.7 microM for Ni2+. The results of the analyses of pond water and a Chinese herbal soup present the advantages of this method, including simplicity, rapidity, reproducibility, and low costs.     

Enhancement of detection sensitivity for indirect photometric detection of anions and cations in capillary electrophoresis

This review focuses on the indirect photometric detection of anions and cations by capillary electrophoresis. Special emphasis has been placed on the sensitivity of the technique and approaches taken to enhance detection limits. Theoretical considerations and requirements have been discussed, including buffering, detection sensitivity, separation of cations, and detector linearity. A series of tables detailing highly absorbing probes and the conditions of their use for indirect photometric detection are included.     

Determination of anions with capillary electrophoresis and indirect ultraviolet detection

A method is validated for the determination of anions with capillary electrophoresis (CE) in combination with indirect UV detection. The method described here is used for the analysis of eight of the most common anions (fluoride, chloride, bromide, sulphate, nitrate, nitrite, thiosulphate and phosphate). Next, the method is compared with a another buffer system for the determination of anions with CE and indirect UV detection. Typical limits of detection are obtained between 1 and 3 mg/l for the above-mentioned compounds. The repeatability and reproducibility of the system differs per compound and is, with the exception of fluoride and phosphate, between 4 and 6% and 5–10%, respectively. Linearity was observed between 1 and 10 mg/l. The method is applied for the determination of anions in drinking water, serum and urine.     

Determination and prediction of transfer ratios for anions in capillary zone electrophoresis with indirect UV detection

Transfer ratios (i.e. the number of moles of the UV-absorbing probe anion displaced by one mole of analyte anion) were determined for the separation of inorganic and organic anions by capillary zone electrophoresis using indirect UV absorbance detection. When the electrolyte was buffered and contained only the probe anion and a single counter-cation, transfer ratios calculated from Kohlrausch theory were found to agree well with values obtained experimentally from accurately determined mobility data. However, these electrolyte systems gave long analysis times and were therefore considered impractical. More useful electrolytes were obtained by the addition of surfactants to suppress or reverse the electroosmotic flow but the co-anion introduced with the surfactant can reduce the value of the measured transfer ratio and hence adversely affect sensitivity. This problem was overcome by the use of a surfactant in the hydroxide from such as cetyltrimethylammonium hydroxide combined with a suitable buffering counter-cation such as protonated 1,3-bis[tris(hydroxymethyl)-methylamino]-propane or tris(hydroxymethyl)aminoethane. Four buffered electrolytes consisting of chromate, benzoate, phthalate, or trimellitate as probes and a suitable surfactant were used to determine transfer ratios. These systems were shown to give transfer ratios that were close to those calculated from Kohlrausch theory, thereby enabling prediction of experimental conditions giving maximum transfer ratios.     

In-channel indirect amperometric detection of nonelectroactive anions for electrophoresis on a poly(dimethylsiloxane) microchip

In the present paper, we describe a microfluidics-based sensing system for nonelectroactive anions under negative separation electric field by mounting a single carbon fiber disk working electrode (WE) in the end part of a poly(dimethylsiloxane) microchannel. In contrast to work in a positive separation electric field described in our previous paper (Anal. Chem. 2004, 76, 6902-6907), here the electrochemical reduction reaction at the WE is not coupled with the separation high-voltage (HV) system, whereas the electrochemical oxidation reaction at the WE is coupled with the separation HV system. The electroactive indicator is the carbon fiber WE itself but not dissolved oxygen. This provides a convenient and sensitive means for the determination of nonelectroactive anions by amperometry. The influences of separation voltage, detection potential, and the distance between the WE and the separation channel outlet on the response of the detector have been investigated. The present detection mode is successfully used to electrochemically detect F-, Cl-, SO4(2-), CH3COO-, H2PO4-. Based on the preliminary results, a detection limit of 2 microM and a dynamic range up to three orders of magnitude for Cl- could be achieved.     

Determination of ethyl sulfate--a marker for recent ethanol consumption--in human urine by CE with indirect UV detection

A CE method for the determination of the ethanol consumption marker ethyl sulfate (EtS) in human urine was developed. Analysis was performed in negative polarity mode with a background electrolyte composed of 15 mM maleic acid, 1 mM phthalic acid, and 0.05 mM cetyltrimethylammonium bromide (CTAB) at pH 2.5 and indirect UV detection at 220 nm (300 nm reference wavelength). This buffer system provided selective separation conditions for EtS and vinylsulfonic acid, employed as internal standard, from urine matrix components. Sample pretreatment of urine was minimized to a 1:5 dilution with water. The optimized CE method was validated in the range of 5-700 mg/L using seven lots of urine. Intra- and inter-day precision and accuracy values, determined at 5, 60, and 700 mg/L with each lot of urine, fulfilled the requirements according to common guidelines for bioanalytical method validation. The application to forensic urine samples collected at autopsies as well as a successful cross-validation with a LC-MS/MS-based method confirmed the overall validity and real-world suitability of the developed expeditious CE assay (sample throughput 130 per day).     

Separation and indirect ultraviolet detection of common fluorine-containing anions by ionic liquids in reversed-phase chromatography

Abstract

A new approach for the simultaneous separation and indirect ultraviolet detection of four common fluorine-containing anions using 1-hexyl-3-methylimidazolium chloride as a mobile phase additive in reversed-phase chromatography was developed. The principle of separation and detection is that fluorine-containing anions and imidazolium cations form ion-pairs with ultraviolet absorption. Under the optimal determining conditions, the limits of detection for tetrafluoroborate, hexafluorophosphate, trifluoroacetate, and trifluoromethane sulfonate were 0.6, 0.6, 1.5, and 3.1?µmol/L, respectively. The approach has been successfully applied to the determination of these anions in ionic liquid samples, and the spiked recovery was between 98.5 and 102.6%. The detection limit, linearity, reproducibility, and accuracy of the approach are suitable for quantitative analysis. The method is simple and easy to popularize because of the use of a common C₁₈ reversed-phase column and ultraviolet detector.     

Ion exchange preconcentration and separation of mercury species by CE with indirect contactless conductometric detection

Capillary electrophoretic separation of four mercury species (inorganic Hg(2+), methyl-, ethyl-, and phenylmercury) was achieved in less than 8 min with an electrolyte consisting of 150 mM 2-amino-2-methyl-propanol (AMP) at pH 11.6. The analytes were complexed with 0.1% mercaptopropionic acid (MPA), separated in counter-EOF as anionic complexes, and detected by a contactless conductometric detector. Ion exchange preconcentration with on-column formation of MPA-mercury complexes was developed. Preconcentration factors of 25-150 were achieved and LODs in the range of 2.9-6.9 ng/mL were feasible. This method may prove to be applicable as a rapid screening method for mercury speciation in environmental samples.     

New preconditioning strategy for the determination of inorganic anions with capillary zone electrophoresis using indirect UV detection

It is widely accepted that preconditioning procedures are indispensable in capillary electrophoresis in order to achieve reproducibility of migration times and peak areas. Several preconditioning strategies have been employed for electrophoretic determinations of inorganic anions using indirect UV detection including simple flushing with buffer or alkaline or acid pre-rinsing followed by flushing with electrolyte. We investigated the influence of various preconditioning strategies on the reproducibility of migration times and peak areas of inorganic anions. The electrolyte systems for indirect UV detection were based on pyromellitic acid and chromic acid respectively as UV absorbing probes and hexamethonium hydroxide as electroosmatic flow modifier. Preconditioning agents under investigation were electrolyte buffer, NaOH, HCl and the free acids of the UV absorbing probes. Investigations showed that reproducibility of migration times and peak areas can be significantly improved by acid pre-rinsing using the corresponding acid of the UV absorbing probes compared to preconditioning by flushing the capillary with buffer. In contrast to acid pre-rinsing using hydrochloric acid no interfering signals within the migration time window of inorganic anions under investigation can be observed. The optimized preconditioning procedure yields relative standard deviations of migration times less than 0.25% (n=10). Relative standard deviations of corrected peak areas were below 5% applying acid preconditioning using pyromellitic acid.     

Method development and validation for the determination of various sulfur-containing anions and other anions in the corrosion process by capillary ion electrophoresis with indirect detection

A method of capillary ion electrophoresis with indirect detection is developed for the simultaneous determination of the sulfur-containing anions S2O4(2-), S2O3(2-), SO4(2-), SO3(2-), and S2- and other anions (Cl-, Br-, NO2-, NO3-, (COO)2(2-), F-, and PO4(3-)) in the corrosion process. The effects of pH, tetradecyltrimethylammonium hydroxide, chromate, 2-[n-cyclohexylamino]-ethane sulfonate, calcium gluconate, and acetonitrile on the migration and resolution of the anions and the stability of sulfur-containing anions are systematically investigated. The detection limits, repeatability, and linearity for the anions are comparatively studied at 374, 274, and 254 nm, and the results show that 374 nm is the optimal length. The simultaneous multiwavelength detection at 374, 254, 214, and 195 nm can assist in confirming the identification of UV-absorbing anions.     

Subphthalocyanines as fluoro-chromogenic probes for anions and their application to the highly selective and sensitive cyanide detection

The use of a subphthalocyanine derivative as a selective chromo-fluorogenic reporter for the anion cyanide in mixed aqueous solutions is reported.     

Enhanced pH-mediated stacking of anions for CE incorporating a dynamic pH junction

A technique has been developed to enhance analyte focusing for CE for the analysis of physiological samples. High-ionic-strength samples are titrated to low-ionic-strength on-line using pH-mediated sample stacking in conjunction with a dynamic pH junction. This method concentrates analytes by reducing their electrophoretic mobility during field-amplification. Parameters responsible for enhanced focusing were investigated, and an enhanced pH-mediated stacking method was optimized for anionic nucleosides. The process results in ultra-narrow peak widths, for example, 0.28 s for thymidine with a 10 min analysis time. Peak width and resolution with the enhanced stacking method were also compared to normal base stacking and electrokinetic injection. With this technique, mass-loading capacity can be increased without degradation in peak shape and resolution is dramatically improved.     

Potential of CE for the determination of inorganic and acidic anions in cyanoacrylate adhesives

In this work, a CZE method with indirect UV detection was developed for the simultaneous determination of the inorganic and acidic anions, chloride, sulfate, nitrate, fluoride, formate, phosphate, diethylphosphate, methyl sulfonate, cyanoacetate, and methacrylate present in cyanoacrylate adhesives. Chromate was employed as the probe ion, and the EOF was reversed by incorporating CTAB into BGE. Detection limits of 0.7-4.6 microg/mL were obtained for all the anions studied. The CE method developed is a significant improvement on traditionally used chromatographic methods such as ion chromatography, as it resulted in shorter analysis times with enhanced separation efficiencies. This method was successfully employed for the analysis of inorganic and acidic anions in cyanoacrylate adhesive samples.     

Online transient micellar phase concentration of anions using CTAB in CE

A transient micellar phase extractor using CTAB was described for the online sample concentration of various anionic analytes (drugs and herbicides) in CE. Stacking and separation was performed at neutral pH in coelectroosmotic flow in a hexadimethrine bromide coated fused‐silica capillary. A micellar plug (e.g. 10 mM CTAB) was injected prior to hydrodynamic injection of the analytes prepared in aqueous organic solvent (e.g. with 30% ACN). In the presence of an electric field, the micelles interacted with the anions inside the capillary. This was followed by selective analyte focusing via the mechanism of micelle to solvent stacking. The micelles acted as transient extractor because the stacking ends when the injected micelles completely migrated through the boundary between the sample and micellar plug. Fundamental studies were performed (effect of surfactant concentration, etc.) and the technique yielded 13‐ to 30‐fold improvements in peak height. A stacking CE method in conjunction with liquid–liquid extraction was also tested for the detection of the herbicides fenoprop and mecoprop in fortified drinking water at analyte concentration levels relevant to Australian Drinking Water Guidelines.     

In-column fiber-optic laser-induced fluorescence detection for CE

A highly sensitive in-column fiber-optic LIF detector for CE has been constructed and evaluated. In this detection system, a 457-nm diode-pumped solid-state blue laser was used as the excitation light source and an optical fiber (40 mum od) was used to transmit the excitation light. One end of the optical fiber was inserted into the separation capillary and was in situ positioned at the detection window. The other end of the fiber was protruded from the capillary to capture the excitation light beam from the blue laser. Fluorescence emission was collected by a 40 x microscope objective, focused on a spatial filter, and passed through a yellow color filter before reaching the photomultiplier tube. The present CE-fluorescence detection is a simple and compact optical system. It reduces the laser scattering effect from the capillary and fiber as compared to the conventional LIF detection for CE. Its utility was successfully demonstrated by the separation and determination of D-penicillamine labeled with naphthalene-2,3-dicarboxaldehyde. The detection limit was 0.8 nM (S/N = 3). The present detection scheme has been proven to be attractive for sensitive fluorescence detection for CE.     

Microcolumn ion chromatography of inorganic anions using bovine serum albumin stationary phase with indirect photometric detection

Summary Microcolumn ion chromatography of inorganic anions has been studied using bovine serum albumin immobilized on silica gel as a stationary phase. Several mobile phase solutions were examined, involving sodium iodide, potassium hydrogen phthalate, 2,6-anthraquinone disulfonate (2,6-AQDS) and sodium salicylate. 2,6-AQDS achieved better separation of the analytes tested. Chloride, nitrate, iodide, thiocyanate and sulphate could be separated within 8 min. Detection limits were in the range of 0.9–2.9 M, corresponding to mass detection limits of 0.18–0.59 pmol. The system was applied to the determination of inorganic anions in environmental water and biological samples.     

Development of a fully buffered molybdate electrolyte for capillary electrophoresis with indirect detection and its use for analysis of anions in Bayer liquor

A new counterion-buffered molybdate electrolyte was developed and optimized for simultaneous quantitative determination of up to eight anions (chloride, sulphate, oxalate, fluoride, formate, malonate, succinate, and acetate) in Bayer liquor by capillary electrophoresis with indirect detection at 214 nm. The separation parameters were optimized in respect to separation of the critical analyte group fluoride-formate-malonate, with the optimal electrolyte prepared from molybdic trioxide containing 5.0 mmol/L MoO3, 1.3 mmol/L cetyltrimethylammonium bromide (CTAB), and buffered with diethanolamine (DEA) to pH 9.2 (ca. 20 mM DEA). Total length of separation capillary was 80 cm, resulting in run time of under 4 min. The method is suitable for a wide concentration range of the analytes (1-50 mg/L) with linear calibration plots (R2 = 0.9983-0.9999). Relative standard deviations were 0.05%-0.07% for migration times and 0.67%-2.04% for peak areas. The detection limits were in the range of 0.17-0.51 mg/L or 2-10 micromol/L (hydrostatic injection of 30 s of 1000 x diluted sample). Due to its good buffering capacity, the electrolyte exhibited an excellent ruggedness and good tolerance to the alkaline samples. Consequently, Bayer liquor samples could be diluted as little as 100 x which allows more sensitive determination of minor components over previous methods. The method was successfully applied to analysis of Bayer liquor samples with recoveries in the range of 95-105%.     

Simultaneous determination of inorganic cations and anions in capillary zone electrophoresis (CZE) with indirect fluorescence detection

Summary An instrumental configuration for the simultaneous determination of inorganic cations and anions in capillary zone electrophoresis (CZE) is described. The sample is injected into the central part of the capillary by hydrostatic injection. Indirect fluorescence detection is used. The electrolyte system consists of 2,5-dihydrobenzoate as fluorescent anion and cerium(III) as fluorescent cation. This method allows the analysis of both cations and anions without changing the electrolyte system in less than five minutes.     

离子色谱法-间接紫外检测离子液体中三种含氟酸根阴离子

建立了离子色谱-间接紫外检测法同时分析离子液体中的三氟乙酸根(CF3COO-)、四氟硼酸根(BF4-)和三氟甲烷磺酸根(CF3SO3-)的方法。用Shodex IC NI-424阴离子交换柱作为分离柱,探讨了影响上述三种离子保留和检测的因素及其保留规律。最终选用色谱条件为:以1.75 mmol·L-1邻苯二甲酸-1.68 mmol·L-1三(羟甲基)氨基甲烷为淋洗液,柱温45℃,流速1.0 m L·min-1,UV检测波长设定在265 nm。在此条件下,可实现上述三种阴离子的同时分析,且色谱峰形良好。所测阴离子的检出限(S/N=3)为0.39~1.57 mg·L-1,保留时间和峰面积的相对标准偏差(n=5)均在1%以下。将此方法应用于测定离子液体中的三氟乙酸根、四氟硼酸根及三氟甲烷磺酸根,加标回收率为99%~102%。该方法简单、准确、可靠,具有较好的实用性。