Electroosmotic flow-balanced isotachophoretic stacking with continuous electrokinetic injection for the concentration of anions in high conductivity samples

An EOF counter-balanced ITP boundary has been used to stack anions from high conductivity samples during continuous electrokinetic injection of the sample. In a polystyrenesulfonate/poly(diallyldimethylammonium chloride) polyelectrolyte coated capillary, the time at which the ITP boundary exited the capillary could be prolonged by balancing the movement of the boundary with the EOF. Using a bis-tris-propane electrolyte, the ITP boundary was removed from the capillary within 7 min, while when using triethanolamine the ITP boundary was still at 30% of the capillary after 2 h of injection. Using these systems, the sensitivity of a mixture of simple organic acids in 100 mM Cl⁻ was improved by 700–800-fold using bis-tris-propane with a whole-capillary injection of the sample and 5 min of electrokinetic injection at +28 kV, and 1100–1300-fold using triethanolamine and 60 min of electrokinetic injection under the same conditions. The potential of the method to be applicable to high conductivity samples was demonstrated by stacking a whole capillary filled with urine spiked with naphthalenedisulfonic acid, with limits of detection 450 times lower than those achievable with a normal hydrodynamic injection.     

Combination of large volume sample stacking and dynamic pH junction for on-line preconcentration of weak electrolytes by capillary electrophoresis in comparison with isotachophoretic techniques

An on-line preconcentration capillary electrophoresis (CE) technique, which combines a large volume sample stacking with a dynamic pH junction technique, is introduced in this paper. This dynamic pH junction with co-electroosmotic migration is formed between sodium borate pH 9.5 and sodium phosphate pH 2.5 with 150 mM sodium dodecylsulfate (SDS). A full capillary based injection allows determination of weak acidic compounds at ppb concentration levels (achieved LOD for benzoic acid was 11 nmol L(-1)). The proposed preconcentration method was compared with ITP/ITP (LOD 120 nmol L(-1)), ITP/CZE (LOD 740 nmol L(-1)) and a simple CZE method (LOD 23,330 nmol L(-1)). The analytical potential of this method was assessed with juice test samples.     

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.     

Sensitive analysis of donepezil in plasma by capillary electrophoresis combining on-column field-amplified sample stacking and its application in Alzheimer's disease

Field-amplified sample stacking (FASS) in capillary electrophoresis (CE) was used to determine the concentration of donepezil, an acetylcholinesterase inhibitor, in human plasma. A sample pretreatment by liquid-liquid extraction with isopropanol/n-hexane (v/v 3:97) and subsequent quantification by FASS-CE was used. Before sample loading, a water plug (0.5 psi, 6 s) was injected to permit FASS. Electrokinetic injection (7 kV, 90 s) was used to introduce sample cations. The separation condition for donepezil was performed in electrolyte solutions containing Tris buffer (60 mM, pH 4.0) with sodium octanesulfonate 40 mM and 0.01% polyvinyl alcohol as a dynamic coating to reduce analytes' interaction with capillary wall. The separation was performed at 28 kV and detected at 200 nm. Using atenolol as an internal standard, the linear ranges of the method for the determination of donepezil in human plasma were over a range of 1-50 ng/mL. The limit of detection was 0.1 ng/mL (S/N=3, sampling 90 s at 7 kV). One female volunteer (54 years old) was orally administered a single dose of 10 mg donepezil (Aricept, Eisai), and blood samples were drawn over a 60 h period for pharmacokinetic study. The method was also applied successfully to monitor donepezil in sixteen Alzheimer's disease patients' plasmas.     

Direct simultaneous determination of eight sweeteners in foods by capillary isotachophoresis

A method for isotachophoretic determination of sweeteners of different character in candies and chewing gums was developed. A capillary of 0.8 mm ID and 90 mm effective length made of fluorinated ethylene-propylene copolymer is filled with an electrolyte system consisting of 10 mM HCl + 14 mM Tris, pH 7.7 (leading electrolyte) and 5 mM L-histidine + 5 mM Tris, pH 8.3 (terminating electrolyte). The analysis is performed at a driving current of 200 microA and for detection current is decreased to 100 microA. Boric acid is added to the aqueous sample solution to form borate complexes with substances of polyhydroxyl nature and make them migrate isotachophoretically. Using conductivity detection, the calibration curves in the tested concentration range up to 2.5 mM were linear for all components of interest: acesulfame K, saccharine, aspartame, cyclamate, sorbitol, mannitol, lactitol, and xylitol. The concentration detection limits ranged between 0.024 and 0.081 mM. Good precision of the ITP method is evidenced by favorable RSD values ranging from 0.8 to 2.8% obtained at the analyte concentration of 1.0 mM (n = 6). The analysis time was about 20 min. Simplicity, accuracy, and low cost of analyses make ITP an alternative procedure to methods used so far for the determination of ionizable sweeteners.     

Novel moving reaction boundary-induced stacking and separation of human hemoglobins in slab polyacrylamide gel electrophoresis

We developed a novel polyacrylamide gel electrophoresis (PAGE) method to stack and separate human hemoglobins (Hbs) based on the concept of moving reaction boundary (MRB). This differs from the classic isotachophoresis (ITP)-based stacking PAGE in the aspect of buffer composition, including the electrode buffer (pH 8.62 Tris–Gly), sample buffer (pH 6.78 Tris–Gly), and separation buffer (pH 8.52 Tris–Gly). In the MRB-PAGE system, a transient MRB was formed between alkaline electrode buffer and acidic sample buffer, being designed to move toward the anode. Hbs carried partial positive charges in the sample buffer due to its pH below pI values of Hbs, resulting in electromigrating to the cathode. Hbs would carry negative charges quickly when migrated into the alkaline electrode buffer and be transported to the anode until meeting the sample buffer again. Thus, Hbs were stacked within a MRB until the transient MRB reached the separation buffer and then separated by zone electrophoresis with molecular sieve effect of the gel. The experimental results demonstrated that there were three clear and sharp protein zones of Hbs (HbA_1c, HbA₀, and HbA₂) in MRB-PAGE, in contrast to only one protein zone (HbA₀) in ITP-PAGE for large-volume loading (≥15 μl), indicating high stacking efficiency, separation resolution, and good sensitivity of MRB-PAGE. In addition, MRB-PAGE was performed in a conventional slab PAGE device, requiring no special device. Thus, it could be widely used in separation and analysis of diluted protein in a standard laboratory.

Base-induced transient isotachophoretic stacking of acidic drugs in capillary zone electrophoresis

Online sample concentration of acidic drugs by transient isotachophoresis (t-ITP) with the injection of a base is described in capillary zone electrophoresis (CZE). A positively coated capillary was conditioned with background electrolyte (ammonium acetate at pH 6). A long plug of sample solution (S) prepared in ammonium acetate was then hydrodynamically injected followed by the base (tetrapropylammonium hydroxide). A negative voltage was applied and caused the hydroxide ions from the base to penetrate the S zone and created a pH junction that swept through the S zone. The analytes stack at the junction where the mechanism of focusing was transient ITP with the acetate and hydroxide ions as leading and terminating ions, respectively. The concentrated analytes separated in co-EOF CZE once the hydroxide was exhausted. The base stacking strategy was tested using hypolipidemic, nonsteroidal anti-inflammatory, and diuretic drugs, and afforded 19-37 improvements in peak height.     

On-line coupling of capillary isotachophoresis and capillary zone electrophoresis for the determination of flavonoids in methanolic extracts of Hypericum perforatum leaves or flowers

Five flavonoids (hyperoside, isoquercitrin, quercitrin, quercetin and rutin) were separated and determined in extracts of Hypericum perforatum leaves or flowers by capillary zone electrophoresis (CZE) with isotachophoretic (ITP) sample pre-treatment using on-line column coupling configuration. The background electrolyte (BGE) used in the CZE step was different from the leading and terminating ITP electrolytes but all the electrolytes contained 20% (v/v) of methanol. The optimal leading electrolyte was 10 mM HCl of pH* approximately 7.2 (adjusted with Tris) and the terminating electrolyte was 50 mM H3BO3 of pH* approximately 8.2 (adjusted with barium hydroxide). This operational system allowed to concentrate and pre-separate selectively the flavonoid fraction from other plant constituents before the introduction of the flavonoids into the CZE capillary. The BGE for the CZE step was 50 mM Tris buffer of pH* approximately 8.75 containing 25 mM N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid as co-ion and 55 mM H3BO3 as complex-forming agent. The ITP-CZE method with spectrophotometric detection at 254 nm was suitable for the quantitation of the flavonoids in real natural samples; kaempferol was used as internal standard. The limit of detection for quercetin-3-O-glycosides was 100 ng ml(-1) and calibration curves were rectilinear in the range 1-10 microg ml (-1) for most of the analytes. The RSD values ranged between 0.9 and 2.7% (n=3) when determining approximately 0.07-1.2% of the individual flavonoids in dried medicinal plants.     

Online stacking of carboxylated magnetite core–shell nanoparticles in capillary electrophoresis

The stacking effect on carboxylated magnetite core–shell nanoparticles using sodium borate buffer pH 9.5 as the background electrolyte is presented. The ionic strength of the background electrolyte ranged from 5 to 100 mM, and the ionic strength of a sample zone ranged from 5 to 100 mM. Moreover, water was used as the sample dispersant. Both stacking and de‐stacking effects were observed when conductivities of the sample zone and the background electrolyte differed. An explanation of carboxylated magnetic core‐shell nanoparticles behavior was suggested based on the Derjaguin–Landau–Verwey–Overbeek theory supposing that the aggregation point is defined by the energetic barrier as the sum of energies given by electrostatic interactions and Van der Waals interactions. Moreover, the stacking conditions were applied for the evaluation of the lowest detectable dilution of magnetic nanoparticles. The carboxylated magnetic nanoparticles were dispersed in 10 mM borate/NaOH pH 9.5 and injected for 60 s to the background electrolyte composed of 100 mM borate/NaOH pH 9.5 that allowed the detection of 100‐fold diluted nanoparticles.     

Analysis of phenolic acids by non-aqueous capillary electrophoresis after electrokinetic supercharging

Electrokinetic supercharging (EKS), a new and powerful on-line preconcentration method for capillary electrophoresis, was utilized in non-aqueous capillary electrophoresis (NACE) to enhance the sensitivity of phenolic acids. The buffer acidity and concentration, leader and terminator length and electrokinetic injection time were optimised, with the optimum conditions being: a background electrolyte of 40 mM Tris-acetic acid (pH 7.9), hydrodynamic injection of 50 mM ammonium chloride (22 s, 0.5 psi) as leader, electrokinetic injection of the sample (180 s, -10 kV), hydrodynamic injection of 20 mM CHES (32 s, 0.5 psi) as terminator, before application of the separation voltage (-25 kV). Under these conditions the sensitivity was enhanced between 1333 and 3440 times when compared to a normal hydrodynamic injection with the sample volume <3% of the capillary volume. Detection limits for the seven phenolic acids were in the range of 0.22-0.51 ng/mL and EKS was found to be 3.6-7.9 times more sensitive than large-volume sample stacking and anion selective exhaustive injection for the same seven phenolic acids.     

Effect of NaOH on large-volume sample stacking of haloacetic acids in capillary zone electrophoresis with a low-pH buffer

Large-volume sample stacking (LVSS) is an effective on-capillary sample concentration method in capillary zone electrophoresis, which can be applied to the sample in a low-conductivity matrix. NaOH solution is commonly used to back-extract acidic compounds from organic solvent in sample pretreatment. The effect of NaOH as sample matrix on LVSS of haloacetic acids was investigated in this study. It was found that the presence of NaOH in sample did not compromise, but rather help the sample stacking performance if a low pH background electrolyte (BGE) was used. The sensitivity enhancement factor was higher than the case when sample was dissolved in pure water or diluted BGE. Compared with conventional injection (0.4% capillary volume), 97-120-fold sensitivity enhancement in terms of peak height was obtained without deterioration of separation with an injection amount equal to 20% of the capillary volume. This method was applied to determine haloacetic acids in tap water by combination with liquid-liquid extraction and back-extraction into NaOH solution. Limits of detection at sub-ppb levels were obtained for real samples with direct UV detection.     

毛细管电泳场放大进样-高压非接触电导检测测定痕量Zn~(2+)

利用高压电容耦合非接触电导检测器(HV-C4D),结合毛细管电泳场放大进样(FASS),以2-N-吗啡啉乙磺酸(MES)/组氨酸(His)为缓冲溶液,电泳分离测定了Zn2+.考察了样品溶液中MES/His的浓度及电动进样时间对场放大浓缩因子及缓冲溶液浓度对检测灵敏度的影响.在10mmol/LMES/His(pH=4.9)的分离缓冲溶液中,FASS对Zn2+的浓缩因子为1.3×103.Zn2+的浓度在10～1000nmol/L范围内与峰面积有良好线性关系(R=0.9995),检测限为5nmol/L(S/N=3).该方法可用于痕量Zn2+的测定.     

Different sample stacking strategies to analyse some nonsteroidal anti-inflammatory drugs by micellar electrokinetic capillary chromatography in mineral waters

Three on-column preconcentration techniques were compared to analyse a group of nonsteroidal anti-inflammatory drugs (NSAIDs) using micellar electrokinetic capillary chromatography (MEKC) under pH-suppressed electroosmotic flow (EOF) in water samples. The analysed drugs were ibuprofen, fenoprofen, naproxen, ketoprofen, and diclofenac sodium. The micellar background electrolyte (BGE) solution was formed by 75 mM sodium dodecyl sulfate (SDS), 40% (v/v) acetonitrile, and 25 mM sodium phosphate at pH 2.5. When this BGE solution was used the applied voltage was reversed, -10 kV, and the drugs were separated within 20 min. The on-column preconcentration modes, characterised all of them for the sample matrix removal out of the capillary by itself under a reverse potential at the same time as the EOF was reduced, were stacking with reverse migrating micelles (SRMM), stacking with reverse migrating micelles-anion selective exhaustive injection (SRMM-ASEI), and field-enhanced sample injection with reverse migrating micelles (FESI-RMM). The sensitivity was improved up to 154-, 263-, and 63-fold, respectively when it was calculated through the peaks height. The optimised methods were validated with spiked mineral water by combining off-line solid-phase extraction (SPE) and the proposed on-line sample stacking strategies. The detection limits (LODs) of NSAIDs in mineral water were at ng/L levels.     

Analysis of tetracycline residues in bovine milk by CE-MS with field-amplified sample stacking

A rapid, sensitive, and robust CE-MS method has been developed for the determination of tetracycline, oxytetracycline, and chlortetracycline in milk. Field-amplified sample stacking with electromigration injection (FASS-EMI) was used for the online concentration of tetracyclines. The conditions of separation, MS detection, and stacking were systematically optimized. The optimum buffer composition was 35 mM Tris, 1.1% formic acid, 5% methanol, and 15% ACN. By using the online concentration method of field-enhanced sample stacking (FESI)-EMI stacking, the sensitivity was increased six- to seven-fold. The RSDs (n=6) of the relative migration time of tetracyclines were 1.1-1.4% for intraday and 2.4-2.9% for interday. The RSDs (n=6) of the relative peak area of tetracyclines were 3.2-4.6% for intraday and 4.7-6.1% for interday. The LODs (S/N=3) were 7.14 ng/mL for tetracycline, 11.4 ng/mL for oxytetracycline, and 14.9 ng/mL for chlortetracycline. The method has been successfully used to analyze tetracyclines residues in bovine milk.     

Anionic microemulsion to solvent stacking for on‐line sample concentration of cationic analytes in capillary electrophoresis

The common SDS microemulsion (i.e. 3.3% SDS, 0.8% octane, and 6.6% butanol) and organic solvents were investigated for the stacking of cationic drugs in capillary zone electrophoresis using a low pH separation electrolyte. The sample was prepared in the acidic microemulsion and a high percentage of organic solvent was included in the electrolyte at anodic end of capillary. The stacking mechanism was similar to micelle to solvent stacking where the micelles were replaced by the microemulsion for the transport of analytes to the organic solvent rich boundary. This boundary is found between the microemulsion and anodic electrolyte. The effective electrophoretic mobility of the cations reversed from the direction of the anode in the microemulsion to the cathode in the boundary. Microemulsion to solvent stacking was successfully achieved with 40% ACN in the anodic electrolyte and hydrodynamic sample injection of 21 s at 1000 mbar (equivalent to 30% of the effective length). The sensitivity enhancement factors in terms of peak height and corrected peak area were 15 to 35 and 21 to 47, respectively. The linearity R² in terms of corrected peak area were >0.999. Interday precisions (%RSD, n = 6) were 3.3–4.0% for corrected peak area and 2.0–3.0% for migration time. Application to spiked real sample is also presented.     

Analysis of glutathione by capillary electrophoresis based on sample stacking

Glutathione is a small peptide, which participates in cellular oxidation-reduction and detoxification. It is present in most biological tissues at different concentrations. The oxidized and reduced forms of the peptide were measured in erythrocytes and myocardial tissue by capillary electrophoresis based on stacking. After tissue homogenization or hemolysis of the red blood cells, the samples were deproteinized with acetonitrile and injected filling about 13% of the capillary volume. The electrophoresis was performed at 10 kV using a separation buffer of 250 mM borate, 50 mM Tris, pH 8.0. Sample stacking increased the sensitivity of detection by 10-20-fold.     

Trace ion analysis of sea water by capillary electrophoresis: determination of strontium and lithium pre-concentrated by transient isotachophoresis

The applicability of capillary zone electrophoresis (CZE) to ions having relatively low natural occurrences in sea water is limited by method's relatively poor concentration detection sensitivity. A combination of CZE with indirect UV detection and transient isotachophoresis (tITP) pre-concentration was developed to evolve the CZE practical utility towards the quantitative determination of the minor sea water cationic components, strontium and lithium. The ITP stacking criterion at the initial stage of a CZE separation was met by taking a highly mobile sodium, the principle matrix cation, to perform the role of a leading ion, whereas the moderately mobile sample macrocomponents, Ca2+ and Mg2+, acted as the terminating ion. The carrier electrolyte, consisting of 10 mM 4-methylbenzylamine and 1.5 mM citric acid at pH 4.8, was found to be optimal to accommodate both analyte cations in the ITP range and then separate them in the CZE mode, with relative standard deviations for migration times from 0.06-0.15% and for peak areas from 4-8%. The limits of detection were 1.3 mg l(-1) Sr2+ and 0.12 mg l(-1) Li+. The developed method was applied to the analysis of a surface sea water sample and a sea water reference material. The results were in good agreement with those obtained by inductively coupled plasma atomic emission spectroscopy (ICP-AES) and electrothermal atomic absorption spectrometry (ET-AAS).     

Determination of trace cationic impurities in butylmethylimidazolium-based ionic liquids: from transient to comprehensive single-capillary counterflow isotachophoresis-zone electrophoresis

Determination of impurities in ionic liquids (ILs) remains a difficult task. In this work, the hyphenation of isotachophoretic (ITP) preconcentration to zone electrophoresis (ZE) has been explored for the trace analysis of the cationic impurities Na(+), Li(+), and methylimidazolium (MI(+)) in butylmethylimidazolium (BMI(+))-based ILs. Simultaneous detection of UV-transparent and UV-absorbing impurities was ensured by a BGE composed of creatinine-acetate buffer. To induce ITP, three different strategies were evaluated: (i) Sample self-stacking ensured by the addition of ammonium acetate (NH(4)Ac) to 25-50-fold diluted IL solution (transient ITP). (ii) Complete ITP-ZE separation performed in a single capillary: ITP was realized in discontinuous electrolytes comprising an 80 mM NH(4)Ac, 40 mM acetic acid, 30 mM alpha-CD, pH 5.05, leading electrolyte (LE) and a 10 mM creatinine, 10 mM acetic acid, pH 4.9, terminating electrolyte (TE). To create the ZE stage, the ITP stack of analytes was moved back toward the capillary inlet by pressure and simultaneously the capillary was filled with the BGE. This protocol made it possible to accommodate a 2.5-times diluted IL sample. (iii) Complete counterflow ITP-ZE with continuous electrokinetic sample supply: the ITP stage was performed in a capillary filled with a 150 mM NH(4)Ac, 75 mM acetic acid, 30 mM alpha-CD, pH 5.0 LE, with 40-times diluted IL at the capillary inlet. BMI(+) from IL acts as the terminating ion. The LODs reached in this latter case were at the 10 and 1 ppb levels for MI(+) and Li(+) in diluted IL matrix, respectively.     

Comparison of two capillary electrophoresis online stacking modes by analysis of polycyclic aromatic hydrocarbons in airborne particulates

Naphthalene, fluorene, pyrene, anthracene, phenanthrene, and chrysene were successfully separated by CD-modified MEKC (CD-MEKC) using 20 mM borate (pH 9.0) containing 90 mM SDS and 75 mM beta-CD. Two online stacking methods, i.e., sweeping and field-enhanced sample injection (FESI), were explored to enhance the detection sensitivity. The influences of some crucial parameters in sweeping and FESI procedures were investigated. For FESI method, a plug of water and low-conductivity sample matrix was used to increase the stacking efficiency. Compared with the sweeping method, FESI can increase the sensitivity in the range of 10-20-fold. The proposed method was used for the analysis of polycyclic aromatic hydrocarbons in airborne particulates.     

Analysis of selected constituents in methanolic extracts of Hypericum perforatum collected in different localities by capillary ITP-CZE

The on-line combination of CZE with capillary ITP (ITP-CZE) was used for the separation and quantification of selected flavonoids and phenolic acids in Hypericum perforatum leaves and flowers collected in six different localities in Slovakia. The leading electrolyte in the ITP preseparation step was 10 mM HCl with Tris as counterion (pH* 7.2). The terminating electrolyte was 50 mM boric acid of pH* 8.2 (adjusted with barium hydroxide). The BGE in the electrophoretic step contained 25 mM beta-hydroxy-4-morpholinopropanesulfonic acid (MOPSO), 50 mM Tris, 65 mM boric acid, pH* 8.3. The content of methanol in all electrolytes was 20% v/v. The total time of the analysis (including the preseparation step) was approximately 35 min. The rectilinear calibration ranges were between 0.125 and 5.0 microg/mL with kaempferol as internal standard. The correlation coefficients ranged between 0.9912 (for quercitrin and chlorogenic acid) and 0.9988 (for isoquercitrin). The RSD values are between 0.86 and 7.78% (n = 6) when determining rutin and quercetin (4 microg/mL). The optimized method was employed for the assay of flavonoids in medicinal plant extract of different collections of Hypericum perforatum haulm. The variability of the content of the active components depending on the place of collection was confirmed.