Optimization of background electrolyte composition for simultaneous contactless conductivity and fluorescence detection in capillary electrophoresis of biological samples

In this article, optimization of BGE for simultaneous separation of inorganic ions, organic acids, and glutathione using dual C⁴D‐LIF detection in capillary electrophoresis is presented. The optimized BGE consisted of 30 mM 2‐[4‐(2‐hydroxyethyl)piperazin‐1‐yl]ethanesulfonic acid, 15 mM 2‐amino‐2‐hydroxymethyl‐propane‐1,3‐diol, and 2 mM 18‐crown‐6 at pH 7.2 and allowed simultaneous separation of ten inorganic anions and cations, three organic acids and glutathione in 20 min. The samples were injected hydrodynamically from both capillary ends using the double‐opposite end injection principle. Sensitive detection of anions, cations, and organic acids with micromolar LODs using C⁴D and simultaneously glutathione with nanomolar LODs using LIF was achieved in a single run. The developed BGE may be useful in analyses of biological samples containing analytes with differing concentrations of several orders of magnitude that is not possible with single detection mode.     

Analysis of inorganic cations in biological samples by the combination of micro-electrodialysis and capillary electrophoresis with capacitively coupled contactless conductivity detection

Micro-electrodialysis (μED) and CE were combined for rapid pretreatment and subsequent determination of inorganic cations in biological samples. Combination of μED with CE greatly improved the analytical performance of the latter as the adsorption of high molecular weight compounds present in real samples on the inner capillary wall was eliminated. Fifty microliter of 80-fold diluted human body fluids such as plasma, serum and whole blood was used in the donor compartment of the μED system requiring less than 1?μL of the original body fluid per analysis. Inorganic cations that migrated through a cellulose acetate dialysis membrane with molecular weight cut-off value of 500?Da were collected in the acceptor solution and were then analyzed using CE-C?D. Baseline separation of inorganic cations was achieved in a BGE solution consisting of 12.5?mM maleic acid, 15?mM L-arginine and 3?mM 18-crown-6 at pH 5.5. Repeatability of the CE-C?D method was better than 0.5% and 2.5% for migration times and peak areas, respectively; limits of detection of all inorganic cations in the presence of 2?mM excess of Na(+) were around 1?μM and calibration curves were linear with correlation coefficients better than 0.998. Repeatability of the sample pretreatment procedure was calculated for six independent electrodialysis runs of artificial and real samples and was better than 11.8%. Recovery values between 96.3 and 110% were achieved for optimized electrodialysis conditions of standard solutions and real samples; lifetime of the dialysis membranes for pretreatment of real samples was estimated to 100 runs.     

Capillary Electrophoretic Separation of Tricyclic Antidepressants Using 1‐Alkyl‐3‐Methylimidazolium‐Based Ionic Liquids as Background Electrolyte

A capillary electrophoretic (CE) method coupled with the use of 1‐ethyl‐3‐methylimidazolium tetrafluoroborate (1E‐3MI‐TFB) ionic liquid as background electrolyte (BGE) has been developed for the simultaneous separation of nine tricyclic antidepressants, viz. amitriptyline (Ami), clomipramine (Clo), desipramine (Des), fluphenazine (Flu), imipramine (Imi), nortriptyline (Nor), promazine (Pro), thioridazine (Thi) and trimipramine (Tri). Resolution of TCAs with similar molecular structures and pK_a values was accomplished by minute manipulation of the electrophoretic velocities of TCAs via reversed electroosmotic flow (EOF) generated by adsorption of 1E‐3MI cations onto the capillary wall. The optimal separation was obtained with a 50 mM 1E‐3MI‐TFB as the sole BGE at pH 3. Symmetric peaks with efficiencies up to 2.4 × 10⁵ plates/m were achieved. RSD values on migration times and peak areas were in the ranges of 0.63–0.95% and 3.41–6.34% (n = 4), respectively. The role of different alkyl groups on the imidazolium cations was also investigated.     

Co‐electroosmotic capillary electrophoresis of basic proteins with 1‐alkyl‐3‐methylimidazolium tetrafluoroborate ionic liquids as non‐covalent coating agents of the fused‐silica capillary and additives of the electrolyte solution

The paper reports the results of a study carried out to evaluate the use of three 1‐alkyl‐3‐methylimidazolium‐based ionic liquids as non‐covalent coating agents for bare fused‐silica capillaries and additives of the electrolyte solutions (BGE) for CE of basic proteins in the co‐EOF separation mode. The three ionic liquids are differentiated from each other by the length of the alkyl group on the imidazolium cation, consisting of either an ethyl, butyl or octyl substituent, whereas tetrafluoroborate is the common anionic component of the ionic liquids. Coating the capillary with the ionic liquid resulted in improved peak shape and protein separation, while the EOF was maintained cathodic. This indicates that each ionic liquid is effective at masking the protein interaction sites on the inner surface of the capillary, also when its adsorption onto the capillary wall has not completely neutralized all the negative charges arising from the ionization of the silanol groups and the ionic liquid is not incorporated into the BGE employed for separation. Using the coated capillaries with BGE containing the ionic liquid employed for the coating, at concentration low enough to maintaining the EOF cathodic, both peak shape and protein separation varied to different extents, based on the particular ionic liquid used and its concentration. Fast and efficient separation of the model basic protein mixture in co‐electroosmotic CE is obtained with the 1‐butyl‐3‐methylimidazolium tetrafluoroborate coated capillary and 100 mM acetate buffer (pH 4.0) containing 4.4 mM 1‐butyl‐3‐methylimidazolium tetrafluoroborate as the BGE.     

Capillary ion electrophoresis-capacitively coupled contactless conductivity detection of inorganic cations in human saliva on a polyvinyl alcohol-coated capillary

Capillary ion electrophoresis–capacitively coupled contactless conductivity detection (CIE-C4D) with a polyvinyl alcohol chemically coated capillary (PVA capillary) was used to analyze inorganic cations (Na⁺, K⁺, NH₄⁺, Mg²⁺, and Ca²⁺) commonly found in human saliva. The PVA capillary, which was made by our laboratory, minimized electro-osmotic flow in the wide pH range of the background electrolyte (BGE), and the PVA layer adsorbed to capillary wall did not affect the conductimetric background level. In this study, we determined an optimized BGE of 30 mM lactic acid/histidine plus 3 mM 18-crown-6 for the CIE-C4D system using the PVA capillary, which could simultaneously improve the separation of Mg²⁺ and Ca²⁺ from Na⁺ and that of K⁺ from NH₄⁺. This system obtained highly reproducible separation of cations in human saliva samples within 8 min at 20 kV without deprotonation. The quantifiability of cations in human saliva samples on the CIE-C4D system was demonstrated through identification by ion chromatography with satisfactory results.     

Simultaneous determination of inorganic cations by capillary ion chromatography with a non-suppressed contactless conductivity detector

A non-suppressed capillary ion chromatographic method with a laboratory-made packed cation-exchange column (100 mm × 0.32 mm i.d.) was developed for the separation and simultaneous determination of five common inorganic cations (sodium, ammonium, potassium, magnesium and calcium). Cation exchangers were prepared by the reaction of the hydroxyl group on the surface of diol-group bonded silica gel with 1,3-propanesultone in methanol. Simultaneous separation of these five common inorganic cations were achieved within 17 min using 1 mM methanesulfonic acid and 0.1 mM 15-crown-5 ether in methanol-water (8:2, v/v) as the eluent. The effects of organic solvents and crown ethers in the eluent on the retention of analytes were investigated. The limits of detection (S/N = 3) of the cations were in the range of 18-124 μg/l, the linear correlation coefficients were 0.9991-0.9998, and the RSD values of retention time and peak height were all smaller than 2.1%. The present analytical method was successfully applied to the rapid and direct determination of inorganic cations in samples of river water and commercial drinks, with satisfactory results.     

Ion association with alkylammonium cations for separation of anions by capillary electrophoresis

A background electrolyte (BGE) containing a 100 mM concentration of an alkylammonium cation with ethyl, propyl or butyl groups provides an excellent medium for separation of anions by capillary electrophoresis (CE). Two major effects were noted. Use of one of a series of alkylammonium cations in the BGE at a selected pH provides a simple and effective way to vary and control electroosmotic flow (EOF) over a broad range. It is believed that the alkylammonium cations are coated onto the capillary surface through a reversible dynamic equilibrium. Secondly, alkylammonium cations modify the electrophoretic migration of sample anions and the electroosmotic migration of neutral organic analytes by association interaction. This selective interaction results in improved anion separations and permits the simultaneous separation of neutral analytes. The degree of association interaction varies with the bulk and hydrophobicity of the alkylammonium cations. Incorporation of an aliphatic amine salt of moderate molecular weight in the running electrolyte provides a valuable new way to vary the migration times of sample anions and to optimize their resolution. The interactions between alkylammonium cations and sample anions or neutral organics appear to take place entirely within the liquid phase and do not require a polymeric or micellar pseudo phase.     

Direct determination of small cations in proteinaceous samples using a flow injection-capillary electrophoresis system.

A method is described for the direct determination of small inorganic cations in samples containing large amounts of proteins, such as milk or blood plasma. The method is based on electrokinetic injection in a flow injection analysis-capillary electrophoresis (CE) system. The selected CE-electrolyte, containing 5 mM 4-aminopyridine and 7 microM cetyltrimethylammonium bromide at pH 4.5, prevents detrimental protein adsorption on the capillary walls. Therefore, no sample pretreatment, except for dilution, is required. Up to 30 repeated injections in one electrophoretic run can be performed, yielding RSD values of the migration time of less than 1 and 2.5% (n=30) for milk and blood plasma samples, respectively.     

Simultaneous separation of basic and acidic proteins using 1-butyl-3-methylimidazolium-based ion liquid as dynamic coating and background electrolyte in capillary electrophoresis

1-Butyl-3-methylimidazolium tetrafluoroborate ionic liquids (1B-3MI-TFB ILs) were employed as a coating material and BGE in CE for simultaneous separation of basic and acidic proteins such as lysozyme, cytochrome C, ribonuclease A, albumin, and alpha-lactalbumin. 1B-3MI-TFB ILs effectively reversed the surface charges on the capillary inner surface, preventing the adsorption of positively charged proteins onto the silica surface, as well as associated with proteins, thus benefiting the separation efficiencies and reproducibility. Consequently, simultaneous baseline separation of five proteins was achieved within 14 min by using 10 mM of 1B-3MI-TFB ILs as dynamic coating and the only running electrolyte at the voltage of +20 kV. The proposed coating technique is simple, less time-consuming, reproducible, and also stable enough for proteins separation without the need of additives. Symmetrical peaks with efficiencies up to 670,000 plates/m were obtained. Recoveries of proteins with RSD (for migration times) of 0.23-0.42% (run-to-run) and 2.5-3.8% (day-to-day) were achieved, respectively. The applicability of the proposed method in proteins separation was evaluated by the separation of egg white samples.     

Use of disposable open tubular ion exchange pre-columns for in-line clean-up of serum and plasma samples prior to capillary electrophoretic analysis of inorganic cations

A simple analytical system using disposable, open-tubular ion exchange clean-up precolumns coupled in-line to capillary electrophoresis for direct injection of biological samples is presented. The clean-up precolumns were prepared from fused silica capillaries by thermally initiated layer-by-layer polymerization of poly(butadiene-maleic acid) (PBMA) directly on the capillary wall. Typically, 6 cm long precolumns with 4-layers of PBMA were used for sample pretreatment. A robust and reproducible coupling between the precolumn (75 μm ID) and the analytical capillary (50 μm ID) was achieved using an inexpensive, commercially available low dead volume union. No extra dispersion of the analyte zones was observed. Proteins and other high molecular weight compounds from biological sample matrices were retained on the cation-exchanger sites of the precolumn, which eliminated their adsorption on analytical capillary walls and ensured stable electroosmotic flow and migration times of target analytes. Unretained small inorganic cations migrated freely into the analytical capillary for separation and detection. Applicability of the sample clean-up procedure was proved by determination of major inorganic cations in blood serum and plasma samples using capillary electrophoresis with contactless conductivity detection. Separations were performed in background electrolyte solution consisting of 15 mM L-arginine, 12.5 mM maleic acid, 3 mM 18-crown-6 at pH 5.5 and repeatabilities of migration times and peak areas were below 1.5% and 7.3%, respectively. Less than 1 μL of biological sample was required for injection.     

Determination of inorganic anions in human saliva by zwitterionic micellar capillary electrophoresis

Capillary electrophoresis (CE) using sulfobetaine-type zwitterionic micelles as the background electrolyte (BGE) has been used to determine inorganic anions in human saliva. The zwitterionic micelles resulted in unique migration behavior for the separation of inorganic anions. They also prevented adsorption of proteins on the inner wall of the capillary. These properties of the zwitterionic micelles enabled the direct determination of inorganic anions in human saliva. Three species of inorganic anions, NO2-, NO3-, and SCN-, were found in real samples and the analysis was achieved within 3 min. Direct UV-absorption was used as the detection method and the detection limits for these anions were 2.0, 1.0, and 5.0 micromol L(-1), respectively (0.09, 0.06, and 0.30 microg mL(-1)).     

Simultaneous capillary electrophoretic separation of small anions and cations after complexation with ethylenediaminetetraacetic acid

A new approach for simultaneous separation of small inorganic and organic anions and metal cations by capillary electrophoresis is demonstrated. Metal cations in the sample are transformed into their chelates with EDTA and are separated together with the anions using an anionic separation mode. Simultaneous separation of 19 common anions and cations was achieved in about 6 min with the electrolyte containing 5 mM K2CrO4, 3 mM boric acid, 35 microM cetyltrimethylammonium bromide and 12 microM EDTA at pH 8. Limits of detection (s/n = 3) were in the range from 4 ppb for Cl- up to 1250 ppb for Cu-EDTA and RSDs of peak areas ranged from 1.4% for Cl- up to 8.5% for Mn-EDTA chelate. The practical applicability of the method was demonstrated on the analysis of anions and cations in various water samples.     

Capillary electrophoresis of organic cations at high salt concentrations

At concentrations of 100 mM or higher the chemical nature of both the cation and anion in the background electrolyte (BGE) can be varied to manipulate the migration times of protonated aniline cations. Significant differences were noted with Li+, Na+ and K+ for capillary electrophoretic runs carried out at pH 3. However, much greater differences in migration times were observed at acidic pH values when the BGE contained protonated cations of aliphatic amines. Analyte migration became progressively slower in the series: methylamine, diethylamine, diethylamino ethanol and triethylamine. A major part of this effect was attributed to an opposing electroosmotic flow (EOF) resulting from a positively-charged coating of the capillary surface with the amine cations in the BGE via a dynamic equilibrium. The amine cations also interact in solution with the analyte ions to reduce their electrophoretic mobilities. Migration times of anilines could be varied systematically over a broad range according to the BGE amine cation selected. Excellent separations of seven closely-related anilines were obtained with the new system.     

Determination of ammonia, creatinine and inorganic cations in urine using CE with contactless conductivity detection

CE with capacitively coupled contactless conductivity detection (C(4)D) was used to determine waste products of the nitrogen metabolism (ammonia and creatinine) and of biogenic inorganic cations in samples of human urine. The CE separation was performed in two BGEs, consisting of 2 M acetic acid + 1.5 mM crown ether 18-crown-6 (BGE I) and 2 M acetic acid + 2% w/v PEG (BGE II). Only BGE II permitted complete separation of all the analytes in a model sample and in real urine samples. The LOD values for the optimized procedure ranged from 0.8 microM for Ca(2+) and Mg(2+) to 2.9 microM for NH(4)(+) (in terms of mass concentration units, from 7 microg/L for Li(+) to 102 microg/L for creatinine). These values are adequate for determination of NH(4)(+), creatinine, Na(+), K(+), Ca(2+) and Mg(2+) in real urine samples.     

An air‐assisted flow‐gating interface for capillary electrophoresis

A new kind of flow gating interface (FGI) has been designed for online connection of CE with flow‐through analytical techniques. The sample is injected into the separation capillary from a space from which the BGE was forced out by compressed air. A drop of sample solution with a volume of 75 nL is formed between the outlet of the delivery capillary supplying the solution from the flow‐through apparatus and the entrance to the CE capillary; the sample is hydrodynamically injected into the CE capillary from this drop. The sample is not mixed with the surrounding BGE solution during injection. The functioning of the proposed FGI is fully automated and the individual steps of the injection process are controlled by a computer. The injection sequence lasts several seconds and thus permits performance of rapid sequential analyses of the collected sample. FGI was tested for the separation of equimolar 50 μM mixture of the inorganic cations K⁺, Ba²⁺, Na⁺, Mg²⁺, and Li⁺ in 50 mM acetic acid/20 mM Tris (pH 4.5) as BGE. The obtained RSD values for the migration times varied in the range 0.7–1.0% and the values for the peak area were 0.7–1.4%; RSD were determined for ten repeated measurements.     

Capillary electrophoresis analysis of inorganic cations in post-blast residue extracts applying a guanidinium-based electrolyte and bilayer-coated capillaries

A new CE method was developed for the identification and quantitation of inorganic cations in post‐blast residues. The simultaneous analysis in 20 min total runtime of eight cations in post‐blast residues (ammonium, potassium, monomethylammonium, calcium, sodium, magnesium, strontium), plus lithium cation as the internal reference, was carried out with a BGE involving a non‐CMR (carcinogenic, mutagenic, and harmful to reproduction) chromophore (guanidinium cation) and a double‐layer modified capillary (hexadimethrine bromide/polyvinylsulfonate). A study of UV detection conditions using guanidinium ion as the probe led us to set the analysis and reference wavelengths and their associated bandwidths as well as the probe concentration fixed at 15 mM. The successive multiple ionic‐polymer layer approach limited the cation adsorption on capillary wall and improved the EOF stability. These caused a significant improvement in method repeatability. Intermediate precisions were 2.4% for corrected areas and 1.3% for normalized migration times. Limits of detection close to 1 mg/L for all cations were obtained. The matrix effects were studied with chemometric approach for different matrices representative of those collected after explosion. Tests with blank matrix extracts of soil, cloth, and with simulated matrix extract containing 800 mg/L Ca²⁺ and 500 mg/L Fe²⁺ were carried out and no significant matrix effects were observed. Finally, analyses of real residues collected after cash dispenser and homemade firework explosions demonstrate excellent correlation between the CE results and those obtained with the ion chromatography method used routinely.     

Optimization of the chiral resolution of baclofen by capillary electrophoresis using beta-cyclodextrin as the chiral selector

The chiral resolution of baclofen was achieved by capillary electrophoresis using a fused-silica capillary (60 cm x 75 microm ID). The background electrolyte (BGE) was phosphate buffer (pH 7.0, 50 mM)-acetonitrile (95:5 v/v) containing 10 mM beta-cyclodextrin. The applied voltage was 15 kV. The values of alpha and R(s) were 1.06 and 1.00, respectively. The electrophoretic conditions were optimized varying the pH and the ionic strength of the BGE, concentrations of beta-cyclodextrin and acetonitrile and the applied voltage.     

Determination of inorganic anions in human saliva by zwitterionic micellar capillary electrophoresis

Capillary electrophoresis (CE) using sulfobetaine-type zwitterionic micelles as the background electrolyte (BGE) has been used to determine inorganic anions in human saliva. The zwitterionic micelles resulted in unique migration behavior for the separation of inorganic anions. They also prevented adsorption of proteins on the inner wall of the capillary. These properties of the zwitterionic micelles enabled the direct determination of inorganic anions in human saliva. Three species of inorganic anions, NO₂ ^–, NO₃ ^–, and SCN^–, were found in real samples and the analysis was achieved within 3 min. Direct UV-absorption was used as the detection method and the detection limits for these anions were 2.0, 1.0, and 5.0 μmol L^–1, respectively (0.09, 0.06, and 0.30 μg mL^–1).     

Determination of salbutamol enantiomers in human urine using heptakis(2,3-di-O-acetyl-6-O-sulfo)-beta-cyclodextrin in nonaqueous capillary electrophoresis

Nonaqueous capillary electrophoresis (NACE) was successfully applied to the resolution and the determination of salbutamol enantiomers in urine samples using heptakis(2,3-di-O-acetyl-6-O-sulfo)-beta-cyclodextrin (HDAS-beta-CD). After optimization of the electrophoretic parameters, namely the background electrolyte (BGE) composition and the HDAS-beta-CD concentration, salbutamol enantiomers were completely resolved using a BGE made up of 10 mM ammonium formate and 15 mM HDAS-beta-CD in methanol acidified with 0.75 M formic acid. Isoprenaline was selected as internal standard. Solid-phase extraction (SPE) was used for sample cleanup prior to the CE separation. Different sorbents involving polar, nonpolar interactions or dual retention mechanisms were evaluated and extraction cartridges containing both nonpolar and strong cation-exchange functionalities were finally selected. Salbutamol enantiomers recoveries from urine samples were determined. The method was then successfully validated using a new approach based on accuracy profiles over a concentration range from 375 to 7500 ng/mL for each enantiomer.