System peaks and optimization of anion separation in capillary electrophoresis with non-reversed electroosmotic flow

We studied system peaks present in the electropherograms obtained in the separation of anions by capillary electrophoresis with indirect spectrophotometric detection and cathode electroosmotic flow (EOF) with a chromate background electrolyte. The system peaks correspond to the zones with changed concentration of the background electrolyte; they formed when the zones of each analyte passed through the outlet of the capillary and then moved towards the EOF detector. It has been revealed that the height and area of the system peaks linearly depends on the concentration of the corresponding anion and the areas of the system peaks can achieve 10% of the anion peak area. An algorithm has been proposed for the determination of the optimal conditions for anion separation using hydrodynamic pressure for the regulation of the EOF flow rate. This algorithm prevents the overlapping of the anion and system peaks.     

The preparation of background electrolytes in capillary zone electrophoresis: golden rules and pitfalls

In this article the methodology of the design of suitable background electrolytes (BGEs) in capillary zone electrophoresis (CZE) is described. The principal aspects of the role of a BGE in CZE are discussed with respect to an appropiate migration behavior of analytes, including the transport of the electric current, the buffering of pH, the Joule heat, the electro-endosmotic flow (EOF) and the principal migration and detection modes. The impact of the composition of the BGE upon migration and detection is discussed. It is shown that the total concentration of the BGE is a principal factor and the adjustment of migrating analyte zones according to the Kohlrausch regulating function (KRF) is the principal effect in most of the sample stacking techniques. The number of co-ions and their properties are of key importance for peak shapes of the analyte peaks and for the existence of system zones. The detection of UV-transparent analytes may advanteously be done in the indirect UV mode, by using UV-absorbing co-ions, however, both peaks and dips may be expected in the UV trace in case of multiple co-ionic BGEs. Properties of BGEs can be predicted applying mathematical models and it is shown that with SystCharts, predictions can be given concerning the existence of system zones, detection modes and the peak shapes of analytes for a given BGE. Practical examples of methodological considerations are given in the design of suitable BGEs for four principal combinations of migration and detection modes. The properties of the BGEs selected are exemplified with experimental results. Golden rules are summarized for the preparation of suitable BGEs in CZE.     

Capillary electrophoretic analysis of anions in a multi-anion background electrolyte: Interference of system peaks

Summary This study deals with the simultaneous analysis of UV-transparent anions by capillary electrophoresis with indirect UV-detection. With a background electrolyte (BGE) based on UV-absorbing chromate and UV-transparent borate, the interference of system peaks with those of sample anions (chloride, sulfate, citrate, phosphate) is shown. The existence of such system peaks, and their position in relation to the peaks of the sample anions, are explained on the basis of the eigenpeak theory proposed by Poppe [1]. With this BGE the system peaks were manifested as a negative peak followed by a positive peak. Their shapes depended on the relative mobilities of the analyte and BGE anions and their areas depended on the amount of sample. The mobility of the system peak depends on the borate/boric acid mobility, which was adjusted by slight variation of the pH close to its pK _a-pH is the key factor governing system-peak mobility. When the locations of the system peaks are optimized, the quantification of citrate can be achieved; this was successfully used for determination of anions in milk.     

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.     

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.     

A fresh look into background electrolyte selection for capillary electrophoresis‐laser induced fluorescence of peptides and proteins

This study reports a reinvestigation of background electrolyte selection strategy for performance improvement in CE‐LIF of peptides and proteins. This strategy is based on the employment of high concentrations of organic species in BGE possessing high buffer capacity and low specific conductivity in order to ensure excellent stacking preconcentration and separation resolution of fluorescently tagged peptides and proteins. Unlike universal UV detection, the use of such BGEs at high concentrations does not lead to degradation of LIF detection signals at the working excitation and emission wavelengths. At the same buffer ionic strength, pH and electric field, an “inorganic‐species‐free” BGE (or ISF BGE) for CE‐LIF of fluorescently labeled beta amyloid peptide Aβ 1–42 (a model analyte) offered a signal intensity and peak efficiency at least three‐times higher than those obtained with a conventional BGE normally used for CE‐LIF, while producing an electric current twice lower. Good peak performance (in terms of height and shape) was maintained when using ISF BGEs even with samples prepared in high‐conductivity phosphate buffer saline matrix. The advantageous features of such BGEs used at high concentrations over conventional ones in terms of high separation resolution, improved signal intensities, tuning of EOF magnitudes and minimization of protein adsorption on an uncoated fused silica capillary are demonstrated using Alexa‐488‐labelled trypsin inhibitor. Such BGE selection approach was applied for investigation of separation performance for CE‐LIF of ovalbumin labelled with different fluorophores.     

Success and failure with phthalate buffers in capillary zone electrophoresis

Phthalate buffers are currently used in capillary electrophoresis as robust electrolyte systems for indirect detection. This contribution demonstrates that these buffers show regularly not only successful regions of mobilities of analytes (sample window) but also regions of failure where the migration of analytes is strongly deteriorated due to the presence of a system zone. System zones in phthalate buffers may be easily detected by UV detection and manifest themselves as peaks or dips. Peak shape diagrams are advantageously used for the prediction of the migration behavior of system zones in phthalate background electrolyte (BGE) systems at various pH. It is shown that the mobility of the system zone varies strongly with pH, is practically zero at pH values below 4 and above 7, and shows a maximum at pH 5. Thus, the system peak may coincide either with the peaks of various analytes or with the electroosmotic flow (EOF) peak. Experiments are given showing the effects of such coincidences as, e.g., zigzag detection patterns, double EOF peaks, and/or unusually broad peaks/dips. The message of this contribution is to show how to understand the electrophoretic properties of phthalate BGEs that, regardless of possible failure regions, may be successfully used in the analytical practice of capillary zone electrophoresis (CZE).     

Nonaqueous capillary electrophoresis with alcoholic background electrolytes: separation efficiency under high electrical field strengths

The effect of high voltage on capillary electrophoresis (CE) separations of anionic analytes in nonaqueous separation media was investigated. Methanol, ethanol, 1-propanol, and 1-butanol were tested as background electrolyte (BGE) solvents. Experiments were carried out with a laboratory-built CE instrument suitable for high-voltage separations. Potentials up to 60 kV were applied with reversed polarity to generate unusually high field strengths (e.g. 2000 Vcm-1) and so achieve fast and efficient separations. Highest separation efficiencies were obtained with propanol as BGE solvent, and the dependency of the efficiency on the separation voltage was more or less linear. With the other alcohols, separation efficiency decreased or remained roughly constant with increasing absolute voltage. The separation efficiencies are discussed in terms of longitudinal diffusion, Joule heating, and analyte interaction with the capillary wall. Capillary preconditioning had a varied effect on the separations in the different BGEs as the BGE and the conditioning process affected the electroosmotic flow (EOF) velocity and direction.     

Off-line integration of CE and MALDI-MS using a closed-open-closed microchannel system

In this work, a new technique for off-line hyphenation between CE and MALDI-MS is presented. Two closed fused-silica capillaries were connected via a silicon chip comprising an open microcanal. The EOF in the system was evaluated using mesityloxide or leucine-enkephalin as a sample and with a running buffer that rendered the analyte neutrally charged. Comparison was made between the EOF in a closed system (first capillary solely included in the electrical circuit) and in a closed-open system (first capillary and microcanal included in the electrical circuit). It was concluded that the experimental values of the EOF agreed with the theory. The influence of the capillary outer diameter on the peak dispersion was investigated using a closed-open-closed system (first capillary, microcanal and second capillary included in the electrical circuit). It was clearly seen that a capillary with 375 microm od induced considerably higher peak dispersion than a 150 microm od capillary, due to a larger liquid dead volume in the connection between the first capillary outlet and the microcanal. Mass spectrometric analysis has also been performed following CE separation runs in a closed-open-closed system with cytochrome c and lysozyme as model proteins. It was demonstrated that a signal distribution profile of the separated analytes could be recorded over a 30 mm long microcanal.     

Separation of organic cations using novel background electrolytes by capillary electrophoresis

A background electrolyte for capillary electrophoresis containing tris(-hydroxymethyl) aminomethane (THAM) and ethanesulfonic acid (ESA) gives excellent efficiency for separation of drug cations with actual theoretical plate numbers as high as 300,000. However, the analyte cations often elute too quickly and consequently offer only a narrow window for separation. The best way to correct this is to induce a reverse electroosmotic flow (EOF) that will spread out the peaks by slowing their migration rates, but this has always been difficult to accomplish in a controlled manner. A new method for producing a variable EOF is described in which a low variable concentration of tributylammonium- or triethylammonium ESA is added to the BGE. The additive equilibrates with the capillary wall to give it a positive charge and thereby produce a controlled opposing EOF. Excellent separations of complex drug mixtures were obtained by this method.     

Determination of free cyanide and zinc cyanide complex by capillary electrophoresis

A capillary electrophoretic (CE) protocol was developed for the separation and quantification of free cyanide and zinc cyanide complex, two key species in gold cyanidation of zinc-bearing sulfidic ores. Several common carrier electrolytes were implemented in an indirect UV detection method. The effect of electric field strength, injection volume, concentration of electro-osmotic flow (EOF) modifier and UV-absorbing agent in background electrolyte (BGE) was examined while peak height, peak area and noise were considered for optimization. The best results were obtained using a BGE that contained 35 mM sodium chromate, 12 mM free cyanide and 0.45 mM hexamethonium bromide at pH 10.5. Free cyanide concentration was compared to that measured with the conventional silver nitrate titration method in solutions containing free cyanides and weak cyano-complexes. The developed CE protocol proved very robust in capturing the concentration of free cyanides (4% error) unlike the titration method which exhibited substantial sensitivity to the interfering weak cyano-complexes (38% error).     

EOF measurement by detection of a sampling zone with end-channel amperometry in microchip CE

A simple method for EOF measurement by detection of sampling zones with end-channel amperometry in microchip CE is developed. This method is based on the principle of the Kohlrausch regulating function (KRF). A dilute electroactive ionic species is added to the BGE as a continuously eluting electrophore which is used as a probe. When a BGE-like sample at a different concentration is injected, a peak of sampling zone appears and the migration time is related to EOF. In a microchip CE with hybrid PDMS/glass channel, a cathodic EOF of the hybrid glass/PDMS microchip was measured by end-channel amperometry; the effects of sample concentration and different probes on EOF rate were discussed. The present method was applied to monitor EOF rates in glass and in PDMS microchips. There was no significant difference between the values of EOF rates measured by the present method and the current-monitoring method. Detection of nonelectroactive analytes K(+), Na(+), and Li(+) can also be accomplished by the indirect amperometric method. Hence, the effective mobility of analyte can be accurately obtained.     

Thermal marks as a signal processing aid for a portable capillary electropherograph

The interpretation of raw signals in capillary CE can be challenging if there are unknown peaks, or the signal is corrupt due to baseline fluctuations, EOF velocity drift, etc. Signal processing could be required before results can be interpreted. A suite of signal processing algorithms has been developed for CE data analysis, specifically for use in field experiments for the detection of nerve agents using portable CE instruments. Everything from baseline correction and electropherogram alignment to peak matching and identification is included in these programs. Baseline correction is achieved by interpolating a new baseline according to points found using all local extremes, by applying an appropriate outliers test. Irreproducible migration times are corrected by compensating for EOF drift, measured with the aid of thermal marks. Thermal marks are small disturbances in the capillary created by punctual heating that move with the velocity of EOF. Peaks in the sample electropherogram are identified using a fuzzy matching algorithm, by comparing peaks from the sample electropherogram to peaks from a reference electropherogram.     

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.     

Use of chiral amino acid ester‐based ionic liquids as chiral selectors in CE

In this study, the applicability of a chiral ionic liquid (CIL) as the sole chiral selector in CE was investigated for the first time. In particular, five amino acid ester‐based CILs were synthesized and used as additives in the BGE in order to evaluate their chiral recognition ability. The performance of these CILs as the sole chiral selectors was evaluated by using 1,1′‐binaphthyl‐2,2‐diylhydrogenphosphate (BNP) as the analyte and by comparing the resolution values. Different parameters were examined, such as the alkyl group bulkiness and the configuration of the cation, the anion type of the CIL and its concentration, and the pH of the BGE, in order to optimize the separation of the enantiomers and to demonstrate the effect that each parameter has on the chiral‐recognition ability of the CIL. Baseline separation of BNP within 13 min was achieved by using a BGE of 100 mM Tris/10 mM sodium tetraboratedecahydrate (pH 8) and a chiral selector of 60 mM l ‐alanine tert butyl ester lactate. The run‐to‐run and batch‐to‐batch reproducibilities were also evaluated by computing the %RSD values of the EOF and the two enantiomer peaks. In both cases, very good reproducibilities were observed, since all %RSD values were below 1%.     

Efficient and reproducible analysis of peptides by capillary electrophoresis using noncovalently bilayer-coated capillaries

The usefulness of a noncovalent capillary coating consisting of two layers of oppositely charged polymers for the separation of peptides with capillary electrophoresis (CE) was studied. Capillaries were coated simply by subsequently flushing with solutions of 1% m/v Polybrene and 1% v/v poly(vinylsulfonate) (PVS) forming a bilayer, which showed to produce a strong and highly reproducible electroosmotic flow (EOF) at low pH. Using this coating in combination with a background electrolyte (BGE) containing sodium phosphate (pH 2.5) and 0.01% v/v PVS, initially broadened and overlapping peaks were obtained for some test peptides. By omitting the PVS from the BGE, the peak width and shape of the peptides improved resulting in baseline separation. A systematic study of the influence of the BGE composition showed that considerable further enhancement of the separation efficiency was achieved by increasing the ionic strength of the BGE. Using a BGE of 200 mM tris(hydroxymethyl)aminomethane (Tris)-phosphate (pH 2.5) plate numbers for the peptides were in the 300 000-600 000 range and the relative standard deviation of the peptide migration times was less then 0.3% (n = 5). The use of Tris-phosphate instead of sodium phosphate allowed the current to stay within acceptable limits when 30 kV was used as separation voltage. Overall, the bilayer coating showed a remarkable EOF repeatability, as well as long-term stability. Compared to bare fused-silica capillaries the intraday and interday repeatability of migration times was very favorable and coated capillaries could be used for over a month performing analyses with low and high ionic strength BGEs without any performance deterioration. The usefulness of the bilayer-coated capillaries for the analysis of positively charged peptides was demonstrated by the fast and efficient separation of various closely related enkephalins and the baseline separation of an isomeric peptide/peptoid couple exhibiting efficiencies of over 550 000 plates.     

Two-dimensional nano-liquid chromatography-mass spectrometry system for applications in proteomics

Transient isotachophoresis-capillary zone electrophoresis with artificial seawater as the background electrolyte (BGE) was improved to further lower the limit of detection (LOD) for determination of nitrite and nitrate in seawater. By lowering the pH of BGE, the difference between effective mobility of nitrite and that of nitrate increased, thereby permitting increased sample volumes to be tolerated and their LOD values to decrease. Artificial seawater with pH adjusted to 3.0 using phosphate buffer was adopted as the BGE. To reverse electroosmotic flow (EOF), a capillary was flushed with 0.1 mM dilauryldimethylammonium bromide for 3 min before the capillary was filled with the BGE. Limits of detection (LODs) for nitrite and nitrate were 2.7 and 3.0 microg/l (as nitrogen), respectively. The LODs were obtained at a signal-to-noise ratio of 3. Values of the relative standard deviation (RSD) of peak area for these ions were 2.0 and 0.75%, respectively, when nitrite concentration was 0.05 mg/l and that of nitrate was 0.5 mg/l. The RSDs of peak height were 4.4 and 2.3%. The RSD values of migration time for these ions were 0.19 and 0.17%. The proposed method was applied to determination of nitrite and nitrate in a proposed certified reference material for nutrients in seawater, MOOS-1, distributed by the National Research Council of Canada. Results agreed with the assigned tolerance interval. This method was also applied to determination of these ions in seawater collected around Osaka Bay. Results nearly agreed with those obtained by a conventional spectrophotometric method.     

Using multiple short-end injections to develop fast electrophoretic separations--applications in iodide analysis

The aim of this study was to develop a fast CE separation method by using multiple short-end injections in a capillary coated with quaternary ammonium chitosan (HACC), in order to determine the iodide content of pharmaceutical formulations. The BGE was composed of 20 mM tris(hydroxymethyl)aminomethane and 11 mM hydrochloric acid, at pH 8. The internal standard used was thiocyanate. Separations were performed in a fused silica capillary (32 cm total length, 8.5 cm effective length and 50 μm i.d.) coated with HACC and direct UV detection at 220 nm. EOF was modified by flushing the capillary with polymeric solution, resulting in a semi-permanent coating of controlled and stable EOF. The EOF was anodic at pH 8. Different strategies, using single and multiple injection short-end configurations, were studied to develop a CE method that resulted in a maximum number of iodide samples analyzed per hour: one plug and flush (Sflush) 35 samples/h, one plug without flush (SWflush) 76 samples/h, four plugs and flush (Mflush) 61 samples/h, and four plugs without flush (MWflush) 80 samples/h. Using the multiple injection configuration, it was possible to inject up to four plugs using spacer electrolytes with good separation efficiency and selectivity. The voltage application time needed to separate the eight peaks (iodide and thiocyanate) with MWflush was only 12s. The method was validated and samples were analyzed using MWflush. Good linearity (R(2)>0.999); a limit of detection 0.4 mg L(-1); intermediate precision better than 3.8% (peak area) and recovery in the range of 99-102% were obtained.     

Low EOF rate measurement based on constant effective mobility in microchip CE

A new method for quickly determining low EOF rates (micro(EOF)) in microchip CE is described. The measurement is based on the notion that the effective mobility (micro(eff)) of an analyte is a constant in a certain BGE. The micro(eff) of an analyte is determined in a reference fast-electroosmosis microchip, and the apparent mobility (micro(app)) of the analyte can be determined in the microchip with unknown low electroosmosis, and then micro(EOF) in the low-electroosmosis microchip can be calculated according to the equation mu(EOF) = micro(app) - micro(eff). By an indirect method or other conventional methods, micro(eff) can be easily measured in the reference microchip. The proposed method is particularly useful for low-electroosmosis measurements in wall-modified microchannels.     

Separation and determination of homologues of linear alkylbenzenesulfonates by nonaqueous capillary zone electrophoresis using alkylammonium salts in ethanol

The separation of linear alkylbenzene sulfonates (LAS) by nonaqueous capillary electrophoresis (NACE) using negative polarity, and a buffer containing acetic acid and an alkylamine in nonaqueous ethanol, has been investigated. Several primary, secondary, and tertiary alkylamines with alkyl chains of different length were compared. The solutes travelled against the electroosmotic flow (EOF), and at the same time were braked by association with the alkylamine molecules or with the alkylammonium ions. The best resolution between adjacent LAS homologues (R approximately 2.1), partial isomer resolution in two peaks, and at the same time an excellent repeatability, was obtained with a small dipentylamine excess over the acetic acid. When the buffer concentration increased, resolution between the homologues increased slightly (R approximately 2.4), and a different isomer group was partially separated. A background electrolyte (BGE) containing 10 mM acetic acid and 20 mM dipentylamine to separate and quantify the homologues within 25 min is recommended. The isomer peak profile with up to three peaks can be estimated using this buffer and another one with 80 mM acetic acid and 90 mM dipentylamine. The former BGE was used to determine LAS in liquid and powder laundry detergents. The detection limit for the determination of total LAS in these products was 2.5 microg mL(-1), and the peak area and migration time interday repeatabilities were below 4.3 and 2.8%, respectively.