Computer-assisted choice of discrete spacers for anionic isotachophoresis separations

In isotachophoresis (ITP), the sample constituents migrate, depending on their concentrations in the loaded sample, either in fully developed zones or in the boundary layers between the zones of constituents of the corresponding effective mobilities. The latter (spike) migration mode is analytically beneficial in selective detections of trace analytes, especially, when appropriately chosen discrete spacers minimize detection interferences due to matrix constituents. To facilitate a search for suitable mixtures of discrete spacers, a two-step calculation procedure was developed in this work. Using a pool of discrete spacers consisting of 42 anionic and zwitterionic constituents, this procedure was shown effective in the anionic ITP separations performed at pH = 6.5-10.0. Besides the predictions of the migration orders, it was helpful in identifying the spacing constituents that could cause resolution problems due to an uncertainty with which pH of the leading electrolyte solution is known. The ionic mobility and pKa data, taken for the spacing constituents from the literature and the ones obtained from the ITP experiments carried out in this work, were used in the calculations performed in a context with the choice of spacers. Although the data obtained from the ITP experiments provided better results, small uncertainties with which they were acquired (attributable to fluctuations in the experimental conditions) set practical limits in the calculation based choice of multi-component mixtures of the spacing constituents.     

Enhancement of analyte detectability in isotachophoresis exploiting spectrophotometric diode‐array detection with chemometric data processing

The potentialities of capillary ITP combined with diode‐array detection (DAD) with subsequent chemometric data processing have been investigated in this work. A series of different migration configurations were created using model analytes, interferents and appropriate spacers. Special attention has been paid not only to constituents migrating in fully developed ITP zones but also to the spike mode of ITP migration. The purity assessment and identity confirmation of model analytes migrating in both modes were performed by means of multivariate curve resolution and target transformation factor analysis (TTFA). Their successful applications have revealed a smart way to increase in the analytical information obtained by ITP separation even in the instance of trace analysis.     

Multiphasic electrophoresis with diversified spacers

The principle and some applications of multiphasic electrophoresis with diversified spacers are described. The method combines previously published concepts of introducing highly diversified spacers into the stack of an isotachophoretic system with the theory of snow- and telescope-electrophoresis. The link between this electrophoretic method and isotachophoresis on the one hand and monophasic zone electrophoresis on the other hand is exemplified. The importance of the nature of the spacers as well as of the pH and ions in the leading and terminating zone is illustrated. Details are given concerning the experimental set-up. This electrophoretic technique could serve as an alternative to isoelectric focusing.     

Immunobinding‐induced alteration in the electrophoretic mobility of proteins: An approach to studying the preconcentration of an acidic protein under cationic isotachophoresis

The objective of this study is to explore an approach for analyzing negatively charged proteins using paper‐based cationic ITP. The rationale of electrophoretic focusing the target protein with negative charges under unfavorable cationic ITP condition is to modify the electrophoretic mobility of the target protein through antigen‐antibody immunobinding. Cationic ITP was performed on a paper‐based analytical device that was fabricated using fiberglass paper. The paper matrix was modified with (3‐aminopropyl)trimethoxysilane to minimize sample attraction to the surface for cationic ITP. Negatively charged BSA was used as the model target protein for the cationic ITP experiments. No electrophoretic mobility was observed for BSA‐only samples during cationic ITP experimental condition. However, the presence of a primary antibody to BSA significantly improved the electrokinetic behavior of the target protein. Adding a secondary antibody conjugated with amine‐rich quantum dots to the sample further facilitated the concentrating effect of ITP, reduced experiment time, and elevated the stacking ratio. Under our optimized experimental conditions, the cationic ITP‐based paper device electrophoretically stacked 94% of loaded BSA in less than 7 min. Our results demonstrate that the technique has a broad potential for rapid and cost‐effective isotachphoretic analysis of multiplex protein biomarkers in serum samples at the point of care.     

Window optimization in isotachophoresis superimposed on capillary zone electrophoresis

A sample stacking procedure to which a specific combination of electrolyte solutions is applied is isotachophoresis (ITP) superimposed on capillary zone electrophoresis (CZE), a so-called ITP/CZE system. In ITP/CZE some components migrate in an ITP fashion on top of a background electrolyte, and the other analytes migrate in a zone electrophoretic manner. For such a system, the leading electrolyte consists of a mixture of an ionic species, L1, of high mobility (the leading ion of the ITP system), an ionic species, L2, of low mobility (the coions of the CZE system), and a buffering counter-ionic species, whereas the terminating solution only contains the ionic species L2 and the buffering counterions. The zones of the components migrating in the ITP/CZE mode are sharp owing to the self-correcting properties of the zones and the concentrations of the L1 ions of the system. Mobility windows can be calculated, indicating which ions can migrate in the ITP/CZE mode. In this article mobility windows are calculated by applying both strong and weak acids as L1 and L2 ions and it appears that mobility windows can be optimized by chosing different ratios of L1 and L2 as well as different pH values. It is possible to construct very narrow mobility windows, and thereby choose which component of a sample solution can be concentrated, and to what concentration, in a very selective way. The big advantage of ITP/CZE compared with applications such as transient ITP and transient stacking is that the stacked sample ionic species migrate in the ITP mode during the whole experiment; furthermore, they do not destack. Experimentally obtained electropherograms validate the calculated mobility windows for the ITP/CZE mode.     

Charge-reduced nano electrospray ionization combined with differential mobility analysis of peptides, proteins, glycoproteins, noncovalent protein complexes and viruses

This study explores the potential of a novel electrospray-based method, termed gas-phase electrophoretic mobility molecular analysis (GEMMA), allowing the molecular mass determination of peptides, proteins and noncovalent biocomplexes up to 2 MDa (dimer of immunglobulin M). The macromolecular ions were formed by nano electrospray ionization (ESI) in the 'cone jet' mode. The multiple charged state of the monodisperse droplets/ions generated was reduced by means of bipolar ionized air (generated by an alpha-particle source) to yield exclusively singly charged positive and negative ions as well as neutrals. These ions are separated subsequently at atmospheric pressure using a nano differential mobility analyzer according to their electrophoretic mobility in air. Finally, the ions are detected using a standard condensation particle counter. Data were expressed as electrophoretic mobility diameters by applying the Millikan equation. The measured electrophoretic mobility diameters, or Millikan diameters, of 32 well-defined proteins were plotted against their molecular weights in the range 3.5 to 1920 kDa and exhibited an excellent squared correlation coefficient (r(2) = 0.999). This finding allowed the exact molecular weight determination of large (glyco)proteins and noncovalent biocomplexes by means of this new technique with a mass accuracy of +/-5.6% up to 2 MDa at the femtomole level. From the molecular masses of the weakly bound, large protein complexes thus obtained, the binding stoichiometry of the intact complex and the complex stability as a function of pH, for example, can be derived. Examples of specific protein complexes, such as the avidin or catalase homo-tetramer, are used to illustrate the potential of the technique for characterization of high-mass biospecific complexes. A discussion of current and future applications of charge-reduced nano ESI GEMMA, such as chemical reaction monitoring (reduction process of immunglobulin G) or size determination of an intact virus, a supramolecular complex, and monitoring of partial dissociation of a human rhinoviruses, is provided.     

Capillary isoelectric focusing-mass spectrometry for shotgun approach in proteomics

We report on capillary isoelectric focusing-mass spectrometry (CIEF-MS) of complex peptide mixtures in the absence of carrier ampholytes. Furthermore, the use of low concentrations of carrier ampholytes as mere spacers is investigated. Carrier ampholytes are complex mixtures of amphoteric compounds with high buffering capacity. Since all peptides are amphoteric compounds by themselves, the use of carrier ampholytes may be superfluous to establish a stable pH gradient in CIEF analysis of protein digests. Our research showed that when carrier ampholytes are omitted, the analyte ions are not focused at their isoelectric point. The analytes are charged, leading to electrophoretic mobility uncharacteristic for CIEF. The method was tested for a five-protein-mixture at 0.02 mg/mL per protein and 0.05 mg/mL per protein. At the lower concentration, the analytes were stacked during the focusing process in only a limited length of the capillary. Therefore, the higher concentration led to better separation efficiency. It was found that at low concentration (0.20%) the carrier ampholytes could work as spacers. Though it led to sensitivity losses of 15-45%, this was compensated by the higher separation efficiencies seen. The method was evaluated with an eight-protein-mixture, of which all could be identified after performing MS/MS.     

A new multiphasic buffer system for sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins and peptides with molecular masses 100,000-1000, and their detection with picomolar sensitivity.

A novel multiphasic buffer system for high resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of dansylated and nondansylated proteins/peptides in the relative molecular mass (Mr) range of 100,000-1000 is described. The system, based on Jovin's theory of multiphasic zone electrophoresis, allows complete stacking and destacking of proteins/peptides within the above Mr range. The buffer system uses Bicine and sulfate as trailing and leading ion, respectively, and Bistris and Tris as counter ions in the stacking and separating phase, respectively. Through selection of two different counter ions--the characteristic feature of the present ionic system--the stacking limits of a multiphasic buffer system can be further widened, thus making it applicable to gel electrophoresis of a larger spectrum of rapidly migrating species, such as sodium dodecyl sulfate-proteins/peptides and nucleic acids, than has been possible previously. Highly sensitive detection methods for proteins as well as for polypeptides down to approximately Mr 1000 are described. Dansylated proteins/peptides were detected by their fluorescence either directly within the gel or following electroblotting into anion-exchange or polyvinylidene difluoride membranes. The latter procedure resulted in detection sensitivities of approximately 1 ng. Nondansylated proteins/peptides were either detected within the gel by colloidal Coomassie staining or by electroblotting into polyvinylidene difluoride membranes, followed by colloidal gold staining. Prior to both staining procedures the proteins/peptides were pretreated with glutardialdehyde in the presence of borate at near neutral pH values to generate protein/peptide polymers of poor solubility. For a given pH the efficiency of the latter procedure was significantly influenced by the nature of the buffer ion used in the fixation buffer. In contrast to conventional fixation procedures even small polypeptides (Mr 1000) were immobilized and approximately 15 ng and 0.75 ng could be detected after colloidal Coomassie and colloidal gold staining, respectively.     

Purification of ovalbumin and lysozyme from a commercial product by recycling isotachophoresis.

The aim of this work was to test the suitability of using recycling isotachophoresis (RITP) for the purification of ovalbumin (OVA) and/or lysozyme (LYSO) from a commercial OVA product containing LYSO and conalbumin (CAL) as major proteinaceous impurities. The search for suitable electrolyte systems and spacers was carried out by capillary isotachophoresis. RITP was performed in a recycling free-flow focusing apparatus in the batch mode with immobilization of the advancing zone structure via a controlled counterflow. Typically 700 mg of the commercial product were processed within 2 h. Enhancement of the sample load was achieved by a feed of sample under counterflow control. The collected fractions were analysed separately for conductivity, pH and ultraviolet absorption, and selected fractions were characterized by analytical capillary electrophoretic methods. All three proteins could be separated and fractionated using suitable spacers. Depending on the chosen conditions either OVA or LYSO could be purified in amounts larger than milligrams per hour (OVA 300 mg/h; LYSO 10 mg/h). The instability of CAL in solution prevented its isolation in the investigated configurations.     

Enantiomeric separation of clenbuterol by transient isotachophoresis-capillary zone electrophoresis-UV detection new optimization technique for transient isotachophoresis

A method for the in-line preconcentration and enantioseparation of clenbuterol by transient isotachophoresis-capillary zone electrophoresis-UV absorbance detection (transient ITP-CZE-UV) has been developed. It implies the use of dimethyl-beta-cyclodextrin as chiral selector and the application of a hydrodynamic counterflow during the ITP step. ITP is used to focus the sample constituents prior to CE whereas a counterpressure counterbalances the electrophoretic migration of the compounds. The sample is then focused and kept stationary in the proximity of the capillary inlet before CZE separation, leading to an extended-volume ITP-CZE system. A new strategy for the fast optimization of the counterpressure has been developed which implies the measurement of the hydrodynamic and electrophoretic velocities of the analyte during ITP. The in-line preconcentration and enantioseparation of clenbuterol selected as model compound was optimized using this method. Salbutamol was chosen as internal reference in order to check the reproducibility of the method. A 173-nl volume of aqueous ample solution was injected which implies an improvement of the injection volume of about 16 and a resolution of 4.8 was obtained for the clenbuterol enantiomers. A concentration detection limit of 10(-6) mol/l was readily achieved for clenbuterol and salbutamol using only 3 min ITP preconcentration in in-line counterflow transient ITP-CZE-UV. Thanks to its fast optimization, the method is applicable to any enantioseparation by means of only five very short preliminary measurements.     

On-line isotachophoretic preconcentration and gel electrophoretic separation of sodium dodecyl sulfate-proteins on a microchip

A microchip for integrated isotachophoretic (ITP) preconcentration with gel electrophoretic (GE) separation to decrease the detectable concentration of sodium dodecyl sulfate (SDS)-proteins was developed. Each channel of the chip was designed with a long sample injection channel to increase the sample loading and allow stacking the sample into a narrow zone using discontinuous ITP buffers. The pre-concentrated sample was separated in GE mode in sieving polymer solutions. All the analysis steps including injection, preconcentration, and separation of the ITP-GE process were performed continuously, controlled by a high-voltage power source with sequential voltage switching between the analysis steps. Without deteriorating the peak resolution, four SDS-protein analyses with integrated ITP-GE system resulted in a decreased detectable concentration of approximately 40-fold compared to the GE mode only. A good calibration curve for molecular weights of SDS-proteins indicated that the integrated ITP-GE system can be used for qualitative analysis of unknown protein samples.     

Direct determination of valproate in serum by zone electrophoresis–isotachophoresis on a column‐coupling chip

A feasibility study was performed using zone electrophoresis (ZE) coupled on‐line with isotachophoresis (ITP) sample pretreatment on a poly(methyl methacrylate) column‐coupling chip with integrated conductivity detection for direct determination of drugs in serum. Valproic acid (an antiepileptic drug), having a therapeutic range of 0.35–0.69 mmol/L (50–100 mg/L), was a test analyte while reference serum samples served as proteinaceous matrices. ITP provided in the ITP‐ZE combination a multitask sample pretreatment: (1) separation of the analyte from the serum matrix and its concentration into a narrow ITP band, (2) removal of the matrix constituents migrating in the ITP stack from the separation compartment of the chip, (3) ITP stacking of the drug released on a continuous electrophoretic decomposition of the drug‐protein complex. A high sample loadability, closely linked with the use of ITP in the first separation stage, made it possible to inject diluted serum samples with the aid of a 0.95 μL sample channel of the chip. Consequently, a 1–2 μmol/L concentration limit of quantitation for valproate from the response of the conductivity detector in the ZE stage of the combination was reached. The drug could be reliably determined in less than 10 minutes also in instances when its concentration in serum was below the lower value of the therapeutic range. 90–94% recoveries of valproate from serum samples were obtained in its direct ITP‐ZE determination when the filtration of the diluted serum (a 0.45 μm pore size filter) was the only pre‐column sample handling operation. No disturbances attributable to the precipitation of proteins from the loaded samples in the chip channels were detected.     

Isotachophoretic analysis of peptides. Selection of electrolyte systems and determination of purity

Capillary isotachophoresis (ITP) was applied to the qualitative and quantitative analysis of both natural and synthetic oligo- and polypeptides. Based on the mathematical model of acid-base equilibria for a general ampholyte, a procedure and a computer program for the calculation of the pH dependence of the effective and specific charge and effective mobility of peptides with known amino acid sequence were developed which allow the selection of electrolyte systems for peptide isotachophoretic analysis to be rationalized. Basic peptides (bovine pancreatic trypsin inhibitor, bull seminal isoinhibitors of trypsin, arginine vasopressin and adamantylamide-alanylisoglutamine) were analysed with a cationic ITP system at acidic pH. Neutral and acidic peptides (insulin, proinsulin, bull seminal isoinhibitors of trypsin, cow colostrum isoinhibitors of trypsin) were analysed with an anionic ITP system, mostly at alkaline pH. Peptide purity (electrophoretic homogeneity) was determined from the ITP degree of purity defined by a peptide itself and the zone length ratio of its admixtures. Enrichment of peptide in the sample during the purification procedure was measured by its zone length relative to unit mass of the amount of sample analysed.     

Effective Pre-Concentration and Analysis of Heavy Metals Using Ligand Step Gradient Focusing in Combination with Isotachophoresis

A capillary electrophoretic method for the pre-concentration of metals based on selective focusing of metal chelates with subsequent online ITP analysis was developed and verified. The selected ions of heavy metals (Pb²⁺ and Cd²⁺) were subjected to pre-concentration from the mixture and analysed. Focusing of the metals was carried out in a ligand step gradient, which was created by the addition of a convenient ligand agent to the regular stationary pH step gradient. The metal ions were continuously dosed into the column, where they were selectively trapped (with the exception of Na⁺) on the stationary ligand step gradient in the form of non-moving zones of citrate complexes with an effective charge of zero. After a detectable amount of analyte was accumulated, the dosing was stopped and the accumulated zones were mobilized to the analytical column, where they were analysed by the ITP method with conductivity or photometric detection. The electrolyte systems for the dosing (mode IEF), mobilizing (mode MBE), and analytical steps (mode ITP) were developed and tested. The cLOD was decreased by approximately two orders of magnitude using an acceptable dosing time of 25 min.     

Recent applications of conductivity detection in capillary and chip electrophoresis

The review provides a comprehensive survey of the recent applications of contact and contactless conductivity detection in capillary electrophoretic and chip electrophoretic analyses of a broad scale of compounds, from low-molecular-mass highly mobile small inorganic and organic ions, via medium-molecular-mass peptides and oligo- and polynucleotides up to high-molecular-mass biopolymers, proteins and nucleic acids fragments. The review presents also the recent developments in the construction of different types of conductivity detectors (detectors with galvanic contact of the sensing electrodes with the BGE and sample components, contactless conductivity detectors with capacitively coupled tubular and semitubular electrodes and combined conductivity/optical detectors) applied in the capillary electromigration methods performed in classical fused silica, polytetrafluorethylene, and polyetheretherketone capillaries or on glass and polymethylmethacrylate microchips. In addition, the principle and theoretical bases of conductivity detection in capillary electromigration techniques, zone electrophoresis, ITP, micellar EKC, and electrochromatography are briefly described.     

Optimization of sequencing conditions using near-infrared lifetime identification methods in capillary gel electrophoresis

We have investigated the sample preparation and electrophoresis conditions necessary to prepare DNA sequencing samples appropriate for use with near-infrared (IR) fluorescent labels with dye identification accomplished via lifetime techniques. It was found that several sample preparation protocols required attention to maximize the fluorescence yields of the labeling dyes, such as thermal cycling conditions, choice of counter ion used for the ethanol precipitation step and also, dye-primer versus dye-terminator chemistries. In addition, several different sieving matrices were investigated for their effects on both the fluorescence properties of the labeling dyes and electrophoretic resolution. Extended times used for the high temperature denaturing of duplexed DNA fragments during cycle sequencing produced cleavage products, in which the covalently attached dye to the sequencing primer was released through attack by dithiothreitol (DTT). Even under optimized thermal cycling conditions, free dye was generated that masked readable data from the sequencing traces. Ethanol precipitation was necessary to remove this free dye with the proper choice of counter ion (sodium). The results using different sieving matrices indicated that linear polyacrylamides (LPAs) were appropriate for any fluorescence measurement, since they could readily be replaced between runs minimizing deleterious memory effects associated with cross-linked polyacrylamide gels. After investigation of several different sieving LPAs, the commercially available POP6 was found to be particularly attractive, since it produced good electrophoretic resolution, single exponential behavior for the near-IR dye series investigated herein, and also, discernible lifetime differences within the dye set. Finally, dye-terminator chemistry was also found to minimize bleeding in the gel matrix produced by large amounts of unextended dye-primer within the gel lane.     

等速电泳-高效液相色谱联用分离复杂样品的研究

将ＯＣＥＰ－１型等速电泳仪和Ｗａｔｅｒｓ高效液相色谱仪主要部件联用,配以自制的接口,建立高效液相色谱－等速电泳新联用系统,成功地研究了含蛋白质和一些金属离子的复杂样品的分离分析。     

Isotachophoresis and isotachophoresis--zone electrophoresis separations of inorganic anions present in water samples on a planar chip with column-coupling separation channels and conductivity detection

The use of a poly(methylmethacrylate) chip, provided with two separation channels in the column-coupling (CC) arrangement and on-column conductivity detection sensors, to electrophoretic separations of a group of inorganic anions (chloride, nitrate, sulfate, nitrite, fluoride and phosphate) that need to be monitored in various environmental matrices was studied. The electrophoretic methods employed in this study included isotachophoresis (ITP) and capillary zone electrophoresis (CZE) with on-line coupled ITP sample pretreatment (ITP-CZE). Hydrodynamic and electroosmotic flows of the solution in the separation compartment of the CC chip were suppressed and electrophoresis was a dominant transport process in the separations performed by these methods. ITP separations on the chip provided rapid resolutions of sub-nmol amounts of the complete group of the studied anions and made possible rapid separations and reproducible quantitations of macroconstituents currently present in water samples (chloride, nitrate and sulfate). However, concentration limits of detection attainable under the employed ITP separating conditions (2-3 x 10(-5) mol/l) were not sufficient for the detection of typical anionic microconstituents in water samples (nitrite, fluoride and phosphate). On the other hand, these anions could be detected at 5-7 x 10(-7) mol/l concentrations by the conductivity detector in the CZE stage of the ITP-CZE combination on the CC chip. A sample clean-up performed in the ITP stage of the combination effectively complemented such a detection sensitivity and nitrite, fluoride and phosphate could be reproducibly quantified also in samples containing the macroconstituents at 10(4) higher concentrations. ITP-CZE analyses of tap, mineral and river water samples showed that the CC chip offers means for rapid and reproducible procedures to the determination of these anions in water (4-6 min analysis times under our working conditions). Here, the ITP sample pretreatment concentrated the analytes and removed nanomol amounts of the macroconstituents from the separation compartment of the chip within 3-4 min. Both the ITP and ITP-CZE procedures required no or only minimum manipulations with water samples before their analyses on the chip. For example, tap water samples were analyzed directly while a short degassing of mineral water (to prevent bubble formation during the separation) and filtration of river water samples (to remove particulates and colloids) were the only operations needed in this respect.     

Highly sensitive tITP‐CZE determination of l‐histidine and creatinine in human blood plasma using field‐amplified sample injection with mobility‐boost effect

2D computer simulation revealed that amino acids and weak electrolytes were cationized because of the migration of counter‐ion from a BGE zone to a sample zone, which encouraged electrokinetic injection (EKI) of these analytes (by the mobility‐boost (MB) effect). To investigate the effects of kinds and concentrations of counter‐ions on the MB effect and the analyte amount injected into the capillary, experiments, and 1D computer simulations were performed. When acetate was used as the counter‐ion, the LODs (S/N = 3) of l ‐histidine and creatinine, respectively, reached 0.10 and 0.25 nM because of the concentration effect by transient ITP (tITP). The concentrations of l ‐histidine and creatinine in human blood plasma obtained using the proposed method were agreed with those obtained using the conventional methods. The proposed method can be applied to the analysis of amino acids and weak bases that have similar pI and pK_a to l ‐histidine and creatinine.     

Solid-phase microextraction based on polypyrrole films with different counter ions

Solid-phase microextraction (SPME) fiber coatings based on polypyrrole (PPY) films were prepared by electrochemical deposition of PPY films on platinum wires. To evaluate the effects of counter ions in PPY films on their performance in SPME, PPY films with different counter ions were prepared using different electrolytes during the polymerization processes. The results showed that these PPY films had different extraction properties to the compounds studied due to the different functional groups introduced into the films by the counter ions. Unlike the PPY films formed with small counter ions (such as perchlorate ion) that had anion exchange property, the PPY films having large counter ions such as poly(styrenesulfonate) (PSS) ion showed cation exchange property. Compared with the PPY films having small inorganic counter ions, the PPY films having large organic counter ions, such as dodecylsulphate (DS) ion, showed better extraction efficiency towards nonpolar compounds due to the increased hydrophobic interactions between the compounds and the films. In addition, PPY films formed with large aromatic counter ions had better mechanical stability compared with PPY films with small inorganic counter ions. These films could be applied for SPME of a range of analytes.