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.     

Electrolyte system strategies for anionic isotachophoresis with electrospray‐ionization mass‐spectrometric detection. 1. Regular isotachophoresis and free‐acid isotachophoresis

The subject of this work is the definition of a simple model based on general ITP theory that allows describing and predicting the behavior of ITP systems compatible with ESI‐MS detection. The model is exemplified by anionic ITP of weak acids that represent an interesting potential application field of ITP‐ESI‐MS. Suitable ESI‐compatible electrolyte systems of very simple composition are proposed including a special free‐acid ITP arrangement. The properties of these systems are discussed using illustrative diagrams of their stacking windows. The use of anionic ITP‐ESI‐MS in negative‐ion ESI mode is reported for the first time and its suitability for sensitive trace analysis is demonstrated. The presented ITP‐ESI‐MS application example comprises a free‐acid ITP system formed of formic and propionic acids and direct injection analysis of ibuprofen and diclofenac in waters with quantitation limits of the order 10^?10 M.     

Application of capillary isotachophoresis in peptide analysis

This paper gives a broad and detailed review of the applications of one of the modern high-performance electromigration separation techniques--capillary isotachophoresis (ITP)--in peptide analysis. Examples are presented of the utilization of capillary ITP for peptide analysis in the fields of chemistry, general and clinical biochemistry, biology, biotechnology, pharmacy and the food industry. The complete composition of all the electrolyte systems used for peptide ITP analyses in both cationic and anionic techniques is given in tabular form. According to the purpose of analysis the applications are divided into several sections: model studies, determination of physico-chemical characteristics, purity control of both intermediate and final peptide preparations, including the determination of low-molecular-mass ionogenic admixtures, and the analysis of peptides in biological fluids and tissue extracts. In addition to the main applications the theoretical and methodological aspects of peptide ITP analysis are discussed. The basic electromigration properties of peptides (their polyampholyte character, effective and absolute mobilities, acid-base equilibria) are explained and the selection of parameters for peptide ITP analysis is described in detail. The advantages and disadvantages of ITP compared with other electrophoretic and chromatographic methods used for peptide analysis are discussed.     

Methodological aspects of an off‐line combination of preparative isotachophoresis and high‐performance liquid chromatography with mass spectrometry in the analysis of biological matrices

This work reports on some methodological aspects of an off‐line combination of preparative ITP and HPLC with mass spectrometric detection (pITP‐HPLC‐MS) and its potential applications to the analysis of high molecular mass compounds present in complex biological matrices from the analytical chemistry perspective. Lysozyme served as the model analyte and human saliva as the complex biological matrix in this study. A mixture of five low‐molecular mass compounds was found and successfully used in the pITP experiments as discrete spacers to isolate the analyte from the interferents present in the complex biological matrix and to minimize their disturbance effect on the final MS analysis. The experiments at the pITP stage were performed in the cationic mode. On‐column conductivity detectors were used for the detection of ITP zones. Lysozyme was found in the human saliva samples using just deconvolution of the MS data after background correction. The MS data obtained from HPLC‐MS analysis of pITP fractions exhibited the great analytical potential of the combination of pITP‐HPLC‐MS resulting from the ITP clean‐up effect as well as the ITP preconcentration of the analyte present at low concentration levels in complex biological matrices.     

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.     

Improving the detection limit in capillary isotachophoresis using asymmetric neutralisation reaction boundary

An online method involving transient electrokinetic dosing and ITP with neutralization reaction boundary (NRB) and/or carrier ampholyte-free isoelectric focusing (CAF IEF) was developed for the preconcentration, preseparation, and analytical determination of glyphosate in aqueous samples containing low concentrations of the analyte of interest. Various parameters were investigated in the framework of an optimization study with the aim of achieving the maximum concentration limit of detection (cLOD) decrease in minimum time. The proposed method used CAF IEF and/or ITP with NRB. The sample was dosed to the column on the stationary reaction boundary (CAF IEF) and/or moving reaction boundary (ITP with NRB), whereat a sharp pH step exists. Here, charge reversal was due to the ampholytes, and/or acid accumulation occurred because of charge loss. Similarly, the accumulated sample was mobilized with TE and analyzed using classical ITP in the second analytical column. Glyphosate (GLY), the analyte of interest, was chosen as a model substance for ITP with NRB and preconcentration as well as focusing preconcentration and CAF IEF using the asymmetric purpose-built NRB. On one side of the asymmetric boundary was the zone of acidic pH; while the opposite side comprised a neutral/basic non-conductive zone of the ampholyte—in this case, GLY. Such an arrangement enables the use of a lower pH on the acidic side, which allows the focusing of strongly acidic ampholytes and the accumulation of weak acids. The electrolyte composition and the dosing time were optimized, and a 14-fold accumulation was achieved in 25 min compared to that by classical ITP and a 180-fold accumulation was achieved through CAF IEF and preconcentration with a glyphosate sample. Both methods are simple and can be conducted using all commercial ITP systems.     

Preconcentration and detection of the phosphorylated forms of cardiac troponin I in a cascade microchip by cationic isotachophoresis

This paper describes the detection of a cardiac biomarker, cardiac troponin I (cTnI), spiked into depleted human serum using cationic isotachophoresis (ITP) in a 3.9 cm long poly(methyl methacrylate) (PMMA) microfluidic channel. The microfluidic chip incorporates a 100× cross-sectional area reduction, including a 10× depth reduction and a 10× width reduction, to increase sensitivity during ITP. The cross-sectional area reductions in combination with ITP allowed visualization of lower concentrations of fluorescently labeled cTnI. ITP was performed in both "peak mode" and "plateau mode" and the final concentrations obtained were linear with initial cTnI concentration. We were able to detect and quantify cTnI at initial concentrations as low as 46 ng mL(-1) in the presence of human serum proteins and obtain cTnI concentrations factors as high as ~ 9000. In addition, preliminary ITP experiments including both labeled cTnI and labeled protein kinase A (PKA) phosphorylated cTnI were performed to visualize ITP migration of different phosphorylated forms of cTnI. The different phosphorylated states of cTnI formed distinct ITP zones between the leading and terminating electrolytes. To our knowledge, this is the first attempt at using ITP in a cascade microchip to quantify cTnI in human serum and detect different phosphorylated forms.     

Electrolyte system strategies for anionic isotachophoresis with electrospray‐ionization mass‐spectrometric detection. 2. Isotachophoresis in moving‐boundary systems

This contribution is the second part of the project on strategies used in the selection of electrolyte systems for anionic ITP with ESI‐mass spectrometric detection. It presents ITP as a powerful tool for selective stacking of anionic analytes, performed in a nonconventional way in moving‐boundary systems where two co‐anions are present in both the leading and terminating zones. The theoretical background is given to substantiate the conditions for the existence and migration of ITP boundaries in moving‐boundary systems and stacking of analytes at these boundaries. The practical aspects of the theory are shown in form of stacking‐window diagrams that bring immediate information about which analytes are stacked in a given system. The presented theory and strategy are illustrated and verified on the example of analysis of a model mixture of salicylic acid, ibuprofen and diclofenac, and comparison of regular and free‐acid ITP with moving‐boundary ITP systems formed by formic and propionic acids and ammonium as counterion.     

Application of capillary isotachophoresis for fruit juice authentication

A method using capillary isotachophoresis (ITP) was developed and applied for the determination of the anionic profile of orange juices with the aim to obtain some useful information on the authenticity or adulteration of imported and native beverage products. An EA 100 electrophoretic analyser (Villa-LABECO, Slovak Republic) was used for capillary isotachophoretic determination of anions in tested samples. More systems of leading and terminating electrolytes were used. Detection conductivity and UV detection at 254 nm were used. Sample injection volume was 30 microl. These systems allow one to determinate inorganic anions, organic acids and some additives--adulterants in anionic forms in orange juices. By capillary isotachophoretic determination the lengths or areas of characteristic zones were established and compared to authentic orange juices of different species and origin and with RSK reference values (Code of Practice). Special emphasis was placed on D-isocitric acid ITP determination as a reliable fruit juice authentication marker. The presented multicomponent analysis of orange juice authenticity according to ITP anionic profiles obtained by capillary isotachophoresis presents an alternative information source necessary for deciding about authenticity of the products.     

Recent progress in capillary ITP

ITP has been attracting constant attention for many years due to its principal capability to concentrate trace analytes by several orders of magnitude. In the current capillary format, it is able to concentrate trace analytes diluted to several microliters of an original sample into concentrated zones having volumes in the range of picoliters. Due to this reason, ITP holds an important position in many current multistage and multidimensional separation schemes. This article links up previous reviews on the topic and summarizes the progress of analytical capillary ITP since 2002. Almost 100 papers are reviewed that include methodological novelties, instrumental aspects, and analytical applications. Papers using ITP and/or isotachophoretic principles as part of multistage and/or multidimensional separation schemes are also included.     

A method to determine quasi-steady state in constant voltage mode isotachophoresis

Identification of the steady state is very challenging in isotachophoresis (ITP); especially in complex microgeometries, such as dog-leg channels or cross-channel junctions. In this work, an elastic matching method is applied to determine the quasi-steady state in microscale ITP. In the elastic matching method, the similarity between two profiles is calculated by comparing intensity distribution of two images or profiles. To demonstrate this similarity-based analysis technique for ITP, a constant voltage mode ITP model is developed and applied to a five-component ITP system. Hydrochloric acid and caproic acid are used as the leader and terminator, respectively, while histidine is used as the counter-ion. Two sample components, acetic acid and benzoic acid, are separated under the action of an applied electric field in both straight and dog-leg microchannels. This analysis shows that conductivity profiles provide a better measure to determine the quasi-steady state in an ITP process. For a straight microchannel, the quasi-steady state is achieved in less than a minute with a total potential drop of 100?V in a 2?cm long channel. In a straight channel, a true steady state can be achieved for ITP with appropriate countercurrent flow where stationary zones are formed, but the time it takes to reach the steady state is much longer than the without counter flow case. The numerical results indicate that a steady state cannot be reached in a dog-leg microchannel because of sample dispersion and refocusing at and near the intersections and at the branch channels. However, the elastic matching method can be used to determine the quasi-steady state in a dog-leg microchannel.     

Challenges and applications of isotachophoresis coupled to mass spectrometry: A review

Electrophoretic separations are of growing interest to tackle complex analytical challenges. Nevertheless, capillary electrophoresis, as the most common mode, still suffers from insufficient detection limits due to low capillary loadability. ITP is of growing interest as preconcentration method for capillary electrophoresis and is also interesting to be applied as an independent analytical method. While mass spectrometric detection is common for capillary electrophoresis, the combination of ITP with MS is still a niche technique. In this work, we want to give an overview on isotachophoretic effects in CE-MS and ITP-MS methods, as well as coupling techniques of ITP with CE-MS. The challenges and possibilities associated with mass spectrometric detection in ITP and its coupling to capillary electrophoresis are critically discussed.     

Separation of carboxylic acids in human serum by isotachophoresis using a commercial field-deployable analytical platform combined with in-house glass microfluidic chips

Portable and field deployable analytical instruments are attractive in many fields including medical diagnostics, where point of care and on-site diagnostics systems capable of providing rapid quantitative results have the potential to vastly improve the productivity and the quality of medical care. Isotachophoresis (ITP) is a well known electrophoretic separation technique previously demonstrated as suitable for miniaturization in microfluidic chip format (chip-ITP). In this work, a purpose-designed ITP chip compatible with a commercial end-used targeted microfluidic system was used to study different injection protocols and to evaluate the effect of the length of the separation channel on the analytical performance. The in-house ITP chips were made from Corning glass and compared to the commercial DNA chip for the ITP separation of anions from the hydrodynamic injection of human serum. Using the in-house ITP chip the isotachophoretic step of lactate from human serum was approximately two times longer. The results of this research suggested that microfluidic ITP with indirect fluorescence detection is a viable technique for separation of organic acids in human serum samples, especially when a chip with suitable design is used.     

Experimental and theoretical description of the isotachophoretic behavior of serum albumin

The isotachophoretic behavior of serum albumin is examined for three anionic and one cationic electrolyte systems by (i) computer simulation, (ii) capillary isotachophoresis (ITP) and (iii) continuous flow ITP. The theoretical relationship between pH of the leading electrolyte and the steady state protein plateau concentration is presented for one of the anionic systems. With leading ion concentrations of the order of 10 mM, experimental protein plateau concentrations of 1.3-2.3% w/v are obtained. The computer predictions are approximately half these values.     

Inverse cationic ITP for separation of methadone enantiomers with sulfated β‐cyclodextrin as chiral selector

Chiral ITP of the weak base methadone using inverse cationic configurations with H⁺ as leading component and multiple isomer sulfated β‐CD (S‐β‐CD) as leading electrolyte (LE) additive, has been studied utilizing dynamic computer simulation, a calculation model based on steady‐state values of the ITP zones, and capillary ITP. By varying the amount of acidic S‐β‐CD in the LE composed of 3‐morpholino‐2‐hydroxypropanesulfonic acid and the chiral selector, and employing glycylglycine as terminating electrolyte (TE), inverse cationic ITP provides systems in which either both enantiomers, only the enantiomer with weaker complexation, or none of the two enantiomers form cationic ITP zones. For the configuration studied, the data reveal that only S‐methadone migrates isotachophoretically when the S‐β‐CD concentration in the LE is between about 0.484 and 1.113 mM. Under these conditions, R‐methadone migrates zone electrophoretically in the TE. An S‐β‐CD concentration between about 0.070 and 0.484 mM results in both S‐ and R‐methadone forming ITP zones. With >1.113 mM and < about 0.050 mM of S‐β‐CD in the LE both enantiomers are migrating within the TE and LE, respectively. Chiral inverse cationic ITP with acidic S‐β‐CD in the LE is demonstrated to permit selective ITP trapping and concentration of the less interacting enantiomer of a weak base.     

Non-aqueous electrolytes for isotachophoresis of weak bases and its application to the comprehensive preconcentration of the 20 proteinogenic amino acids in column-coupling ITP/CE–MS

Isotachophoresis (ITP) has long been used alone but also as a preconcentration technique for capillary electrophoresis (CE). Unfortunately, up to now, its application is restricted to relatively strong acids and bases as either the degree of (de)protonation is too low or the water dissociation is too high, evoking zone electrophoresis. With the comprehensive ITP analysis of all 20 proteinogenic amino acids as model analytes, we, here, show that non–aqueous ITP using dimethylsulfoxide as a solvent solves this ITP shortcoming. Dimethylsulfoxide changes the pH regime of analytes and electrolytes but, more importantly, strongly reduces the proton mobility by prohibiting hydrogen bonds and thus, the so-called Zundel–Eigen–Zundel electrical conduction mechanism of flipping hydrogen bonds. The effects are demonstrated in an electrolyte system with taurine or H⁺ as terminator, and imidazole as leader together with strong acids such as oxalic and even trifluoroacetic acid as counterions, both impossible to use in aqueous solution. Mass spectrometric as well as capacitively coupled contactless conductivity detection (C⁴D) are used to follow the ITP processes. To demonstrate the preconcentration capabilities of ITP in a two-dimensional set-up, we, here, also demonstrate that our non-aqueous ITP method can be combined with capillary electrophoresis–mass spectrometry in a column-coupling system using a hybrid approach of capillaries coupled to a microfluidic interface. For this, C⁴D was optimized for on-chip detection with the electrodes aligned on top of a thin glass lid of the microfluidic chip.     

Recent progress in analytical capillary isotachophoresis

This review brings a survey of studies on analytical ITP published since 2016 until the first quarter of 2018 and includes chapters about theory and principles, instrumentation and techniques, and analytical applications of ITP. It shows the position of analytical ITP among contemporary separation techniques, where particularly its unique concentrating capabilities keep the interest to include it into novel high‐sensitivity analytical procedures. The reviewed papers are considered according to their nature, techniques used, and instrumentation employed. The significance of electrolyte system composition is emphasized by providing explicit values where possible.     

Recycling and screen-segmented column isotachophoresis, two free-fluid approaches for fractionation of proteins.

Recycling and screen-segmented column isotachophoresis (ITP), two approaches for the milligrams to grams preparative-scale purification of proteins, are discussed and compared. Recycling ITP was performed in a recycling free-flow focusing apparatus. In this process, fluid flows rapidly through a narrow channel and the effluent from each channel is reinjected into the electrophoresis chamber through the corresponding input port. The residence time in the cell is of the order of 1 s per single pass, which does not allow complete separation, so recycling is essential to attain the steady state. Immobilization of the advancing zone structure is obtained via a controlled counterflow. Thirty fractions of about 4 ml each are obtained. Column ITP was executed in a Rotofor apparatus and in a similar column operated vertically and without rotation. These instruments feature a screen-segmented annular separation space with twenty subcompartments of about 2 ml each. With both approaches, the collected fractions were analysed separately for conductivity, pH and UV absorbance. Selected fractions were characterized by analytical electrophoretic methods. Examples presented include the cationic and anionic ITP behaviour of model proteins, including bovine serum albumin, ovalbumin and ribonuclease A, and the ITP removal of the major impurities from a commercial ovalbumin sample. These examples revealed that the screen-segmented column is suitable for ITP protein purification and operates optimally in a horizontal rotating mode and without internal cooling. The recycling experiments showed that counterflow improves separation and the steady-state patterns are dependent on the fluid layer thickness in the separation cell but, with a given gap, essentially independent of applied current and recycling pump rate.     

Cationic isotachophoresis separation of the biomarker cardiac troponin I from a high‐abundance contaminant,serum albumin

Cationic ITP was used to separate and concentrate fluorescently tagged cardiac troponin I (cTnI) from two proteins with similar isoelectric properties in a PMMA straight‐channel microfluidic chip. In an initial set of experiments, cTnI was effectively separated from R‐Phycoerythrin using cationic ITP in a pH 8 buffer system. Then, a second set of experiments was conducted in which cTnI was separated from a serum contaminant, albumin. Each experiment took ～10 min or less at low electric field strengths (34 V/cm) and demonstrated that cationic ITP could be used as an on‐chip removal technique to isolate cTnI from albumin. In addition to the experimental work, a 1D numerical simulation of our cationic ITP experiments has been included to qualitatively validate experimental observations.     

10,000-fold concentration increase of the biomarker cardiac troponin I in a reducing union microfluidic chip using cationic isotachophoresis

This paper describes the preconcentration of the biomarker cardiac troponin I (cTnI) and a fluorescent protein (R-phycoerythrin) using cationic isotachophoresis (ITP) in a 3.9 cm long poly(methyl methacrylate) (PMMA) microfluidic chip. The microfluidic chip includes a channel with a 5× reduction in depth and a 10× reduction in width. Thus, the overall cross-sectional area decreases by 50× from inlet (anode) to outlet (cathode). The concentration is inversely proportional to the cross-sectional area so that as proteins migrate through the reductions, the concentrations increase proportionally. In addition, the proteins gain additional concentration by ITP. We observe that by performing ITP in a cross-sectional area reducing microfluidic chip we can attain concentration factors greater than 10,000. The starting concentration of cTnI was 2.3 μg mL?1 and the final concentration after ITP concentration in the microfluidic chip was 25.52 ± 1.25 mg mL?1. To the author's knowledge this is the first attempt at concentrating the cardiac biomarker cTnI by ITP. This experimental approach could be coupled to an immunoassay based technique and has the potential to lower limits of detection, increase sensitivity, and quantify different isolated cTnI phosphorylation states.