Recent progress in analytical capillary isotachophoresis (2018 - March 2022).

This review brings a survey of papers on analytical capillary and microchip isotachophoresis published since 2018 until the first quarter of 2022. Theoretical papers extending fundamental knowledge include those on computer simulations that remain an important research tool useful in the design of electrolyte systems. Many papers are focused on instrumental aspects where new media including microfluidic devices and their hyphenation to various detection techniques bring remarkable results. Papers reporting analytical applications demonstrate the potential of contemporary analytical isotachophoresis. Although it is not being used on a mass scale, its special features are attracting continued interest resulting in applications of isotachophoresis both as a stand-alone analytical method and as a part of multidimensional separation techniques.

Cationic electrophoretic focusing on inverse electromigration dispersion profile with ESI-MS detection. New capillary electrophoretic method for high-sensitivity analysis of 2-hydroxy-s-triazines in waters.

Electrophoretic focusing on an inverse electromigration dispersion profile is a relatively new electrophoretic separation method for the analysis of ionisable analytes. Although it offers alternative selectivity, powerful concentration capability and high sensitivity, so far only several systems for the analysis of anionic compounds were reported. This work describes for the first time a functional electrolyte system for the separation of cationic analytes by this method. Based on computer simulations and calculations, a functional electrolyte system was designed, based on 1-ethylpiperazine and pivalic acid. It is suitable for the analysis of weak bases with pKa values between 4.5 and 6 and due to the volatility of both components, it can be operated in combination with ESI-MS detection. Its practical applicability is demonstrated on direct analysis of four 2-hydroxy-s-triazines in waters. Separation in an uncoated capillary with application of negative pressure at the inlet provides very satisfactory results with good reproducibility of peak areas, peak heights and detection times, and with LODs at the 0.5-1 nM (100 ngL-1) level. The robustness of the method is shown on analyses of real tap and river water samples used without any pretreatment.

System eigenmobilities in zone electrophoresis: A general moving-boundary approach.

System zones in capillary zone electrophoresis represent an important topic, very interesting from the theoretical point but also important for practice. This paper is aimed at contributing to the understanding of system zones as one of the very fundamental properties of electrophoretic systems, by developing an alternative approach to the so far used vector-matrix model of calculation of system mobilities (system eigenmobilities). The presented model is based on the solution of the differential form of the moving-boundary equation. The result for acid-base systems is a single algebraic equation valid universally for a zone comprising any number of constituents (mono- or polyhydric strong or weak acids or bases and/or amphoteric compounds). The value of the described solution against previous models consists in its explicit form, expressing the system eigenmobility of a homogeneous zone of given composition as a function of only known quantities. The obtained equation is shown to be the common source of various simplified equations obtained in the past for particular simple systems. The applicability of the simplified equations is discussed in terms of completeness of the results (number of output system eigenmobilities). For non-buffered systems, the occurrence of a previously unreported non-zero value of system eigenmobility is discussed that is equal to the arithmetic average of mobilities of the solvent ions.

At-line coupling of hollow fiber liquid-phase microextraction to capillary electrophoresis for trace determination of acidic drugs in complex samples.

Direct analysis of complex samples is demonstrated by the at-line coupling of hollow fiber liquid-phase microextraction (HF-LPME) to capillary electrophoresis (CE). The hyphenation of the preparative and the analytical technique is achieved through a 3D-printed microextraction device with an HF located in a sample vial of a commercial CE instrument. The internal geometry of the device guides the CE separation capillary into the HF and the CE injection of the HF-LPME extract is performed directly from the HF lumen. The 3D-printing process ensures uniform dimensions of the devices, their constant position inside the sample vial, and excellent repeatability of the HF-LPME as well as the CE injection. The devices are cheap (∼0.01 €) and disposable, thus eliminating any possible sample-carryover, moreover, the at-line CE analysis of the extract is performed fully autonomously with no need for operator's intervention. The developed HF-LPME/CE-UV method is applied to the determination of acidic drugs in dried blood spot and wastewater samples and is characterized by excellent repeatability (RSD, 0.6-9.6%), linearity (r2, 0.9991-0.9999), enrichment (EF, 29-97), sensitivity (LOD, 0.2-3.4 µg/L), and sample throughput (7 samples/h). A further improvement of selected characteristics of the analytical method is achieved by the at-line coupling of HF-LPME to capillary isotachophoresis (ITP) with electrospray ionization-mass spectrometry (ESI-MS). The HF-LPME/ITP-ESI-MS system facilitates enhanced selectivity, matrix-free analytical signals, and up to 34-fold better sensitivity due to the use of ESI-MS detection and additional on-capillary ITP preconcentration of the HF-LPME extracts.

Capillary isotachophoresis with electrospray-ionization mass-spectrometric detection: Cationic electrolyte systems in the medium-alkaline range for selective analysis of medium strong bases.

This work extends the present working range of isotachophoresis (ITP) with electrospray-ionization mass-spectrometric (ESI-MS) detection and describes for the first time a functional cationic electrolyte system for analyses at medium-alkaline pH. So far no ITP-MS application was published on the analysis of medium strong bases although there is a broad spectrum of potential analytes like biogenic amines, alkaloids or drugs, where this technique promises interesting gains in both sensitivity and specificity. The presented results include a selection of suitable sufficiently volatile ESI-compatible system components, discussion of factors affecting system properties, and recommendations for functional ITP electrolyte systems. Theoretical conclusions based on calculations and computer simulations are confirmed by experiments with a model mixture of beta-blockers. Practical applicability of the method is demonstrated on the example of analysis of sotalol in dried blood spots where direct injection of aqueous extract, ITP stacking and MS detection provide a fast, simple and sensitive technique with limits of quantitation on the sub-nM level.

Theory of electrophoretic focusing on an inverse electromigration dispersion profile.

Electrophoretic focusing on an inverse electromigration dispersion (EMD) profile is based on a principle different from those of other electrophoretic separation methods. It has already proved its applicability in analytical practice by offering competitive separation performance and sensitivity and specific selectivity. It can be classified as an intermediate between field-driven and equilibrium gradient methods and is therefore interesting from the viewpoint of theory of separation methods. This work presents a comprehensive theoretical description of electrophoretic focusing on an inverse EMD profile comprising properties of the electrolyte system, formed gradients, and focused analyte zones. The separation properties are described in terms of resolution and peak capacity and their dependence on system and analyte properties is discussed from the viewpoint of how the counteracting phenomena of electromigration and dispersion are affected by electric current, voltage, and hydrodynamic and electroosmotic flow. The overall performance of the present method is shown to be comparable with other electrophoretic separation methods like zone electrophoresis or isoelectric focusing.

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.

Inverse cationic ITP for separation of methadone enantiomers with sulfated ß-cyclodextrin as chiral selector.

Chiral ITP of the weak base methadone using inverse cationic configurations with H+ as leading component and multiple isomer sulfated ß-CD (S-ß-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-ß-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-ß-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-ß-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-ß-CD in the LE both enantiomers are migrating within the TE and LE, respectively. Chiral inverse cationic ITP with acidic S-ß-CD in the LE is demonstrated to permit selective ITP trapping and concentration of the less interacting enantiomer of a weak base.

Recent progress of sample stacking in capillary electrophoresis (2016-2018).

Electrophoretic sample stacking comprises a group of capillary electrophoretic techniques where trace analytes from the sample are concentrated into a short zone (stack). This paper is a continuation of our previous reviews on the topic and brings a survey of more than 120 papers published approximately since the second quarter of 2016 till the first quarter of 2018. It is organized according to the particular stacking principles and includes chapters on concentration adjustment (Kohlrausch) stacking, on stacking techniques based on pH changes, on stacking in electrokinetic chromatography and on other stacking techniques. Where available, explicit information is given about the procedure, electrolyte(s) used, detector employed and sensitivity reached. Not reviewed are papers on transient isotachophoresis which are covered by another review in this issue.

Electrophoretic focusing on inverse electromigration dispersion gradient. The fundamental resolution equation and pressure-assisted performance enhancement.

Electrophoretic focusing on inverse electromigration dispersion (EMD) gradient is a new analytical technique based on a unique separation principle where weak non-amphoteric ionogenic species are focused, separated and transported to the detector by an EMD profile of suitable properties. The present work extends the theoretical description of this method by introducing the concept of resolution and deriving the fundamental equation expressing resolution as function of basic system parameters. The results indicate that at constant current operation, resolution is proportional to the square root of time. For variable current regimes (e.g. constant voltage), the time variable is replaced by the product of electric current and passed electric charge. Computer simulations for a model pair of substances support the validity of the presented theory and confirm the theoretical conclusion that resolution can be increased by allowing longer electromigration of the gradient in terms of time or passed charge. The experimental example shown comprises an anionic electrolyte system based on maleic acid and 2,6-lutidine, combined with ESI-MS detection and operated in the reverse mode due to strong electroosmotic flow and ESI suction. The practical implementation of the proposed methodology is done by application of negative pressure at the inlet vial, resulting in very substantial resolution enhancement and baseline separation of otherwise unresolved substances. The performance and high sensitivity of the developed technique is demonstrated on the example of simultaneous analysis of four sulfonamides and three dichlorophenols in waters with limits of detection on the 1 nM level.

Methodology of analysis of very weak acids by isotachophoresis with electrospray-ionization mass-spectrometric detection: Anionic electrolyte systems for the medium-alkaline pH range.

This work describes for the first time a functional electrolyte system setup for anionic isotachophoresis (ITP) with electrospray-ionization mass-spectrometric (ESI-MS) detection in the neutral to medium-alkaline pH range. So far no application was published on the analysis of very weak acids by anionic ITP-MS although there is a broad spectrum of potential analytes with pKa values in the range 5-10, where application of this technique promises interesting gains in both sensitivity and specificity. The problem so far was the lack of anionic ESI-compatible ITP systems in the mentioned pH range as all typical volatile anionic system components are fully ionized at neutral and alkaline pH and thus too fast to suit as terminators. We propose an original solution of the problem based on the combination of two ITP methods: (i) use of the hydroxyl ion as a natural and ESI-compatible terminator, and (ii) use of configurations based on moving-boundary ITP. The former method ensures effective stacking of analytes by an alkaline terminator of sufficiently low mobility and the latter offers increased flexibility for tuning of the separation window and selectivity according to actual needs. A theoretical description of the proposed model is presented and applied to the design of very simple functional electrolyte configurations. The properties of example systems are demonstrated by both computer simulation and experiments with a group of model analytes. Potential effects of carbon dioxide present in the solutions are demonstrated for particular systems. Experimental results confirm that the proposed methodology is well capable of performing sensitive and selective ITP-MS analyses of very weak acidic analytes (e.g. sulfonamides or chlorophenols).

Capillary moving-boundary isotachophoresis with electrospray ionization mass-spectrometric detection and hydrogen ion used as essential terminator: Methodology for sensitive analysis of hydroxyderivatives of s-triazine herbicides in waters.

Capillary isotachophoresis (ITP) is an electrophoretic technique offering high sensitivity due to permanent stacking of the migrating analytes. Its combination with electrospray-ionization mass-spectrometric (ESI-MS) detection is limited by the narrow spectrum of ESI-compatible components but can be compensated by experienced system architecture. This work describes a methodology for sensitive analysis of hydroxyderivatives of s-triazine herbicides, based on implementation of the concepts of moving-boundary isotachophoresis and of H+ as essential terminating component into cationic ITP with ESI-MS detection. Theoretical description of such kind of system is given and equations for zone-related boundary mobilities are derived, resulting in a much more general definition of the effective mobility of the terminating H+ zone than used so far. Explicit equations allowing direct calculation for selected simple systems are derived. The presented theory allows prediction of stacking properties of particular systems and easy selection of suitable electrolyte setups. A simple ESI-compatible system composed of acetic acid and ammonium with H+ and ammonium as a mixed terminator was selected for the analysis of 2-hydroxyatrazine and 2-hydroxyterbutylazine, degradation products of s-triazine herbicides. The proposed method was tested with direct injection without any sample pretreatment and provided excellent linearity and high sensitivity with limits of detection below 100ng/L (0.5nM). Example analyses of unspiked and spiked drinking and river water are shown.

Analytical capillary isotachophoresis after 50 years of development: Recent progress 2014-2016.

This review brings a survey of papers on analytical ITP published since 2014 until the first quarter of 2016. The 50th anniversary of ITP as a modern analytical method offers the opportunity to present a brief view on its beginnings and to discuss the present state of the art from the viewpoint of the history of its development. Reviewed papers from the field of theory and principles confirm the continuing importance of computer simulations in the discovery of new and unexpected phenomena. The strongly developing field of instrumentation and techniques shows novel channel methodologies including use of porous media and new on-chip assays, where ITP is often included in a preseparative or even preparative function. A number of new analytical applications are reported, with ITP appearing almost exclusively in combination with other principles and methods.

Recent progress of sample stacking in capillary electrophoresis (2014-2016).

The term "sample stacking" comprises a relatively broad spectrum of techniques that already form an almost inherent part of the methodology of CZE. Their principles are different but the effect is the same: concentration of a diluted analyte into a narrow zone and considerable increase of the method sensitivity. This review brings a survey of papers on electrophoretic sample stacking published approximately since the second quarter of 2014 till the first quarter of 2016. It is organized according to the principles of the stacking methods and includes chapters aimed at the concentration adjustment principle (Kohlrausch stacking), techniques based on pH changes, micellar methods, and other stacking techniques. Not reviewed are papers on transient ITP that are covered by another review in this issue.

New methodology for capillary electrophoresis with ESI-MS detection: Electrophoretic focusing on inverse electromigration dispersion gradient. High-sensitivity analysis of sulfonamides in waters.

This article describes for the first time the combination of electrophoretic focusing on inverse electromigration dispersion (EMD) gradient, a new separation principle described in 2010, with electrospray-ionization (ESI) mass spectrometric detection. The separation of analytes along the electromigrating EMD profile proceeds so that each analyte is focused and concentrated within the profile at a particular position given by its pKa and ionic mobility. The proposed methodology combines this principle with the transport of the focused zones to the capillary end by superimposed electromigration, electroosmotic flow and ESI suction, and their detection by the MS detector. The designed electrolyte system based on maleic acid and 2,6-lutidine is suitable to create an inverse EMD gradient of required properties and its components are volatile enough to be compatible with the ESI interface. The characteristic properties of the proposed electrolyte system and of the formed inverse gradient are discussed in detail using calculated diagrams and computer simulations. It is shown that the system is surprisingly robust and allows sensitive analyses of trace amounts of weak acids in the pKa range between approx. 6 and 9. As a first practical application of electrophoretic focusing on inverse EMD gradient, the analysis of several sulfonamides in waters is reported. It demonstrates the potential of the developed methodology for fast and high-sensitivity analyses of ionic trace analytes, with reached LODs around 3 × 10(-9) M (0.8 ng mL(-1)) of sulfonamides in spiked drinking water without any sample pretreatment.

Capillary isotachophoresis with ESI-MS detection: Methodology for highly sensitive analysis of ibuprofen and diclofenac in waters.

The possibilities of reaching higher sensitivity in capillary electrophoretic analyses of complex samples with ESI-MS detection were investigated on the example of analysis of diclofenac and ibuprofen in waters. The applied separation approach is based on application of isotachophoresis that ensures permanent stacking of analytes until they reach the detector. Investigation of the possibilities of MS detector optimization have shown that optimization of fragmentor voltage and working in the SIM mode with collection of data for multiple fragments both increases the method specificity and approx. doubles its sensitivity. Combination with an offline SPE preconcentration step resulted in very high sensitivity of the described methodology with a reached LOD below 2 × 10(-12) M, corresponding to analyte levels of 0.6 ng L(-1) of diclofenac and 0.4 ng L(-1) of ibuprofen. The results demonstrate that CE-MS, particularly when performed in the ITP mode, has the potential to reach sensitivities comparable to HPLC-MS.

Contemporary sample stacking in analytical electrophoresis.

This contribution is a methodological review of the publications about the topic from the last 2 years. Therefore, it is primarily organized according to the methods and procedures used in surveyed papers and the origin and type of sample and specification of analytes form the secondary structure. The introductory part about navigation in the architecture of stacking brings a brief characterization of the various stacking methods, with the description of mutual links to each other and important differences among them. The main body of the article brings a survey of publications organized according to main principles of stacking and then according to the origin and type of the sample. Provided that the paper cited gave explicitly the relevant data, information about the BGE(s) used, procedure, detector employed, and reached LOD and/or concentration effect is given. The papers where the procedure used is a combination of diverse fragments and parts of various stacking techniques are mentioned in a special section on combined techniques. The concluding remarks in the final part of the review evaluate present state of art and the trends of sample stacking in CE.

Recent progress in analytical capillary isotachophoresis.

This paper brings a survey of papers on analytical capillary ITP published since 2012 until the first quarter of 2014. These papers are ranged according to their nature, the techniques used, and the instrumentation employed. The sequence of the related chapter titles is as follows: Theory and simulations, techniques and instrumentation, single-column and column-switching applications of ITP, ITP in microfluidic systems, on-line ITP-CZE and transient ITP (tITP) techniques and applications. The review shows the position of analytical capillary ITP among contemporary separation techniques and implies the potential future trends.

Electrolyte system strategies for anionic ITP with ESI-MS detection. 3. The ITP spacer technique in moving-boundary systems and configurations with two self-maintained ITP subsystems.

This contribution is the third part of the project on strategies used in the selection and tuning of electrolyte systems for anionic ITP with ESI-MS detection. The strategy presented here is based on the creation of self-maintained ITP subsystems in moving-boundary systems and describes two new principal approaches offering physical separation of analyte zones from their common ITP stack and/or simultaneous selective stacking of two different analyte groups. Both strategic directions are based on extending the number of components forming the electrolyte system by adding a third suitable anion. The first method is the application of the spacer technique to moving-boundary anionic ITP systems, the second method is a technique utilizing a moving-boundary ITP system in which two ITP subsystems exist and move with mutually different velocities. It is essential for ESI detection that both methods can be based on electrolyte systems containing only several simple chemicals, such as simple volatile organic acids (formic and acetic) and their ammonium salts. The properties of both techniques are defined theoretically and discussed from the viewpoint of their applicability to trace analysis by ITP-ESI-MS. Examples of system design for selected model separations of preservatives and pharmaceuticals illustrate the validity of the theoretical model and application potential of the proposed techniques by both computer simulations and experiments. Both new methods enhance the application range of ITP-MS and may be beneficial particularly for complex multicomponent samples or for analytes with identical molecular mass.

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.