Chapter 9 - Capillary Electrochromatography

Analysis of active ingredients and preservatives in creams by capillary electrochromatography (CEC)

Capillary electrochromatography (CEC) has been realised as a combination of high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) allowing the separation of analytes based on their differing adsorption and migration behaviour. There is a considerable interest in developing new methodologies for CEC, which uses chromatographic beads or monoliths as stationary phases or alternatively employs open tubular (OT) capillaries relying on surface modifications. Besides the bulk flat flow profile of the mobile phase in CEC columns allowing an enhanced resolution, due to the influence of the applied electrical field there are other possibilities to provide a higher resolution with better detection. As an additional newly means for peak sharpening and sample enrichment isotachophoresis and a “sweeping mechanism” could be envisaged, which both take advantage of the inhomogeneous electric field pattern in the capillary by choosing appropriate leading and terminating background electrolytes (BGEs). Transient isotachophoresis would be another technique to provide an increased plate height in the separations, using an optimized sample matrix, e.g. containing a relatively high acetonitrile content, with the same leading and terminating BGEs. In this thesis, a new method to generate a “sharpening zone”, which is similar to the concept of transient isotachophoresis, was established using an aqueous sample matrix containing an organic solvent. Target analytes within this zone could be concentrated whilst they migrated through the capillary. A novel technique was developed that used the sharpening zone for sweeping and concentrating several unresolved analyte peaks allowing an enhanced peak resolution. As the aim was to concentrate only some part of the analyte zone containing compounds of interest, the position and width of the sharpening zone needed to be well controlled. Various techniques to tune the width and position of the sharpening zone were thus developed, involving modifying the organic modifier content in the BGE, applying pressure additionally to the electroosmotic flow (EOF) and modifying the inner surface of the capillaries. This work also critically evaluated some related existing knowledge and theoretical approaches in CEC including the concept of tuneable system eigenpeaks (noncomigrating or ghost peaks caused by waves of an electric field in the capillary). Although peak broadening caused by eigenpeaks (which often suppresses resolution in CEC) has been well described for aqueous or pure non-aqueous BGEs, a systematic study of eigenmobility in aqueous/acetonitrile BGEs had not been reported. The experiments related to the eigenmobility led to a new approach to tune the eigenpeak position in chromatograms whereby a selective sample peak sharpening mechanism was elaborated using the sharpening zone. The novel approach was demonstrated for the separation of various peptides in the CE mode and for the resolution of incompletely resolved chiral amino acids in CEC. These investigations furthermore led to the development of an appropriate stationary phase, which was produced using techniques for an in-column synthesis of a molecular imprinted polymer (MIP). Well prepared MIPs are capable of specifically enhancing the retention of target analytes, herein focusing on enantio-selective isolation of chiral molecules. Since such a MIP was rather rigid, which often caused capillary clogging when used as a conventional monolithic stationary phase, this MIP was incorporated into porous layer open tubular (PLOT) capillaries. Numerous experimental difficulties were overcome with respect to the vinylisation of long fused-silica capillaries and for the polymerisation of the MIP within using thermal- and photo-polymerisations. A method for the characterization of such MIP-PLOT capillaries was elaborated involving the Langmuir adsorption theory. The new columns were evaluated using chromatography, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX) and CEC analysis. All experiments were designed according to the principles of Green Chemistry. These concepts involved the use of small scale analysis, simulation studies reducing the number of performed experiments, systematic studies with low amounts of solvents and materials, and optimisation of synthesis methods to reduce time, energy and reagent consumption. The theories, methods and results presented in this thesis will, in the future, allow further targeted developments of novel analytical tools for a green, sensitive, selective and improved resolution analysis of chemical and biological compounds including chiral compounds, peptides and proteins.

Pharmaceutical analysis guarantees patient safety all over the world. Thus, continuous development of existing analytical techniques is still very important. Pressurized capillary electrochromatography and pressure-assisted capillary electrochromatography are hybrid separation techniques that combine the selectivity of liquid chromatography and the high separation efficiency of capillary electrophoresis. They use a smaller amount of reagents and samples, hence, reducing the total cost of analysis. Therefore they have found application in a number of pharmaceutical and biomedical analysis. This review article focuses on the use and importance of pressurized and pressure-assisted capillary electrochromatography in pharmaceutical and biomedical analysis, taking into account types of detectors and capillaries used. Despite the fact that pressurized capillary electrochromatography and pressure assisted capillary electrochromatography offer many possibilities and have been available for over a dozen years, they are still underdevelopment and not fully explored.

The protein modified monolithic column in affinity capillary electrochromatography (CEC) has attracted considerable attention over the past decades because of its great enantioseparation ability. A porous polymethacrylate ester-based capillary monolithic column poly (glycidyl methacrylate-co-ethyleneglycol dimethacrylate) (poly (GMA-co-EDMA)) was prepared by in situ co-polymerization. The process was initiated thermally by azobisisobutyronitrile (AIBN). The polymerization mixture contained GMA as the function monomer and EDMA as the crosslinking agent with 1,4-butanediol and 1-propanol as the binary porogen solvent. Under the optimized reaction conditions, including the proportion of monomer and porogens, reaction temperature etc, the column exhibited a uniform structure, sufficient permeability and excel- lent pressure resistance. The separation of alkyl benzenes on the column was mainly based on typical reversed-phase chromatographic retention mechanism. The reproducibility and stability were good with RSDs less than 9. 0%. A pepsin functionalized organic polymer monolith was prepared by covalently bonded pepsin to poly(GMA-co-EDMA) monolith with glutaraldehyde as a spacer based on the activity of epoxide group. The enantioseparation performance of the pepsin affinity monolith for basic enantiomers has been investigated by CEC. Nefopam, amlodipine, citalopram and chlorpheniramine were resolved, and baseline separations of nefopam, amlodipine, citalopram were achieved. The influences of pH, operating voltage, temperature and sample quantity used on the chiral separation were studied. The chiral recognition mechanism of enantiomers on the monolithic column in CEC is discussed. This work developed a new method for the prepataion and application of protein affinity monolith in CEC.

Chapter 7 - Capillary electrophoresis and electrochromatography of carbohydrates

Capillary electrochromatography (CEC) is a hybrid separation technique of capillary high‐performance liquid chromatography (cHPLC) and capillary electrophoresis (CE). The mobile phase in a capillary column of CEC is driven by electroosmotic flow (EOF), giving advantages such as high efficiency, high resolution, high selectivity, and low sample and solvent consumption. However, the formation of air bubbles hinders the CEC to become a mainstream separation technique. In order to solve this problem, pressurized capillary electrochromatography (pCEC), with EOF combining hydraulic pressure as its driving force, has been developed and gained more attention. In this article, we introduce the development of CEC and pCEC on their theory, instrumentation, sample introduction, column technology, and applications. Besides, gradient elution and two‐dimensional separation based on CEC and pCEC will also be illustrated.

Capillary columns with in situ formed porous monolithic packing for micro high-performance liquid chromatography and capillary electrochromatography

Thein situdetection of organic molecules in space is key to understanding the variety and the distribution of the building blocks of life, and possibly the detection of extraterrestrial life itself. Gas chromatography mass spectrometry (GC-MS) has been the most sensitive analytical strategy for organic analyses in flight, and was used on missions from NASA's Viking, Phoenix, Curiosity missions to ESA's Rosetta space probe. While pyrolysis GC-MS revealed the first organics on Mars, this step alters or degrades certain fragile molecules that are excellent biosignatures including polypeptides, oligonucleotides and polysaccharides, rendering the intact precursors undetectable. We have identified a solution tailored to the detection of biopolymers and other biomarkers by the use of liquid-based capillary electrophoresis and electrochromatography. In this study, we show that a capillary electrochromatography approach using monolithic stationary phases with tailor-made surface chemistry can separate and identify various polycyclic aromatic hydrocarbons, nucleobases and aromatic acids that could be formed under astrophysically relevant conditions. In order to simulate flyby organic sample capture, we conducted hypervelocity impact experiments which consisted of accelerating peptide-soaked montmorillonite particles to a speed of 5.6 km s−1, and capturing them in an amorphous silica aerogel of 10 mg cm−3bulk density. Bulk peptide extraction from aerogel followed by capillary zone electrophoresis led to the detection of only two stereoisomeric peptide peaks. The recovery rates of each step of the extraction procedure after the hypervelocity impact suggest that major peptide loss occurred during the impact. Our study provides initial exploration of feasibility of this approach for capturing intact peptides, and subsequently detecting candidate biomolecules during flight missions that would be missed by GC-MS alone. As the monolith-based electrochromatography technology could be customized to detect specific classes of compounds as well as miniaturized, these results demonstrate the potential of the instrumentation for future astrobiology-related spaceflight missions.

ABSTRACTFlavonoids are compounds which provide many pharmacological activities. In this paper, the literature with regard to the determination of flavonoids using capillary electrophoresis (CE) during 2008–2017 has been reviewed. The aim of the review is to overview the modes and specific determination conditions of CE for flavonoids. The advantages and disadvantages of CE in the determination of flavonoids are analyzed. The applications of the modes of CE for determination of flavonoids are introduced. Capillary zone electrophoresis, micelle electrokinetic capillary chromatography, microemulsion electrokinetic chromatography, and capillary electrochromatography are considered. Specific determination conditions including the pH, buffer, additive, capillary, voltage, temperature, injection, analysis time, detection, and online concentration methods are summarized. Flavonoids were generally separated in alkaline conditions at pH 8–10 in borate buffer. The advantages of CE are the short analysis time and low solvent consumption compared with the most popular high-performance liquid chromatography protocols. CE can be combined with a variety of detectors including ultraviolet spectrophotometry, mass spectrometry, chemiluminescence, electrochemical, potential gradient, dual opposite carbon fiber microdisk electrodes, and laser-induced fluorescence. In CE, short-light path length results in relative low sensitivity when combined with the most widely used ultraviolet spectrophotometry detector. The combination with more sensitive detectors and online concentration methods such as gradient elution, large volume sample stacking, sweeping, and electrokinetic supercharging alleviates this problem and improves the sensitivity. Capillary electrophoresis is an efficient, green, and valuable tool for the determination of flavonoids.

Chapter 15 - Capillary electrophoresis

Kinetic performance evaluation and perspectives of contemporary packed column capillary electrochromatography

A new in-house designed and constructed injection valve for capillary electrochromatography (CEC) based on a rotating injection part with compartments for the eluent as well as for the sample has been coupled to a mass spectrometer via a sheath flow electrospray ionisation (ESI) interface, using short capillary columns of 15 cm length. The CEC columns were packed with 3 microm C(18) bonded silica particles, and a mixture of peptides was analysed using an ammonium acetate/acetonitrile eluent. A significant increase in the signal-to-noise ratio was obtained when the peptides were dissolved in water with the same content of organic modifier as in the eluent with an addition of 0.5% (v/v) acetic acid. When the CEC analysis was performed without any additional pressure, the separation current sometimes dropped tremendously due to bubble formation, caused by different permeability in the first and packed part of the column causing an extremely low electroosmotic flow. The separation current was restored to its original value by applying only 7 bar at the inlet of the CEC column, and the separation performance for the test peptides was recovered. A comparison of the CEC performance of peptides in pure CEC mode and in low-pressure CEC mode is reported.

Capillary electrochromatography (CEC) is a separation method that has gained increasing attention in recent years. It combines the advantages of high performance liquid chromatography (HPLC) selectivity with the high efficiency of electrophoresis. Due to its promise of high peak capacities, CEC appears to be promising for resolution of complex mixtures, such as those generated during protein and peptide mapping. However, direct transfer of HPLC and capillary zone electrophoresis (CZE) methods to CEC is not trivial due to the unique requirements for CEC in terms of stationary phase and operating conditions. In this paper, we discuss the various approaches undertaken for the CEC separation of peptides and proteins.

As one can see from just a few papers dealing with the topic of Enantioseparation in this volume of Electrophoresis, this field appears to have maturated, and the number of research groups still performing innovative research on this topic decreases. There are several objective and subjective reasons for this. One of the reasons is that the separation of enantiomers of chiral compounds, which was a huge challenge 40–50 years ago, is a routine laboratory task currently. The other reason is that research and publications in some trendy areas, such as omics, get better funded, more easily published, and well cited compared with research and publications related to enantioseparations and fundamental aspects of enantioselective molecular recognition. When preparing the previous issue of Electrophoresis on Enantioseparations 2021, we sent about 200 invitations and received numerous submissions, but unfortunately, many of them were rejected due to their low quality. When preparing this special issue, we invited fewer but only high-quality groups, and the rejection rate was lower. However, unfortunately this time, we had fewer manuscripts accepted for publication, even compared with the last issue. Even more troubling is the fact that, of these accepted high-quality manuscripts, only very few deal with capillary electrophoresis (CE), none of them with capillary electrochromatography (CEC), and most of them deal with high-performance liquid chromatography (HPLC). It is absolutely normal that challenging problems drive the research activity of scientists. Such challenges appear, get addressed, disappear, and new challenges arise in time. Thus, one cannot promote any kind of research if there is no real driving reason for that, and, of course, this is also not my task in this short Editorial. Because I have performed research in capillary electromigration techniques over the last 30 years, I just want to share, in a few sentences, my personal opinion about these techniques, their potential, their present, and their future. I remember well the fascination of scientists with both CE and CEC in the 1990s. Both techniques offer something special that is still not offered by any other separative and/or non-separative technique(s), and this makes it very hard for me to accept observing these techniques losing popularity. On a technical note, both techniques are environmentally friendly miniaturized techniques, and with the current fashion about green, sustainable, and circular chemistry, it is just not logical to take CE and CEC out of the repertoire of chemistry. On a fundamental note, both of these techniques offer extremely high separation efficiency with certain positive consequences, which are unmet by other techniques. In addition, electrokinetically driven flow is easier to generate without using mechanics (pumps), to direct, measure, and control. For those who are interested in understanding recognition processes on a molecular level (among them also enantioselective recognition), it must be underlined that CE is the technique with highest sensitivity to detect (inter)molecular recognition. It is true that performing CE and CEC experiments is not as easy and comfortable as the standard HPLC experiment. However, based on my over 30 years of experience, this challenge is very rewarding and worth being taken up. Thus, I sincerely hope that for the next special issue of Electrophoresis on Enantioseparations, we will receive more high-quality submissions based on capillary electromigration techniques. I would like to express my thanks to the Editors-in-Chief of Electrophoresis, Dr. Blanca Lapizco-Encinas and Prof. Hermann Wätzig, as well as the journal publisher, for supporting this special issue and for pleasant and efficient cooperation. My special thanks go also to all authors for submitting their high-quality works for publication in this issue and to all reviewers for their time, efforts, and competent comments. April 29, 2024.

This review summarizes recent developments in chiral separation in capillary zone electrophoresis (CZE), electrokinetic chromatography (EKC), and capillary electrochromatography (CEC) covering literature published since the year 2000. New chiral selectors and innovative approaches for CE and CEC are introduced. Recent progress in column technology for CEC is highlighted and the development of new chiral stationary phases is discussed. This review is not dedicated to list applications but will focus on new developments.