Capillary Electrochromatography for Analysis of Small Polar Molecules Using Etched Chemically Modified Capillaries

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.

Recent applications and chiral separation development based on stationary phases in open tubular capillary electrochromatography (2019–2022)

Capillary electrochromatographic study of the interactions of porphyrin derivatives with amino acids and oligopeptides

Use of nanoparticles in capillary and microchip electrochromatography

Replacing pressure-driven flow with electroosmotic flow in chromatography with packed capillary columns results in a new and powerful analytical technique, combining the advantages of high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE): capillary electrochromatography (CEC). In essence, it involves packing CE capillaries with HPLC stationary phases and applying a voltage across the packed capillary, which generates an electroosmotic flow (EOF) that transports solutes along the capillary toward the detector; on the way, both differential partitioning and electrophoretic migration of the solutes occur, resulting in their separation. This dual separation mechanism can afford unique selectivities. Also, the plug-flow profile of EOF reduces flow-related band broadening, so that separation efficiencies of several hundred thousand plates per meter are often obtained (i.e., one or two orders of magnitude greater than those of present conventional chromatographic systems). There is no back pressure when EOF occurs, so small particle sizes (1–3 μm) and/or long columns can be used. Generally, carrier electrolytes containing high levels (40–80%) of organic solvents such as methanol or acetonitrile are employed, making it useful for water-insoluble compounds, which can be difficult to analyse by CE. CEC can be faster than HPLC, and gradient-elution CEC is being developed as an alternative to gradient-elution liquid chromatography of polymers.

Nine xanthones from a Chinese traditional medicine, Securidaca inappendiculata Hassk, were separated by different capillary electrophoresis (CE) modes, including capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), microemulsion electrokinetic capillary chromatography (MEEKC), and capillary electrochromatography (CEC). The comparison of separation selectivity and efficiency for xanthones by using these CE methods indicated that different CE modes greatly varied in the separation selectivity and efficiency for xanthones; their advantages and disadvantages are discussed. Moreover, compared with traditional chromatographic methods, e.g., high performance liquid chromatography (HPLC), CE shows higher separation ability and analytical speed for these xanthones.

Chiral covalent organic framework-based open tubular capillary electrochromatography column for enantioseparation of selected amino acids and pesticides.

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

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.

The goal of the research reported in this dissertation is to develop packed and open-tubular capillary electrochromatographic methods for improved achiral and chiral separations of various classes of analytes. The first part of this research involves the separation of seven benzodiazepines by the use of the packed mode of capillary electrochromatography (CEC) and a 40 cm packed bed of Reliasil 3 μm C18 stationary phase. Optimal conditions were established by varying the mobile phase, the amount of organic modifier, the buffer concentration, the applied voltage, and the column temperature. The second part of this research focuses on the open-tubular mode of CEC and the polyelectrolyte multilayer (PEM) coating approach. In the first study of this part, poly (diallyldimethylammonium chloride), PDADMAC, was used as the cationic polymer and poly (sodium N-undecanoyl-L-glycinate), poly (L-SUG), was used as the anionic polymer for the construction of the PEM coating. The performance of the modified capillaries as a separation medium was evaluated by use of seven benzodiazepines as analytes. In the second study, the anionic polymeric surfactant poly (sodium N-undecanoyl-L-leucylvalinate), poly (L-SULV), was used as the chiral discriminator for the separations of several drug analytes. Reproducibility of the PEM coating was evaluated by computing the relative standard deviation (RSD) values of the electroosmotic flow (EOF). The PEM-coated capillaries were remarkably robust with excellent reproducibilities and high stabilities against extreme pH values. The stability of the capillary surface was further investigated after exposure to NaOH solutions. The structural changes of these coatings were monitored using laser scanning confocal microscopy (LSCM). These changes were discussed in terms of separations using open-tubular CEC (OT-CEC). In addition, the electropherograms obtained from the chiral separation of 1,1’-binaphthyl-2,2’-dihydrogenphosphate (BNP) in OT-CEC allowed the measurements of both selectivity and electroosmotic mobility changes after long exposure to NaOH.

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.

Evaluation of a composite nanomaterial consist of gold nanoparticles and graphene-carbon nitride as capillary electrochromatography stationary phase for enantioseparation

A novel method based on the adsorption of positively charged compounds on the wall of a fused-silica capillary was applied to prepare stationary phases for open tubular capillary electrochromatography (OTCEC). The positively charged substances including cationic surfactant such as cetyltrimethylammonium bromide (CTAB) and basic chiral selectors such as protein, peptide and amino acid were physically adsorbed onto the capillary wall under specially selected conditions. The adsorbed stationary phase of CTAB was used to separate neutral compounds, while the others were used for chiral separations. The run-to-run reproducibility of retention time was rather good with relative standard deviation (RSD) values of less than 2.3%. The separation efficiency was excellent with the highest theoretical plate number of up to 590000/m and the average one above 250000/m. Stored at 2-8 degrees C in the refrigerator, the adsorbed stationary phase can last at least one month. It was observed that the UV spectra for the enantiomers are significantly different due to the diastereomeric interactions of enantiomers with the chiral stationary phase in the detection window. With the use of the same capillary, the same instrument, and the same mobile phase, the superiority of OTCEC over open tubular liquid chromatography (OTLC) and capillary zone electrophoresis (CZE) was illustrated.

Microorganisms attach to nonliving surfaces in many natural, industrial, and medical environments, enveloped within extracellular polymeric substances. The result is a biofilm. Biofilms are reported to exist in 65-80% of bacterial infections refractory to host defenses and antibiotics therapy and are regarded as a central problem in present-day medical microbiology. Understanding of the parameters governing the interaction of antimicrobials with biofilms is thus of great interest in any attempt to increase biocide efficacy. In this work, study was made of the feasibility of using open tubular capillary electrochromatography (CEC) in bacterial biofilm studies with living cells. Staphylococcus aureus was selected as model bacterium. First, S. aureus was shown, under various conditions, to form a biofilm on the inner wall of a fused-silica capillary coated with poly(L-lysine). Optimal conditions for biofilm formation, such as bacterial concentration, growing time, and the stability of the ensemble, were preliminarily defined with conventional 96-microtiter well plates. Continuous flushing of the capillary with fresh cells meant that no growth medium was needed. The presence of biofilm in the capillary was confirmed by atomic force microscopy. Interactions between S. aureus biofilms and different antibiomicrobial agents were studied by capillary electrochromatography. The effect of five antibiotics (penicillin G, oxacillin, fusidic acid, rifampicin, vancomycin) on biofilms was examined in terms of retention factors and reduced mobilities of the antibiotics. The antibiotic susceptibility profile for S. aureus is similar as the result of minimal inhibitory concentrations registered on the 96-microtiter well plates for both planktonic and biofilm cells. The results show, for the first time, that bacterial biofilms can be studied by CEC. The technique allows highly efficient and easy characterization of interactions between S. aureus biofilms and potentially active antimicrobial compounds under different conditions. Reagent and cell consumption are minimal.

Background Thalassemia and haemoglobinopathies pose a huge burden on Indian population, especially in Eastern India. Several studies indicated that abnormal hemoglobin variants influence Hb A1c estimation by High Performance Liquid Chromatography (HPLC) in patients with haemoglobinopathies. We opted capillary electrophoresis (CE) as an alternative method for Hb A1c measurement and aimed to evaluate Hb A1c test results done by (HPLC) with capillary electrophoresis (CE) to rule out the interference caused by abnormal hemoglobin variants. Methods A total of 3000 patients were tested for haemoglobinopathy in our laboratory from (1st -31th) January, 2025. Haemoglobinopathy were screened by Bio-Rad Variant II hemoglobin testing system. Both HPLC and CE methods were employed for Hb A1C testing in the group of patients (137/3000) with haemoglobinopathy using Bio-Rad Variant II Turbo and Sebia Minicap Flex Piercing respectively. Reference range defined for Hb A1c by HPLC: Non-diabetic: 4-5.7, Pre-Diabetic:5.7-6.4, Diabetic =6.5 and reference range for HbA1C by CE: <6 %. Statistical interpretation by was done by SPSS version 26.0. P value less than 0.05 was considered as significant. Statistical correlation was obtained by using Linear regression study. Results : 4.5% (137/3000) of total patients were reported to have abnormal hemoglobin variants in their HPLC chromatogram. 52% (72/137) Hb E carrier, 7.2% (10/137) homozygous E, 23.3% (32/137) beta thalassemia carriers, 1.4% (2/137) beta thalassemia major, 7.2% (10/137) Hb S carrier, 1.4% (2/137) homozygous S ,5.1% (7/137) Hb D carrier and 1.4% (2/137) HPFH (hereditary persistence of fetal hemoglobin) carrier comprised the group of haemoglobinopathies. We compared Hb A1c test results measured by HPLC and CE techniques. Hb A1c test values measured by using both the techniques were observed to be within reference range (Mean±SD: 5.24±1.05 by HPLC, Mean±SD: 5.19±1.08 by CE) in all patients with haemoglobinopathy. Hb A1c tests conducted in patients with haemoglobinopathies by HPLC and CE methods showed strong correlation in test results (Hb E trait: r=0.75 at p =0.01; beta thalassaemia traits: r=0.89 at p=0.04; Hb D: r=0.94 at p=0.2 Hb S: r=0.78 at p=0.21). In contrast to many reported studies, no observable difference was found in Hb A1c values in persons with haemoglobinopathies. HPLC and CE, both the methodologies failed to detect Hb A1c values in homozygous E, homozygous S and beta thalassaemia major patients. The study is limited by less number (n) of participants in each category which might attribute to high p value for determining level of significance. Conclusion Hb A1c test results done by HPLC and CE were in perfect concordance with each other. No such interference by abnormal haemoglobin variants on Hb A1c test results was observed in our study using HPLC method. Replacement of HPLC by CE as an alternative methodology is not necessitated rather both the methods can interchangeably be used for Hb A1c testing in patients with haemoglobinopathies. However, the present study is limited with small number of patients. An extensive study with larger participants is planned to validate the results claimed by our pilot study.