Capillary Electrophoresis

The complexity and challenges of developing, manufacturing, and controlling cell therapies offer today’s chromatographic scientists new opportunities to join the journey of discovery and innovation needed to develop and commercialize this new drug modality. This article explores the latest developments and highlights the importance of new capillary electrophoresis–mass spectrometry (CE–MS)-based approaches to understand the efficiency of cell therapy production.

This is the second in a series of articles exploring current topics in separation science that will be addressed at the HPLC 2019 conference in Milan, Italy, from 16–20 June.

Analytical separation techniques based on the differential migration velocities of analytes under the action of an electric field are gaining increasing acceptance for the analysis of phenolic compounds in edible and medicinal plants and in plant-derived food products. In Part 2 of this review article the authors discuss the fundamental principles and practical aspects of electromigration techniques, including capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), and capillary electrochromatography (CEC). The development of two-dimensional systems, performed by coupling either liquid chromatography (LC) with an electromigration technique or two electromigration techniques, operated under different separation mechanisms, is also discussed.

The analytical toolbox used in present-day metabolomics encounters difficulties for the analysis of limited amounts of biological samples. Therefore, a significant number of crucial biomedical and clinical questions cannot be addressed by the current metabolomics approach. Capillary electrophoresis–mass spectrometry (CE–MS) has shown considerable potential for the profiling of polar and charged metabolites in volume-restricted or mass-limited biological samples. This article considers advances that significantly improved the performance of CE–MS for in-depth metabolic profiling of limited sample amounts. Attention is also devoted to various technical aspects that still need to be addressed to make CE–MS a viable approach for volume-restricted metabolomics.

Inspired by the work of Jorgenson and Lukacs, 30 years ago, the group of Richard Smith at Pacific Northwest National Laboratory in Washington (USA) reported the first online coupling of the microscale separation technique capillary electrophoresis (CE) to electrospray ionization (ESI) mass spectrometry (MS) using a sheath-liquid interface.

In the biomedical research of molecular bases of both normal and pathological biological processes, it is currently necessary not only to detect, identify, and quantify individual compounds, but also to study their interactions with endo- and exogenous compounds. Obviously, for these purposes it is crucial to develop new advanced high‑performance analytical methods providing high sensitivity, high selectivity, and high throughput. These challenging requirements are well met by capillary electromigration (CE) methods. They have developed in the last three and half decades into high‑performance separation techniques suitable for the analysis of a wide spectrum of both low- and high‑molecular mass bioactive compounds.

In this article, we discuss the use of CE-MS (sheath flow interface) for analysis of intact proteins as well as of protein digests. We discuss the unique aspects that the user needs to be aware of while testing biotherapeutics versus small molecule drugs. We also highlight that the optimization of CE and MS parameters together result in the creation of a more robust and reproducible protein analysis approach. Finally, we list some of the most common errors that are likely to occur during CE-MS analysis and suggest ways to overcome them.

A chiral ionic liquid, namely 1-ethyl-3-methyl imidazole L-tartrate ([EMIM][L-Tar]), was applied as a new chiral ligand for the enantioseparation of tryptophan, tyrosine, and phenylalanine enantiomers by chiral ligand exchange CE. To validate the unique behavior of [EMIM][L-Tar], the performance of L-tartaric acid and 1-ethyl-3-methyl imidazole L-proline as chiral ligands was investigated to make a comparison with [EMIM][L-Tar]. Then the separation mechanism was further discussed. It was proven that [EMIM][L-Tar] was a good chiral ligand and would have good application prospects in separation science.

An analytical methodology for the characterization of the primary structure of biotherapeutic proteins using sheathless CE–ESI-MS-MS instrumentation is presented. For the first time, complete sequence coverage can be achieved using a bottom-up proteomic approach from a single analysis of a tryptic digest. In a biosimilarity assessment, a single amino acid substitution was detected.

October 2006. The authors demonstrate the use of multiple-injection affinity capillary electrophoresis (MIACE) and several variations to MIACE to determine binding constants between the glycopeptide antibotics vancomycin, ristocetin, and teicoplanin from Streptomyces orientalis, Nocardia lurida, and Actinoplanes teichomyceticus, respectively, and D-Ala-D-Ala terminus peptides.

Potentiometry is a new detection method for liquid chromatography (LC) and capillary electrophoresis (CE). The principle behind this method is familiar to chromatographers because the signals depend on the partitioning tendency of analytes over the sensor coating and the eluent. This partitioning provokes a change in the surface potential and the detection of these changes can be classified as "potentiometric". A conversion algorithm is needed to convert the generated signals to concentration-related tracings (chromatograms).

With the threat of terrorism growing, the development of analytical techniques for the detection and identification of chemical warfare agent defradation products has increased. Capillary electrophoresis (CE) presents interesting features for this application.

Capillary extraction (CEx) is used to study the solventless in-tube extraction of naphthalene, acenaphthene, phenanthrene, fluoranthene, chrysene, benzo(a)pyrene and coronene in aqueous samples prepared by analyte spiking into clean waters or, as an alternative, by using the generator–column method of sample preparation. Analysis of laden extractors is conveniently performed by high-resolution gas chromatography (GC), with a flame-ionization detector (FID). Extraction set-ups and main extraction variables are investigated from a practical point of view. For 2- to 4-ring polycyclic aromatic hydrocarbons (PAHs), equilibrium times are within a few minutes, analytical sensitivity is in the parts-per-billion (ppb) range and reproducibility is better than 10% relative standard deviation (RSD) (n = 6). Coronene behaviour is unique and presumably determined by extreme hydrophobicity and thus very negligible aqueous solubility: in-tube extraction of coronene seems possible only if starting from..

Almost 40 years have passed since the concept of capillary electrophoresis (CE) was described by Hjertén (1) in 1967. It emerged as a viable analytical technique after the pioneering work of Jorgenson (2) in the early 1980s, and commercial instruments were first introduced at the end of that decade. It is appropriate at this time to survey the history of CE and to judge its success in the world of analytical instrumentation.

It is hypothesized that in particular cases, conventional planar chromatography provides a more effective and robust system than column chromatography with regard to separation efficiency and peak distribution of mixtures composed of low-retarded analytes. Under similar reversed-phase experimental conditions, a regular distribution of thin-layer chromatography (TLC) spots of four natural estrogens (estetrol, estriol, 17?-estradiol, and estrone) corresponds to strong irregular dispersion of peaks in chromatograms generated by high performance liquid chromatography. In both cases, the efficiency of separation was assessed using simple optimization criteria such as selectivity (?min) and resolution (Rs min). The distribution of chromatographic spots was evaluated using the relative resolution product (r). The results revealed that an excellent separation of the components of interest could be achieved easily using simple nonforced and isocratic TLC. Such an interesting property of planar chromatography is mainly driven by the nonlinear relationship between k and Rf retention factors. This article also reports the practical advantages of TLC for the separation of estrogenic steroid mixtures at different temperatures.

In this article the authors report on a combinatorial natural product discovery methodology that uses a viral vector system to transfer secondary metabolite-related enzymes from C. roseus to tobacco cell cultures. Using high-resolution separation techniques, including HPLC, CE and MS, they describe the analysis of secondary metabolite patterns.

The authors evaluate the performance of multiplexed 96-lane CE with laser-induced fluorescence detection for high-throughput separation–based assays using fluorescein isothiocyanate as a model compound and fluorescein as an internal standard.

The Death Cap mushroom is the cause of most mushroom-related poisonings in the world. The author has developed a highly efficient, sensitive CE technique that toxicologists and forensic analysts can use to determine the poisonous peptides in body fluids of affected patients.

The authors review current strategies for amino acid analysis and explain their novel approach for simplified sample preparation of body fluids for amino acid analysis...

This "CE Currents" column examines the use of capillary electrophoresis (CE) in three important areas of pharmaceutical analysis.

In this article, the authors review recent developments in the research of whole-column-imaging detection for capillary electrophoresis (CE). Whole-column-imaging detection was developed for capillary isoelectric focusing, for which it proved to be an ideal detector. Several whole-column-imaging detectors -- including refractive index gradient imaging, UV-absorption imaging, and fluorescence imaging detectors -- have been studied. The capillary isoelectric focusing UV-absorption imaging technique even has been commercialized. The development of whole-column-imaging detection itself facilitates CE studies in many directions such as in electrophoretic dynamics within narrow channels, new separation modes, and two-dimensional separations. Whole-column-imaging detection also finds application in capillary zone electrophoresis.

Wehr examines developments discussed at "CE in the Pharmaceutical Industry," a symposium sponsored by the California Separation Science Society.

Capillary electrophoresis (CE) is a useful and versatile technique for a large number of applications. Devoting some discussion to the development of these reported methods to illustrate the separation approaches and strategies to obtain reproducible results, they also compare CE's advantages and disadvantages with those of the more typical method for each type of analysis.

This month's installment of "CE Currents" deals with problems that may arise when using capillary electrophoresis for unattended, long-term operation, such as sample carryover, evaporation effects, capillary conditioning, capillary surface changes, buffer handling, capillary breakage, and detector lamp deterioration.

By adopting standard buffers, analysts can gain a great deal of operating efficiency and significantly reduce method development and validation costs.

Despite their different levels of technological maturity, CE and LC coupled with electrospray ionization MS techniques can be operated at the same level of automation. However, they differ in their configuration, selectivity, sensitivity, and method development.

The author describes an ultrathin-layer agarose gel electrophoresis approach that combines the advantages of the multilane format of conventional slab and the performance of capillary gel electrophoresis.