Comparison of Overpressured-Layer Chromatography and High-Performance/High-Pressure Layer Electrochromatography Using the New Prototype Equipment in Various Operational Modes.

In our previous paper we have presented the new prototype equipment and introduced a new analytical technique-high-performance/high-pressure layer electrochromatography (HPLEC), a combination of overpressured-layer chromatography (OPLC) and pressurized planar electrochromatography (PPEC). In this paper, the work of the equipment in various operational modes is investigated. Some difficulties and challenges related to various aspects of separation are discussed. The OPLC and HPLEC techniques are compared in terms of selectivity and performance. The results show that our equipment can be successfully used for singe- and multichannel OPLC and HPLEC separations in various sample application and detection modes. It includes the high-throughput, multichannel, and fully automated online separation of multiple samples simultaneously. The equipment allows for the independent optimization of various operational parameters. HPLEC combines the advantages of column/capillary and planar separation techniques while overcoming their limitations. It also combines the advantages and overcomes the drawbacks of OPLC and PPEC. It provides hydrodynamic flow of the mobile phase, irrespective of the voltage used and/or the mobile phase composition. Thus, any optimization of the composition and the voltage can be used independently. Both can be used to obtain the required selectivity of separation. The voltage can be used to facilitate the mobile phase flow and accelerate the analysis.

The New Omics Era into Systems Approaches: What Is the Importance of Separation Techniques?

Omics sciences have been facing challenges in different fields, especially in life sciences. One of these challenges involves assessing biology into systems interpretation. With the advance of genomics, molecular biology has been projected into the realm of systems biology. In a different direction, systems approaches are making definitive strides toward scientific understanding and biotechnological applications. Separation techniques provided meaningful progress in the omics era, conducting the classical molecular biology to contemporary systems biology. In this introductory chapter, the relevance of these techniques to the development of different omics sciences, within the systems biology context, will be discussed.

Circulating Tumor Cell Enrichment Technologies.

Circulating tumor cells (CTCs) are responsible for the metastatic spread of cancer and therefore are extremely valuable not only for basic research on cancer metastasis but also as potential biomarkers in diagnosing and managing cancer in the clinic. While relatively non-invasive access to the blood tissue presents an opportunity, CTCs are mixed with approximately billion-times more-populated blood cells in circulation. Therefore, the accuracy of technologies for reliable enrichment of the rare CTC population from blood samples is critical to the success of downstream analyses. The focus of this chapter is to provide the reader an overview of significant advances made in the development of diverse CTC enrichment technologies by presenting the strengths of individual techniques in addition to specific challenges remaining to be addressed.

Measurements of Activity Coefficients at Infinite Dilution for Organic Solutes in the Ionic Liquids N-Ethyl- and N-Octyl-N-methylmorpholinium Bis(trifluoromethanesulfonyl)imide. A Useful Tool for Solvent Selection.

In recent years, many papers describing ionic liquids (IL) as promising solvents in separation techniques have been published. The conscious choice of appropriate ionic liquid as absorption media in effective extraction of selected types of analytes requires deeper understanding of the analyte-IL interactions. Therefore, intensive research is conducted to determine the values of activity coefficient at infinite dilution, which allows us to characterize the nature of these interactions. Based on the inverse gas chromatography retention data, activity coefficients at infinite dilution γ 13 ∞ of 48 different organic compounds in the ionic liquids N-ethyl-N-methylmorpholinium bis(trifluoromethanesulfonyl)imide [C2C1Mor][TFSI] and N-octyl-N-methylmorpholinium bis(trifluoromethanesulfonyl)imide [C8C1Mor][TFSI] were determined. The measurements covered a broad range of volatile organic compounds, including n-alkanes, n-alkenes, n-alkynes, alcohols, aldehydes, ketones, aromatic compounds and common polar solvents, representing different types of interactions. Activity coefficients at infinite dilution were measured in the temperature range from 313.15 to 363.15 K. The excess partial molar enthalpies and entropies at infinite dilution were determined. Selectivity at infinite dilution was also calculated for exemplary separation processes in the hexane/benzene system. The obtained results were analyzed and compared with literature data for ionic liquids containing the same anion [TFSI]¯ and different cations. The study results indicate that some potential applications of the investigated ionic liquids in separation problems exist.

Effects of electron beam sterilization on polyethylene terephthalate: Physico-chemical modifications and formation of non-volatile organic extractables.

Our investigations hereby involve the effects of ionizing radiation as a sterilization method, and especially electron beams, on a medical grade Polyethylene Terephthalate (PET). Ionizing radiations are known to induce free radicals formation in the polymer that will then either degrade or crosslink, depending on its chemical nature, ionizing type and irradiation atmosphere (Charlesby, 1967; Dole, 1972 and Dole, 1973). The evaluation of packaging material modifications under radiation sterilization is of great interest and the objective of our paper is to focus on both PET volume modifications and extractable analyses after electron beam sterilization. As regards the polymeric matrix and after sterilization, we did not observe by means of spectral analyses, any single chemical modification whatsoever, whereas by thermal techniques we evidenced polymer chain scissions. As for the non-volatile organic extractables, we further substantiate the presence of numerous compounds, namely oligomers and trimers especially, along with benzoic and terephthalic acids in particular. With respect to pharmaceutical stakes however, we claim herein that the 25kGy sterilization dose used, triggers oligomers and extractables formation, a result which is promising for further risk analysis.

Therapeutic Effect of CD4+CD25+ Regulatory T Cells Amplified In Vitro on Experimental Autoimmune Neuritis in Rats.

BACKGROUND/AIMS: This study aimed to explore whether the adoptive transfusion of autologous CD4+CD25+ regulatory T cells (CD4+CD25+ Tregs) has a therapeutic effect on Experimental autoimmune neuritis (EAN) model rats, and it provides new experimental and theoretical bases for the immunotherapy of Guillain-Barre syndrome (GBS). METHODS: CD4+CD25+ Tregs were sorted from the spleens of rats using immunomagnetic bead separation techniques combined with flow cytometry. Their in vitro inhibitory function was determined using a lymphocyte proliferation inhibition test, and their purity was confirmed by flow cytometry. Cells were stimulated using CD3/CD28 monoclonal antibodies and were cultured in culture medium containing interleukin 2 (IL-2), transforming growth factor-ß (TGF-ß) and rapamycin. After 15 days of amplification, CD4+CD25+ Tregs were collected and transfused into EAN model rats. Changes in the pathology and electron microscopical morphology of rat sciatic nerves in the normal group, untreated group, low-dose group (2 × 107) and high-dose group (4 × 107) were observed, and the expression of CD4+CD25+FOXP3 in peripheral blood in the four groups of rats was detected by flow cytometry. RESULTS: Compared with rats in the untreated group, rats in the treatment groups had significantly reduced infiltration of inflammatory cells in the sciatic nerve, as well as myelin and axonal damage. Additionally, the CD4+CD25+ Tregs levels in peripheral blood were significantly higher than those in the untreated group (P< 0. 05). Moreover, the therapeutic effect became more significant with an increase in the dose of adoptive transfusion. CONCLUSION: Adoptive transfusion of CD4+CD25+ Tregs into EAN model rats has significant therapeutic effects.

Development of an electrophoretic method based on nanostructured materials for HbA1c determination.

Glycosylated hemoglobin (HbA1c) detection is performed routinely in hospitals as it is the most widespread confirmatory diagnosis of diabetes mellitus. Here we present a novel CE method for measuring HbA1c by introducing silica nanoparticles (NPs) modified with a boronic acid derivative (sugar loadings of 51 ± 2 µg/mg) as pseudo-stationary phase. Before the sample injection, SiO2 NP─B(OH)2 were introduced via pressure. Electrophoretic separation was explored through variation of the buffer pH and separation voltage, being the best separation, resolution and shorter separation time achieved with a 25 mM phosphate buffer pH 6.5. The calibration curve obtained was expressed as Area = 182.05%-1 × HbA1c - 377.02; R2  = 0.9826, using a UV/VIS absorbance detector at 415 nm (diode array). No interferences were observed from carbamylated or acetylated hemoglobin and the method shows a noteworthy stability. A paired t-test was applied to compare the developed CE method with a commercial HbA1c test and no significant variations have been observed at a 90% significance level.

Analytical Chemistry: Current Trends in Light of the Historic Beginnings.

Selected current trends in analytical chemistry are discussed and traced back to original work published more than a hundred years ago in the same field. Gravimetric microanalysis has been transformed into a mass-sensitive sensor technology. New developments in molecular spectroscopy are seen in Raman spectroscopy. In the area of chemical sensors, paper-based devices with visual readout (dipsticks, microfluidic pads) celebrate a revival. In "green" applications, the separation of analytes from complicated matrices is often the key to successful analysis. Continuous separation techniques are essential for the 24/7 production of goods with certified quality. Finally, micro- and nanoscale techniques rely on highly selective receptors, and their development is based on detailed understanding of the structure of ligand-receptor complexes.

Mesenchymal stromal/stem cell separation methods: concise review.

Mesenchymal stem (stromal) cells (MSCs) possess unique biological characteristics such as plasticity, long term self-renewal, secretion of various bioactive molecules and ability of active migration to the diseased tissues that make them unique tool for regenerative medicine, nowadays. Until now MSCs were successfully derived from many tissue sources including bone marrow, umbilical cord, adipose tissue, dental pulp etc. The crucial step prior to their in vitro expansion, banking or potential clinical application is their separation. This review article aims to briefly describe the main MSCs separations techniques currently available, their basic principles, as well as their advantages and limits. In addition the attention is paid to the markers presently applicable for immunoaffinity-based separation of MSCs from different tissues and organs.

Approaches for enantioselective resolution of pharmaceuticals by miniaturised separation techniques with new chiral phases based on nanoparticles and monolithis.

This article discusses new developments in the preparation of nanoparticles and monoliths with emphasis upon their application as the stationary and pseudo-stationary phases for miniaturised liquid phase separation techniques, which have occurred in the last 10 years (from 2006 to the actuality). References included in this review represent current trends and state of the art in the application of these materials to the analysis, by EKC, CEC and miniaturised chromatography, of chiral compounds with environmental interest such as pharmaceuticals. Due to their extraordinary properties, columns prepared with these new chiral stationary or pseudo-stationary phases, based on materials such as gold nanoparticles, metal-organic frameworks, ordered mesoporous silicas, carbonaceous materials, polymeric-based and silica-based monoliths or molecularly imprinted materials, can usually show some improvements in the separation selectivity, column efficiency and chemical stability in comparison with conventional chiral columns available commercially.

Pig slurry acidification and separation techniques affect soil N and C turnover and N2O emissions from solid, liquid and biochar fractions.

The combined effects of pig slurry acidification, subsequent separation techniques and biochar production from the solid fraction on N mineralisation and N2O and CO2 emissions in soil were investigated in an incubation experiment. Acidification of pig slurry increased N availability from the separated solid fractions in soil, but did not affect N2O and CO2 emissions. However acidification reduced soil N and C turnover from the liquid fraction. The use of more advanced separation techniques (flocculation and drainage > decanting centrifuge > screw press) increased N mineralisation from acidified solid fractions, but also increased N2O and CO2 emissions in soil amended with the liquid fraction. Finally, the biochar production from the solid fraction of pig slurry resulted in a very recalcitrant material, which reduced N and C mineralisation in soil compared to the raw solid fractions.

A Critical Review on Clinical Application of Separation Techniques for Selective Recognition of Uracil and 5-Fluorouracil.

The most important objectives that are frequently found in bio-analytical chemistry involve applying tools to relevant medical/biological problems and refining these applications. Developing a reliable sample preparation step, for the medical and biological fields is another primary objective in analytical chemistry, in order to extract and isolate the analytes of interest from complex biological matrices. Since, main inborn errors of metabolism (IEM) diagnosable through uracil analysis and the therapeutic monitoring of toxic 5-fluoruracil (an important anti-cancerous drug) in dihydropyrimidine dehydrogenase deficient patients, require an ultra-sensitive, reproducible, selective, and accurate analytical techniques for their measurements. Therefore, keeping in view, the diagnostic value of uracil and 5-fluoruracil measurements, this article refines several analytical techniques involved in selective recognition and quantification of uracil and 5-fluoruracil from biological and pharmaceutical samples. The prospective study revealed that implementation of molecularly imprinted polymer as a solid-phase material for sample preparation and preconcentration of uracil and 5-fluoruracil had proven to be effective as it could obviates problems related to tedious separation techniques, owing to protein binding and drastic interferences, from the complex matrices in real samples such as blood plasma, serum samples.

Solid Phase Extraction as an Innovative Separation Method for Measuring Free and Entrapped Drug in Lipid Nanoparticles.

PURPOSE: Contrary to physical characterization techniques for nanopharmaceuticals (shape, size and zeta-potential), the techniques to quantify the free and the entrapped drug remain very few and difficult to transpose in routine analytical laboratories. The application of Solid Phase Extraction (SPE) technique was investigated to overcome this challenge. METHODS: The separation of free and entrapped drug by SPE was quantitatively validated by High Performance Liquid Chromatography. The developed protocol was implemented to characterize cyclosporine A-loaded 120 nm-sized lipid nanoparticles (LNPs, Lipidot®) dispersed in aqueous buffer. The colloidal stability was assessed by Dynamic Light Scattering (DLS). RESULTS: Validation experiments demonstrated suitable linearity, repeatability, accuracy and specificity to quantify residual free, entrapped and total drug. For the investigated LNPs, the method revealed a very limited shelflife of the formulation when stored in an aqueous buffer at 5°C and even more at elevated temperature. Nevertheless, the DLS measurements confirmed the stability of nanoparticles during SPE in a suitable concentration range. CONCLUSIONS: SPE, when successfully validated, represents a valuable tool for drug development and quality control purposes of lipid-based nanopharmaceuticals in an industrial environment.

PEG-imbedded PEO membrane developed by a novel highly efficient strategy toward superior gas transport performance.

Low-molecular-weight poly(ethylene glycol) (PEG) is deliberately incorporated into synthesized swellable poly(ethylene oxide) (PEO) membranes via a facile post-treatment strategy. The membranes exhibit both larger fractional free volume (FFV) and a higher content of CO2-philic building units, resulting in significant increments in both CO2 permeability and CO2/H2 selectivity. The separation performance correlates nicely with the microstructure of the membranes. This study may provide useful insights in the formation and mass transport behavior of highly efficient polymeric membranes applicable to clean energy purification and CO2 capture, and possibly bridge the material-induced technology gap between academia and industry.

Cells transformed by murine herpesvirus 68 (MHV-68) release compounds with transforming and transformed phenotype suppressing activity resembling growth factors.

In this study, we investigated the medium of three cell lines transformed with murine herpesvirus 68 (MHV-68) in vitro and in vivo, 68/HDF, 68/NIH3T3, and S11E, for the presence of compounds resembling growth factors of some herpesviruses which have displayed transforming and transformed phenotype suppressing activity in normal and tumor cells. When any of spent medium was added to cell culture we observed the onset of transformed phenotype in baby hamster kidney cells (BHK-21) cells and transformed phenotype suppressing activity in tumor human epithelial cells (HeLa). In media tested, we have identified the presence of putative growth factor related to MHV-68 (MHGF-68). Its bivalent properties have been blocked entirely by antisera against MHV-68 and two monoclonal antibodies against glycoprotein B (gB) of MHV-68 suggesting viral origin of MHGF-68. The results of initial efforts to separate MHGF-68 on FPLC Sephadex G15 column in the absence of salts revealed the loss of its transforming activity but transformed phenotype suppressing activity retained. On the other hand, the use of methanol-water mobile phase on RP-HPLC C18 column allowed separation of MHGF-68 to two compounds. Both separated fractions, had only the transforming activity to normal cells. Further experiments exploring the nature and the structure of hitherto unknown MHGF-68 are now in the progress to characterize its molecular and biological properties.

A three-minute synthesis and purification of ibuprofen: pushing the limits of continuous-flow processing.

In a total residence time of three minutes, ibuprofen was assembled from its elementary building blocks with an average yield of above 90% for each step. A scale-up of this five-stage process (3 bond-forming steps, one work-up, and one in-line liquid-liquid separation) provided ibuprofen at a rate of 8.09 g h(-1) (equivalent to 70.8 kg y(-1)) using a system with an overall footprint of half the size of a standard laboratory fume hood. Aside from the high throughput, several other aspects of this synthesis expand the capabilities of continuous-flow processing, including a Friedel-Crafts acylation run under neat conditions and promoted by AlCl3, an exothermic in-line quench of high concentrations of precipitation-prone AlCl3, liquid-liquid separations run at or above 200 psi to provide solvent-free product, and the use of highly aggressive oxidants, such as iodine monochloride. The use of simple, inexpensive, and readily available reagents thus affords a practical synthesis of this important generic pharmaceutical.

A comprehensive review on the enantiomeric separation of chiral drugs using metal-organic frameworks.

Chiral drugs play an important role in modern medicine, but obtaining pure enantiomers from racemic mixtures can pose challenges. When a drug is chiral, only one enantiomer (eutomer) typically exhibits the desired pharmacological activity, while the other (distomer) may be biologically inactive or even toxic. Racemic drug formulations introduce additional health risks, as the body must still process the inactive or detrimental enantiomer. Some distomers have also been linked to teratogenic effects and unwanted side effects. Therefore, developing efficient and scalable methods for separating chiral drugs into their pure enantiomers is critically important for improving patient safety and outcomes. Metal-organic frameworks (MOFs) show promise as novel materials for chiral separation due to their highly tunable structures and interactions. This review summarizes recent advancements in using MOFs for chromatographic and spectroscopic resolution of drug enantiomers. Both the opportunities and limitations of MOF-based separation techniques are discussed. A thorough understanding of these methods could aid the continued development of pure enantiomer formulations and help reduce health risks posed by racemic drug mixtures.

Non-enzymatic methods for isolation of stromal vascular fraction and adipose-derived stem cells: A systematic review.

BACKGROUND: Adipose-derived stem cells (ADSCs) and the stromal vascular fraction (SVF) have garnered substantial interest in regenerative medicine due to their potential to treat a wide range of conditions. Traditional enzymatic methods for isolating these cells face challenges such as high costs, lengthy processing time, and regu-latory complexities. AIM: This systematic review aimed to assess the efficacy and practicality of non-enzymatic, mechanical methods for isolating SVF and ADSCs, comparing these to conventional enzymatic approaches. METHODS: Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, a comprehensive literature search was conducted across multiple databases. Studies were selected based on inclusion criteria focused on non-enzymatic isolation methods for SVF and ADSCs from adipose tissue. The risk of bias was assessed, and a qualitative synthesis of findings was performed due to the methodological heterogeneity of the included studies. RESULTS: Nineteen studies met the inclusion criteria, highlighting various mechanical techniques such as centrifugation, vortexing, and ultrasonic cavitation. The review identified significant variability in cell yield and viability, and the integrity of isolated cells across different non-enzymatic methods compared to enzymatic procedures. Despite some advantages of mechanical methods, including reduced processing time and avoidance of enzymatic reagents, the evidence suggests a need for optimization to match the cell quality and therapeutic efficacy achievable with enzymatic isolation. CONCLUSION: Non-enzymatic, mechanical methods offer a promising alternative to enzymatic isolation of SVF and ADSCs, potentially simplifying the isolation process and reducing regulatory hurdles. However, further research is necessary to standardize these techniques and ensure consistent, high-quality cell yields for clinical applications. The development of efficient, safe, and reproducible non-enzymatic isolation methods could significantly advance the field of regenerative medicine.

Uncertainty propagation from probe spacing to Fourier 3-probe straightness measurement.

Reliable and precise straightness profile measurements are crucial for manufacturing ultra-precision components and are capable of further enhancing their accuracy. The Fourier three-probe (F3P) straightness measurement allows for precise assessment of the workpiece profile on the machine by eliminating the harmful influence of the error motion of the sliding table. However, the probe spacing uncertainty deteriorates the measurement accuracy remarkably; and, the affecting mechanism behind this phenomenon has not yet been studied in detail. In this context, this paper thoroughly investigated the propagation of the probe spacing uncertainty in the F3P measurement. First, the influence of the probe spacing deviation is analyzed. Next, by calculating the partial differential of Laplace transform of the workpiece profile, we algebraically deduce the probe spacing uncertainty propagation law, especially in the harmonic domain. Subsequently, Monte Carlo simulations are carried out to confirm the derived propagation law. To reduce uncertainty propagation, a hybrid approach is presented: (I) F3P measurements are carried out under changing probe spacings to produce several sets of Fourier coefficients; (II) optimal harmonic estimates are selected individually according to the harmonic uncertainty. Finally, simulations and experimental measurements are performed for verification.

Scorpion Venom as a Source of Antimicrobial Peptides: Overview of Biomolecule Separation, Analysis and Characterization Methods.

Scorpion venoms have long captivated scientific researchers, primarily due to the potency and specificity of the mechanism of action of their derived components. Among other molecules, these venoms contain highly active compounds, including antimicrobial peptides (AMPs) and ion channel-specific components that selectively target biological receptors with remarkable affinity. Some of these receptors have emerged as prime therapeutic targets for addressing various human pathologies, including cancer and infectious diseases, and have served as models for designing novel drugs. Consequently, extensive biochemical and proteomic investigations have focused on characterizing scorpion venoms. This review provides a comprehensive overview of the key methodologies used in the extraction, purification, analysis, and characterization of AMPs and other bioactive molecules present in scorpion venoms. Noteworthy techniques such as gel electrophoresis, reverse-phase high-performance liquid chromatography, size exclusion chromatography, and "omics" approaches are explored, along with various combinations of methods that enable bioassay-guided venom fractionation. Furthermore, this review presents four adapted proteomic workflows that lead to the comprehensive dissection of the scorpion venom proteome, with an emphasis on AMPs. These workflows differ based on whether the venom is pre-fractionated using separation techniques or is proteolytically digested directly before further proteomic analyses. Since the composition and functionality of scorpion venoms are species-specific, the selection and sequence of the techniques for venom analyses, including these workflows, should be tailored to the specific parameters of the study.