The linear theory of electromigration, including the first-order nonlinear approximation, is generalized to systems with any equilibria fast enough to be considered instantaneous in comparison with the timescale of peak movement. For example, this theory is practically applied in the electrokinetic chromatography (EKC) mode of the CZE. The model enables the calculation of positions and shapes of analyte and system peaks without restricting the number of selectors, the complexation stoichiometry, or simultaneous acid-base equilibria. The latest version of our PeakMaster software, PeakMaster 6-Next Generation, implements the theory in a user-friendly way. It is a free and open-source software that performs all calculations and shows the properties of the background electrolyte and the expected electropherogram within a few seconds. In this paper, we mathematically derive the model, discuss its applicability to EKC systems, and introduce the PeakMaster 6 software.
A new method has been developed for the preparation of molecular imprinted polymers as porous layers in open tubular (MIP-PLOT) capillary column formats for use in chiral separations by capillary liquid chromatography. The synthesis was based on 'in-capillary' ultraviolet (UV) initiated polymerization using light emitting diodes (LEDs) in conjunction with the continuous delivery of the pre-polymerization reagents into the polymerization zone of the capillary using an automated capillary delivery device. The relationships between exposure times, UV-light intensity and polymer layer thickness have been determined, as well as the effects of reagent delivery rate and multiple LED exposures on the layer thickness for various compositions of pre-polymerization mixtures. The polymer surface morphology was investigated by scanning electron microscopy (SEM). The non-steroidal anti-inflammatory drug S-ketoprofen was used as the template for the preparation of the MIP imprinted PLOT coatings. The separation performance with the ketoprofen racemate was investigated by capillary liquid chromatography. In contrast to alternative methods, which require the use of expensive chiral selectors, the described MIP PLOT stationary phases used non-chiral polymer precursors to create enantioselective nano-cavities through molecular self-assembly processes. The described fabrication methods provide a new avenue to tailor-make chiral MIP-PLOT capillary columns for the separation of chiral compounds present in complex or racemic analyte mixtures of chemical and biological origin.
- MeSH
- chemické bojové látky * izolace a purifikace MeSH
- chromatografie kapalinová metody přístrojové vybavení využití MeSH
- kapilární elektrochromatografie metody přístrojové vybavení využití MeSH
- organofosfáty * analýza MeSH
- plynová chromatografie s hmotnostně spektrometrickou detekcí metody přístrojové vybavení využití MeSH
- spektrofotometrie atomová MeSH
In this work, open-tubular capillary electrochromatography (OT-CEC) method with bare gold nanoparticles (GNPs)-based stationary phase has been developed and applied for separation of tryptic peptide fragments of native and glycated proteins, bovine serum albumin (BSA), and human transferrin (HTF). The GNPs-based stationary phase was prepared by immobilization of bare GNPs, freshly reduced from tetrachloroaurate(III) ions by citrate reduction, on the sol-gel pretreated inner wall of the fused silica capillary. The separation efficiency, peak capacity, and peptide recovery of this open-tubular capillary column were investigated by varying the experimental parameters such as type and concentration of the buffering constituent and pH of the background electrolyte (BGE), temperature, and separation voltage. The best separations of the above tryptic peptides were achieved in the BGE composed of aqueous 100 mmol/L sodium phosphate buffer, pH 2.5, at separation voltage 10 kV per 47-cm long, 50 μm inside diameter capillary thermostated at 25°C. OT-CEC with bare GNPs stationary phase is shown to be a suitable technique for separation of complex peptide mixtures arising from tryptic digestion of native and glycated BSA and HTF, and for investigation of glycation (nonenzymatic glycosylation) of these proteins.
- MeSH
- adsorpce MeSH
- glykosylace MeSH
- kapilární elektrochromatografie přístrojové vybavení metody MeSH
- lidé MeSH
- nanočástice chemie MeSH
- peptidy analýza MeSH
- proteiny chemie MeSH
- trypsin chemie MeSH
- zlato chemie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
In this study, bare gold nanoparticles (GNPs) immobilized in the sol-gel-pretreated fused-silica (FS) capillary as a stationary phase for open-tubular capillary electrochromatography (OT-CEC) are for the first time shown to be able to separate both hydrophobic polyaromatic hydrocarbons (PAHs) as well as hydrophilic cationic antimicrobial peptides. Model mixture of four PAHs, naphthalene, fluorene, phenanthrene, and anthracene, was resolved by OT-CEC in the GNP-modified FS capillaries using the hydro-organic background electrolyte (BGE) composed of 20 mmol/L sodium phosphate buffer, pH 7, modified with ACN at 8:2 v/v ratio. On the other hand, three synthetic analogues of an antimicrobial peptide mastoparan PDD-B, basic tetradecapeptides INWKKLGKKILGAL-NH(2), INSLKLGKKILGAL-NH(2) and NWLRLGRRILGAL-NH(2), were separated in aqueous acidic BGEs, pH 2.1-3.1, composed of weak acids (formic and acetic) or amphoteric amino or imino acids (aspartic or iminodiacetic), utilizing the advantage of a slow reversed (anodic) EOF and slightly positive charge of the GNP-modified FS capillary suppressing the adsorption of cationic peptides on the inner capillary wall and improving their resolution.
- MeSH
- adsorpce MeSH
- aromatické uhlovodíky chemie izolace a purifikace MeSH
- kapilární elektrochromatografie přístrojové vybavení metody MeSH
- nanočástice chemie MeSH
- peptidy chemie izolace a purifikace MeSH
- zlato chemie MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
The review brings a comprehensive survey of the recent developments of high-performance electroseparation methods in capillary and microchip formats: zone electrophoresis, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography and electrochromatography. Applications of these techniques to analysis, isolation, purification and physicochemical and biochemical characterization of peptides are described. Advances in the investigation of electromigration properties of peptides, and in the methodology of their analysis, such as sample preparation, adsorption suppression, EOF control and detection, are presented. New developments, in particular, CE and CEC modes are reported and several types of their applications to peptide analysis are described: conventional qualitative and quantitative analysis, determination in complex (bio)matrices, monitoring of chemical and enzymatical reactions and physical changes, amino acid, sequence and chiral analysis, and peptide mapping of proteins. Some micropreparative peptide separations are shown and capabilities of CE and CEC techniques to provide relevant physicochemical characteristics of peptides are demonstrated.
- MeSH
- elektroforéza kapilární metody MeSH
- kapilární elektrochromatografie metody MeSH
- lidé MeSH
- peptidy analýza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Miniaturization continues to be one of the leading trends in analytical chemistry and one that brings advantages that can be particularly beneficial in biochemical research. Use of a miniaturized scale enables efficient analysis in a short time and requires very small amounts of samples, solvents, and reagents. This can result in a remarkable decrease in costs of enzyme kinetics studies, especially when expensive or rare enzymes and/or substrates are involved. Free zone electrophoresis is without a doubt the most common microscale separation technique for capillary and on-chip enzyme assays. Progress and applications in this field are reviewed frequently whereas other modes of separation, although successfully applied, receive only marginal interest in such publications. This review summarizes applications of less common modes of separation in capillary or chip formats, namely micellar electrokinetic chromatography, liquid chromatography, gel electrophoresis, isoelectric focusing, and isotachophoresis. Because these techniques are based on separation mechanisms different from those of free zone electrophoresis, they can be, and have been, successfully used in cases where zone electrophoresis fails. Advantages and drawbacks of these alternative separation techniques are discussed, as also are the difficulties encountered most often and solutions proposed by different research groups.
- MeSH
- chromatografie micelární elektrokinetická kapilární metody MeSH
- elektroforéza kapilární metody MeSH
- enzymatické testy metody MeSH
- isoelektrická fokusace metody MeSH
- izotachoforéza metody MeSH
- kapilární elektrochromatografie metody MeSH
- kinetika MeSH
- lidé MeSH
- miniaturizace metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Capillary liquid chromatography (cLC) hyphenated with tandem mass spectrometry (MS-MS) was used to separate and quantitate trace concentrations of five estrogens in aqueous samples. New C(18)-based sorption materials bound to the silica support by monomeric and polymeric mechanisms were compared and tested for solid-phase extraction (SPE) of selected analytes with respect to optimization of their preconcentration yield. Application of an endcapped, monomer-bound preconcentration Discovery DSC-18Lt column under the optimized conditions provides yields in the range from 95 to 100% with a high repeatability (n=3, RSD≤7.2%). Using the electrospray ionization in the positive mode (ESI+), the cLC-MS-MS system (the Zorbax SB C18 capillary column and a binary mobile phase of acetonitrile and water containing 0.1% formic acid in both the components) was optimized to attain a sufficient retention of the early eluting estriol, a satisfactory resolution of the analytes and the maximum sensitivity of the determination. Both the isocratic and gradient elution were used and the optimized gradient method permitted analyses of aqueous environmental samples in 14 min within a linearity range from 6.1 to 25.0 (LOQ of analytes) to 500 ng/L and with a very good linearity (r>0.9981) for all the estrogens studied. The detection limits are in the range from 3.0 to 6.8 ng/L (1 μL injection volume). Six environmental water samples were analyzed and the studied estrogens were found in the Vltava river sample collected in Prague (13.2 ng/L for 17β-estradiol) and in the inlet to the wastewater treatment plant in Prague, at an overall concentration of 371.4 ng/L.
- MeSH
- chemické látky znečišťující vodu analýza MeSH
- estrogeny analýza izolace a purifikace MeSH
- extrakce na pevné fázi metody MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- kapilární elektrochromatografie metody MeSH
- lineární modely MeSH
- řeky chemie MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The article brings a comprehensive survey of recent developments and applications of high-performance capillary electromigration methods, zone electrophoresis, ITP, IEF, affinity electrophoresis, EKC, and electrochromatography, to analysis, preparation, and physicochemical characterization of peptides. New approaches to the theoretical description and experimental verification of electromigration behavior of peptides and to methodology of their separations, such as sample preparation, adsorption suppression, and detection, are presented. Novel developments in individual CE and CEC modes are shown and several types of their applications to peptide analysis are presented: conventional qualitative and quantitative analysis, purity control, determination in biomatrices, monitoring of chemical and enzymatical reactions and physical changes, amino acid and sequence analysis, and peptide mapping of proteins. Some examples of micropreparative peptide separations are given and capabilities of CE and CEC techniques to provide important physicochemical characteristics of peptides are demonstrated.
