Saccharides form one of the major constituents of biological macromolecules in living organisms. Many biological processes including protein folding, stability, immune response and receptor activation are regulated by glycosylation. In this work, we optimized a capillary electrophoresis method with capacitively coupled contactless conductivity detection for the separation of eight monosaccharides commonly found in glycoproteins, namely D-glucose, D-galactose, D-mannose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-fucose, N-acetylneuraminic acid, and D-xylose. A highly alkaline solution of 50 mM sodium hydroxide, 22.5 mM disodium phosphate, and 0.2 mM CTAB (pH 12.4) was used as a background electrolyte in a 10 μm id capillary. To achieve baseline separation of all analytes, a counter-directional pressure of -270 kPa was applied during the separation. The limits of detection of our method were below 7 μg/ml (i.e., 1.5 pg or 1 mg/g protein) and the limits of quantification were below 22 μg/ml (i.e., 5 pg or 3 mg/g protein). As a proof of concept of our methodology, we performed an analysis of monosaccharides released from fetuin glycoprotein by acid hydrolysis. The results show that, when combined with an appropriate pre-concentration technique, the developed method can be used as a monosaccharide profiling tool in glycoproteomics and complement the routinely used LC-MS/MS analysis.
- MeSH
- acetylgalaktosamin MeSH
- acetylglukosamin MeSH
- cetrimonium MeSH
- chromatografie kapalinová MeSH
- elektroforéza kapilární metody MeSH
- elektrolyty chemie MeSH
- fetuiny MeSH
- fosfáty MeSH
- fukosa MeSH
- galaktosa MeSH
- glukosa MeSH
- glykoproteiny chemie MeSH
- hydroxid sodný MeSH
- kyselina N-acetylneuraminová * MeSH
- mannosa MeSH
- monosacharidy * analýza MeSH
- tandemová hmotnostní spektrometrie MeSH
- xylosa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Thermodynamic acidity constants (acid or acid-base dissociation constants, sometimes called also as ionization constants) and limiting ionic mobilities (both of them at defined temperature, usually 25°C) are the fundamental physicochemical characteristics of a weak electrolyte, that is, weak acid or weak base or ampholyte. We introduce a novel method for determining the data of a weak electrolyte by the nonlinear regression of effective electrophoretic mobility versus buffer composition dependence when measured in a set of BGEs with various pH. To correct the experimental data for zero ionic strength we use the extended Debye-Hückel model and Onsager-Fuoss law with no simplifications. Contrary to contemporary approaches, the nonlinear regression is performed on limiting mobility data calculated by PeakMaster's correction engine, not on the raw experimental mobility data. Therefore, there is no requirement to perform all measurements at a constant ionic strength of the set of BGEs. We devised the computer program AnglerFish that performs the necessary calculations in a user-friendly fashion. All thermodynamic pKa values and limiting electrophoretic mobilities for arbitrarily charged substances having any number of ionic forms are calculated by one fit. The user input consists of the buffer composition of the set of BGEs and experimentally measured effective mobilities of the inspected weak electrolyte.
We discuss several possible phenomena in electrophoretic systems with complexing agents present in the background electrolyte. In our previous work, we extended the linear theory of electromigration with the first-order nonlinear term, which originally applied to acid-base equilibria only, by generalizing it to any fast chemical equilibria. This extension provides us with a fresh insight into the well-established technique of elecktrokinetic chromatography (EKC). We combine mathematical analysis of the generalized model with its solution by means of the new version of our software PeakMaster 6, and experimental data. We re-examine the fundamental equations by Wren and Rowe and Tiselius in the frame of the generalized linear theory of electromigration. Besides, we show that selector concentration can increase inside the interacting-analyte zone due to its complexation with the analyte, which contradicts the generally accepted idea of a consumption of a portion of the selector inside the zone. Next, we focus our discussion on interacting buffers (i.e., buffer constituents that form a complex with the selector). We demonstrate how such side-interaction of the selector with another buffer constituent can influence measuring analyte-selector interactions. Finally, we describe occurrence and mobilities of system peaks in these EKC systems. We investigate systems with fully charged analytes and neutral cyclodextrins as selectors. Although the theory is not limited in terms of the charge and/or the degree of (de)protonation of any constituent, this setup allows us to find analytical solutions to generalized model under approximate, yet realistic, conditions and to demonstrate all important phenomena that may occur in EKC systems. An occurrence of system peaks in a system with fully charged selector is also investigated.
This work extends the present working range of isotachophoresis (ITP) with electrospray-ionization mass-spectrometric (ESI-MS) detection and describes for the first time a functional cationic electrolyte system for analyses at medium-alkaline pH. So far no ITP-MS application was published on the analysis of medium strong bases although there is a broad spectrum of potential analytes like biogenic amines, alkaloids or drugs, where this technique promises interesting gains in both sensitivity and specificity. The presented results include a selection of suitable sufficiently volatile ESI-compatible system components, discussion of factors affecting system properties, and recommendations for functional ITP electrolyte systems. Theoretical conclusions based on calculations and computer simulations are confirmed by experiments with a model mixture of beta-blockers. Practical applicability of the method is demonstrated on the example of analysis of sotalol in dried blood spots where direct injection of aqueous extract, ITP stacking and MS detection provide a fast, simple and sensitive technique with limits of quantitation on the sub-nM level.
We present a new theoretical approach for calculating changes in the physico-chemical properties of BGEs for measurements by CZE due to the electrolysis in electrode vials (vessels). Electrolysis is an inevitable phenomenon in any measurement in CZE. Water electrolysis, which occurs in most measurements, can significantly alter the composition of the BGE in electrode vials and in the separation capillary and has a negative influence on the robustness and quality of separations. The ability to predict changes in the composition of the BGE is important for evaluation of the suitability of the BGEs for repeating electrophoretic runs. We compared theoretically calculated changes in the physico-chemical properties (pH, conductivity) with those measured using pH-microelectrode and contactless conductivity detection of the BGE after the electrophoretic run. We confirmed the validity of our theoretical approach with a common BGE composed of acid-base pair, where one constituent is fully dissociated while the second constituent is dissociated by only half, and with Good's buffer. As predicted by theoretical approach, the changes in the physico-chemical properties of the Good's buffer after the electrophoretic run were several times lower than in the case of a common BGE composed of a weak acid - strong base pair.
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.
Electrophoretic focusing on inverse electromigration dispersion (EMD) gradient is a new analytical technique based on a unique separation principle where weak non-amphoteric ionogenic species are focused, separated and transported to the detector by an EMD profile of suitable properties. The present work extends the theoretical description of this method by introducing the concept of resolution and deriving the fundamental equation expressing resolution as function of basic system parameters. The results indicate that at constant current operation, resolution is proportional to the square root of time. For variable current regimes (e.g. constant voltage), the time variable is replaced by the product of electric current and passed electric charge. Computer simulations for a model pair of substances support the validity of the presented theory and confirm the theoretical conclusion that resolution can be increased by allowing longer electromigration of the gradient in terms of time or passed charge. The experimental example shown comprises an anionic electrolyte system based on maleic acid and 2,6-lutidine, combined with ESI-MS detection and operated in the reverse mode due to strong electroosmotic flow and ESI suction. The practical implementation of the proposed methodology is done by application of negative pressure at the inlet vial, resulting in very substantial resolution enhancement and baseline separation of otherwise unresolved substances. The performance and high sensitivity of the developed technique is demonstrated on the example of simultaneous analysis of four sulfonamides and three dichlorophenols in waters with limits of detection on the 1 nM level.
- MeSH
- chlorfenoly analýza MeSH
- elektroforéza kapilární MeSH
- elektrolyty chemie MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací * MeSH
- limita detekce MeSH
- maleáty chemie MeSH
- pitná voda analýza MeSH
- pyridiny chemie MeSH
- sulfonamidy analýza MeSH
- tlak MeSH
- Publikační typ
- časopisecké články MeSH
Separations of bare superparamagnetic magnetite nanoparticles (BSPMNPs, approx. 11 nm diameter) was performed using non-complexing (nitrate) and complexing (chloride, citrate and phosphate) electrolyte ions with additions of tetramethylammonium hydroxide (TMAOH), which is commonly applied to control the synthesis of stable iron oxides. The use of TMAOH as a background electrolyte (BGE) additive for capillary electrophoresis (CE) separations provided for the first time electropherograms of BSPMNPs exhibiting symmetrical and highly reproducible peaks, free of spurious spikes characteristic of nanoparticle clusters. Consequently, accurate determination of the electrophoretic effective mobility of BSPMNPs was possible, yielding a value of -3.345E-08 m2 V-1 s-1 (relative standard deviation (RSD) of 0.500%). The obtained mobilities of BSPMNPs in the presence of various electrolyte ions show that the degree of complexation with the surface of BSPMNPs follows the order chloride < citrate < phosphate, correlating with the stabilities of Fe(III) complexes with the respective anions. Finally, bare and carboxylated iron oxide nanoparticles were successfully separated in only 10 min using 10 mM Tris-nitrate containing 20 mM of TMAOH as electrolyte. Our findings show that simple and rapid CE experiments are an excellent tool to characterise and monitor properties and interactions of iron oxide nanoparticles with other molecules for surface modification purposes.
The partial-filling affinity capillary electrophoresis (pf-ACE) works with a ligand present in a background electrolyte that forms a weak complex with an analyte. In contrast to a more popular mobility-shift affinity capillary electrophoresis, only a short plug of the ligand is introduced into a capillary in the pf-ACE. Both methods can serve for determining apparent stability constants of the formed complexes but this task is hindered in the pf-ACE by the fact that the analyte spends only a part of its migration time in a contact with the ligand. In 1998, Amini and Westerlund published a linearization strategy that allows for extracting an effective mobility of an analyte in the presence of a neutral ligand out of the pf-ACE data. The main purpose of this paper is to show that the original formula is only approximate. We derive a new formula and demonstrate its applicability by means of computer simulations. We further inspect several strategies of data processing in the pf-ACE regarding a risk of an error propagation. This establishes a good practice of determining apparent stability constants of analyte-ligand complexes by means of the pf-ACE.
