Cze

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Cze
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Capillary zone electrophoresis (CZE) is a highly efficient separation technique based on differences in the electrophoretic mobilities of analytes, which, in turn, depend on the charge-to-size ratios.
Capillary zone electrophoresis may be performed in the presence or absence of EOF. Separations carried out in the presence of EOF permit the analysis of cations and anions in the same analysis, and the resolution of analytes moving counter to EOF can be enhanced. Uncoated fused silica capillaries exhibit electroosmotic flow toward the cathode, and the magnitude of EOF increases with pH. Capillaries coated with an adsorbed or covalent positively charged coating exhibit reversed electroosmotic flow toward the anode. Reversed-charge CZE can be used for analysis of cationic analytes which would adsorb to bare silica capillaries, and for analysis of high-mobility anions which would not be resolved with normal EOF flow. A disadvantage of performing CZE in the presence of EOF is variation in the magnitude of EOF, which can compromise migration time and peak area reproducibility. Electroosmotic flow can be controlled or eliminated by coating the capillary with an adsorbed or covalent neutral material, usually a hydrophilic polymer. Coated capillaries are often used when analyzing species that have strong affinities for silica, such as proteins.
Mobility and selectivity in CZE are most profoundly affected by analyte charge, and selection of the electrolyte pH is the most effective method of controlling a CZE separation. A wide variety of buffers have been employed in CZE, and a buffer is selected to provide good buffering capacity at the desired pH, low UV absorbance, and low conductivity. In addition to the buffer, other components may be added to the electrolyte to control EOF, reduce solute–wall interactions, or to modulate the mobility or solubility of an analyte. Additives for CZE include neutral salts, organic amines, surfactants, organic solvents, and chiral selectors. Secondary equilibria introduced by additive–analyte interactions are very important for achieving resolution in CZE.
CZE is known for its high detection limits. Correlation techniques, as used in chromatography, can be applied in CZE as well. The goal and basic principles are the same. The main problem is the high demand on the injection system, just as in CC. However, in CCZE the injection system can relatively easy be modified, because CZE is electrically – rather than pressure – driven.
Microchip technology is very well suited for application in CZE systems and particularly in correlation CZE. A high quality injection device on a microchip, connected to a fused silica capillary and particularly usable for correlation CZE, is reported. The speed of separation in a microchip CZE system can be increased due to higher accessible field strengths. Detection is the major problem, because of the smaller channel dimension. The application of correlation techniques drastically reduces the high detection limit in a modest time.
Serban C. Moldoveanu , Victor David , in Selection of the HPLC Method in Chemical Analysis , 2017
Capillary zone electrophoresis (CZE) is a technique successfully used for the separation of proteins, peptides, and nucleic acids. Other applications where CZE may be useful include analysis of inorganic anions and cations, such as those typically separated by ion chromatography. Small charged molecules can also be separated using CZE. The quantitation using CZE is less reliable compared to HPLC. A number of issues such as irreproducibility of the injection, adsorption of the samples on the capillary walls, heating of the capillary during separation, difficulties in assuring a uniform flow of sample when using MS detection, the need of extreme cleanliness of the system, alignment in the detection window when using UV or fluorescence detection, and other similar problems reduce the reproducibility and robustness of this technique [35] .
Capillary zone electrophoresis (CZE) is an effective technique for the definitive verification of the chromatographic purity of the target compound because of the large number of theoretical plates obtainable. The advantages of CZE, such as the possibility of analyzing for relatively labile species because of the absence of chromatographic packing, must be considered given the need for ultrasensitive detection, such as high-resolution ICP-MS because of the small sample amount injected. A commercial interface for CZE–ICP-MS is available that optimizes electrophoretic and nebulizer flows and has minimal dilution and sample consumption.
The potential of CZE–ICP-MS has been particularly realized in the identification and determination of selenoglutathione and differentiation of methionine, selenomethionine, cystamine, and selenocystamine in milk. Se(IV), Se(VI), selenate-carrying glutathione (GSSeSG), selenomethionine, selenocystine, and selenocystamine can be speciated at the 10–50 μg per Se per liter level. A two-dimensional separation approach for selenized yeast speciation, based on size exclusion followed by CZE–ICP-MS, coupling to the ICP via a self-aspirating total consumption nebulizer affords limits of low molecular weight selenium species in the range 7–18 μg l −1 .
J. Havel , D. Fetsch , in Encyclopedia of Separation Science , 2000
CZE has been shown to be the most powerful tool for the separation and characterization of HS due to their ionic and/or polyelectrolyte properties. The CZE separation patterns of HS obtained may, in the future, find a direct application in forensic science.
Nevertheless, even if several different models of HS are proposed and various properties of HS intensively studied, the real structure of humic, fulvic, humin and hymatomelanic acids is still unknown. The latest results obtained by CZE showing separation into 10–30 fractions present an optimistic insight into the humic substances puzzle. On the basis of recent results, fraction collection in order to perform studies on individual fractions by gas chromatography–mass spectrometry, matrix assisted laser desorption–time of flight (MALDI-TOF) mass spectrometry and nuclear magnetic resonance is beginning to appear feasible. It is possible that the problem of HS structure is at last on the way to being resolved.
CZE is the simplest of the CE modes and straightforward to perform ( Figure 1 depicts a typical example of a CZE separation). When employing CZE for protein separation, the choice of capillary (uncoated or with the particular type of coating) and buffer additives should be made carefully depending on sample composition. The uncoated capillaries generally require a prior conditioning step. Detection based on either UV adsorption or laser-induced fluorescence (LIF) is most often employed in the CZE of proteins. Depending on the detection mode, a sample pretreatment may be necessary.
The sensitivity of the detection by UV absorbance is limited since both the optical length (=capillary internal diameter) and sample volumes (typically, a few nanolitres) are very small in CZE. Though the sensitivity can be greatly increased by detecting proteins in the wavelength range of 200–220 nm, UV detection still requires a relatively high concentration of analyte in a sample. That is not always the case and a preconcentration of the sample, often of a volume of a few microlitres, is needed. Several online and offline preconcentration techniques can be employed in CZE. The first and simplest approach to online sample preconcentration is zone sharpening by stacking. Proteins dissolved in a buffer with a conductivity lower than that of the run buffer (commonly, the diluted run buffer) become concentrated at the interface between the sample and the run buffer due to a high voltage drop in the sample zone. Preliminary sample desalting is often necessary for this approach and special methods have been developed for desalting (and concentrating) microlitre volumes of protein samples, using small pore polyacrylamide gels.
Isotachophoresis is the other popular technique to concentrate samples. The preconcentration may be performed either online or in a coupled column, and in the presence of salts. The gain in detection limit is 10- to 100-fold and can be increased up to 1000-fold when a hydrodynamic counterflow is employed. Another efficient method of protein preconcentration is selective accumulation of the proteins on a solid-phase affinity support. This method has been used in both online and offline modes, with several hundred-fold concentration.
After derivatization with a fluorophore, proteins may be detected online by LIF. A number of fluorescent dyes capable of covalently binding to protein molecules (e.g. fluorescein, naphthalenedicarboxaldehyde and fluorescamine) have been used, providing mass detection limits in the attomole range (initial sample concentrations of 10 −8 to 10 −10 mol L −1 ). However, covalent binding of the dyes frequently results in a broadening of protein peaks or even in the formation of multiple peaks due to multiple derivatization.
M. Hamdan , P.G. Righetti , in Encyclopedia of Separation Science , 2000
Capillary zone electrophoresis (CZE) is widely recognized as a powerful analytical technique in its own right, known for its high separation efficiency, short analysis times and low-volume sample requirements. These characteristics made CZE a popular method for the analysis of peptide mixtures, protein digests, drug substances and biotechnological products. The coupling of CZE with electrospray ionization mass spectrometry (ESI–MS), first reported by Olivares et al . in 1887, has added further capabilities, in particular for obtaining molecular mass information and structural details when tandem mass spectrometry (MS–MS) is used. However, it can be said that the major advantage of such coupling is that the migration time is not the only parameter used for identifying the eluted components. These times are subjected to variations between runs, yet such variations become irrelevant when, in the same run, highly diagnostic mass spectra are obtained.
Depending on the ionization method, CZE can be coupled to a mass spectrometer either directly (online) or indirectly (offline). In the latter mode of operation, 252 Cf plasma desorption and matrix assisted laser desorption can be used. The online coupling of CZE is more common and usually performed by electrospray ionization (ESI) or fast atom (ion) bombardment (FAB). Although online CZE/MS is the more common form of application, offline analysis has the advantage of allowing separation in non-volatile buffers, which are highly undesired in ESI.
It goes without saying that every analytical technique has its limitations and CZE/MS is no exception. One of the main limitations of this experimental arrangement is its relatively poor sample concentration/ion sensitivity. Approaches to reduce such limitations included online preconcentration, sample stacking, and the increasing use of time-of-flight (TOF) analysers which use ESI and TOF analysers with and without a quadrupole in between. The innovative feature of this class of instruments is their fast scanning, which allows the acquisition of a number of full spectra per second. Additionally, as all ions in each spectrum are sampled at the same moment in time, spectra are free of mass discrimination or peak skew typical of slow scanning systems that must scan over a narrow chromatographic/electrophoretic peaks.
Capillary electrochromatography (CEC) is another technique which is currently undergoing a rapid phase of advancement and development. This technique was revived by Jorgenson and Lukacs in 1981; these authors used 0.005 mol L −1 phosphate buffer, 170 μm packed column and 30 kV separation voltage to separate 9-methylanthracene and perylene. This technique has recently become more diffuse because of a number of advances in both CE instruments and detection techniques including electrospray mass spectrometry. However, on-column UV detection and in-column laser-induced fluoroscence detection remain the most commonly used methods. Despite its high sensitivity, the latter method is subjected to interferences by buffer fluoroscence. In MS detection, the column is commonly packed right up to the point where the sample is injected into the mass spectrometer. The combination of CEC with mass spectrometry provides reliable molecular weights and in many cases structural information, which makes it highly attractive for a wide range of applications. For more details on this topic, the reader is referred to recent extensive reviews, covering the methodology of CEC and its coupling to MS, by Colòn et al . (1997) and Rentel et al . (1999). Interestingly, packed-CEC offers the possibility of higher sample capacity and the utilization of simpler mobile phases, which are more compatible with MS.
Claudimir Lucio do Lago , ... Zuzana Cieslarová , in Chemical Analysis of Food (Second Edition) , 2020
CZE, also known as free solution capillary electrophoresis, is the simplest and the most used separation mode in CE. In CZE, a capillary is entirely filled with a carrier or background electrolyte (BGE), providing the buffering capacity and conducting the electric current. At one extremity of the capillary, very small zone of the sample is introduced, and the potential is applied. Each component of the sample migrates differentially along the capillary and, after a while, can separate into distinct zones. The migration velocity and the direction of each component are determined by the apparent mobility of the ion, which is related to charge and size of the ion and the EOF velocity at a given pH. All the neutral components of the sample will migrate with the EOF velocity, while charged components can be separated by CZE. Hence, the important condition for successful separation in CZE is to keep the molecule charged by choosing the right composition of BGE. Depending on the p K a values of each functional group in the molecule and the pH of the chosen BGE, the molecule can be ionized.
However, some very important food components contain groups that are difficult to ionize. Such molecules have to be either derivatized or complexed with a substance that can provide ionizable or charged functional group in their structure. The derivatization and complexation can be done during the sample preparation step or on-column during the analysis ( Glatz, 2015 ). In that case, the derivatization or complexation agent is part of the BGE. For instance, saccharides or nucleosides that contain cis -diol groups can be complexed with borates and migrate as anions toward the positive electrode ( Coelho & Jesus, 2016 ; Hoffstetter-Kuhn, Paulus, Gassmann, & Widmer, 1991 ; Landers, Oda, & Schuchard, 1992 ; Quirino & Terabe, 2001 ).
Owing to the simplicity of CZE separation mechanism, one can simulate electropherograms by using computer programs such as PeakMaster, which is available online ( https://web.natur.cuni.cz/gas/peakmaster.html ). Knowing the instrumental parameters—such as capillary length, applied voltage, BGE composition, and analytes to be separated—the electropherogram can be easily simulated. The program contains a large database of ions, enabling the simulation of experimental conditions in order to obtain good separation and signal ( Jaros, Hruska, Stedry, Zuskova, & Gas, 2004 ). In addition, new compounds can be added to the database, by including their p K a values and mobilities.
Hong Heng See , Nurul Adibah Ali , in Encyclopedia of Analytical Science (Third Edition) , 2019
Capillary zone electrophoresis (CZE) is the simplest form of electrophoresis. In CZE, the capillary is filled with run buffer at a constant composition while source and destination vials are filled with identical run buffer. When a sample is injected into capillary filled with buffer and a voltage is applied, solutes will migrate through capillary as zones ( Fig. 4 ). Solutes migrate based on their rates of migration, which depend on the electrophoretic mobilities. EOF moves from anode to cathode in the capillary, so the order of elution of the solutes is cations, neutral compounds, and lastly anions. Both cations and anions are separated based on their charge-to-size ratios. Smaller and highly charged cations will elute first, followed by neutral compounds, which are not separated and move together in the direction of the EOF. Anions, which are strongly attracted to the positive electrode, move opposite the direction of the EOF and are eluted last. If the migration rates of the EOF are greater than the electrophoretic mobilities of the anions, most of the anions will be carried toward the cathode. Unlike cations, anions that are small and highly charged will be eluted last, reversing the charge-to-size ratios. Fig. 4 shows the indications of the elution order of solutes through the capillary accordingly to their charge-to-size ratios.
Fig. 4 . A drawing that depicts the elution order of the capillary zone electropherogram and indicates elution order where small, highly charged cations are eluted first, followed by neutral solutes, and finally small, highly charged anions.
CZE ESI MS is highly efficient in the total analysis of hybrid CS/DS oligosaccharides in either off-line mode, which assumes collection of fractions, or direct coupling, i.e., the on-line CZE ESI MS, since, unlike SEC, CZE is able to separate the species also according to the sulfation content, reducing drastically spectral congestion and eliminating the ion suppression phenomenon occurring for complex mixtures. Due to the anionic nature of CS/DS in solution, CZE ESI MS is usually performed in direct polarity, with sample injection at the anode and detection at the cathode and negative ion mode electrospray, although recent studies involving sheath-flow CZE MS have shown that the reverse polarity separation of GAGs is also feasible as the electrospray process itself is the main contributor to the flow of the sheath liquid [60] .
Certainly, in either off- or on-line mode, a crucial role is played by the choice of the background electrolyte that needs to simultaneously meet a series of requirements for a successful separation, spray, and ionization. First of all, CS/DS must show an excellent solubility in the chosen buffer system. Furthermore, BGE composition and purity, pH, concentration, the sample concentration in BGE, the separation efficiency, and last but not least the performances as a spraying agent contribute each to the success of the complex analysis implying a proper separation, migration of components, a steady spray, ionization of individual CS/DS species, and ultimately their MS detection and fragmentation in tandem MS [36] . On the other hand, ESI MS parameters are to be optimized as well in terms of voltages, desolvation gas type, purity, pressure, collision energy, and collision gas.
The off-line CZE ESI MS is always readily available and more accessible for researchers since, unlike the on-line approach, it does not require the physical coupling of the two
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