Continuous flow electrophoretic separation with continuous sample loading provides the advantage of processing volumes of any sizes, as well as the benefit of a real-time monitoring and optimization of the separation process. In addition, the spatial separation of the sample enables collecting multiple separated components simultaneously and in a continuous manner. The separation is usually performed in mild buffers without organic solvents and detergents (sample biological activity is retained) and it is carried out without usage of a solid support in the separation space preventing the interaction of the sample with it (high sample recovery). The method is used for the separation of proteins/peptides in proteomic applications, and its great applicability is to the separation of the cells, cellular organelles, vesicles, membrane fragments, and DNA. This review focuses on the electrophoretic separation performed in a continuous flow and it describes various electrophoretic modes and instrumental setups. Recent developments in methodology and instrumentation, the integration with other techniques, and the application to the biological sample analysis are discussed as well.
In this work, a novel force equilibrium method called distributed dielectrophoretic cytometry (2DEP cytometry) was developed. It uses a dielectrophoresis (DEP)-induced vertical translation of live cells in conjunction with particle image velocimetry (PIV) in order to measure probabilistic distribution of DEP forces acting on an entire cell population. The method is integrated in a microfluidic device. The bottom of the microfluidic channel is lined with an interdigitated electrode array. Cells passing through the micro-channel are acted on by sedimentation forces, while DEP forces either oppose sedimentation, support sedimentation, or neither, depending on the dielectric (DE) signatures of the cells. The heights at which cells stabilize correspond to their DE signature and are measured indirectly using PIV, which enables simultaneous and high-throughput collection of hundreds of single-cell responses in a single PIV frame. The system was validated using polystyrene micro-particles. Preliminary experimental data quantify the DE signatures of immortalized myelogenous leukemia cell lines K562 and KG1. We show DEP-induced cell translation along the parabolic velocity profile can be measured by PIV with sub-micron precision, enabling identification of individual cell DE signatures. DE signatures of the selected cell lines are distinguishable. Throughput of the method enables measurement of DE signatures at 10 different frequencies in almost real time.
- MeSH
- buňky K562 MeSH
- design vybavení MeSH
- elektrická stimulace MeSH
- elektroforéza přístrojové vybavení MeSH
- laboratoř na čipu MeSH
- lidé MeSH
- počítačová simulace MeSH
- průtoková cytometrie přístrojové vybavení metody MeSH
- stochastické procesy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- amyloidóza * diagnóza klasifikace MeSH
- dospělí MeSH
- elektroforéza metody přístrojové vybavení využití MeSH
- imunologické techniky MeSH
- lehké řetězce imunoglobulinů * analýza krev moč MeSH
- lidé středního věku MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- těžké řetězce imunoglobulinů * krev moč MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- práce podpořená grantem MeSH
Effects of organic solvent type, pH value, and composition of donor/acceptor solution on the efficacy of electromembrane extraction (EME) were examined. For the first time, a comprehensive quantitative study, based also on measurements of electric charge passed through the EME system, was carried out, which demonstrates that apart from the pH value, also the nature of counter-ions in donor and acceptor solution plays a significant role in the electrically induced transfer of charged analytes across supported liquid membranes (SLMs). The EME transfer of model analytes correlated well with electrophoretic mobilities of inorganic cations, which were added to acceptor solutions during their alkalization with alkali metal hydroxides, and were highest for counter-cations with highest mobilities. Up to a 53-fold improvement of extraction efficiency was achieved for EMEs using optimized composition of donor (alkalized with KOH to pH 7) and acceptor (10 mM CsOH, pH 12) solutions. Six chlorophenols (CPs) were selected as model analytes due to the wide range of pH values that are required for their ionization and due to their high environmental relevance; quantitative measurements were carried out by CE with UV detection. Extraction recoveries of the six CPs ranged between 14 and 25% for 5 min EMEs at 150 V and 750 rpm across SLMs impregnated with 1-ethyl-2-nitrobenzene. Calibration curves were strictly linear (r(2) ≥ 0.999) in 0.01-10 μg/mL range, repeatability values of peak areas were between 0.7 and 5.6% and LODs for standard solutions and environmental samples were better than 5 ng/mL.
- MeSH
- chemická frakcionace přístrojové vybavení metody MeSH
- chemické modely * MeSH
- chlorfenoly analýza chemie izolace a purifikace MeSH
- elektroforéza přístrojové vybavení metody MeSH
- limita detekce MeSH
- lineární modely MeSH
- membrány umělé * MeSH
- reprodukovatelnost výsledků MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
This contribution is the third part of the project on strategies used in the selection and tuning of electrolyte systems for anionic ITP with ESI-MS detection. The strategy presented here is based on the creation of self-maintained ITP subsystems in moving-boundary systems and describes two new principal approaches offering physical separation of analyte zones from their common ITP stack and/or simultaneous selective stacking of two different analyte groups. Both strategic directions are based on extending the number of components forming the electrolyte system by adding a third suitable anion. The first method is the application of the spacer technique to moving-boundary anionic ITP systems, the second method is a technique utilizing a moving-boundary ITP system in which two ITP subsystems exist and move with mutually different velocities. It is essential for ESI detection that both methods can be based on electrolyte systems containing only several simple chemicals, such as simple volatile organic acids (formic and acetic) and their ammonium salts. The properties of both techniques are defined theoretically and discussed from the viewpoint of their applicability to trace analysis by ITP-ESI-MS. Examples of system design for selected model separations of preservatives and pharmaceuticals illustrate the validity of the theoretical model and application potential of the proposed techniques by both computer simulations and experiments. Both new methods enhance the application range of ITP-MS and may be beneficial particularly for complex multicomponent samples or for analytes with identical molecular mass.
Intensive research and development of electrophoresis methodology and instrumentation during past decades has resulted in unique methods widely implemented in bioanalysis. While two-dimensional electrophoresis and denaturing polyacrylamide gel electrophoresis in sodium dodecylsulfate are still the most frequently used electrophoretic methods applied to analyses of proteins, new miniaturized capillary and microfluidic versions of electromigrational methods have been developed. High-throughput electrophoretic instruments with hundreds of capillaries for parallel separations and laser-induced fluorescence detection of labeled DNA strands have been of key importance for the scientific and commercial success of the Human Genome Project. Another powerful method, capillary isoelectric focusing with pressurized and pH-driven mobilization, provides efficient separations and on-line sensitive detection of proteins, bacteria and viruses. Electrophoretic microfluidic devices can integrate single-cell injection, cell lysis, separation of its components and fluorescence or mass spectrometry detection. These miniaturized devices also proved the capability of single-molecule detection.
- MeSH
- bakteriální proteiny analýza MeSH
- elektroforéza přístrojové vybavení metody trendy MeSH
- imunoanalýza metody MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- proteom analýza MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza DNA metody MeSH
- stereoizomerie MeSH
- virové proteiny analýza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
A method utilising isotachophoresis and capillary zone electrophoresis in the column coupling configuration with UV detection at 320 nm was developed for separation and determination of five phenolic acids (rosmarinic, p-coumaric, ferulic, caffeic and chlorogenic) and flavonoid quercitrin in a methanolic extract of Melissae herba. The proposed method has been validated with correlation coefficients from 0.9842 to 0.9988, RSD values between 0.39% and 0.83% for migration times and between 0.40% and 2.05% for peak areas.
- MeSH
- antioxidancia analýza chemie MeSH
- elektroforéza kapilární metody přístrojové vybavení MeSH
- elektroforéza metody přístrojové vybavení MeSH
- financování organizované MeSH
- flavonoidy analýza MeSH
- hydroxybenzoáty analýza MeSH
- koncentrace vodíkových iontů MeSH
- meduňka chemie MeSH
- methanol chemie MeSH
- odběr biologického vzorku metody přístrojové vybavení metody MeSH
- reprodukovatelnost výsledků MeSH
- rostlinné extrakty analýza MeSH
- senzitivita a specificita MeSH
- spektrofotometrie ultrafialová MeSH
A method for determination of metabisulfite and hydrosulfite in poultice and decolorant by isotachophoresis was developed. Metabisulfite and hydrosulfite are ionizable oxoanions of sulfur of similar character that can easily be oxidized to sulfates. To protect the analytes from oxidation the solid samples were dissolved in a 1% (w/v) solution of formaldehyde. Hydrosulfite and metabisulfite present in the samples were transformed by the reaction with formaldehyde to stable compounds, hydroxymethanesulfinate and hydroxymethanesulfonate that were determined isotachophoretically without any pretreatment except for sample filtering and degassing. A capillary of 0.4mm i.d. and 100mm effective length made of fluorinated ethylene-propylene copolymer was filled with an electrolyte system consisting of 10 mmol L(-1) HCl+11 mmol L(-1) imidazole, 0.15% (w/v) hydroxyethylcellulose, pH 6.0 (leading electrolyte) and 5 mmol L(-1) benzoic acid+6 mmol L(-1) imidazole, pH 6.5 (terminating electrolyte). Separation was performed at a driving current of 80 microA and for detection current was decreased to 30 microA. Using contactless conductivity detection, the calibration curves in the tested concentration range up to 2.5 mmol L(-1) were linear for both metabisulfite and hydrosulfite complexes. The concentration detection limits for metabisulfite and hydrosulfite were 2.9 and 3.4 micromol L(-1), respectively. For 1 mmol L(-1) concentration, values of R.S.D. (n=6) were 2.6% for hydrosulfite and 0.8% for metabisulfite. Isotachophoretic determination took about 20 min. The elaborated isotachophoretic procedure is simple to perform, sufficiently sensitive and accurate. In addition to this, low cost of analyses makes the method an alternative procedure to methods used so far for the determination of oxoanions of sulfur.
Kapilárna izotachoforéza bola využitá na separáciu a stanovenie enantiomérov dimetindenu v rôznych liekových formách. Bolo preskúšaných niekoľko typov chirálnych selektorov vo viacerých elektrolytových systémoch s rôznym zložením a rôznym pH. Optimálny vodiaci elektrolyt bol tvorený 10 mmol/l octanom draselným a kyselinou octovou do výslednej hodnoty pH 4,8 s prídavkom 4 mmol/l karboxyetyl-β-cyklodextrínu ako chirálneho selektora a 0,2% metylhydroxyetylcelulózy (m–HEC) na potlačenie elektroosmotického toku. Ako zakončujúci elektrolyt sa použil β-alanín s koncentráciou 5 mmol/l. Bola hodnotená presnosť, správnosť, linearita, robustnosť a selektivita vypracovanej ITP metódy. Predúprava vzorky pred analýzou spočívala v rozpustení a nariedení príslušnej liekovej formy s obsahom dimetindenu demineralizovanou vodou na požadovanú koncentráciu. Takto upravená vzorka bola priamo dávkovaná do prístroja.
Capillary isotachophoresis was employed to separate and determine dimethinden enantiomers in various dosage forms. Several types of chiral selectors were tested in various electrolyte systems of different composition and different pH. The optimal leading electrolyte was composed of 10 mmol/l potassium acetate and acetic acid to achieve pH 4.8 with an addition of 4 mmol/l carboxyethyl-β-cyclodextrin as the chiral selector and 0.2 % of methylhydroxyethylcellulose (m–HEC) to suppress the electroosmotic flow. The terminating electrolyte was β- alanine of a concentration of 5 mmol/l. The evaluation included the precision, correctness, linearity, robustness, and selectivity of the elaborated ITP method. The pretreatment of the sample prior to analysis consisted in the dissolution and dilution of the appropriate dimethinden-containing dosage form with demineralized water to achieve the required concentration. Such a pretreated sample was directly dosed into the apparatus.
- MeSH
- elektroforéza metody přístrojové vybavení využití MeSH
- enzymy imobilizované analýza MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody přístrojové vybavení MeSH
- peptidové mapování metody přístrojové vybavení využití MeSH
- proteiny analýza MeSH
- trypsin analýza MeSH
- vazba proteinů genetika MeSH
