electroosmotic flow
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Electroosmotic flow (EOF) plays a pivotal role in optimization of capillary electrophoresis (CE) separations of (bio)molecules and (bio)particles. EOF velocity is directly related to analysis time, peak resolution and separation efficiency. Here, we report a concept of charged polymer coatings of the inner fused silica capillary wall, which allows anodic EOF with mobility ranging from 0 to ∼(30-40) × 10-9 m2V-1s-1. The capillary wall is modified by covalently bound cationic copolymer poly(acrylamide-co-(3-acrylamidopropyl)trimethylammonium chloride) (PAMAPTAC) containing variable ratio of the charged monomer in the 0-60 mol. % interval. The EOF mobility showed minor variability with composition of background electrolyte (BGE) and pH in the 2-10 interval. The coatings were evaluated by CE-UV and nanospray CE-MS in the counter-EOF arrangement for a series of basic drug molecules in acetic acid based acidic BGE. Tunable EOF velocity was demonstrated as a useful tool for optimization of peak resolution, separation efficiency and migration time of analytes. Electrostatic repulsion of positively charged capillary surface was shown as beneficial for suppression of analyte adsorption, notably for hydrophobic cationic analytes.
- MeSH
- adsorpce MeSH
- elektroforéza kapilární * MeSH
- elektroosmóza * MeSH
- kationty MeSH
- polymery MeSH
- Publikační typ
- časopisecké články MeSH
We present a method for finely adjustable electroosmotic flow (EOF) velocity in cathodic direction for the optimization of separations in capillary electrophoresis. To this end, we use surface modification of the separation fused silica capillary by the covalently attached copolymer of acrylamide (AM) and 2-acrylamido-2-methyl-1-propanesulfonate (AMPS), that is, poly(AM-co-AMPS) or PAMAMPS. Coatings were formed by the in-capillary polymerization of a mixture of the neutral AM and anionic AMPS monomers premixed in various ratios in order to control the charge density of the copolymer. EOF mobility varies in the 0 to ∼40 × 10-9 m2 V-1 s-1 interval for PAMAMPS coatings ranging from 0 to 60 mol.% of charged AMPS monomer. For EOF in PAMAMPS-treated capillaries, we observed (i) a negligible dependence on pH in the 2-10 interval, (ii) a minor variance among background electrolytes (BGEs) in function of their components and (iii) its standard decrease with increasing ionic strength of the BGE. Interest in variable cathodic EOF was demonstrated by the amelioration of separation of two kinds of isomeric anionic analytes, that is, monosaccharides phosphates and helquat enantiomers, in counter-EOF mode.
Small neutral organic compounds have traditionally the role of EOF markers in electrophoresis, as they are expected to have zero electrophoretic mobility in external electric fields. The BGE contains, however, ions that have unequal affinities to the neutral molecules, which in turn results in their mobilization. In this study we focused on two EOF markers-thiourea and DMSO, as well as on N-methyl acetamide (NMA) as a model of the peptide bond. By means of CE and all atom molecular dynamics simulations we explored mobilization of these neutral compounds in large set of Hofmeister salts. Employing a statistical mechanics approach, we were able to reproduce by simulations the experimental electrophoretic mobility coefficients. We also established the role of the chemical composition of marker and the BGE on the measured electrophoretic mobility coefficient. For NMA, we interpreted the results in terms of the relative affinities of cations versus anions to the peptide bond.
- MeSH
- acetamidy chemie MeSH
- cesium chemie MeSH
- dimethylsulfoxid chemie MeSH
- elektroforéza kapilární metody MeSH
- elektroosmóza MeSH
- lithium chemie MeSH
- osmolární koncentrace MeSH
- roztoky MeSH
- simulace molekulární dynamiky MeSH
- soli MeSH
- thiomočovina chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Capillary electrophoretic separation of ketamine, norketamine, hydroxynorketamine, and dehydronorketamine was performed in the counter-current regime under the influence of oppositely-directed electroosmotic flow. For this purpose, the fused silica capillaries were covalently coated with the poly(acrylamide-co-3-acrylamidopropyl trimethylammonium chloride) copolymer (PAMAPTAC). The content of the cationic monomer APTAC in the polymerization mixture varied in the range 0-6 mol. % and the generated electroosmotic flow increased continuously in the 0-20 · 10-9 m2V-1s-1 interval. Importantly, it resulted in improved electrophoretic resolution of ketamine/norketamine, which increased from 0.8 for neutral PAM coating (i.e. 0% PAMAPTAC) to 3.0 for 6% PAMAPTAC. The determination of ketamine and its derivates in rat serum was performed in a 4% PAMAPTAC capillary with an inner diameter of 25 μm. The separation was performed in a 500 mM aqueous solution of acetic acid (pH 2.3). The clinical sample was deproteinized by the addition of acetonitrile to the serum and a large volume of the treated sample was injected directly into the capillary. The achieved limit of detection ranged from 2.2 ng/mL for dehydronorketamine to 4.1 ng/mL for hydroxynorketamine; the intra-day repeatability was 1.0-1.5% for the migration time and 2.8-3.3% for the peak area. The developed methodology was employed for time monitoring of ketamines in rat serum after intra venous administration of low doses of anaesthetic at a level of 2 μg per g of body weight.
- Publikační typ
- časopisecké články MeSH
- MeSH
- adenosindeaminasa metabolismus MeSH
- elektroforéza kapilární metody MeSH
- erytrocyty enzymologie MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
