Covalent cationic copolymer coatings allowing tunable electroosmotic flow for optimization of capillary electrophoretic separations.

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.

Micellar electrokinetic chromatography in the determination of triazoles in fruit peel.

Triazole fungicides (TAFs) are frequently used fungicides for various antifungal treatments of crops. Tre treatment is provided foliarly. However, some significant amount of TAFs may remain on/in fruits. We have developed a methodology for the determination of penconazole, tebuconazole and cyproconazole in tomato fruit peel. The extraction of TAFs was provided with chloroform (acidified with 0.1% acetic acid). In the electrokinetic chromatography, the mixed micellar pseudo-stationary phase was composed of anionic detergent sodium dodecyl sulphate (15 mM) and randomly highly sulphated gamma-cyclodextrin (17.5 mg/mL). The background electrolyte consisted of 100 mM phosphoric acid and 100 mM Tris in the mixed hydro-organic solvent water/methanol (80/20 v/v), apparent pH 4.8. Complete separation of penconazole, tebuconazole, and two diastereomers of cyproconazole with resolutions higher than 5.1 were achieved within a relatively short time of less than 17 min in the bare fused silica capillary of 425/500 mm total/effective lengths and 50/375 µm I.D./O.D. at separation voltage -15 kV (cathode at injection capillary end) and at constant capillary cassette temperature of 22°C. The TAFs were detected by a UV-spectrophotometric diode array detector set at 200 nm. The limits of detection and limits of quantification were in the range of 71-92 and 214-278 µg/kg of peel, respectively. Analyses of the peel extracts revealed that even 10 days after the last treatment, TAF concentrations were higher than the recommended maximum residue limits in both application ways, as individual as well as in the TAF binary or ternary mixtures.

Investigating the position of the separation capillary and emitter tube tips in a nanoflow sheath-liquid CE-ESI-MS interface to decouple the ESI potential.

Robust decoupling of the ESI potential from the separation potential in CE-ESI-MS interfaces is very important for the high performance of the CE-ESI-MS devices and their applications for highly sensitive analyses of ionogenic compounds. In this study, we utilize a nanoflow sheath-liquid CE-ESI-MS interface composed of a quartz emitter and a separation fused silica capillary treated by etching, which are threaded to cross coupling for sheath liquid and electrode connection. Specifically, we have tested the ability of the interface to decouple the ESI potential from the separation potential at different positions of the separation capillary and ESI emitter tube tips. The interface with the separation capillary tip protruding the emitter tip by 20 µm did not provide sufficient robustness. The real ESI potential (delivered as 2.0 kV from the independent high voltage power supply HV2) ranged from 2.1 kV to 4.5 kV depending on the applied separation voltage (12.0-20.0 kV, provided by the power supply HV1) and electric conductivity of the background electrolyte (BGE) used. The interface robustness was partially improved when the capillary tip was aligned with the emitter tip. However, the complete decoupling of the spray and separation potentials was achieved only when the capillary tip was retracted 20 µm inside the emitter. In this arrangement, the ESI potential was stable and independent of both the separation potential (voltage) and the BGE conductivity. Moreover, this setting provided better sensitivity for the CE-ESI-MS analysis of selected drugs and benzylpyridinium cations than the setup with the capillary tip aligned with or protruding the emitter tip.

Comparison of two low flow interfaces for measurement of mobilities and stability constants by affinity capillary electrophoresis-mass spectrometry.

Affinity capillary electrophoresis (ACE) is typically used for the determination of stability constant, Kst, of weak to moderately strong complexes. Sensitive detection such as mass spectrometry (MS) is required for extension of ACE methodology for estimation of Kst of stronger complexes. Consequently, an efficient interface for hyphenation of CE with MS detection is necessary. For evaluation of interfaces for electrospray ionization mass spectrometric (ESI/MS) detection in ACE conditions, potassium-crown ether complexation was used as model system. The effective mobilities of the crown ether ligands and the Kst of their potassium complexes were measured/determined by ACE-ESI/MS using two lab-made interfaces: (i) a sheathless porous tip CE-ESI/MS interface and (ii) a nano-sheath liquid flow CE-ESI/MS interface, and, in turn, compared with those obtained by ACE with UV spectrophotometric detection. Apparent stability constant of potassium-crown ether complexes in 60/40 (v/v) methanol/water mixed solvent, pH* 5.5, was about 1300 L/mol for dibenzo-18-crown-6, 1600 L/mol for benzo-18-crown-6 and 5200 L/mol for 18-crown-6 ligands, respectively. It was observed that electrophoretic mobilities from CE-MS experiments differ from reference values determined by UV detection by ∼7% depending on the CE-MS interface used. Good agreement of CE-MS and CE-UV data was achieved for nano-sheath liquid flow interface, in which the spray potential and the CE separation potential can be effectively decoupled. As for sheathless porous tip interface, a correction procedure involving a mobility marker has been proposed. It provides typically only ca. 1% difference of effective mobilities and Kst values obtained from CE-MS data as compared to those received by the reference ACE-UV method.

Study of solvent effects on the stability constant and ionic mobility of the dibenzo-18-crown-6 complex with potassium ion by affinity capillary electrophoresis.

The effect of solvent on the strength of noncovalent interactions and ionic mobility of the dibenzo-18-crown-6 complex with K+ in water/organic solvents was investigated by using affinity capillary electrophoresis. The proportion of organic solvent (methanol, ethanol, propan-2-ol, and acetonitrile) in the mixtures ranged from 0 to 100 vol.%. The stability constant, KKL , and actual ionic mobility of the dibenzo-18-crown-6-K+ complex were determined by the nonlinear regression analysis of the dependence of the effective electrophoretic mobility of dibenzo-18-crown-6 on the concentration of K+ (added as KCl) in the background electrolyte (25 mM lithium acetate, pH 5.5, in the above mixed hydro-organic solvents). Competitive interaction of the dibenzo-18-crown-6 with Li+ was observed and quantified in mixtures containing more than 60 vol.% of the organic solvent. However, the stability constant of the dibenzo-18-crown-6-Li+ complex was in all cases lower than 0.5 % of KKL . The log KKL increased approximately linearly in the range 1.62-4.98 with the increasing molar fraction of organic solvent in the above mixed solvents and with similar slopes for all four organic solvents used in this study. The ionic mobilities of the dibenzo-18-crown-6-K+ complex were in the range (6.1-43.4) × 10-9 m2 V-1 s-1 .

Determination of acid dissociation constants of triazole fungicides by pressure assisted capillary electrophoresis.

Pressure assisted capillary electrophoresis was applied to determination of acid dissociation constants (pKa) of six widely used triazole fungicides (cyproconazole, epoxiconazole, flusilazole, tebuconazole, penconazole and propiconazole) in aqueous medium. The pKa values were determined from the dependence of effective electrophoretic mobility of the triazole fungicides on p[H(+)] of the background electrolyte (BGE) using non-linear regression analysis. The p[H(+)] was used instead of pH to reflect the increased ionic strength of the strongly acidic BGEs (pH<1.75) as compared to the BGEs at pH equal to or greater than 1.75. Prior to the pKa calculation, the measured effective electrophoretic mobilities were corrected to the reference temperature (25°C) and constant ionic strength (25mM). The regression function was modified to allow the determination of pKa in the BGEs of varying ionic strength. The electrophoretic measurements showed that the above triazole fungicides are very weak bases - their pKa values were found to be in the range 1.05-1.97 and were in a good agreement with the values calculated by SPARC online pKa calculator.