Matti Jussila

Non-aqueous capillary electrophoresis

The benefits of non-aqueous capillary electrophoresis have been described in a number of recent publications. The wide selection of organic solvents, with their very different physicochemical properties, broadens our scope to manipulate separation selectivity. The lower currents present in non-aqueous solvents allow the use of high electric field strengths and wide bore capillaries, the latter in turn allowing larger sample load. In many cases detection sensitivity can also be enhanced. The potential of non-aqueous capillary electrophoresis is discussed throughout the paper, and the feasibility of capillary electrophoresis under non-aqueous media is demonstrated with reference to several applications.

Thermal aggregation of bovine serum albumin studied by asymmetrical flow field-flow fractionation.

The use of asymmetrical flow field-flow fractionation (AsFlFFF) in the study of heat-induced aggregation of proteins is demonstrated with bovine serum albumin (BSA) as a model analyte. The hydrodynamic diameter (d(h)), the molar mass of heat-induced aggregates, and the radius of gyration (R(g)) were calculated in order to get more detailed understanding of the conformational changes of BSA upon heating. The hydrodynamic diameter of native BSA at ambient temperature was approximately 7 nm. The particle size was relatively stable up to 60 degrees C; above 63 degrees C, however, BSA underwent aggregation (growth of hydrodynamic diameter). The hydrodynamic diameters of the aggregated particles, heated to 80 degrees C, ranged from 15 to 149 nm depending on the BSA concentration, duration of incubation, and the ionic strength of the solvent. Heating of BSA in the presence of sodium dodecyl sulfate (1.7 or 17 mM) did not lead to aggregation. The heat-induced aggregates were characterized in terms of their molar mass and particle size together with their respective distributions with a hyphenated technique consisting of an asymmetrical field-flow fractionation device and a multi-angle light scattering detector and a UV-detector. The carrier solution comprised 8.5 mM phosphate and 150 mM sodium chloride at pH 7.4. The weight-average molar mass (M(w)) of native BSA at ambient temperature is 6.6x10(4) g mol(-1). Incubation of solutions with BSA concentrations of 1.0 and 2.5 mg mL(-1) at 80 degrees C for 1 h resulted in aggregates with M(w) 1.2x10(6) and 1.9x10(6) g mol(-1), respectively. The average radius of gyration and the average hydrodynamic radius of the heat-induced aggregate samples were calculated and compared to the values obtained from the size distributions measured by AsFlFFF. For comparison static light scattering measurements were carried out and the corresponding average molar mass distributions of solutions with BSA concentrations of 1.0 and 2.5 mg mL(-1) at 80 degrees C for 1 h gave aggregates with M(w) 1.7x10(6) and 3.5x10(6) g mol(-1), respectively.

Comprehensive two-dimensional gas chromatography in the analysis of urban aerosols.

Comprehensive two-dimensional gas chromatography utilising a semi-rotating cryogenic modulator was applied to the analysis of urban aerosols. Samples were collected onto glass fibre filters using a high-volume sampler in Helsinki, Finland. Sample preparation included extraction into n-hexane-acetone mixture and clean-up on silver-impregnated silica column. Analyses were performed with GC x GC-FID and GC x GC-QMS equipment. Linearity of the method was good with R2 values greater than 0.994 for all analytes. Polycyclic aromatic hydrocarbons (PAHs) and oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) were identified and quantified in urban particulates. PAH and oxy-PAH concentrations ranged from 0.5 to 5.5 ng/m3.

Analysis of eleven iridoid glycosides by micellar electrokinetic capillary chromatography (MECC) and screening of plant samples by partial filling (MECC)–electrospray ionisation mass spectrometry

Of ammonium, lithium and sodium salts of dodecyl sulfate studied as surfactants in the separation of iridoid glycosides by micellar electrokinetic capillary chromatography (MECC), the last one gave the best results. Eleven neutral iridoid glycosides were separated by MECC with sodium dodecyl sulfate as surfactant, and the water-micelle partition coefficients of the compounds were calculated. The separation system was coupled via a coaxial sheath flow electrospray interface to a mass spectrometer, and the partial filling technique was used in the on-line analysis. Seven plant species belonging to five genera (Plantago, Veronica, Melampyrum, Succisa and Valeriana) were screened for the iridoid glycosides by the new method that was developed. The findings confirmed those of an earlier study on five of the iridoid glycosides. Some new iridoid glycosides were found in Plantago lanceolata, Veronica spicata and V. chamaedrys.

Use of a partial filling technique and reverse migrating micelles in the study of N-methylcarbamate pesticides by micellar electrokinetic chromatography–electrospray ionization mass spectrometry

This study describes three ways to couple micellar electrokinetic chromatography (MEKC) on-line with electrospray ionization mass spectrometry (ESI-MS) for the analysis of N-methylcarbamate pesticides. The methods involved the use of a partial filling (PF) technique under basic conditions and the use of reverse migrating micelles (RMMs) under acidic and basic conditions. The use of RMMs in basic electrolyte solutions required coated capillaries with low electroosmotic flows, and capillaries coated with anionic poly(sodium 2-acrylamide-2-methylpropanesulfonate) were selected for the purpose. Before the on-line MEKC-ESI-MS coupling, the MEKC and MS conditions were separately optimized under off-line conditions. The methods were compared in terms of detection limits and the stability of the electrospray process. The PF method offered good separation but poorer stability of the electrospray relative to the other methods. A more stable electrospray performance was obtained with use of RMMs in acidic electrolyte solutions, but some of the analytes were protonated and could not be detected due to the increase in their retention factors. However, with the use of anionic polymer-coated capillaries and RMMs at pH 8.5, all analytes were successfully separated. The high-salt stacking method was applied to improve the sensitivity of MEKC-ESI-MS and the detection limits were in the range of 0.04-2.0 microg/ml.

Extremely high electric field strengths in non-aqueous capillary electrophoresis

The influence of high electric field strength on the separation of basic analytes in non-aqueous alcohol background electrolyte (BGE) solutions was investigated. Increasing the separation voltage in capillary electrophoresis (CE) may be advantageous if the conductivity of the BGE solution is low enough to allow fast separations without excessive Joule heating or band broadening. The voltage range tested was 20-60 kV with methanol and ethanol, and 25-60 kV with propanol and butanol as solvent for BGE. The resulting electric field strengths ranged from 660 V cm(-1) to 2000 V cm(-1). Experiments were made with a special laboratory constructed CE instrument. The separation efficiency vs. voltage curve was found to vary with the alcohol BGE solution. The increase in voltage decreased the separation efficiency in the case of methanol BGE solution, but with the other BGEs a clear efficiency maximum was obtained above 30 kV. The highest separation efficiencies were achieved with propanol BGE solution, where the efficiency maximum was reached at 45 kV. However, reasonable efficiency was achieved even at 60 kV. The extent of Joule heating was determined by calculating the temperature inside the capillary and the observed plate heights were interpreted in terms of the Van Deemter equation. The decrease in the separation efficiency with higher voltage was attributed mainly to Joule heating in the case of methanol and ethanol BGE solution and to the analyte adsorption on the capillary wall with propanol and butanol BGE solutions.

Separation, purity testing and identification of cyanobacterial hepatotoxins with capillary electrophoresis and electrospray mass spectrometry

Abstract Capillary zone electrophoretic and micellar electrokinetic chromatographic methods with UV detection were investigated for separation and identification of some microcystins isolated from cyanobacterial Anabaena 90 strains in Finland. The low-sensitivity purity tests with full scan UV–Vis spectra were done with a CZE method. All the test compounds, [ d -Asp 3 ,Dha 7 ]MCYST-LR, MCYST-LR, MCYST-YR, [Dha 7 ]MCYST-LR, MCYST-RR, [Dha 7 ]MCYST-RR, [ d -Asp 3 ,Dha 7 ]MCYST-RR, [ d -Asp 3 ]MCYST-LR and [ d -Asp 3 ]MCYST-RR (where MCYST stands for microcystin) isolated with a preparative HPLC method, could be separated from each other in a MECC method. The detection limits of the microcystins were below ppm level. The repeatability of the MECC technique was tested by comparing the absolute migration times with the indices calculated with “in-laboratory” designed programs operating in MATLAB Mathworks Inc. The identification and the high-sensitivity purity tests of the isolated microcystins were made with an off-line electrospray mass spectrometer. Identification could be done for MCYST-YR, MCYST-LR, MCYST-RR, [Dha 7 ]MCYST-RR, [Dha 7 ]MCYST-RR, [ d -Asp 3 ]MCYST-LR, [ d -Asp 3 ]MCYST-RR, [ d -Asp 3 , Dha 7 ]MCYST-LR and [ d -Asp 3 , Dha 7 ]MCYST-RR on the basis of their [M+H] + or [M+2H] 2+ ions in the MS mode and by using the protonated molecule as precursor in the MS–MS mode.

Integration of a contactless conductivity detector into a commercial capillary cassette: Detection of inorganic cations and catecholamines

A contactless conductivity detector integrated into the capillary cassette of Agilent (3D)CE equipment is described. The detector is user-friendly, compact and easily modified. The UV detector of the (3D)CE equipment is available parallel with the contactless conductivity detector increasing the detection power. Two electrolyte solutions, 2-(N-morpholino)ethanesulfonic acid-histidine solution (20 mM, pH 6.0) and ammonium acetate (10 mM, pH 4.0), were used as the separation media for inorganic cations and organic catecholamines, respectively. The detection limit for all metal cations except barium was under 0.5 mg/l, and that for four catecholamines was ca. 10 mg/l. This last value was the same order of magnitude as achieved with parallel UV detection.

Alcohols and wide-bore capillaries in nonaqueous capillary electrophoresis

The feasibility of using C1‐C5 alcohols as electrolyte solutions in nonaqueous capillary zone electrophoresis was investigated. The separation of basic narcotic analgesics and acidic diuretics was modified by changing the alcohol in an electrolyte solution containing alcohol‐acetonitrile‐acetic acid (50:49:1, v/v) and 20 mM ammonium acetate while other experimental conditions were kept constant. The alcohols studied were methanol, ethanol, 1‐propanol, 2‐propanol, 1‐butanol, 2‐butanol, and 1‐pentanol. The results indicate that even longer‐chain alcohols can be used in nonaqueous capillary zone electrophoresis and, because of the lower currents they allow, they are especially advantageous in wider capillaries. Basic analytes were separated in 200 μm and 320 μm ID capillaries with 1‐butanol‐acetonitrile‐acetic acid (50:49:1, v/v) containing 20 mM ammonium acetate as electrolyte solution. Problems related to the use of wide‐bore capillaries are discussed.

Nonaqueous capillary electrophoresis with alcoholic background electrolytes: Separation efficiency under high electrical field strengths

The effect of high voltage on capillary electrophoresis (CE) separations of anionic analytes in nonaqueous separation media was investigated. Methanol, ethanol, 1‐propanol, and 1‐butanol were tested as background electrolyte (BGE) solvents. Experiments were carried out with a laboratory‐built CE instrument suitable for high‐voltage separations. Potentials up to 60 kV were applied with reversed polarity to generate unusually high field strengths (e.g. 2000 Vcm–1) and so achieve fast and efficient separations. Highest separation efficiencies were obtained with propanol as BGE solvent, and the dependency of the efficiency on the separation voltage was more or less linear. With the other alcohols, separation efficiency decreased or remained roughly constant with increasing absolute voltage. The separation efficiencies are discussed in terms of longitudinal diffusion, Joule heating, and analyte interaction with the capillary wall. Capillary preconditioning had a varied effect on the separations in the different BGEs as the BGE and the conditioning process affected the electroosmotic flow (EOF) velocity and direction.