Electrophoresis In Fused-silica Capillaries Research Articles

Analysis of lipoproteins by capillary zone electrophoresis in microfluidic devices: assay development and surface roughness measurements.

The development of a new assay for lipoproteins by capillary electrophoresis in fused-silica capillaries and in glass microdevices is described in this paper. The separation of low-density (LDL) and high-density (HDL) lipoproteins by capillary zone electrophoresis is demonstrated in fused-silica capillaries with both UV absorption and laser-induced fluorescence detection. This separation was accomplished using Tricine buffer (pH 9.0) with methylglucamine added as a dynamic coating. With UV detection, LDL eluted as a relatively sharp peak with a migration time of approximately 11 min and HDL eluted as a broad peak with a migration time of 12.5 min. Fluorescence detection of lipoproteins stained with NBD-ceramide was used with the same buffer system to give comparable results. Furthermore, fluorescence staining of human serum samples yielded results similar to the fluorescently stained LDL and HDL fractions, showing that this method can be used to quantify lipoproteins in serum samples. The method was also used to detect lipoproteins in glass micro-CE devices. Very similar results were obtained in microdevices although with much faster analysis times, LDL eluted as a sharp peak at approximately 25 s and HDL as a broad peak at slightly longer time. In addition, higher resolution was obtained on chips. To our knowledge, these results show the first separation and detection of lipoproteins in a microfluidic device using native serum samples. Atomic force microscopy was used to characterize the rms surface roughness (Rq) of microfluidic channels directly. Devices with different surface roughness values were fabricated using two different etchants for Pyrex wafers with a polysilicon masking layer. Using 49% HF, the measured roughness is Rq = 10.9 +/- 1.6 nm and with buffered HF (NH4F + HF) the roughness is Rq = 2.4 +/- 0.7 nm. At this level of surface roughness, there is no observable effect on the performance of the devices for this lipoprotein separation.

Repartition effect of aromatic polyaniline coatings on the separation of bioactive peptides in capillary electrophoresis.

The capillary walls of fused-silica capillary electrophoresis (CE) columns were modified with a thin film of polyaniline (PANI), providing open-tubular columns with a stable coating containing aromatic groups and amine functionalities. Fast and efficient separations were observed for small bioactive peptides under acidic conditions on PANI-coated columns. The mechanism of separation is based on hydrophobic interactions between the analytes and the polymeric matrix. Good reproducibility was observed from run-to-run. Due to the simple derivatization procedure, method flexibility, the uniformity of the coating and its stability, conjugated polymers could find practical application in capillary zone electrophoresis (CZE) separations.

The determination of isoflavones and coumestrol by capillary electrophoresis

The separation of six isoflavones (biochanin A, isoformononetin, formononetin, prunetin, daidzein and genistein) and coumestrol on an uncoated fused-silica capillary electrophoresis column was optimised using alkaline borate buffer as electrolyte and DAD detection. A baseline separation of all analytes except a pair, formononetin-biochanin A was achieved at pH 10.5 in 25 min. Detection limits were low (0.1 µg/ml) and the linearity of the detector response was established in the concentration range 0.4–60 µg/ml (180 µg/ml for coumestrol). Coumestrol was synthesized and the carbon signals in 13C-NMR spectrum of both coumestrol and di-O-acetylcoumestrol were assigned for the first time using two-dimensional HMQC technique.

Analyses of phenolic compounds by capillary electrophoresis electrospray mass spectrometry

Several phenolic compounds found in olive mill wastewater were quantitatively and qualitatively analyzed on an uncoated fused-silica capillary electrophoresis column (67 cm × 75 µm i.d.) using 30 mM aqueous ammonium acetate buffer/methanol (90:10) and negative electrospray mass spectrometry detection. Quantitative analysis, with p-chlorophenol as the internal standard, was carried out by single ion monitoring Limits of detection ranged from 1 pg for 4-hydroxybenzaldehyde and protocatechuic acid to 386 pg for vanillic acid. Copyright © 1999 John Wiley & Sons, Ltd.

Analyses of phenolic compounds by capillary electrophoresis electrospray mass spectrometry

Several phenolic compounds found in olive mill wastewater were quantitatively and qualitatively analyzed on an uncoated fused-silica capillary electrophoresis column (67 cm × 75 µm i.d.) using 30 mM aqueous ammonium acetate buffer/methanol (90:10) and negative electrospray mass spectrometry detection. Quantitative analysis, with p-chlorophenol as the internal standard, was carried out by single ion monitoring Limits of detection ranged from 1 pg for 4-hydroxybenzaldehyde and protocatechuic acid to 386 pg for vanillic acid. Copyright © 1999 John Wiley & Sons, Ltd.

Characterisation of inner surface and adsorption phenomena in fused-silica capillary electrophoresis capillaries

Method validation in capillary electrophoresis (CE) is substantial for the routine use in quality control. Thus all phenomena that can influence results in CE must be carefully characterised. Especially the inner surface of the fused-silica capillaries plays an important role. Sometimes there is a big difference in the properties of capillaries from different batches. Inner capillary surface can be pictured by scanning electron microscopy (SEM). Nature, pattern and number of cracks and other surface defects altered from batch to batch. These surface defects were no artefacts of sample preparation, which could be excluded by comparing different preparation techniques and results from literature. Treatments with hydrofluoric acid cannot completely remove the surface cracks and defects in subsurface areas. Hydrofluoric acid treatment not only changes the surface roughness but also the inner capillary diameter. A correlation between etching time and increase of diameter is investigated. SEM also pictures adsorption phenomena, e.g. proteins are not adsorbed as a homogenous layer. Clusters at different regions are observed. Thus the ζ potential differs considerably with space, leading to losses in separation efficiency. SEM can readily be used to analyse the effectiveness of rinsing procedures. There are residuals of adsorbed compounds even after using aggressive rinsing reagents.

Determination of isoflavones using capillary electrophoresis in combination with electrospray mass spectrometry

Various isoflavones were separated on an uncoated fused-silica capillary electrophoresis (CE) column (110 cm × 75 μm I.D.) using 25 m M ammonium acetate buffer and UV and electrospray ionization mass spectrometric (ESI-MS) detection. CE-ESI-MS with negative-ion electrospray ionization has been shown to be a suitable technique for the determination of this type of natural compound. The modest sample loading of the CE technique can be circumvented by coupling it with MS; in addition, the mass-resolving capability and high sensitivity of MS for structural analysis of mixtures can thus be exploited. Furthermore, ESI-MS allows one not only to determine the molecular mass of isoflavones, but also the presence of various functional groups according to observed losses from the [M - H] − ion during collision-induced dissociation by adjusting some MS parameters.

Potentiometric detection of anions separated by capillary electrophoresis using an ion-selective microelectrode

Capillary electrophoresis in fused-silica capillaries of 10 μm I.D. coupled to an anion-selective microelectrode as on-column detector allows inorganic and organic anions, especially lipophilic ones, to be separated with high resolution and sensitivity of detection. For perchlorate, plate numbers of up to 10 7 /h and a detection limit of 5·10 −8 M have been achieved. After delogarithmizing the response function of the microelectrode, quantitative results are obtained as described previously.

Dependence of electroosmotic flow in capillary electrophoresis on Group I and II metal ions

The effect of Group I and II metal ions on electroosmotic flow in capillary electrophoresis in fused-silica capillaries is characterized. The electroosmotic mobility of aqueous mobile phases of lithium, sodium, potassium, calcium and barium acetates in fused-silica capillaries is measured as a function of pH at constant voltage. Cross contamination is avoided by using separate columns for each study and pH control is maintained with the aid of He sparging. The shape of a plot of pH vs. electroosmotic mobility depends on the particular cation used which in turn depends on the surface sorption properties of the ions. Column history is demonstrated to have an effect on electroosmotic flow and therefore retention times. The resolution of a test mixture is optimal in the lithium-based buffer.

Capillary zone electrophoresis with laser-induced fluorescence detection for analysis of methylmalonic acid and other short-chain dicarboxylic acids derivatized with 1-pyrenyldiazomethane

Methylmalonic acid (MMA) and other short-chain dicarboxylic acids react with 1-pyrenyldiazomethane (PDAM) in aqueous matrices and form stable, highly fluorescent 1-pyrenylmethyl monoesters [Schneede and Ueland, Anal. Chem., 64 (1992) 315-319]. We investigated the migration behaviour of these derivatives in capillary zone electrophoresis in fused-silica capillaries. The 1-pyrenyldiazomethane derivatives were detected with a laser-induced fluorescence detector that was connected to a helium-cadmium laser, delivering light at a wavelength of 325 nm, which exactly matched an excitation maximum of the 1-pyrenylmethyl monoesters. These esters have one free carboxylic acid group, which carries negative charge at pH > 4.0. The electrophoretic mobility varied according to pH, and the pH effect was most pronounced at values close to the p K a value (5.2–5.8) of the esters. The effects of the composition of the running buffer (pH, organic modifier concentration, ionic strength) and some operational parameters (voltage, temperature and capillary length) were tested, and were largely found to comply with validated theoretical models for capillary zone electrophoresis. In an optimal system, as judged by high number of theoretical plates, high resolution and short run times, the MMA derivative was separated from related 1-pyrenylmethyl monoesters. The laser-induced fluorescence detection afforded a limit of detection of about 40 nmol/1 (signal-to-noise ratio of 5) for the MMA derivative. Under optimal conditions, we were able to detect <1 μmol/1 endogenous MMA in human serum.

Adsorption on polymer-coated fused-silica capillary electrophoresis columns using selected protein and peptide standards

Three series of basic proteins and peptides were selected for use in studying the adsorption behavior of fused-silica capillary electrophoresis columns. A comparative study of untreated and treated fused-silica columns was done. The polymers used as coating materials ranged from very hydrophobic to very hydrophilic. The average efficiency of weakly hydrophilic Ucon-coated capillaries for basic proteins was over 1 million theoretical plates at a buffer pH of 4.8. For this coating, it was found that the maximum column efficiency was obtained at a film thickness of approximately 100 nm. It was easily seen that both ionic and hydrophobic interaction forces can play important roles in protein adsorption onto the capillary walls. Furthermore, strongly hydrophilic polymer coatings may possibly lead to hydrogen-bonding interactions, which decrease column efficiency. Studies have been conducted using two series of polypeptides with varying chain lengths. One series was used to study the effect of polypeptide chain length on retention behavior. Efficient separations of bioactive peptide drugs on Ucon-coated columns were achieved. The results indicate that high efficiency and resolution of basic peptides tested can be realized on columns modified with weakly hydrophilic polymers at a pH value of about 5.

Electrophoresis in fused-silica capillaries: the influence of organic solvents on the electroosmotic velocity and the .zeta. potential

3390. (44) De Le RkM J. H. Ar-h s ~ b s ~ ~ Nftrm Academic Press Inc.: New York, 1959. (45) mine, H. J. RMCW m n / s m in orgenic ”/sby, A r m & ReanangementS: Monograph 6, Eaborn, C., Chapman, N. E., Eds.: Elsevier Publishing Company: New York. 1967. (46) Mer&, J. ~b~~~ orgenic cmiSw, RMcbbn Mechanism Structure, 3rd. ed.: John Wlley 8 Sons: New York, 1985; p 501. p. D. RECEIVED for review February 11, 1991. Accepted June 7, 1991. The support of the U.S. Environmental Protection Agency (EPA CK-815749-01-0) is acknowledged. The Support provided by the Emil Aaltonen Foundation and Suomen Kulttuurirahasto is greatly appreciated (T.K.).