Nickolaj Jacob Petersen

Monolithic integration of optical waveguides for absorbance detection in microfabricated electrophoresis devices.

The fabrication and performance of an electrophoretic separation chip with integrated optical waveguides for absorption detection is presented. The device was fabricated on a silicon substrate by standard microfabrication techniques with the use of two photolithographic mask steps. The waveguides on the device were connected to optical fibers, which enabled alignment free operation due to the absence of free‐space optics. A 750 νm long U‐shaped detection cell was used to facilitate longitudinal absorption detection. To minimize geometrically induced band broadening at the turn in the U‐cell, tapering of the separation channel from a width of 120 down to 30 νm was employed. Electrical insulation was achieved by a 13 νm thermally grown silicon dioxide between the silicon substrate and the channels. The breakdown voltage during operation of the chip was measured to 10.6 kV. A separation of 3.2 νM rhodamine 110, 8 νM 2,7‐dichlorofluorescein, 10 νM fluorescein and 18 νM 5‐carboxyfluorescein was demonstrated on the device using the detection cell for absorption measurements at 488 nm.

On-chip electro membrane extraction with online ultraviolet and mass spectrometric detection.

Electro membrane extraction was demonstrated in a microfluidic device. The device was composed of a 25 μm thick porous polypropylene membrane bonded between two poly(methyl methacrylate) (PMMA) substrates, each having 50 μm deep channel structures facing the membrane. The supported liquid membrane (SLM) consisted of 2-nitrophenyl octyl ether (NPOE) immobilized in the pores of the membrane. The driving force for the extraction was a 15 V direct current (DC) electrical potential applied across the SLM. Samples containing the basic drugs pethidine, nortriptyline, methadone, haloperidol, loperamide, and amitriptyline were used to characterize the system. Extraction recoveries were typically in the range of 65-86% for the different analytes when the device was operated with a sample flow of 2.0 μL/min and an acceptor flow of 1.0 μL/min. With the sample flow at 9.0 μL/min and the acceptor flow at 0.0 μL/min, enrichment factors exceeding 75 were obtained during 12 min of operation from a total sample volume of only 108 μL. The on-chip electro membrane system was coupled online to electrospray ionization mass spectrometry and used to monitor online and real-time metabolism of amitriptyline by rat liver microsomes.

Drop-to-drop microextraction across a supported liquid membrane by an electrical field under stagnant conditions

Electromembrane extraction (EME) of basic drugs from 10 microL sample volumes was performed through an organic solvent (2-nitrophenyl octyl ether) immobilized as a supported liquid membrane (SLM) in the pores of a flat polypropylene membrane (25 microm thickness), and into 10 microL 10 mM HCl as the acceptor solution. The driving force for the extractions was 3-20 V d.c. potential sustained over the SLM. The influence of the membrane thickness, extraction time, and voltage was investigated, and a theory for the extraction kinetics is proposed. Pethidine, nortriptyline, methadone, haloperidol, and loperamide were extracted from pure water samples with recoveries ranging between 33% and 47% after only 5 min of operation under totally stagnant conditions. The extraction system was compatible with human urine and plasma samples and provided very efficient sample pretreatment, as acidic, neutral, and polar substances with no distribution into the organic SLM were not extracted across the membrane. Evaluation was performed for human urine, providing linearity in the range 1-20 microg/mL, and repeatability (RSD) in average within 12%.

Selective electromembrane extraction at low voltages based on analyte polarity and charge.

Electromembrane extraction (EME) at low voltage (0-15 V) of 29 different basic model drug substances was investigated. The drug substances with logP<2.3 were not extracted at voltages less than 15 V. Extraction of drug substances with logP≥2.3 and with two basic groups were also effectively suppressed by the SLM at voltages less than 15 V. Drug substances with logP≥2.3 and with one basic group were all extracted at low voltages and with a strong compound selectivity which appeared to have some influence from the polar surface area of the compound. For this group of substances, recoveries varied between 0 and 23% at 5 V, whereas, recoveries varied between 5.5 and 51% at 15 V. Based on mass transfer differences related to charge, polarity, and polar surface, highly selective extractions of drug substances were demonstrated from human plasma, urine, and breast milk. An initial evaluation at low voltage (5 V) was compared with similar extractions at a more normal voltage level (50 V), and this supported that reliable data can be obtained under these low-voltage (mild) conditions by EME.

Liquid-phase microextraction in a microfluidic-chip – High enrichment and sample clean-up from small sample volumes based on three-phase extraction

In this work, a microfluidic-chip based system for liquid-phase microextraction (LPME-chip) was developed. Sample solutions were pumped into the LPME-chip with a micro-syringe pump at a flow rate of 3-4 μL min(-1). Inside the LPME chip, the sample was in direct contact with a supported liquid membrane (SLM) composed of 0.2 μL dodecyl acetate immobilized in the pores of a flat membrane of polypropylene (25 μm thickness). On the other side of the SLM, the acceptor phase was present. The acceptor phase was either pumped at 1 μL min(-1) during extraction or kept stagnant (stop-flow). Amitriptyline, methadone, haloperidol, loperamide, and pethidine were selected as model analytes, and they were extracted from alkaline sample solution, through the SLM, and into 10 mM HCl or 100mM HCOOH functioning as acceptor phase. Subsequently, the acceptor phase was either analyzed off-line by capillary electrophoresis for exact quantification, or on-line by UV detection or electrospray ionization mass spectrometry for time profiling of concentrations. The LPME-chip was found to be highly effective, and extraction efficiencies were in the range of 52-91%. When the flow of acceptor phase was turned off during extraction (stop-flow), analyte enrichment increased linearly with the extraction time. After 10 min as an example, amitriptyline was enriched by a factor of 42 from only 30 μL sample solution, and after 120 min amitriptyline was enriched by a factor of 500 from 320 μL sample solution. This suggested that the LPME-chip has great potentials for very efficient analyte enrichments from limited sample volumes in the future.

Nano-electromembrane extraction.

The present work has for the first time described nano-electromembrane extraction (nano-EME). In nano-EME, five basic drugs substances were extracted as model analytes from 200 μL acidified sample solution, through a supported liquid membrane (SLM) of 2-nitrophenyl octyl ether (NPOE), and into approximately 8 nL phosphate buffer (pH 2.7) as acceptor phase. The driving force for the extraction was an electrical potential sustained over the SLM. The acceptor phase was located inside a fused silica capillary, and this capillary was also used for the final analysis of the acceptor phase by capillary electrophoresis (CE). In that way the sample preparation performed by nano-EME was coupled directly with a CE separation. Separation performance of 42,000-193,000 theoretical plates could easily be obtained by this direct sample preparation and injection technique that both provided enrichment as well as extraction selectivity. Compared with conventional EME, the acceptor phase volume in nano-EME was down-scaled by a factor of more than 1000. This resulted in a very high enrichment capacity. With loperamide as an example, an enrichment factor exceeding 500 was obtained in only 5 min of extraction. This corresponded to 100-times enrichment per minute of nano-EME. Nano-EME was found to be a very soft extraction technique, and about 99.2-99.9% of the analytes remained in the sample volume of 200 μL. The SLM could be reused for more than 200 nano-EME extractions, and memory effects in the membrane were avoided by effective electro-assisted cleaning, where the electrical potential was actively used to clean the membrane.

Design and implementation of an automated liquid-phase microextraction-chip system coupled on-line with high performance liquid chromatography.

An automated liquid-phase microextraction (LPME) device in a chip format has been developed and coupled directly to high performance liquid chromatography (HPLC). A 10-port 2-position switching valve was used to hyphenate the LPME-chip with the HPLC autosampler, and to collect the extracted analytes, which then were delivered to the HPLC column. The LPME-chip-HPLC system was completely automated and controlled by the software of the HPLC instrument. The performance of this system was demonstrated with five alkaloids i.e. morphine, codeine, thebaine, papaverine, and noscapine as model analytes. The composition of the supported liquid membrane (SLM) and carrier was optimized in order to achieve reasonable extraction performance of all the five alkaloids. With 1-octanol as SLM solvent and with 25 mM sodium octanoate as anionic carrier, extraction recoveries for the different opium alkaloids ranged between 17% and 45%. The extraction provided high selectivity, and no interfering peaks in the chromatograms were observed when applied to human urine samples spiked with alkaloids. The detection limits using UV-detection were in the range of 1-21 ng/mL for the five opium alkaloids presented in water samples. The repeatability was within 5.0-10.8% (RSD). The membrane liquid in the LPME-chip was regenerated automatically between every third injection. With this procedure the liquid membrane in the LPME-chip was stable in 3-7 days depending on the complexity of sample solutions with continuous operation. With this LPME-chip-HPLC system, series of samples were automatically injected, extracted, separated, and detected without any operator interaction.

Improving the reproducibility in capillary electrophoresis by incorporating current drift in mobility and peak area calculations.

The traditional way of calculating mobility and peak areas in capillary electrophoresis does not take into account the changes in the buffer viscosity at different thermostatic control and that the analytes may accelerate during the individual runs due to Joule heating effects. We present a method for accounting for these changes based on the monitored changes in current during the separation. The calculation method requires measuring the initial resistance of the buffer filled capillary, performed using a 0.2 min voltage ramping at the start of a separation. The mobility calculation corrected for current drift allowed identification of the tested analytes independent from capillary dimensions, electric field strengths and temperature control. Furthermore, the peak areas become less influenced by the experimental conditions, since the velocities of the analytes passing the detector are corrected for the acceleration during the run. The short voltage ramping could be further used to evaluate the heat transfer of the capillary to the surroundings and to estimate the temperature changes during the separation. The temperature was shown to change the ionization of 2‐phenylethylamine in accordance to a pKa dependency of primary amines reported in literature.

Automated coating procedures to produce poly(ethylene glycol) brushes in fused‐silica capillaries

Many bioanalytical methods rely on electrophoretic separation of structurally labile and surface active biomolecules such as proteins and peptides. Often poor separation efficiency is due to surface adsorption processes leading to protein denaturation and surface fouling in the separation channel. Flexible and reliable approaches for preventing unwanted protein adsorption in separation science are thus in high demand. We therefore present new coating approaches based on an automated in-capillary surface-initiated atom transfer radical polymerization process (covalent coating) as well as by electrostatically adsorbing a presynthesized polymer leading to functionalized molecular brushes. The electroosmotic flow was measured following each step of the covalent coating procedure providing a detailed characterization and quality control. Both approaches resulted in good fouling resistance against the four model proteins cytochrome c, myoglobin, ovalbumin, and human serum albumin in the pH range 3.4-8.4. Further, even samples containing 10% v/v plasma derived from human blood did not show signs of adsorbing to the coated capillaries. The covalent as well as the electrostatically adsorbed coating were both found to be stable and provided almost complete suppression of the electroosmotic flow in the pH range 3.4-8.4. The coating procedures may easily be integrated in fully automated capillary electrophoresis methodologies.

Easy peak tracking in CE-UV and CE-UV-ESI-MS by incorporating temperature-correlated mobility scaling.

A simple data reconstruction technique in CE‐UV‐ESI‐MS (where UV stands for ultraviolet) is presented to overcome the drift in mobilities caused by various factors compromising the reproducibility of such data, for example Joule heating effects and the variation in thermostatic control along the capillary, drift in EOF and the suction effect caused by the nebulizing gas in coaxial CE‐MS interfaces. We present here a method to transform the traditional time‐based electropherogram into the corresponding temperature‐correlated mobility scale allowing tracking of analytes independent from capillary dimensions, electric field strengths, temperature control, and distance between the detectors. The main principle of this alignment is based on including the current in the mobility calculations and relating this to the initial electrical resistance of the buffer‐filled capillary. The temperature‐correlated mobility calculation eliminates the peak shifts due to the viscosity changes, improves the precision of peak identification using the observed temperature‐correlated mobilities, and allows a direct comparison of signals from different detection combinations. The method allows peaks from normal CE‐UV separations to be correlated with the corresponding peak obtained by MS detection in CE‐MS even for differences in capillary dimensions and thermostatic control.