Wolfgang Thormann

Head-Column Field-Amplified Sample Stacking in Binary System Capillary Electrophoresis: A Robust Approach Providing over 1000-Fold Sensitivity Enhancement

Effective electrokinetic field-amplified sample injection occurring at the capillary inlet from a sample volume equivalent exceeding that of the capillary up to 10-fold is described and demonstrated to provide over 1000-fold sensitivity enhancement. Successful application of this head-column field-amplified sample stacking approach to the analysis of positively chargeable, hydrophobic compounds in binary system capillary electrophoresis is shown to require an initially introduced low-conductivity zone (water plug) of >1 mm length, a sample injection voltage <20 kV, and an injection time interval <60 s. Following these conditions for more than 1500 runs with capillaries of 50 μm i.d. and about 20 cm effective length, damaging heat production during electroinjection within the low-conductivity zone at the column inlet (boiling of solvent and possible deposition of solutes or fusing of capillary walls) could be prevented. The solute amount injected by head-column field-amplified sample stacking is further shown to be dependent on the organic fraction and the buffer in the sample solution. High content of organic solvent, low conductivity, and the presence of a small amount of H(+) (50-100 μM) provides the highest sensitivity for analysis of positively chargeable model substances, including amiodarone and desethylamiodarone. Solutes present at the nanomolar level can thereby be accumulated from a sample volume equivalent of about 4 μL (with injection of about 20 nL of sample solvent into the capillary) and measured by UV absorption detection. To prevent disturbances caused by electrolysis, sample vials should be employed only once. The data obtained further show that quantitation can be reliably performed using internal calibration based on peak height (RSDs for inter- and intraday determinations are on the 2% level). However, due to variation of the roughness of the capillary walls and cuts, the time interval between operational steps, and trace adsorption onto the capillary walls, the length of the water zone drawn by capillary action on the inlet side is not constant, and external calibration therefore cannot be employed for quantitation.

Sample self-stacking in zone electrophoresis: Theoretical description of the zone electrophoretic separation of minor compounds in the presence of bulk amounts of a sample component with high mobility and like charge

Abstract A theoretical description of the sample self-stacking effect in zone electrophoresis is given. This effect applies to samples that contain a high concentration of an ionic component with high mobility and like charge. After current application, the sample components are stacked at the rear of the zone of the major component by an isotachophoretic mechanism. Later, the sample substances gradually destack in order of increasing mobility and continue to migrate and separate by the normal mechanism of zone electrophoresis. Simple relationships are derived for model systems allowing an explicit description of the properties of such systems and the zone parameters such as detection time, variance and resolution. The reliability of the model presented is verified and illustrated by computer simulation of selected examples. It is shown that the described sample self-stacking effect provides at least the same concentrating power as is obtained with conventional zone electrophoretic sample stacking.

Enantioselective determination of drugs in body fluids by capillary electrophoresis.

During the past decade, chiral capillary electrophoresis (CE) emerged as a promising, effective and economic approach for the enantioselective determination of drugs in body fluids, hair and microsomal preparations. This review discusses the principles and important aspects of CE-based chiral bioassays, provides a survey of the assays developed and presents an overview of the key achievements encountered. Applications discussed encompass the pharmacokinetics of drug enantiomers, the elucidation of the stereoselectivity of drug metabolism and bioanalysis of drug enantiomers of toxicological and forensic interest.

Determination of substituted purines in body fluids by micellar electrokinetic capillary chromatography with direct sample injection

Many substituted purines (theobromine, caffeine, paraxanthine, theophylline and uric acid, as well as other methylated xanthines and uric acids) can easily be separated and analysed in one run using micellar electrokinetic capillary chromatography with a boratephosphate buffer containing 75 mM sodium dodecyl sulphate (pH approximately 9). Serum, saliva and urine samples collected after the self-administration of caffeine and serum samples from patients receiving theophylline or caffeine pharmacotherapy were screened for substituted purines. The data presented show the ease of using on-column multi-wavelength detection for investigating the feasibility of direct sample application, the characterization of sample pretreatment procedures and peak confirmation by comparing absorption spectra. It is shown that the determination of purines in serum and saliva samples, including therapeutic concentrations of caffeine and theophylline, can be accomplished without any sample pretreatment, whereas sample extraction is required for the determination of purines in urine. Quantitative data for the determination of micromolar amounts of theophylline (samples from adult patients) and caffeine (samples from infants born prematurely) in serum samples compared well with data obtained by non-isotopic immunoassays. Micellar electrokinetic capillary chromatography with the direct injection of serum or saliva samples requires only microlitre volumes of sample and several different compounds can be determined within a few minutes.

Strategies for the monitoring of drugs in body fluids by micellar electrokinetic capillary chromatography

Electrokinetic capillary techniques can exploit numerous separation principles, making them flexible and easily applicable to a variety of separation problems. In recent publications, this emerging technology has been shown to be well suited for monitoring drugs and metabolites in body fluids, including serum, saliva and urine. Most attention has been focused on micellar electrokinetic capillary chromatography (MECC) because it permits the separation and determination of drugs with discrimination being largely based on differences in hydrophobicity. An overview of literature data on the MECC of drugs in body fluids and recent data obtained with antiepileptics in serum and saliva, with model mixtures of illicit drugs, and with extracts from urine specimens that tested positively for opiates and cocaine metabolites are presented. Emphasis is focused on buffer selection and simple sample preparation procedures, including direct injection of body fluids, ultrafiltration and solid-phase extraction.

Capillary isoelectric focusing with electroosmotic zone displacement and on-column multichannel detection

Abstract Isoelectric focusing (IEF) of proteins in uncoated, open-tubular fused-silica capillaries of 75 μm I.D. with on-column multiwavelength detection is reported. Small amounts of hydroxypropylmethylcellulose added to the catholyte are shown to provide column conditioning which allows rapid and high-resolution IEF analysis of proteins to be performed in the presence of an electroosmotic flow along the separation axis. The latter process displaces the developing zone pattern towards and across the point of detection. On-column multichannel zone detection is shown to be an efficient method for the simultaneous monitoring of the eluting proteins and carrier ampholytes. The absorbance profiles monitored at one location towards the capillary end and the temporal behaviour of the current under constant voltage conditions are shown to provide information on the degree of focusing at the time of detection.

Advances of capillary electrophoresis in clinical and forensic analysis (1999–2000)

In this paper, capillary electrophoresis in clinical and forensic analysis is reviewed on the basis of the literature of 1999, 2000 and the first papers in 2001. An overview of progress and relevant examples for each major field of application, namely (i) analysis of drug seizures, explosives residues, gunshot residues and inks, (ii) monitoring of drugs, endogenous small molecules and ions in biofluids and tissues, (iii) general screening for serum proteins and analysis of specific proteins (carbohydrate deficient transferrin, α1‐antitrypsin, lipoproteins and hemoglobins) in biological fluids, and (iv) analysis of nucleic acids and oligonucleotides in biological samples, including oligonucleotide therapeutics, are presented.

Modeling of the Impact of Ionic Strength on the Electroosmotic Flow in Capillary Electrophoresis with Uniform and Discontinuous Buffer Systems

A new dynamic computer model permitting the combined simulation of the temporal behavior of electroosmosis and electrophoresis under constant voltage or current conditions and in a capillary which exhibits a pH-dependent surface charge has been constructed and applied to the description of capillary zone electrophoresis, isotachophoresis, and isoelectric focusing with electroosmotic zone displacement. Electroosmosis is calculated via use of a normalized wall titration curve (mobility vs pH). Two approaches employed for normalization of the experimentally determined wall titration data are discussed, one that considers the electroosmotic mobility to be inversely proportional to the square root of the ionic strength (method based on the Gouy-Chapman theory with the counterion layer thickness being equal to the Debye-Hückel length) and one that assumes the double-layer thickness to be the sum of a compact layer of fixed charges and the Debye-Hückel thickness and the existence of a wall adsorption equilibrium of the buffer cation other than the proton (method described by Salomon, K.; et al. J. Chromatogr. 1991, 559, 69). The first approach is shown to overestimate the magnitude of electroosmosis, whereas, with the more complex dependence between the electroosmotic mobility and ionic strength, qualitative agreement between experimental and simulation data is obtained. Using one set of electroosmosis input data, the new model is shown to provide detailed insight into the dynamics of electroosmosis in typical discontinuous buffer systems employed in capillary zone electrophoresis (in which the sample matrix provides the discontinuity), in capillary isotachophoresis, and in capillary isoelectric focusing.

Options in electrolyte systems for on-line combined capillary isotachophoresis and capillary zone electrophoresis

Abstract A theoretical description of the electrolyte systems that can be used in the on-line combination of isotachophoresis and zone electrophoresis is given. A classification of these systems is presented, based on the type of electrolyte used for the zone electrophoretic separation step. It is shown that transient sample stacking effects always persist from the isotachophoretic step to the beginning of the zone electrophoretic step and that they may negatively influence the zone electrophoretic separation and detection of the sample components. A mathematical description of these effects is given that allows the calculation of their magnitude and consequently the selection of operating conditions such that the stacking is decreased to an acceptable extent. In order to verify the reliability of the theoretical model, a modified PC simulation pack was prepared and used for investigating the behaviour of some model systems.

Application of surface biopassivated disposable poly(dimethylsiloxane)/glass chips to a heterogeneous competitive human serum immunoglobulin G immunoassay with incorporated internal standard

A microfluidic platform for a heterogeneous competitive immunoassay of human immunoglobulin G (IgG) employing Cy5‐human IgG as tracer and Cy3‐mouse IgG as internal standard was developed. The device consisted of microchannels made of poly(dimethylsiloxane) and glass which were patterned with antibodies against human IgG and mouse IgG. Electrokinetic sample transport was employed in order to exploit the small difference between the net mobilities of analyte and tracer, thereby achieving favorable conditions for the performance of the competitive immunoreaction. The overall quality of the disposable chip and performance of the immunoassay were controlled by monitoring the fluorescence of bound tracer and bound internal standard. Analyses with an insufficient internal standard response were discarded, and immunoassay data evaluation was based on the ratio of tracer and internal standard fluorescence. Using synthetic samples in the range from 0 to 80 νg/mL IgG and alkaline running conditions, a concentration‐dependent response with reproducible Cy5/Cy3 signal ratios (average relative standard deviation of 6.8%) was obtained. Chips stored with solution in the channels at 4°C over a two‐month period were found to perform like freshly prepared chips, whereas chips stored dry at –20°C and rehydrated prior to use could not be employed. The analysis of patient sera showed that the immunoassay platform behaved differently in the presence of serum‐based samples. Using the same conditions as for the synthetic samples, no concentration dependence was noted. With a large excess of tracer, however, an IgG concentration dependence was observed, permitting distinction of samples of patients with normal IgG serum levels (8–16 mg/mL) from those with elevated IgG concentrations (> 16 mg/mL).