Hermann Wätzig

Control of the electroosmotic flow by metal-salt-containing buffers

New possibilities to suppress electroosmotic flow (EOF) were investigated. Buffers prepared from salts of zinc (up to 50 mmol/l), aluminium (up to 0.5 mmol/l) and barium (up to 250 mmol/l) lead to an effective reduction of EOF in Tris buffer, pH 7.4, 50 mmol/l. The dependence of EOF on different metal-salt concentrations is documented. The usefulness of barium-salt-containing buffers is demonstrated for pH values between 5 and 9.2. If the suppression is too strong, the EOF can be adjusted by mixtures of sodium and barium salts.

Possibilities to improve automation, speed and precision of proteome analysis: A comparison of two‐dimensional electrophoresis and alternatives

Proteome analysis requires fast methods with high separation efficiencies in order to screen the various cell and tissue types for their proteome expression and monitor the effect of environmental conditions and time on this expression. The established two‐dimensional gel electrophoresis (2‐DE) is by far too slow for a consequential screening. Moreover, it is not precise enough to observe changes in protein concentrations. There are various approaches that promise faster, automated proteome analysis. This article concentrates on capillary (CT isoelectric focusing coupled to mass spectrometry (CIEF‐MSn) and preparative IEF followed by size‐exclusion chromatography, hyphenated with MS (PIEF‐SEC‐MS). These two approaches provide a similar separation pattern as the established 2‐DE technique and therefore allow for the continued use of data based on this traditional approach. Their performances have been discussed and compared to 2‐DE, evaluating 169 recent articles. Data on analysis time, automation, the detection limit, quantitation, peak capacity, mass and pI accuracy, as well as on the required sample amount are compared in a table.

Precise quantitative capillary electrophoresis: Methodological and instrumental aspects

Abstract The separation efficiency of capillary electrophoresis (CE) is said to be unrivalled by liquid chromatography for a number of applications. However, the quantitative precision, which is essential for its use in routine analysis, has been commented upon more critically. The reproducibility of CE is dependent on a number of parameters. The relative standard deviation is predominantly dependent on the absolute sample concentration. Its optimization is very important, and the use of higher concentrations is found to be more favourable. The problem of the non-linear relationship between concentration and peak data, caused by column overload, is less critical. The buffer concentrations are adjusted to the optimum sample concentrations. Validated buffer recipes and rinsing steps will lead to more stable conditions, and the trends that occur can be diminished by the stepwise use of an external standard. Much of the precision is dependent on instrumental aspects. Seven set-ups from different manufacturers were tested. Thermostating proved to be the most important parameter in achieving a reproducible dosage. Ion mobility and buffer viscosity are controlled by the temperature. These parameters influence the amount of sample that is injected by electrokinetic or hydrodynamic injection. The recording of temperature, current, voltage and power is necessary for proper documentation. Short capillaries should be usable for achieving a short analysis time and thus a large number of repetitions per unit time, which is important for statistical certainty.

Recent advances in capillary electrophoretic migration techniques for pharmaceutical analysis (2013-2015)

This review updates and follows‐up a previous review by highlighting recent advancements regarding capillary electromigration methodologies and applications in pharmaceutical analysis. General approaches such as quality by design as well as sample injection methods and detection sensitivity are discussed. The separation and analysis of drug‐related substances, chiral CE, and chiral CE‐MS in addition to the determination of physicochemical constants are addressed. The advantages of applying affinity capillary electrophoresis in studying receptor–ligand interactions are highlighted. Finally, current aspects related to the analysis of biopharmaceuticals are reviewed. The present review covers the literature between January 2013 and December 2015.

Quantitation of acetaminophen and salicylic acid in plasma using capillary electrophoresis without sample pretreatment improvement of precision

Capillary electrophoresis has become one of the most attractive techniques in the analysis of biological samples. Pharmaceuticals in human plasma can easily be determined on uncoated fused-silica capillaries without any sample pretreatment. Intra- and inter-day precision values of about 1-2% R.S.D. (n = 20) and 2-3% R.S.D. (n > 80) respectively are obtained using a sodium dodecyl sulfate-containing borate buffer, pH 10 and acetonitrile as a between-run rinsing reagent. This method is highly robust, no breakdowns of the current or capillary blockings were observed for several weeks. The general applicability is demonstrated for several model drugs. The effectiveness of other rinsing procedures including enzyme-containing solutions, different organic solvents and hydrofluoric acid is discussed.

Quantitation of insulin by capillary electrophoresis and high-performance liquid chromatography. Method comparison and validation

Abstract Two validated high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) assays for insulin are compared. Both methods are selective and robust, with best reproducibility in a 0.9% sodium chloride solution, pH 7–8, as the sample solvent. It is shown that, besides sample solvent and buffer pH, acetonitrile volatility is a crucial point for reproducibility. Its high importance for selectivity and ruggedness is also stressed. Trends have been extensively investigated and characterized. The separation efficiency is better for the CE method. Furthermore, the analysis time of the CE method is up to four times shorter than the respective parameter in HPLC and the acetonitrile consumption is more than 100-fold less. Earlier works stated that all relevant precision data, such as relative signal standard deviation (1.6% R.S.D. for peak areas, n=20), precision of the analytical result and the limit of quantitation, were about a factor of two worse than for corresponding HPLC data (0.8% R.S.D. for peak areas, n=19). This CE precision was further improved using relative instead of absolute peak areas, which compensate for the injection error (1.3% R.S.D. for relative peak areas, n=20). If acetonitrile evaporation is avoided, by covering the buffer with mineral oil, reproducibility is even better than with the HPLC assay (0.5% R.S.D. for relative peak areas, n=60).

Performance of instruments and aspects of methodology and validation in quantitative capillary electrophoresis. An update

The trial of capillary electrophoresis (CE) instruments from 1993 has been updated. The test procedure which was used there could be applied with few modifications. The data of 10 instruments are presented in a detailed table. Additional instruments are included this time. Aspects of method and instrument validation and robustness are discussed and listed in tables. Relevant instrumental features are deduced. Two-dimensional detection and a protocol of the current are very helpful for method development and validation. Some very useful methods that improve the limit of detection or resolution require pressure-driven counter-current and the possibility to control polarity and voltage during runs. Intermediate precisions corresponding to R.S.D. values of below 1% have become state of the art during the last few years. An overall day-to-day precision of 0.1% R.S.D. seems already possible if the multiple injection mode can be used. The detector technology has been improved, therefore injection became the main error source. This error source can be decreased by using internal standards or relative peak areas. In the future CE will be superior to other separation techniques not only in terms of performance, but also in terms of precision.

Precision in affinity capillary electrophoresis for drug–protein binding studies

In order to achieve excellent precision in the estimation of binding constants by affinity capillary electrophoresis (ACE), electroosmotic flow (EOF) stability is the key parameter, especially when using proteins in binding assays. Appropriate rinsing protocols are mandatory. In our study, the capillary was rinsed after each run with 0.1 mol/L sodium hydroxide for 2.0 min, with water for 2.0 min followed by running electrolyte (phosphate buffer at pH 7.4) for 3.0 min (pressure=3000 mbar each). Tryptophan-human serum albumin, warfarin-bovine serum albumin and quercetin-beta-lactoglobulin were used as ACE models. Further improvements in precision have been obtained by avoiding a complete standstill of liquid within the capillary and flushing the capillary with buffer for 25 min after each 30 consecutive runs. The precision of measurements is further improved by the use of mobility ratios to report mobility changes (RSD% less than 0.5% in a long-term measurement, n=300-600). Apart from the importance of a stable EOF, other ACE key parameters include protein concentration, drug plug length, applied voltage, and the choice of the regression method. In the present work, useful protocols and templates are provided in order to allow users a quick and efficient start with ACE methods. The comprehensive experimental part can serve as a checklist, which parameters need to be addressed for successfully applying ACE. Here, the suggested experimental design allows for the determination of binding constants within a couple of hours using standard instrumentation. This time could still be decreased by orders of magnitude using capillary arrays or miniaturized systems.

Appropriate calibration functions for capillary electrophoresis I. Precision and sensitivity using peak areas and heights

Calibration functions for CE can be calculated by using peak heights or areas. The relationship between peak heights and concentrations is non-linear, but can well be approximated by a parabolic function. The precision of peak heights is often better than of areas. However, the sensitivity of the calibration function decreases at higher concentrations. Thus calibration functions calculated by using areas lead to a better precision of the analytical result, which is estimated by the slope-normalized standard deviation.

Capillary electrophoresis-a high performance analytical separation technique.

Abstract Capillary electrophoresis (CE) is often one of the preferred techniques in pharmaceutical quality control and in clinical chemistry, particularly considering the high selectivity and lower costs compared to HPLC. The precision of CE is as good as in liquid chromatography (LC). The sample-throughput is high due to short analysis times. Efforts for sample pre-treatment are usually minor in CE. Urine and even blood plasma can be directly injected without further pre-treatment. After summarising the basic principles of CE, general strategies for method development are described to achieve selective, efficient, precise, fast, sensitive, and validated methods. Sample pre-treatment requirements are discussed. Standard buffer recipes, surfactants used in micellar electrokinetic chromatography (MEKC), chiral selectors, useful buffer additives, actions to deal with complex matrices, and aspects of validation have been collected. Other techniques that can be performed with CE instruments, such as capillary isoelectric focusing (CIEF), capillary isotachophoresis (CITP), and capillary electrochromatography (CEC), are briefly discussed.