Petr Jandik

Optimization of inorganic capillary electrophoresis for the analysis of anionic solutes in real samples

Abstract Inorganic capillary electrophoresis (ICE) is a separation technique which offers many advantages for the analysis of anionic solutes in real samples. Parameters which influence ICE separations configuration, choice of electrolyte anion, electrolyte pH and the addition of electroosmotic flow modifier were investigated and a number of electrolytes of varying mobilities were studied. Optimized conditions were established for the separation of inorganic anions, organic acids and alkylsulfonates and the technique was applied to the analysis of a variety of anionic solutes in sevaral complex sample matrices.

Factors affecting the separation of inorganic metal cations by capillary electrophoresis

Abstract Various alkali metals, alkaline earth metals, transition metals and lanthanides were separated by capillary electrophoresis, and factors influencing the separations were studied. The reproducible separation of fifteen metal cations was completed in 8 min. The detection system showed a linear relationship between peak area and analyte concentration. To permit the use of indirect photometric detection and to ensure symmetrical peak shapes, a highly UV-absorbing amine having an electrophoretic mobility similar to those of the analyte cations was chosen as the major component of the electrolyte. Complexing compounds were added to the electrolyte to maximize selectively the differences in the apparent mobilities of the cations and enhance the separations.

Various approaches to analysis of difficult sample matrices of anions using capillary ion electrophoresis

Abstract Capillary ion electrophoresis is a capillary electrophoretic technique optimized for the analysis of low-molecular-mass inorganic and organic ions. Sensitive detection is based predominantly on indirect UV since the majority of the ions lack specific chromophores. Rapid separations with efficiencies approaching 1 000 000 theoretical plates are a result of directing the electroosmotic flow towards the detector in combination with an appropriately mobile background electrolyte co-ion. Selectivity of separations can easily be predicted with the aid of readily available ionic equivalent conductances. Described in this paper are the optimized separations of several types of samples considered until recently to be beyond the capabilities of capillary electrophoresis. These include samples of disparate concentration levels, reactive anions and trace ion analysis with detection limits in the low parts per trillion (1012) concentration range.

Simultaneous determination of amino acids and carbohydrates by anion-exchange chromatography with integrated pulsed amperometric detection.

A direct, sensitive, simple and practical method for simultaneous determination of amino acids and carbohydrates by anion-exchange chromatography with integrated pulsed amperometric detection was developed. The retention behavior of amino acids and carbohydrates on the anion-exchange column and the detection of amino acids and carbohydrates at different integrated pulsed amperometric detection waveforms were investigated. The optimized gradient eluent conditions for analysis of 17 amino acids and nine carbohydrates were obtained. Separation time was 100 min. Detection limits for amino acids and carbohydrates were 5.2-207.1 nM under injection volume of 25 microl. The RSDs of peak area were 1.2-3.3%. The calibration graphs of peak area for the analytes were linear over about three orders of magnitude with a correlation coefficient of 0.9950-0.9999. The method was applied to determine amino acids and carbohydrates in a liquid condiment with satisfactory results.

Capillary electrophoretic estimation of sulfate stability constants of metal ions and determination of alkali and alkaline earth metals in waters

We studied the influence of sulfate concentration on the effective mobilities of mono-, di- and tervalent ions and developed a method for the determination of sulfate stability constants. Relative migration times of several cations were determined. Capillary electrophoresis with indirect detection using 4 mmol/l copper(II) sulfate, 18-crown-6 and formic acid in background electrolyte was then applied to the determination of the metal ions in mineral and tap waters samples. The results were compared with those of ion chromatography. The CE detection limits were estimated [signal/noise ratio (S/N)=3] in the range from 0.59 (for Li) to 12.9 mg/l (for Ba).

Analyzing mixtures of amino acids and carbohydrates using bi-modal integrated amperometric detection

Described in this work is a new detection methodology - bi-modal integrated amperometric detection - for identifying peaks and as a tool for solving difficult separation problems. Bi-modal integrated amperometry makes it possible to selectively detect amino acids, amino sugars, and carbohydrates following their separation by anion-exchange. Selectivity is gained by two different methods of integrating anodic current on an otherwise identical waveform. As with the single-mode integrated amperometry reported previously, the limits of detection are in the femtomole range and linear calibration plots are possible over three orders of magnitude. This new detection method does not require analyte derivatization. The practical utility of this new technique is demonstrated in the analysis of amino acids and sugars in a recombinant mammalian cell culture medium.

Simplified in-line sample preparation for amino acid analysis in carbohydrate containing samples.

This report describes a new, automated chromatographic procedure eliminating carbohydrates from amino acid samples prior to their analysis by anion-exchange chromatography and integrated amperometric detection. In the first step, a sample is brought onto a short cation-exchange column (trap column) in hydrogen form. Carbohydrates are passing through this column, while only amino acids are retained. Subsequently, the cation-exchange column, holding the amino acid fraction, is switched in-line with the gradient pump and separator column. The mobile phase used at the beginning of the separation (NaOH; pH 12.7) transfers amino acids from the trap column onto the anion-exchange column and the amino acid separation is completed without any interference by carbohydrates. All common amino acids are recovered following the carbohydrate removal step. The average value of their recovery is 88.1%. The calibration plots were tested between 12.5 and 500 pmol (amounts injected). The mean value of correlation coefficients of calibration plots was calculated as 0.99. The mean value of relative standard deviations from five replicates was 3.9%. The usefulness of the method is illustrated with two chromatograms of a carrot juice sample obtained before and after the in-line removal of carbohydrates.

Use of disposable gold working electrodes for cation chromatography-integrated pulsed amperometric detection of sulfur-containing amino acids

We have prepared disposable thin-film gold working electrodes on polymeric substrates. Our microfabrication process allows for inexpensive and reproducible mass production of such electrodes. We utilize this new type of electrode in flow-through electrochemical cells to replace the conventional non-disposable gold working electrodes for integrated pulsed amperometric detection (IPAD) of compounds separated by high-performance cation-exchange chromatography. Using two S-containing amino acids (homocysteine and cysteine) as test compounds, we have modified a previously reported waveform for optimum performance with disposable gold electrodes. With the help of the same two test substances we have characterized the analytical performance of disposable gold electrodes under the new conditions. Compared to non-disposable working electrodes, the disposable working electrodes generated equal or better results in the limit of detection, linearity of calibration and reproducibility. When used with a new IPAD waveform, the disposable electrodes functioned reproducibly for 3 days. At the end of the specified usage period of 3 days, the disposable electrodes are simply replaced. Reconditioning by polishing is thus no longer required.

Simultaneous analysis of homocysteine and methionine in plasma.

The new isocratic cation exchange method separates up to eight different amino thiols. The separated sample components are detected electrochemically using a gold electrode and the integrated pulsed amperometry. The eluent composition is, for example, 0.15 M sodium perchlorate, 0.02 M perchloric acid and 5% acetonitrile. The report describes the optimization of chromatographic parameters such as column diameter and eluent composition. Quantitative performance is discussed for eight different amino thiols using standards. Also presented is a long term quantitative study for homocysteine and methionine in plasma samples. The preparation of plasma samples is simpler than with the previously reported version of the method. Only a reduction step is required, and neither column switching nor derivatization are necessary.

Peak splitting observed during capillary electrophoresis of α- and β-naphthols in borate buffer

Abstract β-Naphthol is an undersirable by-product in the synthesis of α-napthol for various uses in the pharmaceutical and dyestuff industries. We have developed a new procedure for the determination of traces of β-naphthol in the presence of a large excess of α-naphthol. The new method utilizes direct UV detection at 230 nm in a 44.5 cm × 75 μ m I.D. fused-silica capillary. The separation is carried out at 15 kV in a 50 m M borate electrolyte and pH of 9.3. During the research for optimum conditions, we were able to observe an occasional splitting of the peak belonging to β-naphthol. Under certain conditions, we could see up to three peaks for that compounds. The realization that the peak splitting could be observed reproducibly under alkaline conditions and with increased borate concentration (higher than 50 m M ) helped us to elucidate the mechanism behind the peak splitting phenomena. The artifact was caused mainly by complexation reactions between β-naphthol and borate polymers.