New type of microfabricated carbon electrodes for high-performance liquid chromatography--amperometric detection of fat-soluble vitamins and antioxidants.

This study evaluates performance of thin-film carbon electrodes prepared by physical vapor deposition of the electrode material on a polyetheretherketone substrate and compares their performance with that of a standard type of glassy carbon electrodes for chromatographic detection cell of thin-layer type. Kaempferol, retinol, retinyl acetate, cholecalciferol, alpha-tocopherol and gamma-tocopherol were selected for the study and their respective detection limits were found to be 0.63, 5.84, 5.91, 28.19, 16.80 and 16.11 pg. The calibration plots were linear for at least three orders of magnitude for all of the six analytes selected for evaluation. Hydrodynamic voltammograms are shown for both types of electrodes for the range of working electrode potentials between +0.40 and +1.40 V. Also shown and discussed are plots of signal-to-noise vs. detection potential. The working electrode potential of +1.30 V was found to be required for an improved long-term stability of detection performance and was suitable for the detection of all six analytes.

Scaling up a microbore separation for semipreparative analysis: differential recoveries of radiolabeled amino acids.

Application of a high-sensitivity microbore system designed to separate and quantify nonderivatized amino acids by anion exchange chromatography and amperometric detection for determination of amino acid-specific activities in biological samples requires high capacity to recover sufficient labeled material for adequate count statistics. Scale up from a low (25-1000 pmol) to a high (500-15,000 pmol) working range was achieved by use of a thick working electrode gasket to reduce sensitivity and eliminate peak splitting and tailing and by modification of the wash procedure to eliminate carryover. Analysis of recoveries of labeled amino acids revealed that specific amino acids are either selectively retained on the column or partially degraded during analysis and that assessment of purities of labeled compounds and metabolic labeling patterns requires careful analysis of recoveries of labeled compounds in the appropriate eluate fraction.

Analysis of amino acid-carbohydrate mixtures by anion exchange chromatography and integrated pulsed amperometric detection.

A review is presented of recent developments in the area of analysis of amino acid-carbohydrate mixtures. Based on its broad selectivity, the AminoPac PA10 column exhibits a remarkable capability to perform simultaneous separations of amino acids and carbohydrates. This ability is further enhanced by the equal sensitivity for carbohydrates and amino acids exhibited by the "amino acid" integrated pulsed electrochemical detection (IPAD) waveforms. Equimolar levels of carbohydrates and amino acids are separated either by optimized elution gradients alone or by a combination of modified gradients and a bi-modal IPAD waveform. Samples containing large amounts of carbohydrates may be analyzed after suitable sample preparation. Both a manual off-line method and a fully automatic on-line method are discussed. In addition, we will review the application of these methods to various types of samples, including cell culture media, glycoprotein hydrolysates, beverages, condiments and soil extracts.

CarboPac PA20: a new monosaccharide separator column with electrochemical detection with disposable gold electrodes.

This report documents the development of a new monosaccharide separator column (CarboPac PA20, 3x150 mm) that allows fast, efficient monosaccharide separations with good spacing. It is based on a new chemistry with a reduced resin particle size (from 10 to 6.5 microm). Faster, more efficient separations of glycoprotein monosaccharides with better spacing were achieved across a range of isocratic NaOH concentrations at lower flow rates. Detection sensitivity was improved, enabling routine low to sub pmol monosaccharide determinations. Glycoprotein monosaccharides eluted in less than 10 min at a flow rate of 0.5 ml/min. Furthermore, when used with an AminoTrap guard column, the protein matrix consisting of amino acids and peptides (released by acid hydrolysis of glycoprotein) did not interfere with monosaccharide analysis. Compared to previous CarboPac columns (CarboPac PA1 and CarboPac PA10), the CarboPac PA20 has improved selectivity with respect to glycoprotein monosaccharides. The improved selectivity results in better separation of glucosamine and galactose, enabling the accurate determination of monosaccharide ratios for undergalactosylated glycoproteins. Finally, disposable gold working electrodes that eliminate the possibility of working electrode recession affecting peak area response were used.

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.

Development and characterization of microfabricated disposable gold working electrodes for high-performance ion chromatography and integrated pulsed amperometric detection.

We have developed a new type of microfabricated thin-film electrode on polymeric substrates. The microfabrication process allows for inexpensive and reproducible mass production of disposable working electrodes for high-performance ion chromatography and integrated pulsed amperometric detection (IPAD). These microfabricated electrodes are disposable and have been optimized for use in flow-through low-dead-volume electrochemical cells. The analytical performance of microfabricated gold electrodes was characterized with the help of the IPAD method for amino acid detection under alkaline conditions required for anion-exchange separations. When used with a new optimized six-potential IPAD waveform, the electrodes functioned properly for weeks. Compared to nondisposable working electrodes, the disposable working electrodes generated equal or better results in the limit of detection, linearity of calibration, and reproducibility. Disposable electrodes make it possible to avoid polishing and reconditioning, which are required with nondisposable electrodes.

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.