A comparison of pulsed amperometric detection and conductivity detection for carbohydrates

Pulsed amperometric detection (PAD) at a gold electrode and conductivity detection are compared for glucose, as a representative of the general challenge of the chromatographic detection of carbohydrates. In alkaline solutions of barium hydroxide, which are useful for anion-exchange separation of carbohydrates, PAD is significantly more sensitive than the conductivity detector but the latter provides linear response to higher concentrations than those observed for PAD. The combined technique of conductivity in series with PAD is linear for glucose over the range 6×10^?7?1×10^?2 M (ca. 6 ng-10 mg per 50-μl sample injection).     

Pulsed amperometric detection of carbohydrates on an electrophoretic microchip

The first report of pulsed amperometric detection (PAD) on an electrophoretic microchip is presented. A hybrid poly-(dimethylsiloxane)/glass device was coupled with a platinum working electrode for the electrochemical detection of glucose, maltose, and xylose. Under optimized detection conditions, glucose was found to respond linearly from 20 to 500 microM with a measured detection limit of 20 microM. The coupling of PAD with a microchip provides a straightforward approach to the analysis of a wide range of carbohydrates using microfluidics.     

Comparison of pulsed amperometric detection and integrated voltammetric detection for inorganic sulfur compounds in liquid chromatography

A Variety of potential–time waveforms are useful in pulsed electrochemical detection (PED) when applied for the amperometric detection of numerous polar organic compounds following their separation by liquid chromatography (LC). Here, we compare the waveforms for pulsed amperometric detection (PAD) and integrated voltammetric detection (IVD) applied for detection of organosulfur compounds at Au electrodes in acidic media. In PAD waveforms, electrodes response is measured at a constant detection potentials. In IVD waveforms, electrodes current is integrated throughout a fast cyclic scan of the detection potential. As a consequence of this difference in detection strategy, the background signal for IVD is significantly smaller for PAD in the detection of organosulfur compounds whose response mechanisms require the concomitant formation of surface oxides on Au electrodes. Furthermore, in comparison to Pad, IVD has a larger sensitivity and a diminished system peak from 0₂ dissolved in the sample. Use of a preadsorption step increases detection sensitivity in both PAD and IVD. The limit of detection (S/N=3)for cysteine in LC-IVD is ca. 6 nM for a 50-μl injection (i.e., 300 fmol) using a detection waveform that includes a 1000-ms preadsorption period.     

Simplified current decoupler for microchip capillary electrophoresis with electrochemical and pulsed amperometric detection

There is a need to develop broadly applicable, highly sensitive detection methods for microchip CE that do not require analyte derivatization. LIF is highly sensitive but typically requires analyte derivatization. Electrochemistry provides an alternative method for direct analyte detection; however, in its most common form, direct current (DC) amperometry, it is limited to a small number of easily oxidizable or reducible analytes. Pulsed amperometric detection (PAD) is an alternative waveform that can increase the number of electrochemically detectable analytes. Increasing sensitivity for electrochemical detection (EC) and PAD requires the isolation of detection current (nA) from the separation current (muA) in a process generally referred to as current decoupling. Here, we present the development of a simple integrated decoupler to improve sensitivity and its coupling with PAD. A Pd microwire is used as the cathode for decoupling and a second Au or Pt wire is used as the working electrode for either EC or PAD. The electrode system is easy to make, requiring no clean-room facilities or specialized metallization systems. Sensitive detection of a wide range of analytes is shown to be possible using this system. Using this system we were able to achieve detection limits as low as 5 nM for dopamine, 74 nM for glutathione, and 100 nM for glucose.     

Determination of phytosiderophores by anion-exchange chromatography with pulsed amperometric detection

Phytosiderophores of the mugineic acid family are separated by anion-exchange HPLC using NaOH gradient elution. Separation of mugineic acid (MA), 2'-deoxymugineic acid (DMA), 3-hydroxymugineic acid (HMA) and 3-epi-hydroxymugineic acid (epi-HMA) is obtained within 15 min. Detection of the underivatised phytosiderophores is performed using pulsed amperometric detection (PAD) at pH 13. The sensitivity of the detection increases in the order DMA < MA < HMA < epi-HMA and respective detection limits of 5 microM (DMA), 1 microM (MA) and < 0.5 microM (HMA, epi-HMA) are achieved. PAD is discussed in comparison with the well-established fluorimetric detection method after post-column derivatisation with ortho-phthaldialdehyde. The main advantage of PAD is the simplicity of the method (no derivatisation) and the high sensitivity for hydroxylated mugineic acids. The method is used for the determination of phytosiderophores in root washings of iron-deficient and non-deficient wheat and barley plants.     

Pulsed amperometric detection of furan compounds in transformer oil

The failure of high voltage transformers can result in significant cost and supply implications to both power supplier and consumer alike and in extreme cases may result in explosion, serious injury or death. Transformer failure can be predicted by measuring furanics present in the oil, produced by the thermolytic breakdown of cellulosidic insulators. Failing units can have furanic levels of up to 10 μg ml⁻¹. The use of pulsed amperometric detection (PAD) to measure furanics in transformer oils in real time is reported here. Oils were examined by pre-extraction or direct suspension in aqueous measurement solution or by solubilisation and direct PAD measurement in organic solvents. Linear relationships between PAD response and furanic concentration was found for 2-furaldehyde and furfuryl alcohol (F-OH) across the range of 0–10 μg ml⁻¹, with PAD proving most sensitive to the latter compound. PAD was performed directly in the organic phase in t-butanol with 0.1 M tetramethyl ammonium hydroxide, with aged oils containing >2 μg ml⁻¹ of 2-furaldehyde yielding data within close agreement (<9%) of a standard chromatographic method. The simplicity and rapidity of this method offers the power transmission industry a means of monitoring furanic levels in transformers in real time, thereby reducing the risk of uncontrolled transformer failure.     

Pulsed electrochemical detection of penicillins using three and four step waveforms

Aided by the construction of a custom potentiostat, a series of different PAD waveforms were compared to find the optimum detector for penicillin oxidation. The waveforms included standard 3-step direct and indirect PAD in addition to reverse-PAD and 4-step PAD. Two new waveforms, the indirect reverse-PAD and the 4-step indirect PAD were examined in the study. Under the solvent conditions of the study (0.01M acetate buffer, pH 4.6) the indirect waveforms yielded the best detectability for penicillin G while the reverse-PAD waveforms yielded the worst performance. The 4-step PAD methods did not improve detectability when compared to the 3-step types, but they did provide output peak profiles with better shapes and less tailing. Although indirect waveforms gave better detectability than direct detection in the 0.01M acetate buffer solution, the limits of detection for each were found to be differing functions of ionic strength. At higher acetate concentrations, direct PAD was more favorable than indirect detection.     

A consideration of electrode polishing prior to application of pulsed amperometric detection

An examination was made of the effect of electrode polishing on the direct pulsed amperometric detection (PAD) and indirect PAD response to penicillin G. The polishing procedure produced a temporary loss of sensitivity for both types of PAD, which over time relaxed back to higher levels. A temporary increase in the electrode kinetics can be observed as well, particularly for a PAD waveform in the crossover region between direct and indirect PAD. In extreme cases, the penicillin G response can undergo a complete polarity reversal. By observing post-polish equilibration times, it was recommended that the direct and indirect PAD user observe 3 and 5 h delay times, respectively, to insure stable electrode response following an electrode polish.     

Polysaccharides analysis of sinorhizobial capside by on-line anion exchange chromatography with pulsed amperometric detection and mass spectrometry coupling

High performance anion exchange chromatography (HPAEC)-pulsed amperometric detection (PAD) is a performing technique for carbohydrate analysis, due to the selectivity and sensitivity of the detection. The identification occurs through retention times. In absence of standards, structural characterization of complex polysaccharides requests the coupling of HPAEC-PAD with electrospray ionization (ESI)-MS. This is a technological challenge, due to the non-volatility and high conductance of the eluents. Therefore, a desalting device has been installed on-line between the PAD and the MS. On-line HPAEC-MS has only been rarely described. We report here successful analysis of biological acidic oligosaccharides, allowing for the first time to demonstrate that membrane anchored 3-deoxy-D-manno-2 octulosonic acid (Kdo) homopolymers are consensus sinorhizobial capsular polysaccharide (KPS).     

Pulsed coulometric detection of carbohydrates at a constant detection potential at gold electrodes in alkaline media

A significant increase in the signal-to-noise ratio for the pulsed amperometric detection (PAD) of carbohydrates at gold electrodes is obtained by increasing the length of the current integration period (t_i) from the traditional value of 16.7 ms (i.e., $\text{1}\text{60}$ Hz). For t_i > 16.7 ms, the integrated response (q, coulombs) is plotted as the signal. This pulsed coulometric detection (PCD) is applied in a flow-injection system. For t_i = 500 ms, the detection limit with the instrumentation used is 1 μM (S/N = 2) for glucose which is a significant improvement on the value 35 μM found with PAD. The absolute detection limits for glucose and sucrose are ca. 50 pmol and 125 pmol, respectively, in 50-μl samples. Calibration plots (q_p vs. C^b) for PCD are linear over significantly larger dynamic ranges than those observed for PAD because of the lower detection limits.     

Speciation of iron coordinated by phytosiderophores by use of HPLC with pulsed amperometric detection and AAS

Phytosiderophores of the mugineic acid family, and the respective iron species, have been separated by anion-exchange chromatography with NaOH gradient elution. Two different detection methods were used in parallel, pulsed amperometry (PAD) for phytosiderophores and atomic absorption spectrometry (AAS) for iron. This combination enables identification of separated iron species. Up to five different iron species were separated and detected within 30 min - two different phytosiderophore species, two amino acid species, and one species which has not yet been identified but which is most probably a decomposition product of phytosiderophores. The detection limit was in the low micro mol L(-1) concentration range, which is sufficiently low for determination in real plant samples, even after dilution. The method has been applied to root washings of iron-deficient wheat and barley plants and to a xylem exudate of non-deficient maize.     

Pulsed amperometric detection of DNA with an ssDNA/polypyrrole-modified electrode

Pulsed amperometric detection (PAD) of target DNA with platinum electrodes modified by single-stranded DNA (ssDNA) entrapped within polypyrrole (ssDNA/Ppy) is reported for the first time. Single-stranded DNA 20-mers complementary to the target DNA were used to construct the DNA biosensors. Polymerase chain reaction (PCR) amplified bovine leukaemia virus (BLV) provirus DNA was used as target DNA. Electrochemical impedance spectroscopic (EIS) investigation of ssDNA/Ppy before and after incubation in target DNA-containing sample revealed significant changes in terms of an imaginary (Z) vs. a real (Z) component. The PAD results were in good agreement with EIS investigations. The PAD method was selected, because it does not require such sophisticated equipment as it is used to perform EIS and the results obtained can be more easily estimated. Optimum conditions for performing PAD and evaluating an analytical signal were elaborated. No label-binding step was necessary for detection of target DNA in PCR-amplified amplicons and detection time was reduced by as much as 30–35 min. The changes of PAD signals were at least 6–7 times higher if ssDNA/Ppy-modified electrodes instead of blank Ppy-modified electrodes were incubated in the target DNA solutions. If ssDNA/Ppy modified electrodes were incubated in non-complementary (control) DNA solution changes in PAD signals were smaller than those detected after incubation in complementary (target) DNA-containing solution by a factor of at least 6–8.     

Direct determination of hydrogen cyanide in cigarette mainstream smoke by ion chromatography with pulsed amperometric detection

The determination of hydrogen cyanide in cigarette mainstream smoke has been achieved by ion chromatography (IC) with pulsed amperometric detection (PAD). The proposed method of totally trapping whole cigarette mainstream smoke by Cambridge filters, which are treated with sodium hydroxide/ethanol solution, possesses the advantage of fast analysis time over the widespread used solution absorption method. The possible co-existing interferents are evaluated under the optimized detection conditions and excellent recoveries of cyanide are obtained. The cyanide content of absorption solution can be directly determined by the optimized IC-PAD method without any pretreatments. The linear range is 0.0147-2.45 μg/mL with R2 value of 0.9997. The limit of the detection is 3 μg/L for a 25 μL injection loop. The overall relative standard deviation of the method is less than 5.20% and the recovery range from 94.3% to 101.0%. The results obtained from the developed method are in good agreement with that of continuous flow analyzer (CFA) method.     

Platinum particles-modified electrode for HPLC with pulsed amperometric detection of thiols in rat striatum

A new chemically modified electrode based on the immobilization of Pt particles is fabricated and exhibits electrocatalytic oxidation for L-cysteine (L-Cys), glutathione (GSH) and penicillamine (PEN) with relatively high sensitivity. It is also adaptable to HPLC for pulsed amperometric detection (PAD) of these thiols. PAD largely improves the detection sensitivity because the alternated polarizations can effectively clean and reactivate the electrode surface. It is shown that the peak currents of L-Cys, GSH and PEN are linear to their concentrations, with the calculated detection limit of 1.1 x 10(-7), 1.8 x 10(-7) and 3.8 x 10(-7) mol L(-1), respectively (S/N = 3). The method has been successfully applied to assess the contents of L-Cys and GSH in rat striatal microdialysates. The average contents of the two analytes in rat striatum are 2.6 x 10(-6) and 2.8 x 10(-6) mol L(-1), respectively.     

The application of flow programming with pulsed amperometric detection for liquid chromatography

A mixture of seven penicillins was separated on a C-8 column and detected using pulsed amperometric detection (PAD). Due to the polarity range of the penicillin mixture, a gradient program was necessary to produce a reasonable separation, causing some baseline shifting. Application of a flow program, where the solvent flow rate is varied within the separation, was also examined, and found to cause only a small shift in baseline response for PAD. Further examination of a variety of different flow programs was undertaken to characterize the baseline and analyte response under these conditions. Notable was that the shift in baseline from the flow program could be altered to give a shift toward either increased or decreased current, depending on the waveform and solvent combination in use. By manipulating these two parameters, one can customize the PAD response to a flow program, which may allow flow programs to be designed that only produce a minimal impact on the detector response.     

Analysis of delta-L-alpha-aminoadipyl-L-cysteinyl-D-valine by ion chromatography and pulsed amperometric detection

A novel high-performance liquid chromatography (HPLC) method is presented for the detection and trace level determination of the tripeptide delta-L-alpha-aminoadipyl-L-cysteinyl-D-valine (ACV). The tripeptide, an intermediate in penicillin production, is derived from fungal fermentation. The technique relies on ion-exchange separation of the tripeptide on an anion-exchange column followed by detection by reduction on a gold electrode using pulsed amperometry. The sensitivity of direct determination of ACV is increased by employing pulsed amperometric detection (PAD) over direct ultraviolet detection. Choice of the working electrode and optimisation of electrode potentials was based on cyclic voltammograms recorded for the tripeptide in the mobile phase. A linear regression equation was obtained over the range 0-100 micrograms ml-1. The detection limit in fermentation broths was found to be 0.1 micrograms ml-1 whereas in buffer the detection limit was found to be 10 ng ml-1. A good correlation coefficient was observed when ACV concentrations determined by ion chromatography-PAD were compared with measurements obtained by pre-column derivatisation with fluoromethylorthochloroformate followed by HPLC separation on a reversed-phase C18 silica column with UV detection. The procedure has been applied to the measurement of natural levels of ACV in fermentation broths of selected strains of Aspergillus nidulans and Penicillin chrysogenum.     

Determination of saccharides by high performance anion-exchange chromatography with pulsed amperometric detection

Summary High performance anion-exchange chromatography (HPAC) coupled with pulsed amperometric detection (PAD) under alkaline conditions (pH 13) separates neutral saccharides based upon their molecular size, saccharide composition, and glycosidic linkages. Carbohydrates were detected by oxidation with a gold-working electrode. HPAC-PAD was compared to high performance liquid chromatography (HPLC) with refractive index (RI) detection in terms of selectivity and sensitivity of saccharides. The results indicate that HPAC-PAD was more precise, two orders of magnitude more sensitive (pmol range) and gives better resolution of saccharides than HPLC-RI. HPAC-PAD required less sample preparation and was not subjected to matrix interferences. The use of HPAC-PAD was applied to the analysis of organic materials (plant residues, animal wastes and sewage sludge) and soil.     

糖的高效阴离子交换色谱-脉冲安培检测法分析

高效阴离子交换色谱-脉冲安培检测法是新近发展的一种分析糖的有效方法。本文讨论了糖的高效阴离子交换色谱分离和脉冲安培法检测的原理,色谱条件的选择,包括分离糖的固定相、流动相以及检测糖的脉冲电位波形,并对方法的应用进行了较为详细的叙述。     

Direct detection of renal function markers using microchip CE with pulsed electrochemical detection

Creatinine, creatine, and uric acid are three important compounds that are measured in a variety of clinical assays, most notably for renal function. Traditional clinical assays for these compounds have focused on the use of enzymes or chemical reactions. Electrophoretic microchips have the potential to integrate separation power of capillary electrophoresis with devices that are small, portable, and have the speed of conventional sensors. The development of a microchip CE system for the direct detection of creatinine, creatine, and uric acid is presented. The device uses pulsed amperometric detection (PAD) to detect the nitrogen-containing compounds as well as the easily oxidizable uric acid. Baseline separation of creatinine, creatine and uric acid was achieved using 30 mM borate buffer (pH = 9.4) in less than 200 s. Linear calibration curves were obtained with limits of detection of 80 microM, 250 microM and 270 microM for creatinine, creatine and uric acid respectively. An optimization of the separation conditions and a comparison of PAD with other amperometric detection modes is also shown. Finally, analysis of a real urine sample is presented with validation of creatinine concentrations using a clinical assay kit based on the Jaffé reaction.     

Improvement of Pulse Amperometric Detection Integrated Automated Flow Injection Analysis of Ethylenethiourea Determination

Improvement of pulse amperometric detection (PAD) method is demonstrated in determination of ethylenethiourea (imidazolidine‐2‐thione, ETU). The anodic detection of ETU will produce polymeric film on an electrode leading to an inactive electrode surface. Here, the PAD method was used to remove the polymeric film formed on the electrode surface between ETU detection. Further, the scheme was integrated with automated flow injection analysis (AFIA) for determining ETU. The operational parameters of PAD in the AFIA system were discussed thoroughly. The analytical characteristics of the system were evaluated at optimum conditions. The linear range of calibration plot was between 20 to 300 μM (the correlative coefficient, r = 0.999) and the detection limit was 0.9 μM (S/N = 3). The relative standard deviations of detection of 50 μM ETU were 0.82% with and 9.07% without PAD scheme. The results indicate the system is a very promising tool for ETU determination. Finally, the matrix effects of two water samples that were collected from a campus and a farm show good recoveries of 92% and 96%.