Ratio Accuracy with Coulometric Array
Obtaining qualitative information from a Coulometric array
A superior approach directly examines the voltammetric behavior of an analyte after it is resolved chromatographically. This approach uses an array of coulometrically efficient porous graphite electrodes to generate an HDV on "the fly." This technique is the electrochemical equivalent of the PDA where compounds can now be resolved based upon their retention time and by their voltammetric behavior (instead of their spectral behavior).
Not all commercially available electrodes can be used in an array. Inefficient thin layer amperometric electrodes when used in an electrode array are incapable of voltammetrically resolving compounds. As these sensors typically show <5% conversion efficiency, analytes oxidizing at lower potentials at the start of the array will "spill over" onto subsequent downstream electrodes. Furthermore, the silver/silver chloride reference electrodes typically used with these amperometric sensors would not be able to withstand the back pressure generated by such an array.
Coulometrically efficient electrodes (measuring signal arising from close to 100% of an analyte capable of undergoing electrolysis) are uniquely suited to the formation of an electrode array. If a single coulometric electrode has an applied potential equal to the oxidation (or reduction) maxima of an analyte, then a signal equivalent to 100% oxidation (or reduction) will be produced. Although this results in an improved response, it still lacks qualitative information. An alternative approach uses an array of coulometric electrodes. If these electrodes have incrementally applied potentials such that they now span the potential window of the analyte, signal from the oxidation (or reduction) of that analyte can be spread over several adjacent sensors. This response for an analyte across the array contains valuable qualitative information. A given analyte will always give the same voltammetric response across the array as long as the chromatographic conditions are not changed. This response is independent of concentration. See Figure 2. In most cases the potentials across the array are configured such that analytes are found to respond over three adjacent sensors, termed the preceding, dominant and following. Software compares the voltammetric response of an unknown compound to that of an external standard and assigns two "ratio accuracies". Derivation of a ratio accuracy is described in greater detail in the legend to Figure 2.
How an analyte responds across adjacent sensors is a function of its hydrodynamic voltammetric behavior (see Figure 2 upper right-hand quadrant). The array, therefore, spontaneously generates an HDV for every analyte in the sample capable of undergoing electrolysis. Because the potentials are not being altered manually to generate the HDV there is no time delay. Ratio accuracies yield quick and highly accurate information regarding peak identity, presence of co-elutions or misidentification.
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Figure 2. Obtaining qualitative information from the coulometric array. With a typical oxidative array the potentials are configured such that compounds respond at three consecutive electrodes termed the preceding, dominant and following channels. The voltammetric response of an analyte across these three channels is a characteristic of that analyte and is independent of the analyte's concentration. For example the daidzein standard (top left) has a dominant response on channel 5 (+500mV) with less response on the preceding channel, 4 (+440mV), and following channel, 6 (+560mV). Although the coulometric electrodes are 100% efficient, the analyte still responds across three electrodes. The reason for this is that the electrodes are not set to the analyte's maximal oxidation potential but at voltages along its potential axis. This is illustrated in the upper right figure. Here a plot of peak height vs. applied potential shows the analyte's response across the array. The cumulative peak height vs. potential is the analyte's hydrodynamic voltammogram (HDV) and shows why the analyte is expected to respond over more than one channel. Qualitative information is obtained by comparing the standard analyte's HDV to that of the unknown in the sample (in this case daidzein in urine; bottom left quadrant). The system software splits the analyte's HDV into two segments. The analyte's response ratio on the preceding to dominant channels is compared to the unknown's response ratio for the same channels. As shown in the bottom right quadrant, division of the unknown's response ratio by that of the standard produces the first ratio accuracy. The second ratio accuracy is obtained by comparing the response ratio for the following and dominant channels. When expressed as a percentage this gives a numerical indicator as to the authenticity and purity of the unknown peak in the sample. When a peak is positively identified in the sample, both ratio accuracies will approach 100%.
If a series of coulometric detectors is employed, the response for each compound typically occurs across more than one electrode, due to oxidation along the C/V curve of the analyte. As an example, if Emax for a given compound is 750 mV, it is likely that some oxidation will be observed at the electrodes set at 660 mV, 720 mV and 780 mV.
With a series of coulometric sensors, the proportion of the signal between adjacent electrodes for a particular analyte remains constant (e.g., the E 720 / E 660 signal ratio is 2.43). This provides additional information to indicate that the eluted compound is indeed the compound of interest. As an example, if the ratio is 1.66, it is likely that the compound that eluted at a given time is not the compound of interest (or two compounds co-eluted).
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