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Four Channel Liquid Chromatography/Electrochemistry Four Channel Liquid Chromatography/Electrochemistry Bruce Peary Solomon, Ph.D. The new epsilon family of electrochemical detectors from BAS can Hong Long, Ph.D. Yongxin Zhu, Ph.D. control up to four working electrodes simultaneously. There are several Chandrani Gunaratna, Ph.D. advantages to using multiple detector electrodes. By using four different Lou Coury, Ph.D.* applied potentials with electrodes placed in a parallel arrangement, a Bioanalytical Systems, Inc. hydrodynamic voltammogram can be generated quickly through Corporate R&D Laboratories 2701 Kent Avenue acquisition of four data points for every analyte injection. This speeds West Lafayette, IN method development time. In addition, co-eluting compounds in complex 47906-1382 mixtures can be resolved on the basis of their observed half-wave * corresponding author potentials by using the same arrangement of electrodes, also in parallel. This article presents a few examples of four-electrode experiments performed with epsilon detectors in the BAS R&D labs during the past few months, using both radial-flow and cross-flow thin-layer configurations. The epsilon Platform the past fifteen years, our contract These instruments are fully network- research division, BAS Analytics, able and will be upgradable over the BAS developed and introduced the has provided analytical data of the Internet. New techniques and fea- first commercial electrochemical de- highest quality to the world’s leading tures may initially be ordered àla tector for liquid chromatography pharmaceutical companies using carte, or added at any time when the over twenty-five years ago. With this state-of-the-art products from BAS, need arises. In the coming months, issue of Current Separations,BAS as well as other leading vendors. Un- we will unveil additional capabilities continues this tradition of innovation like our competitors, we do not need of the epsilon platform through our by introducing another new family to guess what instrumental features web site and through articles in Cur- of electrochemical instruments and might be of interest to users (and rent Separations. In this article, we chromatographs called epsilon. what features are not of interest) will discuss just one specific feature These instruments have been de- since BAS scientists use BAS prod- of the epsilon family of instruments: signed and built on an entirely new ucts on a daily basis in the most the ability to perform amperometric platform that takes into account the demanding applications. measurements using four inde- increasingly regulated environment We have leveraged this com- pendent working electrodes for de- in which analytical chemistry is per- bined, corporate experience to make tection in liquid chromatography formed, the rise of intranets and the instruments available to our external experiments. Internet, and the need to detect ever customers that truly meet the de- decreasing amounts of more potent mands of modern analytical chemis- 4-Electrode Hydrodynamic substances in complex media. PM- try. Through optical isolation Voltammograms 91e and PM-92e pumps have been circuitry and the use of up to 24-bit developed with epsilon forming the resolution during data conversion, When using an electrochemical de- basis of new families of LC systems we have produced instruments that tector in liquid chromatography including the BAS 200e, BAS 480e display high signal-to-noise charac- (LCEC), perhaps the most important and BAS 500e. teristics and the ability to measure instrumental parameter controlled Unlike every other participant on low currents in the typical laboratory by the user is the potential applied the instrument industry playing environment. The epsilon instru- between the working and reference field, BAS has diversified across the ments can be configured in 1-chan- electrode (“detector potential”). In user-manufacturer boundary. Over nel, 2-channel or 4-channel versions. an oxidative application, the detector 113 Current Separations 18:4 (2000) potential should be set to a value well LCEC). One unique feature of the F1 E (volts) positive of the observed half-wave epsilon instrument family is that cy- A “blast from the past.” 1.0 0.8 0.6 0.4 0.2 0 0 Four hydrodynamic potential of the analyte of interest to clic voltammetry can be performed voltammograms obtained obtain maximum sensitivity (current using an epsilon LC detector (and an using different 0.2 carbon-based working per unit change in concentration). epsilon purchased to perform CV ex- electrodes. Data were One way to determine half-wave periments can be used for LCEC) obtained over time by sequential injections. 0.4 potentials for the selection of detec- simply by purchasing the appropri- Modified from: “Choosing Ø tor potential(s) is to perform a cyclic ate software package from BAS. the Right Electrode is Important,” Current 0.6 voltammetry (CV) experiment for Another way to determine the Separations 1979, 1(1), 5. each analyte of interest. In the past, appropriate detector potential during 0.8 this has required access to both an methods development efforts is to electrochemical instrument (for CV) generate a hydrodynamic voltam- 1.0 and an amperometric detector (for mogram using a flow cell and an amperometric detector. This proce- F2 dure involves selecting an initial de- Hydrodynamic tector potential, injecting a standard voltammogram (HDV) obtained with only 3 solution of the analyte(s), and re- injections using an epsilon cording the response (height or area four-electrode system. Experimental data from of the chromatographic peak ob- Dr. Bruce Solomon. See served). The detector potential is text for details. then incremented to a new value, another analyte injection made, and the response again recorded. By re- peating this process, a plot of detec- tor response (peak current) versus applied potential is gradually built up. F1 is a reprint of a hydrodynamic F3 voltammogram obtained for nor- Inlet Various four electrode epinephrine that appeared in the in- cells used in the augural issue of Current Separations experiments described in this paper. in 1979. The figure compares the response obtained using three differ- Outlet ent formulations of carbon paste and a glassy carbon electrode. The data show that the current responses re- Quad Square corded become maximal and Locking Series, Crossflow (roughly) independent of the applied Collar potential for all but one of the elec- Reference trodes once a sufficiently positive Electrode potential is reached. However, as many as nineteen separate injections Auxiliary were made for a single electrode ma- Electrode O-ring Block terial, and the amount of time in- vested in collecting such data was Quad Square considerable. Few people have the Parallel, Radial-flow time (or patience) to do such an ex- Gasket periment today, when companies merge and create spin-offs seem- ingly as frequently as people used to publish papers in Analytical Chem- istry! Working Electrode By comparison, F2 shows a hy- Block drodynamic voltammogram for nor- Quick Release Quad Arc epinephrine, generated using only Mechanism Parallel, Crossflow three injections and an epsilon four- electrode system equipped with four Current Separations 18:4 (2000) 114 F4 NE a true (not pseudo-) reference elec- Chromatogram for an trode for all measurements. injection of 5 ng norepinephrine and 5 pg epinephrine. A detector Electronics Optimized for sensitivity of 100 nA full scale was employed. Top Demanding Analytical and bottom panels show Problems the same data set displayed on two different scales. Experimental data By virtue of optical isolation of the obtained by Dr. Bruce detector circuitry as well as high- Solomon. resolution analog-to-digital conver- sion, the epsilon family of LC E detectors has been optimized for the most demanding analytical situ- ations. F4 shows two chromato- grams obtained for a mixture of 5 ng of norepinephrine (NE) and 5 pg of epinephrine (E). The detector sensi- F5 tivity was selected to a relatively low Chromatogram obtained gain setting (viz., 100 nA full scale) by Dr. Hong Long for a in order to keep the peak for NE on mixture of six phenolic acids. scale. In the upper panel, the peak due to E cannot be discerned. How- ever, by using the zoom function in the BAS epsilon ChromGraph soft- ware, the scale can be adjusted to reveal and quantitate the E peak (bot- tom panel). Note that it was not nec- essary to select a higher gain setting and to re-inject the sample. The data are of such high signal-to-noise qual- ity that the display only needs to be re-scaled to examine the E peak. This sort of situation commonly arises in F6 pharmaceutical analysis, where an active ingredient must be quantified Chromatogram obtained by Dr. Hong Long for a along with an impurity. honey extract using a four-electrode epsilon system. See text for Multi-Potential Detection: An details. Additional Separation Dimension The ability to acquire chroma- tographic data using four parallel de- tectors set at different potentials provides an additional separation di- mension (oxidation potential) that is orthogonal to the time axis (retention time). That is, the applied potential can be used to resolve different com- pounds in a mixture in a way analo- glassy carbon working electrodes in and 850 mV for the final injection. gous to the use of retention times to the arc configuration (see F3). For The electrodes were allowed to discriminate between compounds. the first injection, the detectors were equilibrate for fifteen minutes after Of course, all four working elec- set to 300, 350, 400, and 450 mV (vs. each change in potential. These were trodes need not be constructed of the Ag/AgCl). The detectors were reset ten-minute chromatographic runs, same material; thus, selectivity on to 500, 550, 600, and 650 mV for the so the complete HDV took only sev- the basis of kinetic and/or surface second injection, and 700, 750, 800, enty-five minutes to generate, using catalytic effects is also possible.
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