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Simultaneous Multiple Electrode Liquid Chromatography/Electrochemistry of Phenolic Acids in Honey

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Simultaneous Multiple Electrode Liquid Chromatography/Electrochemistry of Phenolic Acids in Honey LC•GC Europe - June 2001 Long, Zhu, Coury, Duda, Kissinger and Kissinger 1 Simultaneous Multiple Electrode Liquid Chromatography/Electrochemistry of Phenolic Acids in Honey Hong Long, Yongxin Zhu, Lou A. Coury, Chester T. Duda, Candice B. Kissinger and Peter T. Kissinger, Bioanalytical Systems Inc., West Lafayette, Indiana, USA. A scheme using liquid chromatography with four-channel electrochemical detection for the identification and quantification of phenolic acids is reported. After a preliminary identity assignment based on spiking samples with standard compounds, an electrochemical detector was employed to obtain a voltammetric characterization of eluting compounds. Peak height ratios from simultaneously generated chromatograms were calculated and a comparison made between standards and sample to confirm identities. As an illustration of the merits of the described approach, determination of phenolic acids in several honey samples is presented. Introduction and analytes. The scheme is simple, were collected locally, clover and Chinese Liquid chromatography/electrochemistry economical and compatible with common honey were purchased from retail stores. (LCEC) has become a popular technique in liquid chromatographic procedures. The the assay of natural phenolic compounds. methodology described here is useful in Procedures Dual-electrode detection is now widely the study of phenolic acids in a wide Extraction: The honey samples (1.0 g) were used. It can be used in the parallel or series variety of natural products. Honey samples thoroughly mixed with 10 mL deionized configuration (1). The parallel are analysed as a representative example. water by ultrasonication for 10 min. Then, configuration has been employed to 1 mL of the solution was acidified to pH 2, monitor eluting compounds at two Experimental Section saturated with NaCl, and extracted with potentials simultaneously, while the series Apparatus: The LCEC system was 2 mL of ethyl acetate for 5 min on a vortex configuration has been used to modify the composed of a chromatographic pump shaker. The extraction was repeated twice analyte at the upstream electrode to allow (PM-92e, BAS, West Lafayette, Indiana, more. Combined ethyl acetate collections more selective detection at the USA) coupled with a Rheodyne injection were dried under a stream of nitrogen at downstream electrode. In this work, a four- valve (Model 7125), an ODS 3 µm column room temperature. The residue was electrode detection scheme is employed, (PEEK 100 ϫ 2.0 mm, BAS), a multichannel dissolved in 500 µL of mobile phase and which illustrates several advantages over amperometric detector (epsilon, BAS) filtered through a 0.2 µm Nylon-66 dual-electrode detection schemes. coupled to four glassy carbon working membrane before injection into the liquid A frequently encountered problem electrodes and referenced to an Ag/AgCl chromatography system. A 20 µL sample during the analysis of complex samples electrode. Data were acquired and was injected throughout the study. with liquid chromatographic techniques is integrated through BAS ChromGraph Liquid chromatography: Mobile phase the reliability of sample constituent (version 9.35) chromatography software. composition was 20 mM NaH2PO4, identification. Preliminary identification is Reagents: Gallic acid, protocatechuic acid, pH ϭ 3.03, containing 4% methanol. The based on the comparison of retention vanillic acid, caffeic acid, syringic acid and flow-rate was 0.7 mL/min. Detector factors (k’) for sample components and p-coumaric acid were purchased from potentials were selected over the range standard compounds. Reliable assignment Sigma (St Louis, Missouri, USA). Methanol ϩ550 mV to ϩ900 mV versus Ag/AgCl. of peak identity generally requires and ethyl acetate were of HPLC grade additional characterization, which can be, (Burdick & Jackson, Muskegon, Michigan, Results and Discussion for example, optical and/or mass spectra. USA). Reagent-grade water was prepared Preliminary identification of sample In this article we present an alternative: the by in-house deionization using a constituents: Honey is rich in phenolic use of LCEC for comparison of the NANOpure system (Barnstead/Thermolyne, acids (2, 3). These reducing agents can be current/potential behaviour of standards Dubuque, Iowa, USA). Spring and fall honey detected sensitively by anodic (oxidation) 2 Long, Zhu, Coury, Duda, Kissinger and Kissinger LC•GC Europe - June 2001 electrochemical detection. Figure 1 presents that an interference is coeluting with parallel and series detection may be hydrodynamic voltammograms (HDV) of the vanillic acid. performed for the same injection. This cross- phenolic acids studied. Four simultaneous The four-electrode detector (Figure 3(a)) flow configuration, depicted in Figure 3(c), current/potential data points were acquired can be arranged in another flow tests not only the energy of the oxidation for each analyte in an injection. configuration (Figure 3(c)) that has proven to process, but also the stability of the oxidation Using isocratic reversed-phase liquid be useful for confirming compound identity. products. By this scheme, we have identified chromatography, six phenolic acids present This is an extension of earlier work on dual- six phenolic acids in all four honey samples. in honey were separated within 30 min. series electrodes (4, 5). The upstream Except for vanillic acid, the other five phenolic Figure 2 presents chromatograms of four electrode oxidizes the analyte and the acid peaks represent single compounds. honey samples monitored at a potential of product is reduced at the downstream For different configurations, different ϩ700 mV. Preliminary identity assignment electrode, mimicking fluorescence detection. gaskets and auxiliary electrodes are used. of each peak was made by spiking the With the four-electrode cell, both dual One has the flexibility to choose a suitable sample with standard compounds. Confirmation by Voltammetric 1.0 Characterization of Sample Constituents 0.9 After preliminary assignment, the four- channel electrochemical detector was 0.8 employed to confirm the identification. This was achieved by comparing peak height 0.7 ratios at different potentials for standards with sample peaks of equal retention. First, 0.6 the four electrodes were set in a parallel 0.5 flow configuration. A schematic depiction is shown in Figures 3(a) and 3(b). With these 0.4 configurations, an equal flow with the same concentration of analyte passes over Normalized response (Ø) 0.3 each of the four electrodes in the thin-layer channel. Thus, four potentials can be 0.2 monitored simultaneously, analogous to diode array ultraviolet detection. From each 0.1 injection, three peak height ratios are derived. Tables 1 and 2 show the 0 comparison of ratio values between 0 ϩ200 ϩ400 ϩ600 ϩ800 ϩ1000 ϩ1200 ϩ1400 standards and samples for peaks with E (mV) equal retention times. Except for vanillic acid, the ratios of samples match well with Figure 1: Hydrodynamic voltammograms for the oxidation of six phenolic acids present in those of standards. There are two possible honey. N ϭ gallic acid, I ϭ protocatechuic aicd, L ϭ vanillic acid, N ϭ caffeic acid, reasons why the ratio for vanillic acid does L syringic acid, I p-coumaric acid. not match the standard. One is that some other compound is coeluting with it; the other is that the peak may represent a different compound. By using the extraction method 12 mentioned in the experimental part of our 1 procedure, the constituents extracted from 10 honey samples included acidic and neutral compounds. To isolate neutral compounds 8 from acidic compounds, the following 2 modification was employed. The combined nA 6 6 mL ethyl acetate layers were extracted 4 with 1.5 mL of 10 mM, pH 7.5, phosphate 3 buffer four times. The acidic compounds 4 5 6 2 (a) were extracted into buffer solution. The (b) ethyl acetate was evaporated to dryness (c) 0 under a stream of nitrogen, and the (d) residue was reconstituted in 500 µL of 0 5 10 15 20 25 30 mobile phase and filtered prior to injection. Time (min) Figure 4 presents the chromatograms of both extracts. In Figure 4(b), a small peak is Figure 2: Chromatograms of honey samples at a potential of ϩ700 mV: (a) ϭ fall honey, observed in the position of vanillic acid. It is (b) ϭ clover honey, (c) ϭ spring honey, (d) ϭ Chinese honey. Peaks: 1 ϭ gallic acid, 2 ϭ smaller than that in Figure 4(a), indicating protocatechuic acid, 3 ϭ vanillic acid, 4 ϭ caffeic acid, 5 ϭ syringic acid, 6 ϭ p-coumaric acid. LC•GC Europe - June 2001 Long, Zhu, Coury, Duda, Kissinger and Kissinger 3 configuration according to the analytes and the purpose of the experiment. In all experiments the four electrodes are controlled and monitored by software via the amperometric detector. Furthermore, one can access the system remotely over the Internet. If the instrument is coupled with an autosampler, the entire automated process can be controlled remotely, even at home. (a) (b) (c) Quantitative Determination of Figure 3: A depiction of the working electrode configuration for four-channel electrochemical Phenolic Acids in Honey detection (working electrode diameter ϭ 2 mm): (a) ϭ radial flow, (b) ϭ arc cross flow, Another advantage of multichannel (c) ϭ cross flow. electrochemical detection is that it allows detection of different compounds at different potentials, simultaneously. Thus a concentrations. Correlation coefficients (r2) confirming peak identity and purity and suitable potential may be selected for each were greater than 0.9991 in all instances. increasing selectivity. All of these merits analyte, which results in the highest signal- The concentration of six phenolic acids in make this methodology suitable for the to-noise ratio for each compound. Figure 5 four varieties of honey is presented in Table 3. assay of complex natural compounds in shows the chromatograms of a honey From these results, it can be seen that fall complex matrices. We have also used this sample monitored at four different potentials honey contains more phenolic acids than scheme to determine resveratrol in wine in the same injection.
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