Anal. Chem. 1981, 53, 1695-11699
Table IV. Results of Analyses on Samples with Simultaneous Chloride and Sulfate Determinations sample ppb c14/3/81 mixed bedu 4.0 4/9/81 mixed beda 13.4 Catex precolumn was not needed. date
PPb
so,*-
to be regenerated as in previous ion chromatographic methods. On-line dedicated instrumentation would be inexpensive because of this simplicity. Automation would require the control of only one valve: the load/inject valve. LITERATURE CITED
6.5 7.2
Small, H.; Stevens, T. C.; Bauman. W. C. Anal. Chem. 1975, 47, 1801. Gjerde, D. T.; Fritz, J. S.; Schmuckler, 0. J. Chromatogr. 1979, 186, 509. Gjerde, D. T.; Schmuckler, G.; Fritz, J. S. J. Chromatogr. 1980, 787,
Analytical results art? given in Tables I11 and IV. Boiler blowdown water contains approximately10 ppm of phosphate, which overloads the concentrator column and prevents the analysis of this type of simple. No difficulty was encountered in analyzing the other simples. Two pairs of samples (boiler feed 17, mixed bed 10 rind boiler feed, mixed bed 8/15/80) were actually the same water taken at two different sampling points. The closeness of the values of chloride and sulfate for the pairs shows the rleproducibility of the chromatographic method and the sampling procedure.
CONCLUSIONS The method of singlle-column ion chromatography is a valuable technique for the analysis of parts-per-billion levels of chloride and sulfate. The instrumen tation is extremely simple. There is no standard suppressor column to be regenerated and then 50% depleted or small1 suppressor column
1895
35.
Stevtms, T. S.;Turkelson, V. T.; Albe, W. R. Anal. Chem. 1977, 49, 1176. Wetzel, R. A.; Anderson, C. L.; Schlelcher, H.; Crook, 0. D. Anal. Chen?.1979,57, 1532. Fulmor, M. A.; Penkrot, J.; Nadalln, R. J. In "Ion Chromatographlc of Environmental Pollutants"; Mullk, J. D., Sawlcki, E., Eds.; Ann Arbor Sclerice: Ann Arbor, MI, 1979; Chapter 32. Barran, R. E.; Fritz, J. S., unpublished results. Gjerde, D. T.; Fritz, J. S. J . Chromatogr. 1979, 176, 199. Gjerde, D. T.; Fritz, J. S., unpubllshed results. Gjerde, D. T. Ph.D. Dlssertatlon, Iowa State Unlverslty, 1980.
RECEIVED for review December 1,1980. Accepted June 5,1981. Ames Laboratory is operated for the US. Department of Energy by Iowa State University under Contract No. W7405-Eng-82. Partial financial support for this research was provided by the Director of Energy Research Office of Basic Energy Sciences, WPAS-KC-03-02-03.
Identification of Phenolic Constituents in Commercial Beverages by Liquid Chromatography with Electrochemical Detection Daryl A. Roston and Peter T. Kisslnger" Department of Chemistry, ipurdue University, West Lafayette, Indiana 47907
A scheme Is presented for the ldentlfication of phenollc constituents in alcoholic belverages and fruit Juices wlth llquld chromatography wlth electrochemical detection (LCEC). Acldlc, neutral, and basic: compounds are isolated Into one or more classes using llquitl-liquid extractions at controlled pH. Initial Identity asslgnments are based on comparlson of k' values of standard compounds and sample components. Atter the prellmlnary ldentlty assignment, the electrochemlcal detector Is employed to obitaln a voltammetrlc characteriratlon of eluting compounds. Comparison of the Current-potential responses of standards and sample components provldes conflrmatlon of the Initial Identity asslgnment. Addltional confirmation Is provided by the sequentlal performance of thin-layer chromatography and LCEC on the sample. As an Illustration of the merlts of the described approach, ldentlflcatlon of plant phenolics lin several commerclal beverages has been completed.
A frequently encountered problem during the analysis of complex samples with modern liquid chromatographic techniques is the reliable identification of sample constituents. 0003-2700/61/0353-1695$01.25/0
Prelimiisly identification with liquid chromatography is based on the comparison of capacity factors (k') for sample components and standard compounds. Reliable assignment of peak identity requires the determination of additional component characteristics. This often involves the collection of fractions eluting from the column and obtaining conventional optical anld/or mass spectra. Verification of sample component identification in this manner is awkward and difficult, especially if confirmation is needed for several sample constituents. For trace work at the picomole level obtaining further information on collected fractions is all but impossible using present day instrumentation. Previously reported work from our laboratory has briefly described a unique approach to sample identity confirmation which involves the sequential performance of thin-layer chromatography (TLC) and liquid chromatography with electrochemical detection (LCEC) (1-4). As an extension of this work we have completed constituent identification studies on ethyl acetate extracts of phenolic compounds in beer, wine, and other alcoholic beverages. Utilization of LCEC for sample component identification involves the voltammetric characterization of eluting compounds to allow comparison of the current-potential behavior of standards and analytes. Additional identity confirmation is provided by the sequential 0 1981 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 11, SEPTEMBER 1981
performance of TLC and LCEC on the sample. The strength of this identification scheme lies in its relative simplicity, economy, and compatibility with liquid chromatographic procedures. In the present paper we describe methodology which will be useful in the study of beverage production and quality control and which is generally useful for the determination of phenolic acids in a wide variety vegetable material. EXPERIMENTAL SECTION Apparatus. The liquid chromatographic system was a Bioanalytical Systems LC-154 (BioanalyticalSystems, Inc., West Lafayette, IN). A Biophase C18column (25 cm X 4.6 mm) was employed. Detection was achieved with a Bioanalytical Systems LC-2A electrochemical detector equipped with a glassy carbon electrode. Dual-electrode LCEC experiments were carried out using two modified BAS LC-4A controllers and a thin-layer cell containing two parallel 3-mm glass carbon electrodese across the channel from a single auxiliary electrode. The technical details of this apparatus will be published elsewhere (5). TLC was performed with 20 cm X 0.25 mm EM Reagent fluorescent silica gel plates (60F-254) (MCB Manufacturing Chemists Inc.). Elution of components from the TLC silica was completed with Bioanalytical Systems MF-1 Centrifugal Microfilters. Reagents. Benzoic, cinnamic, and phenylacetic acid derivatives were purchased from the following sources: gallic acid, vanillic acid, vanillin, ferulic acid, sinapic acid, and 3,4-dihydroxyphenylacetic acid, Aldrich Chemical Co.; gentistic acid, caffeic acid, salicylic acid, and p-hydroxyphenylacetic acid, Sigma Chemical Co.; 2-hydroxy-5-methoxybenzoicacid and p-coumaric acid, Eastman Kodak Co.; p-hydroxybenzoic acid, Matheson, Coleman and Bell. Sinapic and p-coumaric acids were recrystallized once from warmed water. The other standards were used without further purification. With the exception of methanol, reagent grade organic solvents were used without purification. Methanol was distilled once prior to use. Procedures. Extractions. Two milliliters of the beverage was acidified to pH 2 and saturated with NaC1. The sample was then extracted with 2 mL of ethyl acetate for 10 min on a reciprocal shaker. The extractionwas repeated twice more. Combined ethyl acetate layers were dried over anhydrous sodium sulfate. Residual sodium sulfate was washed with 1mL of ethyl acetate. Next, the ethyl acetate was evaporated under a stream of nitrogen at ambient temperature. The extraction residue was dissolved in 600 pL of mobile phase prior to injection on the liquid chromatograph. Thin-Layer Chromatography. The residue from the extraction procedure described above was redissolved in 100 pL of ethyl acetate. A 50-pL portion of the reconstituted extract was spotted on the TLC plate and developed 18 cm with the upper layer of a 100:150:50 benzene/acetic acid/water mixture. A standard mixture was developed in parallel with the sample thin-layer chromatogram. Silica corresponding to the R f range of interest was removed from the sample TLC plate with a razor blade. Sample components were eluted from the silica with the Bioanalytical Systems MF-1centrifugal microfiter using a Teflon filter membrane. The eluting solvent was 1 mL of methanol. After evaporation of the methanol eluent with a stream of nitrogen, the residue was redissolved in 50 pL of mobile phase. Liquid Chromatography. Pertinent experimental parameters were as follows: detector potential, +950 mV, vs. Ag/AgCl reference electrode unless otherwise specified; flow rate, 1mL/min; injector loop volume, 20 wL; mobile phase composition, 2.18% 1-propanol,1.98% acetic acid, 8.71% methanol, 87.13% deionized distilled water, 0.018 M ammonium acetate. RESULTS AND DISCUSSION Preliminary Constituent Identification. Figure 1shows the liquid chromatograms of constituents extracted from beer, wine, and whiskey samples. Two steps were taken to complete a preliminary identification of the chromatographic peaks shown in Figure 1A. The first step was a comparison of the k’values of sample constituents and standards of compounds that are suspected sample components. Phenolic derivatives of cinnamic, benzoic, and phenylacetic acid are frequently found in plant materials (6). Figure 2 is the liquid chromatogram of a mixture of several such derivatives obtained with
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Flgure 1. Chromatograms of ethyl acetate extracts of commercial beverages: (A) Olympia beer, (B) Christian Brothers wine, (C) Canadian
Club whiskey. Conditions: 25-cm Biophase CIBcolumn; flow rate, 1.0 mL/min; detector potential = +950 mV.
the conditions employed in Figure 1. The k’values of four of the standards matched those of eluant peaks contained in the beer extract chromatogram shown in Figure 1A. Liquid-Liquid ExtraCtions. Table I tabulates the matching k’value of the standards and sample components. Because the compounds identified in the beer extract were weak acids, a second preliminary step was employed to c o n f i i their acidic functionality. TO complete the functionality confiimation, ethyl acetate extractions were completed on two aliquots of the same sample after adjustment of the pH to 2. However, prior to the pH 2 extraction a preliminary extraction was completed on one of the aliquots after adjustment of the pH to 7. Adjustment to the neutral pH deprotonates the acidic components and decreases their extraction efficiency. This neutral extraction removes relatively small portions of the acidic beer constituents. In contrast, significant fractions
ANALYTICAL CHEMISTRY, VOL. 53, NO. 11, SEPTEMBER 1981
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Hydrodynamic voltammograms of standard compounds: (B) sinapic acid, ( 0 )ferulic acid, (A)vanillic acid, (0)p-coumaric acid. Flgure 3.
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Flgure 2. Chromatogram of cinnamic, phenylacetic, and benzoic acid derivatives: (A) gallic acid, 0.054nmol; (B) geritistic acid, 0.154 nmol; (C) 3,4-dihydroxyphenylacetic acid, 0.109 nmol; (D) p-hydroxyphenyl acetic acid, 0.142 nmol; (E) vanillic acid, 0.064 nmol; (F) caffeic acid, 0.086 nmol; (G) vanillin, 0.135 nmol; (H) salicylic acid, 0.269 nmol; (I) 2-hydroxy-5-methoxybenzoic acid, 0.088 nmol; (J) p-coumaric acid, 0.150 nmol; (K) ferulic acid, 0.168nmol; (L) sinapic acid, 0.235nmol.
Detector potential = +1100 mV.
~
Table I. Preliminary Comparison of Standard and Sample Component Ckiaracteristics % reduction in peak
k' value
vanillic acid beer component A p-coumaric acid beer component B ferulic acid beer component C sinapic acid beer component D 2-hydroxy-5-methoxybenzoic acid vanillin -___
38 3.8 8.9 8.9 11.3
11.3 12.5
height from neutral extraction 6
12
4 5
12.5 7.9
6
6.2
68
of the neutral components are removed from the beer at neutral pH since their extraction efficiency is relatively pH independent. Internal standards were employed to evaluate the effect of the pH 7 extraction on acidic and neutral compounds. Prior to extraction, vanillin and 2-hydroxy-5-methoxybenzoic acid were added to the beer sample. Comparison of the internal standard peak heights obtained with the different extraction schemes allowed assessment of the relative effect of the pH 7 extraction on neutral and acidic components. The neutral extraction resulted in a 68% reduction in the peak height of the neutral internal standard, vanillin. Only a small decrease (6%) in the acidic internal standard peak height occurred. Table I shows the percentage decrease in the peak height produced by the preliminary pH 7 extractlion on the identified chromatographicpeaks. In each case the change was relatively small in comparison to the change induced in the peak height of the neutral internal standard. Thus, the presence of an acidic functional group on the identified constituents is verified.
Voltammetric Characterization of Eluting Compounds. After the preliminary identity assignment, the electrochemicaldetector is employed to evaluate the voltammetric behavior of the components of interest. Identification of compounds during hydrodynamic voltammetry experiments is based on two characteristics of the current-voltage curve of the analyte. Generally, the most important feature is the Ellz value of the compound, the potential where the current response is half of its diffusion limited value. The E l p value earmarks5 the potential region where the magnitude of the current response is markedly dependent on the electrode potential, An additional identifying feature is the general shape of the current-voltage curve. Some compounds (with rapid electron transfer kinetics) exhibit a dramatic increase in current response for a small change in potential. For others, the change in current response is more gradual. The Ellz values and general shape of the current-potential curve obtained with a hydrodynamic system are dependent on several experimental parameters, including flow rate, cell geometry, and electrode surface properties. The dependence of the current-]potential response on the experimental conditions underlines the importance of obtaining current-potential curves for compounds of interest under conditions identical with thocie employed for analysis. Voltammetric characterization of eluting compounds can be achieved during a series of LCEC experiments in which the detector potential is incrementally increased. The current-potential response of the chromatographic peak is additional information that can be compared to standards and used to confirm identity. Although it is usually possible to obtain a complete current-pott?ntial curve of the compounds of interest, often, this is not practical, If a relatively long period of time is required to elute all1 of the components in the studied matrix, then it is advantageous to implement a partial voltammetric characterizatilon to minimize the time involved. Because the elution OF all the ethyl acetate phenolic extracts required in excess of 40 min, an abbreviated voltammetric characterization is most appropriate. If a partial voltammetriccharacterization is emplo:yed, it is necessary to choose detector potential changes hased on consideration of the current-voltage curves of the coyresponding standards. Figure 3 shows the hydrodynamic voltammograms of the representative compounds that are suspected beer components. The current-potential curves were obtained with the chromatographic conditions employed for the beer extracts. Consideration of the voltammetric curves indicate that detector potential changes between +lo00 and 4-600 mV would alter the current responses of all the standards. Because
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 11, SEPTEMBER 1981
Table 11. Voltammetric Characterization of Sample Constituentsa
a
detector potential, mV
vanillic acid
component p-coumaric component component component A acid B ferulic acid C sinapic acid D
t 950
0.61
0.59
0.51
t 900 t 850 t 800
0.26
0.26
0.19
0.08
0.09
0.00
0.00
0.04 0.00
0.54 0.21 0.05
0.88 0.75 0.59
0.85 0.71 0.56
0.00
0.44
0.41
0.93 0.87 0.73 0.60
0.91 0.83 0.70 0.57
Current responses are normalized to the value observed at t 1000 mV.
potential changes in this region affect all of the standards, the voltammetric characterization can be completed for the compounds of interest in a single series of experiments. Table I1 is a summary of the normalized current responses of the standards and corresponding beer constituents observed at four different detector potentials. Good agreement exists between standards and unknowns. Thus, the voltammetric characterization provides further evidence that the initial identification is correct. A few important considerations concerning the voltammetric characterizations with LCEC deserve mention. Electrode oxidation mechanisms of aromatic compounds often involve reactive intermediates that react yith and decrease the sensitivity of the electrode. Because sample matrices contain more compounds than standard solutions, the extent of electrode passivation is more pronounced during a series of sample elutions than during a series of standard solution elutions. Hence, the electrode passivation phenomenon renders the current-potential responses matrix dependent. This matrix dependence is largely overcome by obtaining the liquid chromatograms for a voltammetric characterization in a standard-mmple-standard-sample sequence. Data shown in Table I1 were obtained with an alternating injection scheme. If the alternating injection scheme is not employed, electrode passivation phenomenon can render the reliability of the comparison questionable. For example, differences in the current responses of the standards in Figure 3 and Table I1 are due to matrix effects. Additional factors that can affect day-to-day precision are slight changes in the reference electrode potential and mobile phase composition. Problems from both of these factors are also overcome by employing a standard-sample injection scheme. Another important factor is the resolution of the compounds of interest. Current-potential responses of compounds that are not well revolved are less accurate. Dual-Electrode Detector Experiments. A negative aspect of performing voltammetric characterizations with LCEC is the large number of experiments required. With a single working electrode detector, only one point on the currentpotential curve can be obtained from each chromatogram. In response to this shortcoming,we have completed preliminary experiments with a dual working electrode detector to implement dual potential monitoring of the eluant. Chromatograms shown in Figure 4 were obtained with an electrochemical detector consisting of two glassy carbon electrodes postitioned in parallel in the thin-layer channel. A depiction of the electrode configuration is shown in Figure 4, The potential of one of the electrodes was +lo00 mV while the potential of the other was +850 mV. Dual potential monitoring halves the number of experiments required to perform a given voltammetric characterization. A more explicit description of the dual electrode electrochemical detector and associated techniques will be given in a subsequent publication (5). TLC Confirmation. If the retention characteristics and current-potential behavior of a sample chromatographicpeak compare well with those of a standard compound, additional peak identity confirmation can be achieved by the sequential performance of TLC and LCEC. The initial experimental step
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Flgure 4. Chromatograms obtained simultaneously with dual electrode detector: (A) detector potential = +lo00 mV, (B) detector potential
= +850 mV.
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Chromatogram of beer components eluted from the TLC silica encompassing the &of p-coumaric acid. The peak at 27.7 min confirms the presence of p-coumaric acld in beer. Flgure 5.
in the TLC verification is the development of the thin-layer chromatogram of an organic sample extract in parallel with a mixture of the standard compounds that are suspected sample constituents. The section of the sample thin-layer chromatogram encompassingthe R, value of the standard (or standards) of interest is removed from the glass plate. After the elution of the components from the silica with a relatively
ANALYTICAL CHEMISTRY, VOL. 53, NO. 11, SEPTEMBER 1981
1099 _.
Table 1I:I. TLC Recovery Study vanillic acid
sinapic acid
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Flgure 0. Chromatogram of components eluted from the TLC silica encompassing the R,of viinilllc and ferulic acids. Peaks at 13.3 rnin and 34.4 min conflrm the presence of vanllllc and ferulic acids in beer.
polar organic solvent and evaporation of the solvent, the residue is dissolved in the mobile phase. If the injection of tlhe reconstituted TLC extract in the liquid chromatograph results in the appearance of a peak at the retention time of the corresponding standard compound, the peak identity assignment is further verified. After characterization of the appropriate standards with the TLC system employed, the above procedure was used to confirm the beer constituent identity assignments. Figure 5 is the chromatogram of the compounds eluted from the silica comprising the section of the TLC plate corresponding to Rf values of 0.37-0.41. The Rf vdlue of p-coumaric acid is 0.39. Clearly, the dominant peak is ti compound with the same retention time as p-coumaric acid. Figure 6 is the chromatogram of the compounds eluted from the section of the TLC plate encompassing the Rf vrdue of vanillic and ferulic acid. Both compounds had R f values of -0.51. The major peaks have retention times that match those of vanillic and ferulic acid. Detection of the phenolic beer constituents in the section of TLC plate corresponding to the Rf values of the appropriate standards further confirms the accuracy of the identity assignment. An attempt at the verification of the Binapic acid identity assignment with the preliminary TLC step proved unsuc-
absolute amt spotted, absolute
aml, spotted, Pg
recovery
Pg
recovery
0.24 0.49 0.92 1.71 5.15 10.30
0.00
0.20
0.29 0.26 0.31 0.41 0.55
0.41
0.51 0.46 0.59 0.59 0.55 0.56
0.79 1.45 4.35 8.70
cessful. The Rf value of the cinnamic acid derivative was 0.44. The failure to detect sinapic acid emphasizes an experimental requirement concerning sequential TLC-LCEC. Performance of TLC prior to LCEC necessitates a careful evaluation of the behavior of the compounds of interest in terms of recovery from the TLC step. Table I11 is a summary of the recovered fractions of vanillic and sinapic acid when varying quantities of the phenolic derivatives were spotted on the TLC plate. Not surprisingly, sinapic acid exhibited erratic behavior, with the fraction recovered being dependent on the quantity spotted. In addition, when 1500 ng of material was spotted, none of the cinnamic derivative could be detected after elution of the silica with methanol. The failure to detect sinapic acid with sequential TLC-LCEC does not preclude its presence in the beer matrix. To the contrary, the retention characteristics and voltammetric data strongly suggest that the chromatiDgraphic peak is sinapic acid. However, the erratic recovery of sinapic acid from the TLC step deems its identity verification with sequential TLC-LCEC unreliable. Vanillic acid exhibited more predictablebehavior during the TLC step. The recatvery was consistent in the range of quantities evaluated. The difference in the recovery behavior of sinapic and vanillic ticid emphasizes the need to evaluate the recoveries of all compounds of interest prior to sequential TLC-LCEC experiments.
LITERATURE CITED (1) Felice, L. J.; King, W. P.; Klssinger, P. T. J . Agric. FoodChem. 1976, 24, 380-382. (2) Felice, L. J.; Kissinger, P. T. Anal. Chem. 1976, 48, 794-796. (3) Kenyhercz, T. M.; Kissinger, P. T. J. Agric. Food Chem. 1977, 25,
959. (4) Kenyhercz, T. M.; Kissinger, P. T. Lloydia 1978, 47, 130-139. (5) Roston, D. A.; Kissinger, P. T.; Bruntlett, C. S.; Evans, D. A., manu-
scrlplt in preparation.
(6) Geissman, T. A.; Crout, D. H. G. “Organic Chemistry of Secondary Plani Metabolism”, 1st ed.; Freeman, Cooper, and Co.: San Francisco, CA,kgS9; Chapter 5.
RECEIVED for review March 30,1981. Accepted June 8,1981.
