374
Anal. Chem. 1901, 53, 374-377
(11) Honocks, W. D., Jr.; Sudnlck, D. R. Sclence 1979, 206, 1194. (12) Haw, Y.; Stein, G. J . Phys. Chem. 1971, 75, 3868. (13) Sawada, T.; Oda, S.; Shlmlzu, H.;Kamada, H. Anel. Chern. 1979, 57, 888. (14) Stone, J. Appl. Phys. Lett. 1975, 26, 183. (15) Stone, J. Appl. Opt. 1978, 77, 2876. (18) Long, M. E.;Swofford, R. L.; Albrecht. A. C. Sclencs 1976, 797. 183. (17) Swofford, R. L.; Long, M. E.; Albrecht, A. C. J. Chem. Phys. 1976, 65. 179. B U ~ ~ M ~M.Y s.; , w e l l , J. A.; Albrecht, A. C.; Swofford, R. L. J. Chem. Phys. 1979, 70, 5522.
Moses, E. I.; Tang, C. L. Opt. Lett. 1979, 7, 115. Tam, A. C.; Patel, C. K. N.; Kerl, R. J. Opt. Lett. 1979, 4 , 81. Patel, C. K. N.; Tam, A. C. Appl. phys. Lett. 1979, 34, 467. Patel, C. K. Cn.; Tam, A. C. Chem. Phys. Lett. 1979. 62, 511. Patel, C. K. N.; Tam, A. C.; Kerl, R. J. J. Chem. Phys. 1979, 77, 1470. (24) Tam, A. C.; Patel. C. K. N. Opt. Lett. 1980, 5 , 27. (25) Kaye, W.; McDaniel, J. B. Appl. Opt. 1974, 13, 1934. (19) (20) (21) (22) (23)
Received for review May 1,1980. Accepted October 10,1980.
Gel Chromatography for the Isolation of Phenolic Acids from Tobacco Leaf M. E. Snook,' P. J. Fortson, and 0. T. Chortyk Tobacco Safety Research Unit, Sclence and Education Administratbn/Agricuitural Research, United States Department of Agriculture, P.O. Box 5677, Athens, Georgia 30613
Phenolic acids are known to occur widely in plants. Generally, paper or thin-layer chromatography is used for its isolation and identification in plant extracts (1-3). However, individual acids are difficult to quantitate by these methods, and only the major components can be determined. Our recent Sephadex LH-20 gel chromatographic work on the isolation of dihydroxybenzenes of cigarette smoke ( 4 ) suggested to us the applicability of this procedure to the isolation of leaf phenolic acids. Our interest in leaf phenolic acids arises from their possible role as precursors of the tumorigenic tobacco smoke catechols. Since phenolic acids are similar in polarity to dihydroxybenzenes (catechols, resorcinols, hydroquinones), they should be adsorbed and separated by the gel in a similar manner. To our knowledge, the application of gel chromatography to the isolation of plant phenolic acids has not been previously reported.
EXPERIMENTAL SECTION Extraction of Tobacco Leaf. NC 2326 flue-cured tobacco was ground to pass a 32-mesh screen. The tobacco (200 g dry wt) was ground with 2.5 L of 1.0 N NaOH for 5 min in a Waring blendor. The resulting slurry was filtered through no. 2 Whatman filter paper by vacuum, and the residue was washed with 500 mL of HzO. The total filtrate was filtered again and extracted with 600 mL of ethyl acetate (EtOAc). The aqueous solution was acidified to pH 1.0 (6 N HCl), saturated with NaCl, and extracted with EtOAc (3 X 600 mL). The EtOAc solution was dried over anhydrous MgSO,, filtered, and evaporated to yield 6.9 g of a leaf acids extract (3.4% yield). The acids extract was dissolved in 25 mL of MeOH/CHC13 (l:l,v/v) in preparation for gel chromatography. Gel Chromatography, The gel column was a 1.25-cm i.d. X 55cm LC-type column (Laboratory Data Control, Riviera Beach, FL), packed with Sephadex LH-20 in CHC13. Solvent flow was 2 mL/min and 5-mL fractions were collected. One-milliliter aliquots of acids extract were introduced on the column with a loop injection valve. For the standard phenolic acids, the initial CHC13 solvent was programmed from CHC13 to 10% MeOH/ CHC13from gel fractions 40 to 55 and subsequently held at 10% MeOH/CHC13. The solvent program for the tobacco acids extract was similar to that for standards, but to hasten elution of the dihydroxy aromatic acids, the solvent was programmed from 10 to 40% MeOH/CHC13 from gel fractions 110 to 115. Eluted materials were monitored at 280 nm, and gel fractions (GF) were pooled to yield fraction A (GF 65-84) and fraction B (GF 85-122). Gas Chromatography (GC). The pooled fractions A and B were concentrated under house vacuum on a rotary evaporator to a small volume (about 250 pL), Aliquota of fractions A and B were added to an equal volume of N,O-bis(trimethylsily1)trifluoroacetamide (BSTFA) in microreaction vials fitted with Teflon-lined caps. The trimethylsilyl (Me3Si)derivatives were prepared by heating the mixtures at 86 "C for 15 min. The derivatives were analyzed with a Hewlett-Packard 5830 gas
chromatograph,equipped with a 183-cm x 2-mm i.d. g h column packed with 6% OV-17 on 100/120 mesh Chromosorb G-HP (temperature program, 90-250 OC at 2 "C/min; He flow, 20 mL/min; injector, 290 "C; flame detector, 300 "C). Compounds were identified by comparison of GC retention times to those of standards, by coinjection with standards, and by GC-mass spectral (MS) data. MS data on the Me3Si derivatives were obtained on a 5930A Hewlett-Packard mass spectrometer, interfaced to an HP 5710 gas chromatograph equipped with an identical OV-17 column. W-Recovery Studies. Sali~ylic-~~C acid (ICN Pharmaceuacid (California ticals, Inc., Irvine, CA) and p-hydroxyben~oic-~~C Bionuclear Corp., Sun Valley, CA) were purified by silicic acid column chromatography (100-gcolumn of Mallinckrodt, 100-mesh silicic acid). The columns were eluted successively with 1L each of 4% diethyl ether (E)/petroleum ether (PE), 10% E/PE, and 50% E/PE. The 50% E/PE fractions were evaporated and dissolved in 1 L of MeOH. Aliquota of the purified labeled compounds were diluted 1:l with CHC13and chromatographed on the gel column. Recovery of the 14C acids was measured by standard liquid scintillation counting techniques. Aliquota (5 mL, 100OOO dpm) of each standard were added to additional NaOH extracts of tobacco. The mixture was acidified and extracted with EtOAc. The extract was subjected to gel chromatography, and the GF were counted.
RESULTS AND DISCUSSION Sephadex LH-20 gel with chloroform as solvent will separate compounds on the basis of hydrogen bonding effects. Phenolic materials are strongly adsorbed by the gel and require the addition of MeOH in order to be eluted in a reasonable time. In contrast, hydrocarbons, ketones, aldehydes, alcohols, and amines are not retained and are eluted with the void volume of the column or shortly thereafter. This property of LH-20 is ideally suited to the separation and purification of phenolic materials from complex mixtures. The elution characteristics of several phenolic acids from a Sephadex LH-20 column are shown in Figure 1. Acids with internal hydrogen bonding (such as sinapic, ferulic, salicylic, and vanillic) were eluted earlier than hydroxyphenylacetic, coumaric, and monohydroxybenzoic acids. Dihydroxybenzoic acids were eluted much later. Interestingly, cinnamic acids were eluted before the benzoic acids. Other compounds, containing two or more hydroxyl groups, were also found to elute in the same GF as the phenolic acids. These included dihydroxybenzaldehydes and both dicarboxylic and tricarboxylic aliphatic acids. The gel elution characteristics of the dihydroxybenzoic acids were investigated further and are shown in Figure 2. The observed elution order of the dihydroxybenzoic acids appears to depend strongly on steric, electronic, and internal hydrogen bonding factors. I t was also noted that, except for the 2,6-
This artlcle not subject to U S . Copyrlght. Publlshed 1981 by the Amerlcan Chemlcal Society
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Flgure 4. Gel chromatographic elution of the total acids extract of tobacco leaf on Sephadex LH-20. (Same solvent program as in Figure 1, except after GF-100, solvent programmed to 40% MeOH/CHCI,.) and not some impurity. This may be a result of the elution of dimers and trimers of the solutes. These elution irregularities did not affect the isolation of the phenolic acids of tobacco. Indeed, the elution properties of LH-20 were successfully utilized to isolate the phenolic acids from tobacco leaf extracts. The developed extraction scheme for tobacco leaf acids is shown in Figure 3. Flue-cured tobacco leaf was extracted with aqueous sodium hydroxide to separate tobacco acids as their water-soluble salts. Acidification and extraction with ethyl
376
ANALYTICAL
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acetate gave an acids extract representing 3.4% of leaf weight. A portion of the acids extract was chromatographed on Sephadex LH-20 gel and the resulting elution curve (280 nm) is shown in Figure 4. The elution of the phenolic acids was quite apparent and appropriate gel fractions were combined to give fractions A and B. Fractions A and B were analyzed by GC on an OV-17 glass column as their Me3% derivatives. Identifications were made by coinjection of standards, GC retention times, and GC-MS analyses of the separated peaks. Mass spectra of Me3Si standards were compared to those of the isolated compounds. Most of the aromatic acids yielded good Me& molecular ions (M+) and M+ - 15 ions. Aliphatic compounds gave M+ - 15 ions with virtually no molecular ions, unless a double bond was present in the molecule. The purification of the phenolic acids, achieved by the single gel chromatographic step, is evident from comparisons of the gas chromatogram of the total acids extract (Figure 5) and those of fractions A and B (Figures 6 and 7). The phenolic acids (other than caffeic) appeared as minor components in the total acids extract but were greatly enriched by the gel chromatography step. This purification greatly facilitated identification and quantitation. Phenolic acids identified in fraction A (Figure 6) included salicylic, m- and p-hydroxybenzoic, p-hydroxyphenylacetic, vanillic, cis- and trans-p-
coumaric, ferulic, caffeic, and sinapic acids. Also found were 3,4- and 3,5-dihydroxybenzaldehydes.Isolated aliphatic acids included malonic, succinic, fumaric, malic, and adipic acids. Several broad peaks were observed to elute very late in the gas chromatogram (not shown) and were identified as chlorogenic acid and two of its isomers. Apparently, the dilute sodium hydroxide solution, used to extract the tobacco, did not hydrolyze all of the chlorogenic acids to caffeic acid. The major components of fraction B (Figure 7) were malic and cis- and trans-caffeic acids, with minor amounts of citric, p-hydroxybenzoic, and both 2,s- and 3,4-dihydroxybenzoic acids. The characterized phenolic acids in fractions A and B are summarized in Table I. On examining standard acids, we found that the six isomeric dihydroxybenzoic acids eluted in three peaks from the OV-17 column. The 2,3-isomer eluted first, followed closely by a peak consisting of the 2,5- and 2,6-isomers, which was followed by a peak consisting of the 2,4-, 3,4-, and 3,5-isomers. However, MS data of the MeBSiderivatives can be used to differentiate between coeluting isomers (5). One can determine which of the 2,5- or 2,6-isomers is present by the presence or absence of an ion a t m / e 269 in the mass spectrum. The absence of the 269 ion in GFs of fraction B confirmed that only the 2,5-dihydroxybenzoic acid was present. MS also confirmed that only the 3,4-dihydroxybenzoic acid was present in the
377
Anal. Chem. 1981, 53, 377-381
i Figure 7. Gas chromatogram of Me,SMerivatized acMs in fraction 6.
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peak where the 2,4-, 3,4-, and 3,5-isomers would have coeluted. This was shown by the presence of an ion at mle 193, which was also the base peak of the spectrum. The other isomers have virtually no 193 ion in their mass spectra. This ion is a result of a cyclic rearrangement containing a silyl group bridged by oxygens at ortho positions of an aromatic ring (5). Horvat (6) has shown that the ion appears to be universally formed whenever adjacent hydroxyl groups exist on an aromatic nucleus. This cyclic ion was demonstrated in the mass spectra of MegSi derivatives of dihydroxybenzaldehydes, caffeic acid, gallic acid, and even catechol.
The quantitative recovery of phenolic acids by the above method was determined with I4C-labeled salicylic and p hydroxybenzoic acids. Aliquots of the purified compounds (in methanol) were added to the sodium hydroxide leaf extract. The extract was acidified and extracted with solvent, and the recoveries of sali~ylic-'~C and p-hydroxybenzoic-14Cacids were determined to be 93.1 and 95%, respectively. The recovery of the 14C-labeledcompounds from the gel chromatographic step could not be determined in the presence of leaf extract material, due to interfering fluorescent material. However, when chromatographed on the gel column by themselves, ~alicylic-'~C and p-hydroxyben~oic-'~C acids were recovered in yields of 90.0 and 99.3%,respectively. Therefore, the entire procedure was quantitative for these two compounds and probably also for all of the phenolic acids. This gel chromatography procedure is now being applied to the characterization of the phenolic acids of cigarette smoke, contained in a tumorigenic, acidic fraction of cigarette smoke condensate. This acid fraction has already been shown to contain numerous alkylated phenols and catechols, and any dihydroxy phenolic acids that are present may also contribute to the biological activity of tobacco smoke. This developed procedure should be applicable to the characterization of phenolic acids in any plant extract or organic mixture.
LITERATURE CITED (1) Rlbereau-Gayon, P. "Plant Phenolics"; Oliver and Boyd: Edinburgh, Great Britain, 1972; Chapter 4. (2) Sugano, N.; Iwata, R.; Nishi, A. phvrochemlsfry 1075, 74, 1205-1207. (3) Fritz, 0. J.; Andresen. B. D. Phytochemistry 1078, 77, 581-582. (4) Snook. M. E.; Fortson, P. J. Anal. Chem. 1070, 57, 1814-1819. (5) Morita, H. J. J. Chromatogr. 1072, 77, 149. ( 8 ) Horvat, J. R.; Senter, S. D. J. Agric. F w d Chem. 1080, 28, 1292-1295.
RECEIVED for review July 21,1980. Accepted November 17, 1980. Reference to a company or product name does not imply approval or recommendation by the USDA.
Differential Pulse Anodic Stripping Voltammetry Control Device With Digital Voltmeter E. B. Buchanan, Jr.,. and D. D. Soleta Chemistry Department, Unlverstty of Iowa, Iowa Ctty, Iowa 52242
The determination of heavy metals, Zn, Cd, Pb, and Cu, a t the parts-per-billion (ppb) level by use of differential pulse anodic stripping voltammetry (DPASV) requires that ex0003-2700/81/0353-0377$01 .OO/O
perimental parameters such as deposition time, drop size, temperature, cell geometry, and stirring rate, if stirring is used, be carefully controlled to achieve reproducible results. Some, 0 1981 American Chemical Society
