ANALYTICAI APPROACH
Hamptm H. Hairfield, &., and ~iizabethM. Hairfield
John Baker Daffin Labaatoy of Chemistw Mary Baldwin College Staunton. VA 24401
A late Bronze Age vessel, shipwrecked at Ulu Burun near southern Turkey, has been called the archaeological find of the century. Found in 1982, it is the oldest ship discovered (dating back to the fourteenth century B.C.) and ita diverse cargo spans seven civilizations (1, 2). Among the numerous items found were approximately 100 amphorae, or jars filled with a yellow resin. This resin was one of the most ahundant commodities in the cargo. The precise identification of the Bronze Age resin is important for two 0003-270019010362-041A1$02.5010 @ 1989 American Chemlcai Society
reasons. It is the fnst archaeological discovery in which resin has been found in abundance, and identifying the resin can provide information about the ship's point of departure and ita destination. Resins, the solid or sticky semisolid secretions from various plants and trees, have been used since ancient times to prepare incense, medicine, cosmetics, and perfumes. Today they are also used to prepare varnish and cement. Of the many resins that have been important since antiquity, terebinth, mastic, and frankincense (olibanum) are likely candidates for the Bronze Age resin because of their yellow color. The source of the terebinth resin is Pistacia terebinthus, a tree that grew abundantly in ancient times in the
graphical origin (6) and odor, it would he possible to confuse one resin for the other. Frankincense, one of the gifta of the Magi, came from Arahia and the Horn of Africa. It is secreted by trees of the genus Boswellia and could have made its way to Egypt via trade routes that date back to the fifth dynasty (2800 B.C.). However, there is no official record of the resin in Egypt until the 18th dynasty (4). ldentmcatlond resills For Complex mixtures such as resins, IR spectroscopyhas limited usefulness; the spectra only provide information about which functional groups are present in the substances. Because these resins contain both acids and esters, their spectra are virtually identical in the 4000-1250-cm-' region. However, resins often afford a characteris-
ANALYTICAL CHEMISTRY, VOL. 62, NO. 1. JANUARY 1. 1990
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tic band in the 1200-700-cm-' region. For example, amber can be identified by succinate absorption (a sharp peak preceded by a broad shoulder in the 1200-1150-cm-' region) (7). Unfortunately, no characteristic IR bands were found in the Bronze Age resin. Gas chromatographylmass spectrometry (GC/MS) has been used successfully to identify triterpenoids in resins. Sample preparation involves either steam distillation of the essential oil fraction (which is volatile and can be analyzed by GC or GC/MS) or extraction of the acidic fraction followed by esterification with diazomethane. John Mills and Raymond White of the National Gallery in London studied the Bronze Age resin using GC/MS. In the methano1:ether (L9)extract of the resin, they identified four acids as their methyl esters: oleanoic acid, moronic acid, masticadienonic acid, and ha-masticadienonic acid (Figure 1). Because these acids are characteristic of resins from the genus Fistacia, Mills and White concluded that the Bronze Age resin is from this genus (8). Although they did not carry out further analyses, they used an historical argument to speculate that the un-
Methyl momnate
Methyl isemasticadienonate
Methyl oleanate
Methyl masticadienonatf
:@re 1. Major acid constituents of the genus Plstacla resins (R
known resin is terebinth. This theory was based solely on their belief that mastic resin was in short supply during the Bronze Age. A large terebinth tree could easily yield up to 2 kg of resin, whereas the more viscous mastic resin
could only be collected one drop at a time. Thus Mills and White believed it was unlikely that the 100 jars discovered on the shipwrecked vessel could be faed with mastic resin. We felt that it was essential to pro-
m.
GCMS anaiyses were pafamedon a 15 m x 0.25 mm b o w capii~arycolumn i n w e d inm B gas Ohmnn-h equipped wlth a m wimlve nsilum gaa flaw rate d -1 RLImin was wed. The injector and me bansfer line were b t e d m 280 O C : hs colunn was tempsrahrs -am& k m 280 QC m 264 O C at 0.1 oClmln. men r a i d m 280 OC and held fw 7 mln.
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ANALYTICAL CHEMISTRY. VOL. 82, NO. 1, JANUARY 1, 1990
cient information for determining the precise source of the Bronze Age resin, In addition, the total ion chromatograms of the four major esters showed that the percent of each ester in the unknown resin does not match the percent of each ester in either mastic or terebinth resin (Table I).
duce chemical evidence to either corroborate or refute their theory. A chemical distinction between the resins was needed to confirm from which species the resins originated. Both terebinth and mastic contain the four acids that are found in the Bronze Age resin. The mass spectra of the four predominant acid constituents (as their methyl esters) of the Bronze Age resin are shown in Figure 2. Unfortunately, the relative intensity patterns of the esters from the acids do not match one resin more than another. Thus GC/MS did not provide suffi-
Thin-layer dvomatography A method that could differentiate these resins was needed to confirm from which species of Pistacia the unknown resin originated. Because we had distinguished commercialvarieties
Table 1. Percent of the four major esters in each regin determkrea by lon dvomatogaphy Rwln
oleanate
M W moronale
Molhyl maeIlcadlenona1e
bmutloadlenon.te
%wueM
10.7 17.5 20.7
34.4 23.6 42.1
334
21 4
Terebinth Mastic
Methyl
of frankincense using thin-layer chromatography (TLC) (9), we also used this method on the unknown resin. TLC can be highly selective for separating compounds with small differences in polarity or potential for hydrogen bonding. It is sometimes possible to distinguish compounds by color, even when they have identical mobilities. TLC can he used to analyze solids and liquids; for a complex mixture such as resin, TLC of fractions containing both solid and liquid componenta can provide more definitive identification. Once the resin has been identified, the chromatogram can serve as a figerprint for future analysis. Determination d the species Three known resins from the genus Pistacia: mastic (P. lentiscw), terebinth from Chioa (P.terebinthus), and terebinth from Cyprus (P.terebinthw, var. atlantica) and olibanum, from the genus Boswellia (B. papyrifera) were used for this study. A 20-mg chip of each resin was extracted with ethanol; a second 20-mg sample was extracted with ether, then separated into neutral and acidic frac-
I
in MS and SEM.
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CIRCLE 45 ON READER SERVICE CARD ANALYTICAL CHEMISTRY, VOL. 62. NO. 1. JANUARY 1, 1990
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ANALYTICAL APPROACH
CareinogeniCiEy
md Pesticides 'rinciples, Issues, and {elationships
J
usrpublishedA current perspechw on tne relationship between pesticides and cancer. A fimirf.its.md approach. rh6 oldme summarizes the l a w thlnlung on tne Nemcides-carcinogenesis redtionsnip. seen in he Ihght of new findings in cancer r e a r c h In a b.end of thearetical ana prachcal Infor. nanon. this timely work emmines the protess f carcinogenesis lWlf and wxierjs m m p b o strike a balance wtween increased agncbl. ural prod&" and the risk of cancer. Beiinning w t h an overview that p a the topic 1 a historical penpenre. Carcinqen,myand 'esdooes unfo.ds 10 target e y iss.es s.chas b the unrertainq of dab Merpmation In predim
tions; and an aliquot of the acidic fraction was esterified with diazomethane. rhe chromatograms of the methyl esters of the Bronze Age resin and of the Pistacia resins coniirm their close relationship. In both daylight and under UV light, the unknown resin can be dis;inguished from olibanum. However, ;he Bronze Age and the two terebinth :esins produced a series of spots with Rf :retardation factor) values greater than ;hat of the strong spots at 0.57. The absence of these spots from the mastic ?xtracts suggests that the Bronze Age resin is structurally similar to the tereKith resin. TLC of the acidic fractions provides widenee that the Bronze Age resin IKW179)favors the Cyprus terebinth resin (Figure 3). The small, faint spots above Rf= 0.60 (which are dark in daylight and pink under UV) are more pronounced in the Chios terebinth chromatogram than in the Bronze Age or 2yprus terehinth chromatograms. The acidic fraction of the Bronze Age resin dso produces spots at R( = 0.54 and Rf = 0.42. The intensity and color of these ipots resemble the stronger spots in ilibanum; consequently, we have wonjered whether the resin from Ulu Burun could be contaminated with
traces of olibanum. The TLC data from the neutral fractions also support the hypothesis that the Bronze Age resin is a terebinth resin. In addition, the chromatogramfrom the Bronze Age resin favors the Cyprus terehinth resin-the spot at Rf = 0.43 in the Chios terehinth chromatogram is absent. Finally, TLC of the ethanol extract shows that the Bronze Age resin is neither olibanum nor mastic resins (Figure 4). Moreover, the Chios chromatogram has a spot at Rf= 0.46 that is not seen in the Bronze Age or Cyprus terebinth chromatograms. Future dlrectiols From the TLC data, it is clear that the Bronze Age resin originates from P. terebinthus. However, several questions still remain unanswered. For example, is atlantica a distinct variety? Botanists disagree ahout whether there are different varieties of terebinth, yet our TLC data support the theory that at least two different varieties do exist. Furthermore, do the differences between the Bronze Age resin and the terebrinth resins result from evolutionary changes or chemical decomposition? We are continuing to study terebinth resin samples from different
Ing whnkr a peruclae will cause cancer in 1 b b
1 1
hU" W m a i t a m that anPn srawgonerlr
m w " s of &muI cardncqelllcm NnurpanlwIy reiallonrhlps rkh asessmem ap!dmloicg ofmmr and ps+Icide exposure
Iruce Ames. onginator of the Ames Test far arcinogenrity. conc.udes the book wth a hodghrhl dixussmn on current research indings and how the, relate to decisions onceming mdespreaa &e of pemcides in
aciety. IfyoJ are a xienhn. policy maker, or Y J . mor concerned m h the effecrs of pemcides in the environment. yo.'l find Canrnogenioty mdPertiodeaaralJabesynth~ofcontem. orary tninMng on a topic of cnhtal importance. laniy N. Ragsdale. Editor, U.S. D e p a r U " of \grKuhure loberl E. Memr. Edmr, University of vlaryland )eueopedfroman~posumsponsaredoymeOmion of&3mhemwairoftne Amencan Chemra Wev ~ C Symposium S Sertes NO. 414
E47 pages (IWS) Clothbound ISBN 04412-1703-3 U: 8918052 h r i w Chemkal Sam DirtrbuYmn Mke. Oept. 53
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Figure 3.
Thin-layer chromatography of the acid fraction.
nc was carried art on gi-
plates -I& wim a 250-em byer of silica pi developed to a height of 15 om wMI memylene chiorMe wmeining 2% acetone and vIsu811zed with ChiomSUifonlC acid (30% in acetic acid). Len: in daylight rlght: under 354-nm UV
4 4 A * ANALYTICAL CHEMISTRY, VOL. 62. NO. 1, JANUARY 1. I990
iim.
merit: C. William Saunders for assistance in operating the GCiMS instRImenti Martha N. Evans for asaistaneewith French translations;and ,James 9.Patrick. Lundy H. Pentz, Eric N. Jones,Joseph M. Garrison. Jr., and Ramona J. Bosserman for helpfulwmments. Theauthorsah thank the Virsinis Foundation for Independent Colleges for partial support of this work.
(1) Bans, G.
269-96.
(2)
F. Am. J. Archaeol. 1986. XI,
P W ,C. Am. J . Archaeol. 1988,92,
1-
I?
V. Recherches dArchOolo8ie de Philologie et d’Histoire. Tome 1 9 Lo ROsine de TMbinthe (Sonter)Chez lea An. ciens Egyptiens; Imprimerie de I’Institut Francais d‘Areh6alogie Orientale: Cairo,
&ioret,
Egypt, 1949.
(4) Groom,M.
Frankincense and Myrrh;
Longman: New York,1981. (5) Reutter, L.De l‘Embaumement Avant et Apr.?s J6sw Christ; Vigot Fr&res:Paris. 1912. ~., (6) Fluekinper, F.A,; Hanbury, D. Pharmocogmphia: A History of the Principal Drugs of Vegetable Origin Met with in Great Britain and British India; Mamillan:London, 1819. (I) Beck,C.W.Appl. Spectrose. Rev. 1986, 22,51-110. (8) Milla. J. S.; White, R Archaeometry 1989,31,31-44. (9) Hairtield,E. M.; Hsirfield,H. H.;Pmtz, L. H.Perfum. Flavor. 1984,9,33-36.
trees in various locations in an effort to resolve these questions. Is the unknown resin contaminated with olibanum? This question is not easily answered. We attempted to identify the compounds whose Rf values suggest olibanum by scraping these spots from the TLC plates followed by extraction with methylene chloride, esterification with diazomethane, and GC analysis. However, these results were not conclusive. The peaks obtained had retention times that were substantially shorter than the retention times in the original sample. It is possible that acidity caused the sample to deteriorate, so we also extracted some methyl ester spots from pure ,9-hweUic acid and &acetyl boswellic acid (the compounds that would be present if the sample were contaminated with olibanum) and performed GC analysis. This procedure was also unsuccessful. We concluded that deterioration occurs whenever these materials are run on a TLC plate. Repetition of this procedure in an inert atmosphere
has not yet been tried. Determining the precise origin of the unknown resin can help archaeologists to better understand the Bronze Age civilization. The fact that the ship was probably traveling to Egypt sheds light on ancient trade routes. The quantity of terebinth found supports Egyptian records regarding the vast quantities of incense that were burned. However, a more extensive comparative study of the terebinth resins is needed to elucidate mechanisms of decomposition and to help botanists better understand the history and structure of the terebinth species. We hope that this aspect of the mystery can be solved in the near future through the application of known analytical techniques. k Curt Beck of Vassar College for providing the Bronze Age resin aampleq John S. Mills of the National Gallery,London, and Rupert Hastings of the Royal Botanical Gardens for providing the known botanicals;Harold McNair and Larry Teylorof V i i n i s PolytechnicInstituteand State University for use of their GCiMS imtru-
The authors h
B.S. iegree in eduiation f r o m the University of Virginia i n 1963 and. until his retirement in 1988, was a member of the faculty of Wilson Memorial High School (Fishersuille, VA).His interest in resins stems from childhood, when he was intrigued by the ceremonial use of incense in t h e Catholic Church. Elizabeth M. Hairfield received a B.S. degree from Wheaton College (IL) in 1961 and a Ph.D. in chemistry from Bryn Mawr College in 1970. S h e then joined the faculty o f Mary Baldwin College, where she is a professor of analytical and physical chemistry.
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