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Talanta 51 (2000) 727 – 734 www.elsevier.com/locate/talanta

MCF fast derivatization procedure for the identification of resinous deposit components from the inner walls of roman age amphorae by GC-MS M. Dolores Petit-Dominguez a,*, Julio Martinez-Maganto b a

Departamento de Quı´mica Analı´tica y Ana´lisis Instrumental, Facultad de Ciencias, Campus de Cantoblanco, Uni6ersidad Auto´noma de Madrid, 28049 Madrid, Spain b Departamento de Arqueologı´a, Facultad de Filosofı´a y Letras, Uni6ersidad Auto´noma de Madrid, 28049 Madrid, Spain Received 12 July 1999; received in revised form 26 October 1999; accepted 8 November 1999

Abstract Resinous deposits from the inner walls of Roman age amphorae have been analysed by GC-MS. A simple dissolution of the sample in chloroform was carried out without any previous treatment of the sample. A new method for derivatization of carboxylic groups in these type of compounds was tested using methylchloroformate (MCF) in the optima conditions, giving good results with a short derivatization time. Some diterpenoid components of conifer resin have been identified, as well as other monoterpenoid components of essential oils. Results obtained allowed to know the nature and origin of these resinous deposits and the early Roman age technological practices in Spain. © 2000 Elsevier Science B.V. All rights reserved. Keywords: MCF derivatization; GC-MS; Resinous deposits; Archaeometry

1. Introduction The nature and purposes of resinous deposits and pitches found covering the inner walls of Roman age amphorae have been subject of controversial discussion in the last years [1–6]. Among the purposes suggested by several authors are: (a) their use as water-proofing agents due * Corresponding author. Tel.: +34-91-3974041; fax: + 3491-3974931. E-mail address: mdolores.petit@uam.es (M.D. PetitDominguez)

their hydrophobic properties, (b) wines flavoured with liquid pitch were very popular among the Romans and (c) pitches were used to seal terracotta amphorae for storage and export of wines and other food products. Concerning the nature and composition, resin is the name given to a series of products exuded spontaneously by a large number of trees and plants. The chemistry of resins is diverse but most are composed of compounds belonging to the extensive chemical class known as the terpenoids, made up of units of the C5 compound isoprene [7,8]. Natural resins contain mono- (C10 com-

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pounds), sesqui- (C15 compounds), di- (C20 compounds) and triterpenoids (C30 compunds), but interestingly, di- and triterpenoids are not found together in the same resin [8]. According to the classical authors (Plinio, Vitrubio, Escribonio and Columena), natural resins used to be heated to obtain liquid pitches. In this way, it was able to cover the inner walls by rotatory movements of the amphora. After this, pitch was cooled down, with the amphora in vertical position, and converted into a shiny brown solid, remaining an important amount of pitch at the bottom of the amphora [9]. Different analytical techniques have been applied to the analysis of amphora inner walls (resinous deposits and other organic residues absorbed and retained by the clay), such as TLC, HPLC, IR and GC for the identification of diterpenoid compounds and food traces [10 –12]. Recently, the GC-MS technique has been applied to the identification of terpenoid compounds in different environmental samples. [13 – 18]. The aim of this work was to determine the chemical composition of the organic constituents of the resinous deposits obtained from the inner walls of two types of amphorae. To reach our objetive, we needed to perform a method of analysis that allowed to use a low amount of sample, with a simple handling and where no major energy and time were involved. For these reasons, we have chosen a new method for derivatization of carboxylic groups in these type of compounds in a microscale level using methylchloroformate (MCF) as derivatization agent [19 – 22]. On the other hand, reference materials were not available

for this kind of sample. Therefore the analytical method should not required these substances. In this sense, the GC-MS was choosen as instrumental analytical technique that allowed to obtain the mass spectra of the different components, previously separated on a chromatographic column. Results obtained for the two types of amphorae and for a natural pine resin were compared, in order to identify the nature of the samples. From the chemical composition of the samples, we could deduce if the resins have been subjected to a heating process or an adulterant addition; establishing or corroborating early technological practices of Romans in Spain.

2. Experimental The archaeological samples investigated as well as the natural pine resin for comparison are described in Table 1.

2.1. Chemicals Care was taken to keep the contamination to a minimum and ultrapure water was used throughout. Chloroform, methanol, acetonitrile, pyridine and sodium bicarbonate were obtained from Merck and methyl chloroformate (MCF) from Aldrich.

2.2. Sample treatment All fragments of the resinous deposits were taken from the inner part of Roman age am-

Table 1 Description of investigated samples Sample

Appearance

Origin

Natural Pine Resin Resinous deposit 1

Shiny and light brown solid Shiny and dark brown solid

Resinous deposit 2

Shiny and dark brown solid

Pine resin from Avila (Spain) Amphora. Dresell 11 variant. Number 503 Museum of Ceuta (Spain). Aquatic environment. Amphora type Beltran IV. Number 15882. Museum of Almeria (Spain). Aquatic environment


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phorae. Normally, these resinous deposits were formed by a variable thickness layer of about several millimeters in the wall of the amphora and even 1–2 cm at the bottom of this. Samples were obtained taking a minimum part, to prevent a damage of archaeological object, of these layers with the aid of a scalpel. Samples obtained in this way were converted into powder and about 10 mg were dissolved in 5 ml of chloroform. A dark brown solution was obtained in all the cases except for the natural pine resin where a light yellow solution was obtained.

2.3. Deri6atization procedure A mixture of acetonitrile/water/methanol/ pyridine in a 7:1:1:2 ratio was used as solvent, to provide the reaction medium. One hundred microlitres of this solvent and 5 mL of MCF were shaken during about 10 s and mixed with 100 mL of sample (dissolved in chloroform). Finally, 100 mL of 1 M NaHCO3 were added and the reaction mixture was thoroughly shaken. The reaction was completed after 1 min. The chloroform phase was separated, dried with anhydrous sodium sulphate and subjected to GC-MS analysis.

2.4. GC-MS analyses The GC-MS analyses were carried out on a Hewlett–Packard 5890, series II, system; under the following conditions: 25m×0.28mm i.d. fused silica column coated with 0.25 mm 5% phenylmethylsilicone; carrier gas, helium (1 mL/min); column temperature, programmed from 70°C (1 min) to 210°C (20 min) at 20°C/min; injector block temperature, 180°C; detector temperature, 280°C; and sample size, splitless 1 mL. The mass spectrometer was run in the scan mode, where a series of mass spectra were acquired in a continuous mode by the spectrometer. A total ion chromatogram (TIC) was obtained in each analysis. Every point of this chromatogram represented a mass spectrum that could be analysed by the system and compared with those kept in the library of the data base.

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3. Results and discussion

3.1. Deri6atization procedure Using GC, most of the active groups in a molecule have to be chemically converted to inactive species to supress adsorption onto the analytical column and to increase the volatility and/or temperature stability of the sample. The preparation of sample derivatives is usually a time-consuming part of an analytical procedure and it is desirable that it could be carried out as quickly as possible with a minimum number of reaction steps. Many compounds that cannot be analyzed directly using GC are derivatized by silytation or diazometane reactions. Among the disadvantages of silytation produces are the need of silylate under anhydrous conditions, heating the sample for a given period and having to inject the reactive mixture onto chromatographic column. Methylation of the acids with diazometane is easy to perform but double peaks and side-products are obtained. Alkyl chloroformates are a serie of compounds that react with many functional groups, even in aqueous solution, such as mono- and dicarboxylic acids, keto and hydroxy carboxilic acids, amino acids, aromatic acids, biogenic amines, etc. Taking this into account, we propose the use of methylchloroformate (MCF) to obtain an rapid reaction where the methyl esters of carboxilic acids groups, present in the samples, are formed at room temperature. In this derivatization procedure, both organic base and solvent composition had to be optimized. Pyridine was preferred as the organic base because the results of carboxilic acid esterification on a microscale were better and, in addition, an optimum amount of pyridine led to methyl esters formation and to the absence of other side-products as mixed anhydrides. When an insufficient amount of pyridine was used, the derivatization reaction did not take place or led to side-products formation, and when an excesive amount of this was used an interferent peak of pyridine appeared in the chromatograms. The ratios among solvent components had to be optimized too, in order to


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Table 2 Identified compounds found in resinous deposit samples by GC-MS Sample

Natural pine resin

Retention time (min) 3.18 7.68 11.48 12.19 17.21

19.91 Resinous deposit 1

5.30 5.77 7.85 13.66 16.98 19.80

Resinous deposit 2

11.77

12.49 13.05

14.84 17.41 19.79

Compound

a-Pinene C10H16 Cadinene C15H24 Phenoxybenzyl acetate C15H14O3 3H-Phenothiazinone C12H7NOS 1,4-Cyclohexadiene 3,3,6,6-tetramethyl C10H16 Abietic acid, methyl ester C21H30O2 Camphor C10H16O Endoborneol C10H18O Cadinene C15H24 Molecular sulphur S8 (E,E) 2,5 Diphenil-2,4hexadiene C18H18 Abietic acid, methyl ester C21H30O2 Naphtalene 1,2,3,4.tetrahydro1,1,6-trimethyl C13H18 Phenanthrene 2,5dimethyl C16H14 10-Methoxybenzen [a] azulen- 1,4-dione C15H10O3 Phenanthrene 2,3,5trimethyl C17H16 (E,E) 2,5 Diphenil-2,4hexadiene C18H18 Abietic acid, methyl ester C21H30O2

reduce side-product formation, as well as the molar ratio of base to MCF, which determined whether the mixed anhydrides or esters were formed. Taking all these aspects into account, different experiments carried out with the pine resin showed the adequate solvent to provide an optimum reaction medium was a mixture of acetonitrile/water/methanol/pyridine in a 7:1:1:2 ratio, using 100 ml of this solvent per 5 ml of MCF and

100 ml of the dissolved sample. In these conditions only 1 min was necessary to complete the derivatization reaction. An additional advantage of this derivatization procedure was the simple dissolution of the sample in chloroform, without any previous treatment.

3.2. Total ion chromatograms TIC obtained for the three considered samples are shown in Fig. 1. In Table 2 are the retention times of the compounds which allowed the component identification by comparing with those kept in the library of the data base. The corresponding mass spectra of some of these compounds can be seen in Fig. 2. Results of GC-MS analyses suggest that both deposit 1 and deposit 2 are of similar generic origin, been abietic acid the common constituent. This diterpenoid is the more abundant component of resin from Pinus species where laevopimaric, palustric, abietic and neoabietic acids are interconvertible and the equilibrium among them contains predominately abietic acid. Monoterpenes are relatively volatile components when crude pine resin is subjected to a heating-treatment. In this way, the a-pinene monoterpene appear in the natural pine resin extract while deposit 2 has a total absence of monoterpenes. The dark brown colour of the extract in deposit 2 indicates a probable heatingtreatment of this sample, which can originate three probable effects: 1. terpenes volatilization; 2. thermal dehydrogenation; and 3. decarboxylation of part of the abietic acid. Both, (2) and (3) can lead to a rise of other phenanthrene derivatives. Although the dark brown colour of extract in deposit 1 suggests a heating-treatment of this sample too, two different monoterpenes are present (camphor and endoborneol). An explication for this can be that some essential oils could be added, after the heating-treatment, to the resin in order to alter its physical properties (e.g. consistency and softening point or to obtain flavoured wines). In the same way, the presence of sulphur can be taken as an


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Fig. 1. Total Ion Chromatograms: (A) natural pine resin; (B) resinous deposit 1; (C) resinous deposit 2.

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adulterant (e.g. to prevent microorganism growth in wines). A sesquiterpene (cadinene) is present too in natural pine resin and in the deposit 1 samples. Its structure is shown in Fig. 3, as well as other mono- and diterpenoid compounds found in the analysed samples.

4. Conclusions A method for derivatization and identification of resinous deposit components has been performed. Derivatization step was carried out using MCF, in the optima conditions, as derivatization agent. A low amount of sample with a minimum

Fig. 2. Mass spectra of some terpenoid compounds and other components identified in the considered samples: (A) camphor; (B) cadinene; (C) endoborneol; (D) molecular sulphur; (E) (E,E) 2,5.diphenil-2,4-hexadiene; (F) abietic acid, methyl ester.


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Fig. 3. Structures of same diterpenoid and monoterpenoid components of resin from Pinus species.

handling, as well as a short time for the reaction were required in this step. This novel derivatization procedure for these type of archaeological samples, gave good results in the formation of the methyl ester of abietic acid, principal component of pinus resin. The GC-MS system allowed us to identify components of the samples in absence of reference materials. In this way, a total absence of monoterpenes are present in deposit 2 and other components appear such as diverse phenanthrene derivatives, probably due a heating-treatment of the samples, something corroborated too by the dark brown colour of extracts obtained from archaeological samples. On the other hand, some components normally not found in these type of samples seem to have been added to the resin as adulterants, to reduce the cost of the pitch or to alter its physical properties. The presence of abietic acid in all the samples suggested a similar generic origin of the resins, probably obtained from Pinus species in the Mediterranean area.

Acknowledgements Authors are grateful to Junta de Andalucia for the financial support and to Dr Rucandio-Sa´ez and Dr Garcı´a-Gime´nez for many useful suggestions.

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R13_Análisis MCF Resina Ánfora (Talanta 51_ 2000)