Applied methods 1. Archaeological samples

3.2.1.1. Sample preparation

The main objective of the analysis of organic residues is the characterization of lipids eventually preserved in the ceramic matrix and which have survived degradation. This technique is destructive for the archaeological material, since the ground of pottery is extracted.

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As described in Chapter 2, the samples were taken from the lip or base of the containers, since the edge is more susceptible to contamination (Regert 1999). The samples taken are representative of the diversity of shapes of each of the ceramic sets studied, which implies a previous ceramic analysis.

In order to avoid modern contamination, sample preparation was done in the cleanest environment posible: using nitrile gloves, safety vessels and samples were stored in al-uminium foil in a cold environment.

In this work, 200 samples from 9 sites in the Iberian Peninsula have been analysed: Coro Trasito, Cova del Sardo, Camp del Colomer, Carrer Llinàs 28, Feixa del Moro, La Draga, Mines de Gavà, Cabecicos Negros and Cueva de El Toro.

3.2.1.2. Sample extraction 3.2.1.2.1. Extraction strategy

A part of the lipids is easily extractable from the use of organic solvents. However, an-other part of these lipids remains insoluble due to strong bonds or polymerization be-tween the organic molecules and the ceramic wall (Aillaud 2001, Correa-Ascencio &

Evershed, 2014, Craig et al. 2014, Regert et al 2001b).

This raised the need to apply new extractive methods, alternatives to the conventional extraction of diclormethane or chloroform/methanol, in order to increase the efficiency in the extraction of organic molecules from archaeological ceramics, such as extraction by hydrolysis (Craig et al. 2004), extraction by alkaline hydrolysis (Aillaud 2001, Copley et al. 2005e, Craig et al. 2004, Regert et al. 1998, 2001b) or by TMTFTH extraction (Stern et al. 2010). These complementary methods have favored the recovery of identifiable lipids in contexts where acidic soil conditions and atmospheric conditions most affect the degradation of organic compounds (Correa-Ascencio & Evershed, 2013; Goldenberg et al. 2014; Papakosta et al. 2015).

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In this thesis, microwave-assisted extraction has been used (Gregg et al. 2009, Gregg and Slater 2010). The use of microwaves favours the mixing of organic solvents with ceramic powder, applying temperature and magnetic stirrers, in order to recover or-ganic compounds in contexts where they are affected by high degradation.

Furthermore, in cases where the lipid concentration is lower than 5μg, samples are reex-tracted by acid extraction (Correa-Ascencio and Evershed, 2013; Goldenberg et al. 2014;

Heron et al. 2015; Papakosta et al. 2015; Stern et al. 2000). Lipid extractions with meth-anol acid have a significant effect on some of the lipid residues found in archeological ceramics that must be kept in mind. The main residues identified are fatty acids, which are esterified in fatty acid methyl esters (FAMEs), whereas compound lipids, such as acylglycerols or wax esters, are hydrolyzed in their constituent acids - converted into methyl esters - and in alcohols (Correa-Ascencio & Evershed 2014). Other residues, such as alkanes or ketones, remain unaltered, however, derivatization with BSTFA is neces-sary to protect alkanes as TMS esters in total lipid extracts.

Figure 3.2. Extraction protocol followed in this thesis.

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In order to increase the efficiency in the extraction of lipids and the interpretation of the results, a methodological strategy has been followed that starts from the storage of ce-ramic fragments in the refrigerator wrapped in aluminium foil, to avoid degradation (Fig-ure 3.2). Once the sample is prepared, it is extracted following the microwave-assisted organic solvent extraction protocol and analysed by chromatographic gas analyses (GC-FID). In cases where the samples present interpretable organic residues, they are ana-lysed by gas chromatography-mass spectrometry analyses (GC-MS) to identify the com-pounds. Finally, samples with sufficient concentrations of C16:0 and C18:0 are analysed for the isotopic value of δ¹³C of both by analysis in a chromatographic gas stable isotope mass spectrometer (GC-IRMS).

In case sufficient lipid concentrations cannot be identified for interpretation in GC-FID, the ceramic powder is re-extracted by acid extraction, cases of Coro Trasito, Camp del Colomer, Carrer Llinàs 28, Feixa del Moro, Cueva de El Toro and Cabecicos Negros. The sample is re-analysed in GC-FID and, if sufficient lipid concentrations are identified, it is rederivatised with BSTFA in order to detect alcohols and alkanes and then analysed by GC-IRMS. This method is used in case extraction by organic solvents fails, since this tech-nique involves a loss of information on the conservation and nature of the heavy com-pounds, such as esters or triacilglycerides.

3.2.1.2.2. Organic solvent extraction

For lipid extraction of potsherds at the ICTA-UAB (Barcelona, Spain), the surface of a sub-sample of the archaeological potsherds was cleaned with manual modelling drill (Dremel 4000) to remove exogenous lipids (Heron et al. 1993, Stacey 2009); ca. 2 g of ground from the inner surface was taken with the cleaned drill and then stored in a vessel tube previously muffled. The powdered samples were extracted using a mixture of 10mL dichloromethane and methanol (3:1 v/v) after addition of 50 μL of internal standard (IS) (n-tetratriacontane). In addition to the archaeological samples, four blanks were analysed, two with the same internal standard (IS) and mixture of solvents and two only with the mixture of DCM and MeOH. Lipids were extracted using a microwave (MarsX, CEM) at 70 °C for 10 min. The resulting extract was decanted, in each case, into

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vessel tubes and dried by exposure to a gentle nitrogen stream. Next, each sample was dried by eluting them through vessel columns filled in with anhydrous sodium sulphate.

After removing the solvent, the dry extracts were redissolved in 50 μL of DCM and deri-vatised with 50 μL of BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide) and the mixture heated at 70 °C for 60 min. After removing the derivatisation mixture and dry it with a gentle nitrogen stream, the sample was redissolved with 50 μL of isooctane prior to their analysis by gas chromatography.

For lipid extraction at the CEPAM (Nice, France), a 2g of the inner surface was removed by manual modelling drill (Dremel 4000) and the surface was discarded to avoid exoge-nous lipids. The powdered samples were extracted using a mixture of 10mL DCM/MeOH (2:1 v/v). After 15 minutes sonication and a 15 minutes centrifugation at 3000 rpm/mi-nute the supernatant was extracted. This procedure was repeated three times and the samples were dried under a gentle nitrogen stream and heated at 40ºC. Next, they were redissolved with 500 μL of DMC/MeOH and derivatised with 100 μL of BSTFA at 70ºC for 1 hour. After cooling, samples were dried under gentle nitrogen stream and redissolved with 20 μL of cyclohexane prior to their analysis by gas chromatography.

3.2.1.2.3. Acidified methanol extraction

1-2g of ground pottery is mixed with 4 mL of MeOH and 50 μL of IS and sonicated in an ultrasonic bath for 15 min. Then, 200 μL of sulphuric acid (H2SO4) was added, and the mixture was heated at 70 °C for 4 h. After cooling, lipids were extracted three times by adding 2 mL of hexane and vortexing. The hexane phase was removed with the help of a Pasteur pipette, taken to dryness under a nitrogen stream. Finally, samples were re-dissolved in isooctane prior to gas chromatographic analysis. After screening for the presence of interpretable lipids, selected samples were trimehtylsilylated and submitted to GC-MS.

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3.2.2. Modern samples

3.2.2.1. Sample preparation

Ca. 5g of modern animal fats (Sample selection described in chapter IV), were taken with a scalpel from the thoracic fat of the selected animals using nitrile gloves. The sample was stored in a previously muffled vessel pot and frozen. Once the fat is frozen, it is freeze-dried for 36 hours under vacuum in order to remove all moisture and traces of H2O. Once dry, a sample of ca. 0.1g is extracted and the rest is stored in a drying heated in a heating plate to 70ºC and after 1 hour it is left to cool. Next, a liquid-liquid extraction is made, shaking it with the help of a vortex and discarding the hexane phase with a Pasteur pipette to eliminate contaminating compounds and traces of H2O. To acidify the lipids, hydrochloric acid (HCl) (0,5M, 32%) is added to obtain a pH3 and then 3ml of hexane. The mixture is stirred in the vortex and transferred with a Pasteur pipette the first phase from hexane to another tube, this process is repeated three times to ensure all lipids are extracted.

A 2ml of a mixture of 40ml of MilliQ water and 500µl of sodium chloride (NaCl) is added.

Then, the hexane phase is extracted. The solvent is evaporated under a soft ray of ni-trogen and, once dry, the acidified fraction is derivatised with 500µl of a boron trifluo-ride complex with methanol (BF3-methanol) and heated in a heating plate to 75ºC for 1 hour.

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Once cooled, the reaction is stopped by adding 3ml of MilliQ water and the lipids are extracted adding 3ml of hexane. The hexane transferred to vial is evaporated under nitrogen and we add 50µl of isooctane to inject the sample in the GC-IRMS to know the value δ¹³C of the released fatty acids.

3.3. Analytical techniques