In archaeology, Organic Residue Analysis (ORA) refers to the study of micro-remains trapped in or adhered to artifacts from the past. These organic residues can include lipids, proteins, starches, and sugars. By analyzing these residues, ORA can reveal insights into ancient dietary behaviors, agricultural practices, housing organization, technological advancements, and trade interactions. Furthermore, it provides information on the use of cosmetics, arts, crafts, medicine, and burial preparations in ancient societies.
ORA's broad applicability encompasses a variety of amorphous materials such as substances used in mummification, pastes, glues, binders, and colorants. These materials can be preserved in pottery, stone tools, the mineral matrix of bones, dental calculus, as well as in habitation floors or pits. The unique value of ORA lies in its ability to provide direct evidence of the materials and substances utilized by ancient peoples, often offering insights that other archaeological techniques cannot.
For instance, analyzing organic residues in pottery can disclose specific dietary components, such as animal and plant fats, shedding light on ancient dietary habits and food sources. Similarly, the study of ancient adhesives and pigments can enhance our understanding of the production techniques and materials used in ancient art and craftsmanship.
Moreover, ORA plays a crucial role in uncovering ancient medical knowledge, cosmetic usage, and the processes involved in creating artworks and handicrafts. Utilizing modern chemical analysis techniques, ORA offers archaeologists a useful tool to directly explore and understand the daily lives, cultural practices, and technological progress of ancient societies.
Organic residues The makeup of an organic residue depends on the organic components that got absorbed, trapped, or attached to unglazed and porous materials, like pottery, crusts, wood, and stones. The formation of organic residues can occur during a wide range of pre- and post-depositional events, including food preparation, cooking, storage, transportation, reparation and sealing. Given the complexities of organic residues, they can be defined at five nested scales: tissues, cells, macromolecules (e.g. lipids, proteins, metabolites, DNA and starches), molecules (e.g. fatty and amino acids) and atoms (e.g. carbon, nitrogen and hydrogen). We're especially interested in the residues at the level of biomolecules, particularly fats and metabolic products, because of how well they can be preserved and their importance to archaeology. Discussions about proteins are covered elsewhere under the topic of Paleoproteomics.
Lipids Lipids (lipos, gr. fat) are a class of biomolecules that mostly fall within the ester family and include fats and oils, waxes, terpenes, steroids, triglycerides (TAGs), free fatty acids, ketones, alcohols, dicarboxylic acids, bituminous substances and resins.
By definition, lipids are hydrophobic biomolecules. Fatty acids consist of long carbon chains with various lengths and double bonds, ending with a carboxyl group. In biological systems, most fatty acids have an even number of carbon atoms, mainly between C14 and C24, maximising at C16 and C18.
Waxes are simple esters of long-chain carboxylic acids and alcohols, fats and oils have a more complex structure. These are formed from triglycerides. Each triglyceride contains three long-chain carboxylic acids attached to a triester of 1,2,3-propantriol (glycerin). Typically, the fatty acids are unbranched and have an even number of carbon atoms. TAGs are important storage lipids and are the main constituent (~ 99%) of vegetable oils and food.
Lipids are hydrophobic and therefore cannot be 'washed out' or accidentally 'washed into' the matrix of an analysed material. The ceramic matrix serves as a protective environment which favours significant lipid preservation.
Metabolites Metabolites are small organic molecules usually involved in metabolism either as a substrate or product. They can also refer to endogenous compounds, which includes organic acids, lipids, sugars, amino acids, and phenolic compounds, produced during metabolic processes. The field of metabolomics (Section 6) generally revolves around these molecules with a molecular weight between 50 – 1500 daltons (Da). Recently, the application of metabolomics in ancient residue analysis has been established where it was successfully used to determine distinct molecular residue markers in archaeological pottery.
Development of the method During the 1950s and 1960s, the emergence of chromatographic methods, especially those linking Gas Chromatography (GC) and Mass Spectrometry (MS), resulted in a methodology used to resolve and recognise molecules. One of the earliest papers using GC analysis applied to archaeological material was published by Thornton et al. (1970) and, investigated the composition of ancient bog butter.
Scientists have developed different methods to study the various materials stuck in ancient pottery. A popular way to get these materials out involves using special liquids like chloroform and methanol to pull out fats that aren't tightly attached to the pottery. Then, they treat the samples with a process to prepare them for further analysis. They can also treat the pottery powder a second time to get fats that are more firmly attached, through strong chemical bonds. By doing this, they can learn even more about substances that break down differently. This helps them understand more about what ancient people might have stored or cooked in these pots.
A high-throughput protocol to extract both 'unbound' and 'bound' lipids was developed and published in 2014, using a direct acidified methanol extraction. This method results in a higher recovery of lipids. Compositional information is lost to hydrolysis of complex lipids, such as TAGs and wax esters.
Nowadays, it is possible to identify animal fats, plant waxes, resins and tars, wine, and beeswax. Modern studies of plant resins, consisting of di- and triterpernoids, show confident identification of these sources to the genus level and, sometimes even the botanic species. Other classes of specific plant lipids, such as hydrocarbons, ketones, alcohols and/or fatty acids, can also reveal the origins of resources.
When using selected ion monitoring (SIM) methods, a higher sensitivity of the GC-MS can be achieved, and thus, known compounds can be detected, e.g. cereal biomarkers (alkylresorcinols) and specific marine biomarkers.
Alkylresorcinols (ARs) are amphiphilic phenolic lipids characterised by a non-polar odd-numbered alkyl side chain attached to a polar resorcinol (1,3-dihydroxybenzene) ring. In the case of cereals, the alkyl chain ranges from 15 to 25 carbon atoms. These cereal biomarkers were previously found in a well-preserved Bronze Age wooden container from Switzerland, and coarse ware vessels from a Roman cavalry barrack at Vindolanda. The latter study demonstrated that the survival of ARs is highly dependent on the cooking procedures and burial conditions. However, if recoverable, analysis of these phenolic lipids in archaeological contexts is valuable as it can help explain the uptake and spread of cereal processing of past communities in particular regions.
Diagnostic compounds for marine resources, including ⍵-(o-alkylphenyl)alkanoic acids (APAAs) C18, C20 and C22, were recently observed in numerous archaeological potsherds and are degradation products of unsaturated fatty acids. These biomarkers were recently detected in Narva ceramics confirming the exploitation of marine resources during the Late Mesolithic period in Eastern Baltic as well as in Korean hunter-gatherer pottery demonstrating the process of marine resources to e.g. rendering aquatic fats.
Identification becomes more complex when dealing with animal fats and plant oils. Some consist mainly of polyunsaturated fatty acids, which rarely survive in the archaeological record. Through the degradation processes of hydrolysis, undiagnostic n-alkanoic acids form. By combining lipid biomarkers and compound specific isotope analysis of individual fatty acids, it becomes possible to differentiate between ruminant and non-ruminant adipose, and ruminant milk, but the method can also help differentiate between wild ruminants, chicken and detect maize.
Radiocarbon dating is a way to figure out how old things are by measuring a type of carbon found in materials. Compound-Specific Radiocarbon Analysis (CSRA) has been developed since the end of the 1990s and lately there has been some developments in the approach that contributed to address some dating questions with more reliability, and to shed light on the importance of pottery as a proxy for chronological assessments. CSRA has been applied by Casanova et al. to directly date palmitic (C16:0) and stearic (C18:0) fatty acids extracted from archaeological pottery belonging to different Neolithic sites in Europe, Africa, Anatolia and Saharan Africa. This study revealed that CSRA of pottery can shed light on multiple archaeological questions such as when the pottery was used, and thus understanding pottery typochronologies, and how old the organic residues are, allowing to discuss the beginning of the consumption of a specific resource. CSRA, used together with analysis of faunal assemblages has also been applied to investigate the consumption of marine products.
Development of analytical approaches Since the 1950s, mass spectrometry (MS)-based techniques have been used to characterise lipids, proteins and metabolites. The underlying principle of MS is that charged molecules (ions) are characterised by their mass-to-charge ratios in electric or magnetic fields. Generally, there are four key components of any MS workflows: extraction, separation (gas or liquid chromatography), sources of ionisation (e.g. electron, electrospray (ESI) and chemical (CI) ionisation) and types of analysers (e.g. Quadrupole and Orbitrap). For hydrophobic lipids, they are extracted from biological or archaeological material with nonpolar solutions. In order to analyse the extracted organic compounds, analytical techniques that target the specific molecules are required. To identify possible mixtures of organic compounds, the use of chromatographic and/or mass spectrometric methods is imperative. This is achieved using Gas Chromatography-Mass Spectrometry (GC-MS) alongside other methods, including isotope ratio mass spectrometry (IRMS), various spectroscopies, such as infrared (FT-IR), Raman, nuclear magnetic resonance (NMR), and ultraviolet (UV), scanning electron microscopy (SEM) for imaging and elemental analysis by energy dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), X-ray fluorescence (XRF), and high magnification light microscopy
The main chromatographic technique utilised for the identification of lipid molecules from food crusts and absorbed residues is GC-MS. In GC, the liquid sample is converted into vapour and the targeted lipids are separated on a capillary column with a specific polarity. The separation is based on the molecule's interaction with the mobile and stationary liquid phases. Larger lipids, such as triacylglycerols (TAGs) and wax esters, are analysed using high-temperature GC-MS (HTGC-MS) without any prior hydrolysis step. Meanwhile, a GC system coupled to a flame ionisation detector (FID) is widely used for the quantification of preserved lipids. One observed limitation of GC-based analysis is its inability to identify polar lipids (e.g., glycerophospholipids and D-galactosyl diacylglycerols), which are mainly found in cereals. To mitigate this drawback liquid chromatography (LC)-MS can be used.