Click here to go back to frontpage

Carbon and nitrogen isotope analysis


Nitrogen and carbon isotope analysis uses an isotope ratio mass spectrometer to determine stable 13C/12C and 15N/14N ratios. They are related to the corresponding isotope ratios of standard materials and displayed as δ13C and δ15N values, respectively.

The method is applicable to all materials containing one or both of these elements. The main field of application for this type of analysis at CEZA is diet reconstruction of humans and animals, for which collagen extracted from bone or dentine is usually examined. Carbon isotope analysis can also be carried out on apatite, the inorganic component of bones and teeth.


Diet reconstruction

Delta13C and δ15N values in bone or dentine collagen reflect the composition of the diet of humans and animals. They may reveal differences within and among burial communities regarding the access to food depending on age, sex, and social status, or changes in dietary habits over time. Serial analyses of tooth dentine as well as changes in the isotope ratios of bone collagen, or fingernails of infants of different ages, may inform on the duration of breastfeeding. Analyses of samples from animal bones contribute to the reconstruction of food webs and provide information about husbandry and feeding strategies of domestic animals. Such data can reveal long-term changes in landscape history, such as deforestation. Sampling of crop remains, in particular charred cereal grains, testifies to the use of animal manure for fertilization as a central aspect of agricultural history.

Other applications

Beyond the use in archaeometry, stable isotope analyses of carbon and nitrogen offer numerous applications in biology, soil science, forensics and food analysis.


Diet reconstruction

Stable carbon and nitrogen isotope ratios in collagen, the organic fraction of bone and dentine, relate to the kind of proteins consumed during life. Since collagen itself is a protein, the carbon bound in it mainly originates from the carbon in dietary protein. Nitrogen occurs exclusively in proteins and cannot be metabolized from fats or carbohydrates.

Delta13C values can also be determined for the carbon which makes up the structural carbonate in the inorganic fraction of bones and teeth (hydroxyapatite). These values reflect an average of the carbon from all macronutrients, i.e. fats, carbohydrates and proteins. For technical reasons, the analysis also provides a δ18O value for the same sample.

Changes in the isotope composition of nitrogen and carbon result from stable isotope fractionation during biochemical reactions, such as photosynthesis, metabolic processes and protein biosynthesis.

The δ13C values mainly indicate the proportion of C3 and C4 plants in the average diet. Plants with a C3 photosynthesis cycle include most of the wild and cultivated species of plants in Central Europe, whereas millet, corn and sugar cane are the most important Cfood plants. The consumption of sea fish is also reflected in elevated δ13C values. Variable habitats produce slight changes to the resulting carbon signatures recovered, such as those under forest cover compared to open landscapes. Differences in water availability also lead to smaller variations within the data spectrum of C3 plants.

Delta15N values change primarily along food chains. These values increase from plants to herbivores to carnivores, and thus reflect the proportion of animal proteins in the average diet. Due to the longer food chains, particularly high δ15N values can be achieved in marine environments and reflect the consumption of sea fish in bone collagen. Furthermore, the fertilization of cultivated areas with animal manure can lead to increased δ15N values in plants at the base of the food webs. This observation may reveal valuable insights into agricultural management strategies, but also obscure data interpretation regarding diet composition.

Other applications

Isotope fractionation also plays a central role for other applications of stable nitrogen and carbon isotope analysis. The methodological background for differentiating source or target materials using isotope compositions may differ, depending on the materials and the research questions.


Diet reconstruction

The determination of δ13C and δ15N values is a routine analytical procedure with high accuracy and precision. Both isotope ratios in collagen as well as carbon isotope ratios of the structural carbonate, reflect an average value of the food consumed over the course of years. Individual meals, short-term diet changes, or restricted periods of starvation cannot be identified from the data.

Basically, the isotopic compositions of foodstuffs depend on environmental conditions and climate. They are also subject to anthropogenic influences and means of soil fertilization, such as the application of manure. Comparative analyses of plant remains and animal bones contribute significantly to our understanding of these effects, which may occasionally lead to ambiguities in data interpretation.

In principle, light stable isotope analyses reflect overall trends of the internal differentiation of burial communities or dietary changes over time, rather than the consumption of certain kinds of foodstuff by specific individuals.

Sample properties

The actual determination of C and N isotope values often requires only a few micrograms or milligrams of the respective sample material. However, most samples require extraction or cleaning procedures in order to isolate or purify the fraction that is required for analysis. We recommend the submission of the following quantities of the different sample types:

Diet reconstruction

Bone samples for collagen extraction

Ribs are preferred as sample material for diet reconstruction based on C and N isotope compositions of bone collagen. They remodel constantly and reflect an average of the diet consumed over the last years of life. If no ribs are available, other kinds of bones may be used. Certain research questions may require a specific selection of other or several different skeletal elements per individual. The samples should be individualised and determined by skeletal element.

Optimum sample size: bone fragment of approx. 5 cm length

Tooth samples for collagen extraction

Collagen for C and N isotope analysis can be extracted from dentine. Such investigations may be performed on bulk material that is preferentially taken from the root of a tooth. The dentine from the complete tooth may also be cut up and analyzed sequentially. Such sub-samples allow tracing changes of the dietary composition over the time of tooth formation.

For C- and N-isotope analysis on dentine, the entire tooth should be submitted and the sampling strategy should be coordinated.

Comparative samples

In order to make statements about the composition of the human diet, the analysis of comparative samples is necessary. Ideally, bones from animals with different food sources (herbivores, omnivores, carnivores, domestic and wild animals) from the same temporal and spatial context are used.

Optimum sample size: bones of approx. 5 cm length, determined according to animal species and skeletal element.

For temporal and spatial contexts of arable farming communities, sampling of plant macro remains (especially charred cereal grains) provides important data for characterizing the human food base. Stable isotope data of charred plant remains may also be informative on their own regarding fertilization, irrigation and agricultural strategies.

Optimum sample size: Each sample should include at least 5 charred grains of the same species and the same archaeological context.

Bone and tooth samples for δ13C determinations on apatite

A few milligrams of sample material are sufficient for carbon isotope analysis on apatite. In the interest of handling and sample pre-treatment, we recommend submitting bone samples with an edge length of at least approx. 1 cm.

For carbon isotope analysis on enamel, the entire tooth should be submitted including information on its anatomic position and species. Especially for high-crowned teeth, the extraction of sequential samples is also possible. They are taken from parallel, horizontally alligned grooves that are evenly distribued along the tooth crown and inform on diet changes over time.

Please contact us for the submission of samples that are already extracted and possibly pre-treated.

Other applications

Please contact us for other types of samples.