Overview of Stable Isotope Research

Last revised July 22, 1997

Page contents: (click to skip down)

* [Available analytes]
* [Introduction]
* [Measurement notation]
* [Tracing and Fractionation]
* [Standard materials and calibrants]
* [Applications and current research]

(IMAGE: Peak scans, anca06a.gif)

Available analytes

* Plant [nitrogen-15 assay]
* Soil [nitrogen-15 assay]
* Plant [carbon-13 assay]
* Soil [carbon-13 assay]

Since nucleons (protons and neutrons) weigh approximately 1 unit on the scale used to measure such things, the atomic weight of an atom can be treated as the same as the number of its nucleons. That the atomic weights of many of the elements listed in tables are not neat whole numbers shows that these weights are averages of the differing atomic weights of two or more forms.

A chemical element's atomic number is the number of positive charges (the number of protons) in the nucleus of each of its atoms. This number is the defining characteristic of a given element, invariant for all atoms of that element. Thus if some atoms of an element have a different atomic weight from others, the difference must lie in the number of neutrons. Atoms of the same atomic number but different atomic weights are called isotopes.

Elements can exist in both stable and unstable (radioactive) forms. Most elements of biological interest (including C, H, O, N, and S) have two or more stable isotopes, with the lightest of these present in much greater abundance than the others. Among stable isotopes the most useful as biological tracers are the heavy isotopes of carbon and nitrogen. These two elements are found in the earth, the atmosphere, and all living things. Each has a heavy isotope (13C and 15N) with a natural abundance of ~1% or less and a light isotope (l2C and 14N) that makes up all of the remainder, in the case of nitrogen, or virtually all in the case of carbon (carbon also has a radioactive isotope, 14C.)

Table 1.1. Average Terrestrial Abundances of the Stable
           Isotopes of Major Elements of Interest in
           Ecological Studies

Element    Isotope  Abundance (%)

Hydrogen     1H         99.985
             2H          0.015
Carbon       12C        98.89
             13C         1.11
Nitrogen     14N        99.63
             15N         0.37
Oxygen       16O        99.759
             77O         0.037
             18O         0.204
Sulfur       32S        95.00
             33S         0.76
             34S         4.22
             36S         0.014

Measurement Notation
Atom Percent:

Results from environmental and agricultural studies using isotopically enriched tracers are usually reported in units of atom percent (At%). This value gives the absolute number of atoms of a given isotope in 100 atoms of total element:

(IMAGE: At%15N equation, eq02.gif)

(IMAGE: At%13C equation, eq01.gif)

(N.B., for the At%13C calculation the amount of naturally present 14C is usually treated as negligible and the sum of 12C and 13C taken to be total C).

Atom Percent Excess:

Medical tracer studies of human physiology are most often reported in units of atom percent excess (APE). This specifies the level of isotopic abundance above a given background reading, which is considered zero. The background reading in At% is subtracted from the experimental value to give APE.


Studies examining stable isotopes at or near natural abundance levels are usually reported as delta, a value given in parts per thousand or per mil ("o/oo").

Delta values are not absolute isotope abundances but differences between sample readings and one or another of the widely used natural abundance standards which are considered delta = zero (e.g. air for N, At%15N = 0.3663033; Pee Dee Belemnite for C, At%13C = 1.1112328). Absolute isotope ratios (R) are measured for sample and standard, and the relative measure delta is calculated:

(IMAGE: Delta equation, eq05.gif)


(IMAGE: R equation, eq03.gif)

For instance, if a leaf sample is found to have a 15N/14N ratio R greater than the standard's by 5 parts per thousand, this value is reported as delta15N = +5 delta o/oo.

The transformation of absolute At% values into relative (to a certain standard) delta values is used because the absolute differences between samples and standard are quite small at natural abundance levels and might appear only in the third or fourth decimal place if At% were reported.

Tracing studies
For biological tracing, labelled compounds are available with the heavy isotope making up 99% of the tracer element (e.g. 99 At% 13C-amorphous carbon, Sigma Chemical Co. 27,720-7; 99 At% 15N-ammonium sulfate, Sigma 29,928-6). To biologists the principal advantage of stable isotopes over other tracers is that they are not radioactive. 14C is somewhat hazardous and subject to a great many regulations and licensing requirements; for N there is no convenient radioactive nuclide.
Isotopic fractionation
Isotopes of the same element take part in the same chemical reactions, but because the atoms of different isotopes are of different sizes and different atomic weights they react at different rates. Physical processes such as evaporation discriminate against heavy isotopes; and enzymatic discrimination and differences in kinetic characteristics and equilibria can result in reaction products that are isotopically heavier or lighter than their precursor materials.

The naturally occurring delta13C values for biologically interesting carbon compounds range from roughly 0o/oo to ~-110o/oo relative to the Pee Dee Belemnite (PDB) standard. C3 plants, those using the Calvin-Benson photosynthetic pathway, fractionate carbon differently from C4 plants that use the Hatch-Slack pathway. The different 13C/12C ratios that result can be used to distinguish C3 from C4 plants. The tissues of animal grazers reflect the plants on which they feed, and this can be used to make inferences about diet both at present and in the archaeological record.

Natural 15N levels in biological materials typically range from ~-5o/oo to ~+10o/oo. Grazing animals show 15N enrichment relative to the plants they consume; predators show further 15N enrichment relative to their prey species. Atmospheric N is isotopically lighter than plant tissues, and soil 15N values tend to be higher still, suggesting that microbes discriminate against the light isotope during decomposition. Non-nitrogen-fixing plants, which derive all their N from the soil N pool, can thus be expected to be isotopically heavier than nitrogen-fixing plants, which derive some of their N directly from the atmosphere.

Standard materials and calibrants
The five principal light elements of biological interest are measured against four widely accepted standards:

H, O      Standard Mean Ocean Water (SMOW)
C, O      Pee Dee Belemnite (PDB)
N         atmospheric air
S         the Canyon Diablo meteorite (CD)

The natural abundance of 15N in air is a constant 0.3660%; air, being thus suitable as well as omnipresent and free, is used as the standard for nitrogen analyses.

The common reference for delta13C, the Chicago PDB Marine Carbonate Standard, was obtained from a Cretaceous marine fossil, Belemnitella americana, from the PeeDee formation in South Carolina. This material has a higher 13C/12C ratio than nearly all other natural carbon-based substances; for convenience it is assigned a delta13C value of zero, giving almost all other naturally-occurring samples negative delta values.

All original supplies of both SMOW and PDB have been used up and replaced by secondary standards prepared by the U.S. National Bureau of Standards (for instance NBS-21 graphite, having a carbon isotope ratio of -28.10o/oo compared to PDB) and the International Atomic Energy Agency, including V-SMOW (Vienna SMOW), which has an isotopic composition nearly duplicating original SMOW, and SLAP (standard light antarctic precipitation). The supply of air has not yet been exhausted (but stay tuned.)

Applications and uses of stable isotopes
I. Tracing studies:

Nitrogen cycling: To investigate nitrogen cycling in crop plants, 15N-labelled fertilizer (urea, ammonium nitrate, and so on) either 2-5% enriched or 0.36% depleted in 15N is applied. Following the tracer yields data with which one can quantify the fate of the added fertilizer N as it passes into various partitions: the portion taken up by the plants, the portion remaining in the soil N pool, the portion lost by denitrification into the atmosphere, and the portion leached into runoff waters. Such data leads to recommendations for fertilization that yield the greatest benefit to food crops and the least possible pollution of drinking water by nitrate runoff. 15N levels in the soil and water can also be an indication of the origin of the N, pinpointing its source.

Physiological tracing: Medical researchers use 13C as a noninvasive alternative to 14C for analyzing metabolic processes. 13C-labeled compounds metabolize to 13CO2, which is detectable in the breath.

II. Fractionation studies:

Nitrogen fixation: Since soil N is often more abundant in 15N than the atmosphere, and non-N-fixing plants must obtain all their nitrogen from the soil while N-fixing plants have an alternative N source in the form of (isotopically lighter) air, it is expected that N-fixing and non-fixing plants will differ in their 15N values. The lighter (more negative delta) the plant material is found to be with respect to soil N, the better its N-fixing ability. This difference forms the basis for the 15N natural abundance technique of estimating symbiotic N. N-fixation can also be quantified using tracer methods, and tracer techniques are more popular for examining N-fixation in crop plants. The natural abundance method has found an increasing number of applications by ecologists studying natural, nonmanaged ecosystems.

Photosynthesis and carbon cycling: Terrestrial plants fix atmospheric CO2 by two main photosynthetic reaction pathways: the Calvin-Benson, or C3, and the Hatch-Slack, or C4. C3 plants convert atmospheric CO2 to a phosphoglycerate compound with three C atoms while C4 plants convert CO2 to dicarboxylic acid, a four-C compound. Carbon isotopes are strongly fractionated by photosynthesis and the C3 and C4 processes involve different isotopic fractionation, with the result that C4 plants have higher delta13C values ranging from -17o/oo to -9o/oo with a mean of -13o/oo relative to PDB, while C3 plants show delta values ranging from -32o/oo to -20o/oo with an average value of -27o/oo. Most terrestrial plants are C3, all forest communities and most temperate zone plant communities of all kinds being dominated by C3 plants. The native plant populations of North America and Europe are almost exclusively C3. Over 80% of crop plants are C3.

C4 plants are characteristically found in hot, arid environments: a selective advantage of C4 photosynthesis is more efficient use of water. Some crops of immense importance are C4 plants: maize, sorghum, millet, and sugar cane. The 13C value is a standard method for distinguishing the C3 and C4 plant groups and is used by plant physiologists to determine drought resistance in C3 plants, as well as to breed for improvement in this increasingly vital characteristic.

III. Archaeological investigations:

As mentioned above, the characteristic isotope-ratio "signatures" of food species are passed on to consumers. Though there may be further fractionation during metabolic processing of food by the consumers, the mean delta13C values of the two main groups, C3 and C4, of primary producers can remain visible through many trophic levels to the top of the food chain. It is possible to determine the proportion of C3 and C4 plant species in the diet of herbivores and to make inferences about the prey species selected by carnivores. A remarkable application of this fact depends on the further observation that original North American plant communities were composed almost exclusively of C3 species (mean delta13C = -27o/oo), while maize (Zea mays) is a C4 plant with a delta13C value of -14o/oo. It has proven possible to determine the time of introduction of maize agriculture in the New World, and the rate at which it was adopted, by examining the delta13C values of skeletons and carbonized deposits in cooking pots. During the period A.D 1000-1200, the delta values of human collagen recovered from skeletal material changed from -21.4o/oo to -12o/oo as the isotope content of the diet was altered by the introduction of maize. This in turn can now be correlated with the great changes in population density and levels of civilization that resulted from the abandonment of the hunter-gatherer mode of life and the substitution of long-term agricultural settlements.

IV. Correction of carbon-14 dates:

Since carbon is strongly fractionated by biological processes, it is not possible to date ancient carbon-bearing material by the carbon-14 method without taking this fractionation into account. If biological samples selectively accumulate heavy C isotopes, this will make them appear spuriously young. It has been found that rates of 13C stable isotope fractionation are doubled for 14C. Stable isotope analysis gives an independent measure of fractionation such that if, for instance, a sample is 1.5% heavier in 13C than "modern standard carbon" through the effects of fractionation, then it will be 3% heavier in 14C than it would have been had fractionation not taken place. Since the average lifetime of a 14C atom is ~8000 years, a 3% increase in 14C content through fractionation will make it appear too young by 3% of 8000 years, or 240 years.


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