Overview of Stable Isotope Research
Last revised July 22, 1997
Page contents: (click to skip down)
[Tracing and Fractionation]
[Standard materials and calibrants]
[Applications and current research]
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
Element Isotope Abundance (%)
Hydrogen 1H 99.985
Carbon 12C 98.89
Nitrogen 14N 99.63
Oxygen 16O 99.759
Sulfur 32S 95.00
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:
(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:
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.
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.
Standard materials and calibrants
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
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.
Applications and uses of stable isotopes
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
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.)
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,
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
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
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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