12. Assessing Ecosystem-Level Water Relations Through Stable Isotope Ratio Analyses
189
oD Analysis of Organic Matter
All carbohydrates contain hydroxyl groups that can
exchange their hydrogens with those in water, and
thus in order to determine the oD of organic matter
at the time of formation, only nonexchangeable hydrogens (C-H bonds) can be analyzed for isotopic
composition. The most common process is nitration
in which each hydroxyl group is exchanged for a
nitrate (N0 3 ). The purified organic material (acellulose, see above) is placed in a flask with a solution of nitric acid and acetic anhydride on ice due
to the exothermic nature of the reactions (Bennett
and Time1l1955; DeNiro 1981; Yapp and Epstein
1982). The material is then washed, dissolved in
acetone and then precipitated in water to obtain purified tri-nitrated cellulose (all three hydroxyls per
glucose are nitrated). The material is then freezedried for storage.
The nitrated material (9 to 14 mg) is then placed
into a tube with cupric oxide (1 g), evacuated,
sealed, and combusted for 3 hr at 520°C. Following
combustion, the gases are separated on the same
type of vacuum line (see Figure 12.3) as used for
organic o 13 C, only instead of trapping CO2, water
vapor is trapped by using the ethanol/dry ice slush
to control the water movement while evacuating the
other gases. The water is then transferred to a tube
containing a Zn catalyst (see section on oD analysis
of water above) for reduction to diatomic hydrogen
and input into the mass spectrometer. Although this
procedure yields good precision «2%0), it is also
labor intensive and requires relatively large sample
sizes for the nitration.
0 18 0 Analysis of Organic Matter
Conversion of organic matter to CO2 for 0 18 0 analysis involves a pyrolytic reaction. For off-line conversion, the a-cellulose (see above) is placed in a
tube with mercuric chloride, evacuated, sealed, and
then heated for 6 hr at 550°C to form carbon monoxide (CO), CO 2 , and hydrochloric acid (HCI)
gases. The CO is converted to CO 2 in an ionization
chamber and the HCI is reacted with Zn. After a
distillation step, the CO2 is then ready for the mass
spectrometer. This procedure, especially the conversion of CO to CO 2 in the spark chamber, is time
consuming and along with the toxicity of mercuric
chloride makes this technique much less appealing
than newer on-line techniques.
Similar to on-line analysis of 013C (see above),
the use of continuous flow and an elemental analyzer as an automated pyrolysis device has greatly
simplified the estimation of 0 18 0 in organic matter
(Saurer et al. 1998). Dried a-cellulose (1 to 2 mg)
contained in a silver cup is dropped into a hot
(1l00°C) quartz combustion furnace containing
glassy carbon catalyst in a stream of He. The excess
C from the catalyst facilitates the conversion at high
temperatures to the thermodynamically favorable
products of CO and N2• These two gases are then
separated by a chromatographic column and sent
into the mass spectrometer for analysis. A typical
cycle time is around 20 minutes and many samples
can be run by using an autos ampler. If care is taken
to reduce memory effects (repeated sampling is recommended) then this method has proven to be robust and precise «0.4%0).
Short-Term, Ecosystem
Process-Level Applications
Our understanding of ecosystem-level water use on
a short-term basis has been significantly advanced
with the application of stable isotope methods. This
is particularly true if the isotopic methods and data
collected are linked with other methods at the plant,
catchment, and atmospheric levels (Dawson and
Ehleringer 1998). If chemical and/or physical reactions result in differential representation of the
heavy and light isotopes (Figure 12.4), the isotopic
distributions of the reactants and products can inform us about "processes" (i.e., reaction conditions)
(Peterson and Fry 1987). The different distributions
of stable isotopes, in water for example, can also
help to identify and then trace the origin of different
water resources in the ecosystem as well (Ehleringer and Dawson 1992). The isotopic values (signatures) of source pools then provide a baseline
with which to assess process-level information by
looking for changes in the isotopic signature along
a reaction pathway (termed isotope fractionation or
discrimination) (Peterson and Fry 1987; Hoefs
1987; Lajtha and Michener 1994).
Partitioning of Water Resources Among
Plants Within Ecosystems
There are now a number of very good examples of
how different plant species may partition soil water
resources that have used H or 0 stable isotope anal-
189
oD Analysis of Organic Matter
All carbohydrates contain hydroxyl groups that can
exchange their hydrogens with those in water, and
thus in order to determine the oD of organic matter
at the time of formation, only nonexchangeable hydrogens (C-H bonds) can be analyzed for isotopic
composition. The most common process is nitration
in which each hydroxyl group is exchanged for a
nitrate (N0 3 ). The purified organic material (acellulose, see above) is placed in a flask with a solution of nitric acid and acetic anhydride on ice due
to the exothermic nature of the reactions (Bennett
and Time1l1955; DeNiro 1981; Yapp and Epstein
1982). The material is then washed, dissolved in
acetone and then precipitated in water to obtain purified tri-nitrated cellulose (all three hydroxyls per
glucose are nitrated). The material is then freezedried for storage.
The nitrated material (9 to 14 mg) is then placed
into a tube with cupric oxide (1 g), evacuated,
sealed, and combusted for 3 hr at 520°C. Following
combustion, the gases are separated on the same
type of vacuum line (see Figure 12.3) as used for
organic o 13 C, only instead of trapping CO2, water
vapor is trapped by using the ethanol/dry ice slush
to control the water movement while evacuating the
other gases. The water is then transferred to a tube
containing a Zn catalyst (see section on oD analysis
of water above) for reduction to diatomic hydrogen
and input into the mass spectrometer. Although this
procedure yields good precision «2%0), it is also
labor intensive and requires relatively large sample
sizes for the nitration.
0 18 0 Analysis of Organic Matter
Conversion of organic matter to CO2 for 0 18 0 analysis involves a pyrolytic reaction. For off-line conversion, the a-cellulose (see above) is placed in a
tube with mercuric chloride, evacuated, sealed, and
then heated for 6 hr at 550°C to form carbon monoxide (CO), CO 2 , and hydrochloric acid (HCI)
gases. The CO is converted to CO 2 in an ionization
chamber and the HCI is reacted with Zn. After a
distillation step, the CO2 is then ready for the mass
spectrometer. This procedure, especially the conversion of CO to CO 2 in the spark chamber, is time
consuming and along with the toxicity of mercuric
chloride makes this technique much less appealing
than newer on-line techniques.
Similar to on-line analysis of 013C (see above),
the use of continuous flow and an elemental analyzer as an automated pyrolysis device has greatly
simplified the estimation of 0 18 0 in organic matter
(Saurer et al. 1998). Dried a-cellulose (1 to 2 mg)
contained in a silver cup is dropped into a hot
(1l00°C) quartz combustion furnace containing
glassy carbon catalyst in a stream of He. The excess
C from the catalyst facilitates the conversion at high
temperatures to the thermodynamically favorable
products of CO and N2• These two gases are then
separated by a chromatographic column and sent
into the mass spectrometer for analysis. A typical
cycle time is around 20 minutes and many samples
can be run by using an autos ampler. If care is taken
to reduce memory effects (repeated sampling is recommended) then this method has proven to be robust and precise «0.4%0).
Short-Term, Ecosystem
Process-Level Applications
Our understanding of ecosystem-level water use on
a short-term basis has been significantly advanced
with the application of stable isotope methods. This
is particularly true if the isotopic methods and data
collected are linked with other methods at the plant,
catchment, and atmospheric levels (Dawson and
Ehleringer 1998). If chemical and/or physical reactions result in differential representation of the
heavy and light isotopes (Figure 12.4), the isotopic
distributions of the reactants and products can inform us about "processes" (i.e., reaction conditions)
(Peterson and Fry 1987). The different distributions
of stable isotopes, in water for example, can also
help to identify and then trace the origin of different
water resources in the ecosystem as well (Ehleringer and Dawson 1992). The isotopic values (signatures) of source pools then provide a baseline
with which to assess process-level information by
looking for changes in the isotopic signature along
a reaction pathway (termed isotope fractionation or
discrimination) (Peterson and Fry 1987; Hoefs
1987; Lajtha and Michener 1994).
Partitioning of Water Resources Among
Plants Within Ecosystems
There are now a number of very good examples of
how different plant species may partition soil water
resources that have used H or 0 stable isotope anal-