Primary formation dynamics of peroxynitrite following photolysis of nitrate

Article abstract of DOI:10.1021/jp906354c

The photolysis of nitrate, NO₃^-, in D₂O solution has been investigated by femtosecond infrared spectroscopy. In accordance with previous investigations, we observe that the peroxynitrite ion, ONOO^-, is the domin

Full text of DOI:10.1021/jp906354c

10488
J. Phys. Chem. A 2009, 113, 10488–10494
Primary Formation Dynamics of Peroxynitrite Following Photolysis of Nitrate
Jan Thøgersen, Ane Gadegaard, Jakob Nielsen, Svend Knak Jensen, Christian Petersen,† and
Søren R. Keiding*
Department of Chemistry, UniVersity of Aarhus, Langelandsgade 140, DK-8000 Aarhus C, Denmark
ReceiVed: July 6, 2009; ReVised Manuscript ReceiVed: August 12, 2009
The photolysis of nitrate, NO-3 , in D2O solution has been investigated by femtosecond infrared spectroscopy.
In accordance with previous investigations, we observe that the peroxynitrite ion, ONOO-, is the dominant
photochemical product following the excitation of nitrate at 200 nm. Moreover, we are able to identify the
cis/trans isomers of peroxynitrite and the dynamics of their formation in solution. We observe that the trans-
ONOO- isomer is formed directly and solely from the excited NO-3 ion within the first two picoseconds after
excitation. Subsequently, about half of the trans-ONOO- isomerizes to cis-ONOO- in 25 ps; thereafter, the
ratio between the two isomers remains constant for the 300 ps duration of the experiment. The observed
vibrational frequencies of the terminal OdN bonds are at 1515 and 1580 cm-1 for trans- and cis-peroxynitrite,
respectively. The detailed analysis of the infrared bands of cis- and trans-peroxynitrite is facilitated by electronic
structure calculations on the conformers in a cluster of 11 D2O molecules and by steady-state infrared
spectroscopy of ONOO- in D2O. In addition to the formation of ONOO-, the experiments also reveal a slow
∼50 ps formation of NO2 following the photolysis of nitrate.
Introduction
Therefore, the time resolution ensures that only the primary
photodynamics are investigated and that secondary reactions
can safely be neglected. For the nitrate-peroxynitrite system,
in particular, the ability to perform a “collision and impurity
free” experiment is very important because of the high reactivity
of peroxynitrite.2
This work describes one of the most common photochemical
transformations in aqueous environments: the photoisomeriza-
tion of the aqueous nitrate into peroxynitrite and the subsequent
isomerization of trans-peroxynitrite into cis-peroxynitrite.1 The
nitrate-peroxynitrite system has thus been studied in great detail
as a model system for aqueous chemical reactions2-10 because
it displays all key concepts in aqueous chemistry: charge
stabilization, electron detachment, high mobility, contact pairs,
solvent caging, and energy dissipation through the hydrogen
bond network. In addition, peroxynitrite plays a very important
role as a biochemical oxidant and nitrating agent formed in vivo
from nitric oxide, NO, and superoxide, O-2 .11-15 The presence
of elevated concentrations of peroxynitrite has been linked to a
broad range of cardiovascular and other pathologies. Peroxyni-
trite leads to oxidative stress in many organisms and inflicts
damage to blood vessels, skin, heart, lung, kidney, and brain.
Consequently, there are numerous papers published on the
fundamental properties of the nitrate-peroxynitrite system, and,
in particular, on the biomedical implications of peroxynitrite.
Contrary to most of the previous work on the nitrate-peroxynitrite
system, the work presented here is performed with femtosecond
time resolution. Apart from the deservedly celebrated possibility
of observing chemical transformation in real time, the short time
resolution actually presents another advantage of similar propor-
tions: The diffusion coefficient of a small molecule in water is
typically D ≈ 2 × 10-9 m2 s-1 () 0.2 Å2 ps-1), and the diffusion
length is LD ) (6Dt)1/2. Consequently, the volume of the solution
covered by the diffusing molecules is very small on femto- and
picosecond time scales. If, for example, an excited NO3-
molecule were to encounter a molecule other than water, in 50
ps, the concentration of this molecule should exceed 1 M.
We have previously investigated the nitrate-peroxynitrite
system with femtosecond time resolution using ultraviolet laser
pulses. Unfortunately, the specificity of the ultraviolet absorption
spectra of the involved photoproducts is low because they all
have rather broad and featureless absorption spectra in the range
between 200 and 300 nm. Therefore, although we could
determine the primary quantum yields of peroxynitrite from the
photolysis of NO-3 , we could not directly determine which of
the isomers of peroxynitrite was formed. In this work, we extend
our previous studies of the nitrate-peroxynitrite system by
utilizing femtosecond transient absorption spectroscopy in the
infrared spectral region. This allows us to determine the
distribution of peroxynitrite isomers formed after the excitation
of nitrate. In our previous work,5 we assigned the transient UV
spectrum of peroxynitrite solely to the cis isomer, and this was
recently questioned by Rabani et al.,6 who suggested that the
trans isomer should also be present after photolysis of nitrate.
Using Raman spectroscopy, Tsai et al. have found that only
the cis isomer is observed under ambient conditions.16 Using
femtosecond IR transient absorption spectroscopy, we can
distinguish the trans and cis isomers and thus reveal how they
are generated from nitrate and their rate of conversion.
Furthermore, our experiments also reveal the slow (50 ps)
formation of NO2 following the photolysis of nitrate. This
observation resolves the current uncertainty6 concerning the third
channel in the primary photolysis of nitrate. Internal conversion
of excited nitrate and isomerization into peroxynitrite constitutes
∼75-85% of the yield following the photolysis of nitrate, and
from our data, we see that the formation of NO2 constitutes the
last ∼15-25%.
* Corresponding author. E-mail: keiding@chem.au.dk. Tel: +45 2899
2061/+45 8942 3861. Fax: +45 8618 6199.
† Present address: FOM Institute for Atomic and Molecular Physics
(AMOLF), P.O. Box 41883, 1009 DB Amsterdam, The Netherlands.
10.1021/jp906354c CCC: $40.75  2009 American Chemical Society
Published on Web 09/08/2009

Primary Formation Dynamics of Peroxynitrite
J. Phys. Chem. A, Vol. 113, No. 39, 2009 10489
TABLE 1: Vibration Frequencies and Intensitiesa
ωcalcd (cm-1
)
0.9664 × ωcalcd
ωobsd (cm-1
)
1370c/1350d
1400
Icalcd (km/mol)
b
NO-3 , 11 D2O
1417 ( 12
1499 ( 14
1425 ( 19
1504 ( 22
1701 ( 18
1369
1449
1377
1453
1644
421 ( 46
439 ( 46
442 ( 77
391 ( 46
321 ( 33
NO-3 , K+, 11 D2O
cis-ONOO-, 11 D2O
1370
1400
1560c/1564d
1580e
trans-ONOO-, 11 D2O
NO2, 11 D2O
1605 ( 37
1726 ( 3
1551
1668
1515e
232 ( 39
333 ( 23
1610e
a ωcalcd is the harmonic normal mode frequency with a dominant contribution from the terminal NdO vibration calculated at the B3LYP/
6-31G(d) level of theory. The two frequencies in the case of NO-3 (aq), 11 D2O correspond to the highest E′ frequency, ν3 of NO-3 (aq). Icalcd is
the calculated intensity of the corresponding transition. ωobsd is the experimental transition frequency assigned to scaled calculated frequency
using the scale factor of 0.9664.19 b Ref 19. c This work, pD > 11. d Ref 16. e This work, pD ) 7.
Experimental and Theoretical Methods
configurations. The average value and the rms uncertainty are
listed in Table 1. The calculated vibrational frequencies were
multiplied with a scaling factor of 0.9664 before comparison
with the experimental transition frequencies.19
The laser pulses are produced by an amplified titanium-
sapphire laser system. The laser pulses are 100 fs long and have
a center wavelength of 800 nm and pulse energy of 1 mJ. The
laser beam is divided into two equally powerful beams. The
beam of 200 nm excitation pulses is generated by frequency
doubling and sum-frequency-mixing the 800 nm laser pulses
in three ꢀ-barium borate (BBO) crystals. The resulting pump
beam is focused onto the sample by a concave mirror. The beam
of probe pulses is generated by difference frequency mixing
the signal and idler pulses from an optical parametric amplifier,
which is pumped by the second beam from the titanium-sapphire
laser system. The probe beam is divided into a signal and a
reference beam. The signal beam is probing the sample inside
the volume excited by the pump pulse, whereas the reference
beam bypasses the sample. The signal and reference beams are
subsequently sent through a grating spectrometer and measured
by a 2 × 32 channels cooled dual-array HgCdTe detector. The
accuracy of the energy scale calibration is (10 cm-1, corre-
sponding roughly to 1 pixel in the HgCdTe detector.
The geometry of the nitrate anion requires a comment. The
isolated ion has a three-fold axis of symmetry, and the
considered N-O stretch vibration (E′) is two-fold degenerate.
In the solution, this symmetry is broken and the degeneracy is
lifted, causing the vibrational line to split into two.20 In nitrate
salts, the magnitude of the splitting depends on the counterion.21
We have also calculated the N-O stretch frequency for clusters
that include the potassium ion. (See Table 1.) The calculated
splitting of about 80 cm-1 is roughly the same in the three cases,
confirming that the splitting observed in NO-3 (aq) is caused by
the solvent. Table 1 also shows that the NdO bands in cis- and
trans-ONOO- are separated by about 100 cm-1, with the cis
isomer assigned to the highest vibrational transition frequency,
which is in agreement with most previous studies.22-24 For the
potassium and hydrogen salts of peroxynitrite in matrices, this
tendency is reversed.25,26
To obtain the steady-state infrared spectrum of peroxynitrite,
we synthesized peroxynitrite. The setup for the synthesis of
peroxynitrite in H2O was as described by Robinson and
Beckman.27 We note that by lowering the temperature to ∼5
°C, the yields are increased. Using the UV extinction coefficient
of ONOO- (ε ) 1670 M-1 cm-1 at λ ) 302 nm),28 we
determined the concentration of ONOO- to be 0.3 M. The
bending mode of liquid H2O is at 1644 cm-1 and thus precludes
the identification of typical NdO vibrations in the range from
1400 to 1700 cm-1. The D2O bending frequency is at 1210 cm-1.
Therefore, to facilitate the observation of the vibrational
transition frequencies in the infrared, we removed the H2O
solvent by repeated vacuum distillation (35 °C at 20 mbar) and
addition of D2O to the solution. After three to four cycles, most
H2O was either removed or exchanged to form HDO. Interest-
ingly, we were not able to synthesize any ONOO- using the
procedure described by Robinson and Beckmann27 using fully
deuterated solvents and reagents.
The sample consists of 0.07 M aqueous KNO3. Thin liquid
films of the samples are suspended in a constant flow between
two parallel, 50 µm thick titanium wires separated by 4 mm.17
No sample degradation is observed in the measurements, but
to avoid the potential buildup of photoproducts, we replace the
sample on a daily basis. This also reduces the accumulation of
HDO resulting from the gradual exchange of D2O with HDO
caused by the absorption of H2O by the thin liquid film and
subsequent H-D exchange in the sample reservoir. The
measurements presented below are taken with the polarizations
of the excitation and probe beams oriented at 90° with respect
to each other. This reduces the strong coherence spike at zero
delay present with collinear polarizations of the two beams.
To facilitate the experimental identification of the various
reactants and products, we performed electron structure calcula-
tions on the species solvated by 11 water molecules (D2O) using
the Gaussian03 software.18 A potential minimum was found that
allows the calculation of the harmonic vibration frequencies for
normal and heavy water. The chosen level of theory is the
B3LYP implementation of the density functional theory with
the basis set 6-31G(d). Inspection of the various frequencies
showed that the normal mode with a dominant contribution from
the terminal NdO stretch was a useful fingerprint in D2O
because its absorption is well separated from other bands. When
NO-3 is converted into ONOO-, the rearrangement of solvent
molecules around the anion changes because of differences in
geometry and charge distribution. To obtain some measure for
the sensitivity of the frequencies to the solvent rearrangement,
we have repeated the calculations for a number of different
The observed infrared spectrum of ONOO- is shown in
Figure 1. A drop of the solution is placed between two CaF2
windows to obtain a sample thickness of ∼5-10 µm. The cutoff
observed below 1000 cm-1 is caused by the CaF2 window
material. The D2O bending mode is observable at 1210 cm-1
,
but the corresponding bending modes of H2O and HDO at 1644
and 1460 cm-1 are not observed. The broad double peak at 1370/
1400 cm-1 corresponds to nitrate, which is a known byproduct
of the synthesis of ONOO-. Finally, we assign the line at 1560
cm-1 to cis-ONOO-. The solution of ONOO- is strongly basic
(pD > 11) to stabilize the ONOO-, and we believe that the slight

10490 J. Phys. Chem. A, Vol. 113, No. 39, 2009
Thøgersen et al.
Figure 1. Infrared absorption spectrum of ONOO- in D2O. The sample
is contained between two CaF2 windows spaced by ∼5-10 µm. The
low frequency cutoff is caused by the windows. The NO-3 signal is a
byproduct of the synthesis. The H2O solvent was replaced by D2O by
vacuum distillation, and no traces of either HDO or H2O are observed
in the spectrum.
difference between the observed cis-ONOO- line position at
1580 cm-1 in the femtosecond experiments (pD ≈ 7) and 1564
cm-1 in Raman and 1560 cm-1 in infrared absorption (pD >
11) is caused by the difference in basicity.
Figure 2. (a) Transient infrared absorption following the photolysis
at 200 nm of 0.07 M NO-3 in D2O. The transient absorption reveals the
strong bleaching of the nitrate vibrational transition at 1350 and 1400
cm-1 and induced absorption at higher frequencies. (b) Infrared
absorption frequencies of NO-3 in H2O. The two bars indicate the
position of the calculated frequencies. The bleaching in Figure 2a
recovers in a few picoseconds, and the similar line shapes in the
transient (a) and steady state (b) spectra are indicative of vibrational
relaxation not involving the NdO stretching vibrations.
Results
We have divided the presentation of the results into four
subsections. First, we briefly recapitulate the results of the UV
pump UV probe experiments. Then, we address the presence
of the strong background signal from the water solvent, and in
the last sections, we describe the assignment of the transient
infrared spectrum and the temporal dynamics of the observed
infrared peaks.
most of the infrared region with just a few narrow windows
with low absorption. Second, the infrared spectrum of water is
very temperature-dependent.29,30 When working in pump-
probe mode, where a strong pump initiates a chemical change,
there will be a large transient signal due to water that tends to
obscure signals from solute molecules. This is, in particular,
the case when the solute is hydrogen bonded to the water
solvent. To circumvent these challenges, we use heavy water,
D2O, which is relatively transparent in the range from 1300 to
1900 cm-1. Furthermore, we use a thin wire-guided film of
liquid water with a thickness between 10 and 20 µm to allow
for sufficient transmission of the infrared probe. Finally, we
have conducted a series of control experiments using nitrite,
NO-2 in H2O and D2O and used the results to estimate the
background response from the water solvent. However, true
background subtraction cannot be obtained because the specific
hydrogen (deuterium) bonding of both nitrite and nitrate is
different.
In our previous work,5 we have shown that when photolyzed
at 200 nm, 48% of the nitrate molecules will form peroxynitrite,
44% will return to the ground state of nitrate through fast internal
conversion, whereas the remaining 8% was ascribed to a solvent
separated pair of [O2 + NO]-, where the exact location of the
negative charge could not be determined. The formation of these
reaction products is in all three cases observed within the 300
fs time resolution of the experiment. The nitrate, formed through
internal conversion, is vibrationally hot, and within 2 ps, this
excess energy relaxes and the NO-3 molecule is again in the
electronic and vibrational ground state.
-1
k g 3 ps
NO-3 + pω (200 nm)
8 ONOO- (48%)
Figure 2a shows the transient infrared absorption in the region
from 1300 to 1600 cm-1 following photolysis of a 0.07 M
solution of NO-3 at λ ) 200 nm. Figure 2b shows the infrared
absorption of NO-3 in aqueous solution obtained from an FTIR
spectrometer. The characteristic double peak in Figure 2b comes
from the degenerate ν3 vibrations where the degeneracy has been
lifted by interaction with the solvent.8,20,21 The two gray bars
are the calculated vibrational frequencies listed in Table 1 for
the nitrate molecules microsolvated by 11 D2O molecules. The
transient signal in Figure 2a shows a strong bleaching caused
by the removal of nitrate molecules by the pump pulse. The
nitrate signal recovers during the first 10 ps because of internal
conversion and vibrational relaxation according to the reaction
schemes in eq 1. The line shape of the transient absorption is
very similar to that of the absorption spectrum in Figure 2b,
indicating a very fast vibrational relaxation mostly taking place
through low-frequency vibrations and not the NdO stretching
-1
-1
k g 3 ps
kvib rel ≈ 0.5 ps
8 [NO-3 ]X
8 NO-3 (44%)
(1)
-1
k g 3 ps
8 [NO + O2]- (8%)
In this work, we focus on the product channel where
peroxynitrite is formed, and we use femtosecond infrared pulses
to follow the reaction products.
Infrared spectroscopy of aqueous systems, in particular, with
femtosecond time resolution is inherently difficult. First of all,
the infrared absorption of water is very strong and spreads over

Primary Formation Dynamics of Peroxynitrite
J. Phys. Chem. A, Vol. 113, No. 39, 2009 10491
Figure 4. Transient infrared spectra obtained by subtracting the D2O
background obtained from Figure 3b. Amplitude scaling of the
background was obtained from the frequency range between 1650 and
1700 cm-1, where only the solvent contributes to the signal. At short
delays, the broad trans-ONOO- absorption from 1500 to 1550 cm-1
dominates. After 10 ps, the cis-ONOO- absorption appears at 1580
cm-1, and half of the trans-ONOO- absorption remains at 1515 cm-1
.
The absorption at 1610 cm-1, assigned to NO2, rises with a time constant
of ∼50 ps and remains constant after 80 ps.
Figure 3. (a) Transient infrared absorption following the photolysis
at 200 nm of 0.07 M NO-3 in D2O at selected delays showing the
absorption lines of trans-ONOO-, cis-ONOO-, and NO2 and the high
frequency wing of the NO-3 bleaching. The data were offset for clarity.
The red dotted line is the background signal from the thermally excited
D2O solvent obtained from the measurements of the solvent response
following the photolysis of NO-2 shown in part b. (b) Background
obtained from the photolysis of nitrite (NO-2 ) in both D2O and H2O,
where results from H2O was frequency scaled by ꢀ2.
modes. This is in good agreement with the notion that the
excited state populated in nitrate by the 200 nm pump pulse
has a pyramidal geometry giving rise to strong excitation of
bending modes upon internal conversion to the planar nitrate
ground state.
We now turn our attention to the transient infrared spectrum
of peroxynitrite observed following the photolysis of nitrate at
200 nm. Guided by the infrared spectrum in Figure 1, the Raman
spectra of peroxynitrite,16 showing a broad absorption line at
1564 cm-1, and our calculations of the microsolvated peroxy-
nitrite isomers listed in Table 1, we focus our attention on the
transient spectrum in the range from 1500 to 1700 cm-1 shown
in Figure 3a. In Figure 3b we also show the transient spectrum
observed 50 ps after the photolysis of nitrite.30 In nitrite, most
of the transient absorption in this region can be assigned to the
solvent alone, and the spectrum in Figure 3b can thus serve as
an approximate background spectrum for the photolysis of
nitrate. We show both the spectrum obtained in D2O (filled
circles) and the spectrum obtained in H2O (full line). The H2O
spectrum was spectrally shifted by dividing the frequency axis
with ꢀ2. In D2O, the dominant solvent mode in this frequency
range is the combination band containing the bending mode
and the low-frequency libration mode. The factor ꢀ2 thus
approximately shifts the H2O spectrum to the corresponding
position in D2O. The overall shapes of the background spectra
are similar, reflecting a temperature increase in the solvent
causing a red shift of the combination band.30 The transient
absorption of the nitrate-peroxynitrite system is shown in
Figure 3a. At the lowest frequencies, we observe the nitrate
bleaching also shown in Figure 2a, and at higher frequencies,
we observe a modulated signal caused by the combined signals
from the solvent background and the transient signal from the
photolysis of nitrate. We have measured the transient signals
to a delay of 300 ps and show only selected time delays in
Figure 3a.
Figure 5. 2D representation of the full set of time delay versus probe
frequency. The estimated solvent response from D2O was subtracted
from the data. The experimental data show the initial broad features
assigned to trans-ONOO- gradually relaxing to the trans- and cis-
ONOO- absorption at 1515 and 1580 cm-1. Isolated from these events,
the NO2 signal at 1610 cm-1 appears on a 50 ps time scale.
To facilitate the analysis and assignment of infrared spectral
features to cis- and trans-peroxynitrite, we show in Figure 4
the transient signals at five delay times from 2 to 100 ps. At
each delay, we have normalized the background estimated from
Figure 3b to give a “zero baseline” of the residual transient
absorption. We note that in the region above 1675 cm-1, the
time dependence of the transient absorption in both nitrite and
nitrate is similar, reflecting the same solvent response. The
transient signals are vertically displaced for visual clarity. We
observe three distinct spectral features in Figure 4. There are
two absorption lines at 1515 and 1580 cm-1, which we assign
to trans- and cis-peroxynitrite, respectively, guided by the IR
and Raman spectra and the calculated vibrational frequencies.
In addition, we observe at long delays appearing after ∼50 ps
a prominent infrared peak at 1610 cm-1. We also observe this
peak in the photolysis/photodetachment of nitrite and assign it
to NO2. The full set of transient absorption at all probe
frequencies is shown in a 2D plot in Figure 5. As in Figure 4,
the background caused by the heating and subsequent thermal-
ization of the solvent has been subtracted.

10492 J. Phys. Chem. A, Vol. 113, No. 39, 2009
Thøgersen et al.
indicates that only the trans-ONOO- isomer is formed directly
from the photoexcited nitrate molecules. Referring to the our
previous study of the UV-pump UV probe experiments,5 we
note that the UV absorption spectra of cis- and trans-ONOO-
are very similar. The observation of solely trans-ONOO-
immediately after photolysis of nitrate and the gradual conver-
sion of half of the molecules into cis-ONOO- is thus in
accordance with our observations in the UV region. From the
previous experiments,5 we know that the concentration of
peroxynitrite at pH ∼7 is constant from just after the photolysis
pulse up to the longest delays measured (300 ps). The assign-
ment of trans- and cis-ONOO- at 1515 and 1580 cm-1 is further
corroborated by lowering the pH (pD) of the solution and
monitoring the decay of the amplitudes of the 1515 and 1580
cm-1 signals because of diffusive protonation of ONOO-.4
Comparing the bleaching of the NO-3 signal at 1350 and 1400
cm-1 with the induced absorption of ONOO- and taking into
account the line strength calculated also provides a rough
verification of the quantum yields previously measured in the
UV region.5
Figure 6. Transient absorption traces at selected probe frequencies
showing the dynamics of the solvent (1688 cm-1), NO2 (1610 cm-1),
cis-ONOO- (1580 cm-1), and trans-ONOO- (1515 cm-1) and the
bleaching of NO-3 at 1413 cm-1. The data are shifted for clarity. The
estimated solvent background was not subtracted from the data.
Discussion
The infrared frequencies pertaining to the terminal OdN bond
in both cis- and trans-peroxynitrite is central to this work.
Experimentally, the only observation of peroxynitrite vibrational
frequencies in aqueous phase is the Raman spectra of Tsai et
al.16 Most prominent lines are a very broad line at 642 cm-1
assigned to torsional motion of the central -N-O- bond and
a broad line at 1564 cm-1assigned to the terminal NdO
stretching frequency. On the basis of comparison with ab initio
calculations, it was concluded that only the cis isomer is stable
in aqueous solution. In a recent QM/MM simulation by LeBrero
et al.,23 the Raman line at 642 cm-1 was reassigned to a -NO-
stretch, and it was shown how explicit inclusion of the aqueous
solvent produced a significant red shift and broadening of this
-NO- stretching frequency. Depending on the specific theo-
retical model used, they obtained a terminal NdO frequency
in the range of 1475 to 1621 cm-1. In conjunction with the
calculated stabilities and the isomerization barrier,31 it was again
concluded that peroxynitrite in aqueous solution solely exists
in the cis form. Additional information on cis- and trans-
peroxynitrite can be obtained from low-temperature spectra of
the species in an argon matrix,22,24 where the terminal NdO
vibrations of cis- and trans-peroxynitrite were observed close
to 1460 and 1435 cm-1, respectively. Therefore, in the majority
of experimental and theoretical calculations, the terminal NdO
vibration in cis-peroxynitrite is predicted to have the highest
frequency. This is also in good agreement with our own
calculations shown in Table 1. The values observed in the
present work for cis- and trans-ONOO- are 1515 and 1580
cm-1, which are in good agreement with the calculated values
from Table 1 and in agreement with the Raman and infrared
line position of the cis-ONOO- at 1564 cm-1 from Tsai et al.16
As mentioned, there is a slight shift of the cis-ONOO- frequency
when comparing the transient infrared spectrum in neutral
solution with the steady-state infrared spectra observed in
alkaline solutions by Tsai et al.16 and in the present work. The
simulations of Lebrero et al.,23 Tsai et al.,32 and our own
calculations show that hydration predominantly takes place by
hydrogen bonding to the peroxy oxygen atom (ONOO- · · ·H),
which results in a slightly shorter NdO bond and a correspond-
ingly higher NdO vibrational frequency. We observe that the
frequency is shifted from 1580 cm-1 in neutral solution to 1560
cm-1 when pH is increased. Assuming that peroxynitrite is
In Figure 6, we show the time dependence of the transient
absorption at five selected probe frequencies close to representa-
tive transitions: the solvent response at 1688 cm-1, the nitrate
bleach at 1413 cm-1, the absorption signals corresponding to
cis- and trans-peroxynitrite at 1562 and 1527 cm-1, and the
1610 cm-1 feature assigned to NO2. We have displaced the five
curves slightly to emphasize visually their time dependencies.
The trace at 1688 cm-1 is dominated by the combination band
of water (D2O) composed of the bending mode and the
librational band. Following the photolysis of nitrate, the solvent
heats up, causing the combination band to red shift, which gives
rise to a strong negative transient absorption (bleaching) at the
probe frequencies from 1625 to 1740 cm-1 in Figure 5.30 The
time dependence of the solvent signal reflects the dissipation
of heat from the reaction site and is well represented by a double
exponential decay with decay times of 1.8 and 36 ps. This
temporal behavior of the signal at 1688 cm-1 is similar to the
transient absorption observed in nitrite at probe frequencies far
from nitrite resonances.30 We know from previous work that
the spectral profile of the solvent relaxation is unchanged after
∼2 ps, so any time dependence different from the double
exponential decay at 1688 cm-1 is indicative of the dynamics
of the reaction products.
The 1515 cm-1 signal, corresponding to the trans-ONOO-
peak, appears within the time resolution of the experiment. In
Figures 4 and 5, it is seen that the trans-ONOO- absorption at
short delays is broader and shifted to lower wavenumbers. The
signal gradually blue shifts and decays. The time constant for
this decay is difficult to entangle from the strong solvent
response, but we estimate that the trans-ONOO- absorption
decays to half of its initial value in 25 ps. The decay of the
trans-ONOO- absorption is followed by a similar rise of the
cis-ONOO- absorption at 1580 cm-1. If the decay times of
the solvent are taken into account, then the signals from trans-
and cis-ONOO- exhibit the same decay and risetime of about
25 ps. After 50 ps, both the trans- and cis-ONOO- signals
remain largely unchanged. We estimate that about half of the
trans-ONOO- isomerizes to cis-ONOO- and that the initial
concentration of the cis-ONOO- isomer is close to zero. The
estimate is based on the calculated intensities, shown in Table
1, for the infrared transitions in the cis and trans isomers. This

Primary Formation Dynamics of Peroxynitrite
J. Phys. Chem. A, Vol. 113, No. 39, 2009 10493
hydrated to a lesser degree at high pH, this is in accordance
with the suggested hydration structures of the peroxynitrite-water
system. A similar but opposite trend is seen in the infrared
spectrum of nitrate. In this case, hydration leads to a red shift
in the NO frequency as the water molecules on the average
hydrogen bond equally to the oxygen atoms of nitrate, thus
lengthening the NO bond slightly and lowering the vibrational
frequency.
Cis and Trans Conversion. The photolysis of aqueous nitrate
is initiated by the excitation of the π f π* transition with a
maximum near 205 nm. The strong transition accesses two
nearly degenerate excited electronic states.21 Resonance Raman
experiments indicate that the excited states have pyramidal
geometry33 with a single longer NO bond compared with the
ground state. Both of these properties facilitate the isomerization
of nitrate into trans-peroxynitrite.
tolyzed NO-3 under acidic conditions (pH ∼1) and observed
the rapid decay of the peroxynitrite peaks due to the formation
of peroxynitrous acid (ONOOH), but the 1610 cm-1 was
unaffected. Finally, we photodetached the electron in NO-2 (aq)
and observed NO2 at 1610 cm-1, again with a relatively slow
appearance time similar to that observed in the photolysis of
NO-3 (aq). We can thus conclude that the signal at 1610 cm-1 is
caused by NO2 and is not associated with the formation and
isomerization of peroxynitrite. To our knowledge, this is an
unusually slow solvation process for NO2, and one could instead
speculate whether we are monitoring a slow dissociation of a
solvent-separated contact pair [O--(aq)-NO2]. A similar
solvent-separated contact pair was recently shown in simula-
tions34 to play a role in the homolysis of the peroxynitrous acid
(ONOOH) into OH + NO2. If this assignment is valid, then
our results thus provide a direct measurement of the lifetime of
the solvent-separated contact pair. The observed lifetime is,
based on the risetime of the 1610 cm-1, ∼50 ps. This relatively
long lifetime indicates that the contact pair must be considered
to be an important intermediate in secondary reactions following
the photolysis of concentrated nitrate solution. In particular,
when the solvent-separated contact pair breaks up, the O- moiety
will be protonated (pH ∼7) and thus serve as a source of
hydroxyl radicals. Furthermore, experimental and theoretical
studies are underway to confirm the assignment of the 1610
cm-1 peak to NO2. The slow appearance of the NO2 signal is
also the reason we did not observe it in our previous UV
pump-UV/vis probe experiments.5 In that work, we scanned
only to delays shorter than 18 ps at selected wavelengths not
including the region where NO2 absorbs. Furthermore, given
the very low UV extinction coefficient of NO2(aq), the estimated
signal strengths would be close to the detection limit. Given
the observation of a slowly emerging NO2 signal in this work,
we reconsider the assignment and yield of the third channel in
eq 1. The third channel was assigned to [NO + O2]-, but with
the present observation, we find that both the UV and IR
transient data are consistently described by [NO2 + O-]. The
UV spectra of O-2 , NO-, and O- are very similar apart from
their extinction coefficient. The smaller extinction coefficient
of O- in UV and the strength of the 1610 cm-1 line observed
in this work led us to increase the yield of the third channel
from 8 to 15-25%. We also note that reassigning the third
channel to NO2 + O- is in agreement with recent work from
Goldstein and Rabani.6 We have considered if the solvent
separated contact pair of [NO2+O-] could be formed from trans-
peroxynitrite along with cis-peroxynitrite. However, because of
the low signal strengths and the inherent problem of the large
background signal caused by the heated water solvent, we are
not able to answer this question presently.
Only a few high-level ab initio calculations are available for
the water-nitrate system, and they primarily address the
solvent-solute interactions in the ground state. Simple geometric
considerations suggest that the trans isomer of peroxynitrite is
formed from the pyramidal geometry of the excited states in
nitrate. However, experimental evidence5,6,16 indicates that only
the cis form is present in aqueous solution. This is in good
agreement with ab initio calculations,31 suggesting that the cis
isomer is more stable by ∼20 kJ/mol with a rotational barrier
of ∼100 kJ/mol around the ON-OO bond caused by the
partially π-character of the planar peroxynitrite molecule. Our
observation of trans-ONOO- immediately after the photolysis
pulse is in accord with the notion of a pyramidal transition state,
and the subsequent isomerization is also in agreement with the
notion that the cis form is the more stable. We do, however,
see only a partial isomerization, where about half of the trans
isomerizes on a 25 ps time scale. This can be explained by
considering the microenvironment in which the trans-ONOO-
is formed: Nitrate is photolyzed by a 200 nm pulse, and a
substantial amount of excess energy is dissipated to the solvent
during the formation of ONOO-. This excess energy rapidly
(∼1 ps) thermalizes, resulting in a very hot solvent sphere
surrounding the trans-ONOO- molecule. The local temperature
increase causes the broadening and slight shift of the trans-
ONOO- peak at 1515 cm-1 observed at short time delays. The
hot solvent is also driving the isomerization of trans- into cis-
ONOO-, thereby converting about half of the trans- into cis-
peroxynitrite. Simultaneously, the excess heat diffuses away
from the reaction site, causing the temperature of the solvent
sphere and, consequently, the rate of trans-to-cis isomerization
to drop. Our experimental observations are limited to a delay
time of 300 ps, and we do not observe a slow decay of the
remaining trans-ONOO-. However, the acceleration of the trans-
to-cis isomerization caused by the heated solvent, estimated to
be ∼30-60 degrees above room temperature30 during the first
tens of picoseconds, is in accordance with an isomerization
barrier of ∼100 kJ/mol.
Conclusions
We have observed the formation of trans-ONOO- following
the photolysis of NO-3 in D2O solution at 200 nm. Using infrared
probe frequencies, we observed the trans isomer at 1515 cm-1
and subsequently its partial isomerization to the cis-isomer on
a 25 ps time scale. After 25 ps, the ratio between cis- and trans-
ONOO- is unchanged out to the longest delay (300 ps) observed
in this work. The cis-ONOO- is observed at 1580 cm-1, which
is in good agreement with infrared and Raman experiments.16
We have also observed a prominent infrared absorption at 1610
cm-1, which is assigned to NO2 following the slow (∼50 ps)
decay of a solvent-separated contact pair between O- and NO2.
Observation of NO2. In addition to the transient absorption
pertaining to peroxynitrite, we also observe a relatively strong
infrared peak at 1610 cm-1 that we tentatively assign to NO2.
On the basis of the observed and calculated line strength of the
1610 cm-1 vibration, we estimate that the yield of this third
channel is between 15 and 25%. It is very interesting that the
vibrational transition assigned to NO2 appears with a large delay
of ∼50 ps. We performed several experiments to test the validity
of the assignment of the 1610 cm-1 peak to NO2. We used
different concentrations of nitrate and observed the 1610 cm-1
absorption to scale linearly with NO-3 concentration. We pho-
Acknowledgment. We acknowledge the support from the
Danish Natural Science Research Council and the Carlsberg

10494 J. Phys. Chem. A, Vol. 113, No. 39, 2009
Thøgersen et al.
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Foundation. We wish to thank Professor Koppenol and his
colleagues at ETH, Zu¨rich, for useful discussions.
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