Identification of Fleeting Electrochemical Reaction Intermediates Using Desorption Electrospray Ionization Mass Spectrometry

Article abstract of DOI:10.1021/jacs.5b03862

We report a new method for the mass spectrometric detection of fleeting reaction intermediates in electrochemical reactions utilizing a "waterwheel" working electrode setup. This setup takes inspiration from desorption electrospray ionization (DESI) mass spectrometry, where the sampling time is on the order of milliseconds, to sample directly from the surface of a working electrode for mass spectrometric analysis. We present data that show the formation of a diimine intermediate of the electrochemical oxidation of uric acid that has a lifetime in solution of 23 ms as well as data that provide evidence for the formation of a similar diimine species from the electrooxidation of xanthine, which has not been previously observed.

Full text of DOI:10.1021/jacs.5b03862

Communication
pubs.acs.org/JACS
Identification of Fleeting Electrochemical Reaction Intermediates
Using Desorption Electrospray Ionization Mass Spectrometry
Timothy A. Brown,‡ Hao Chen,*,† and Richard N. Zare*,‡
‡Department of Chemistry, Stanford University, Stanford, California 94305-5080, United States
†Center for Intelligent Chemical Instrumentation, Department of Chemistry and Biochemistry, and Edison Biotechnology Institute,
Ohio University, Athens, Ohio 45701-2979, United States
S
* Supporting Information
As depicted in Figure 1, the experimental design employs a
round rotating platinum working electrode with a width of 0.64
ABSTRACT: We report a new method for the mass
spectrometric detection of fleeting reaction intermediates
in electrochemical reactions utilizing a “waterwheel”
working electrode setup. This setup takes inspiration
from desorption electrospray ionization (DESI) mass
spectrometry, where the sampling time is on the order
of milliseconds, to sample directly from the surface of a
working electrode for mass spectrometric analysis. We
present data that show the formation of a diimine
intermediate of the electrochemical oxidation of uric acid
that has a lifetime in solution of 23 ms as well as data that
provide evidence for the formation of a similar diimine
species from the electrooxidation of xanthine, which has
not been previously observed.
n situ analysis of electrochemical reactions is traditionally
1
Icarried out using cyclic voltammetry, spectroelectrochem-
istry,2,3 and/or surface scanning probe techniques.4 However,
these methods lack high chemical specificity. In this regard,
mass spectrometry (MS) can serve as a powerful detector for
electrochemical reactions with high sensitivity and selectivity
because MS can provide molecular weight information for
products and/or intermediates of redox reactions.5−8
Online coupling of electrochemistry (EC) with MS has a
history of over 40 years9 that includes ionization techniques
such as electrospray ionization (ESI),6,10,11 desorption ESI
(DESI),7,11 thermospray,12−14 nanoDESI,15 and differential
electrochemical mass spectrometry (DEMS).16,17 The chal-
lenge in acquiring faster response times exists in transferring
electrochemically generated species quickly from solution to
the gas phase for MS detection. DEMS has been shown to have
response times as fast as 0.1 s,17 but most electrochemistry
coupled ambient ionization mass spectrometry methods
typically have response times on the order of seconds.18,19
The development of DESI20−22 and the subsequent easy
ambient sonic-spray ionization (EASI),23−25 allows analyses to
be made on surfaces under atmospheric pressure at room
temperature with a sampling time on the order of milli-
seconds.26,27 The technique presented below is an ambient
ionization mass spectrometric approach that utilizes DESI-MS
with no high voltage applied to the spray solution. The
electrode surface is sampled in situ by fast transfer of
electrogenerated species to the gas phase for analysis by the
mass spectrometer on the time scale of milliseconds.
Figure 1. (top) Apparatus used to detect electrochemical reaction
intermediates, employing a rotating waterwheel electrode. The
direction of rotation is indicated by the arrow. (bottom) Zoomed-in
area showing sample ionization.
cm (0.25 in) and a diameter of 3.81 cm (1.50 in) that is
partially immersed in an aqueous electrolyte solution. As the
working electrode rotates, a thin layer of liquid film
(approximately 1 mm thick) develops on the electrode surface,
forming a “waterwheel”. A platinum wire counter electrode and
a Ag/AgCl reference electrode are immersed in the reservoir of
electrolyte solution. A metal contact (not shown) rests against
the platinum working electrode to complete the three-electrode
system. Above the rotating waterwheel system, a custom spray
probe directs a spray of analyte microdroplets to the surface of
the working electrode. To ensure that any electrochemical
Received: April 14, 2015
Published: June 1, 2015
© 2015 American Chemical Society
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Journal of the American Chemical Society
Communication
oxidation or reduction is constrained to take place at the
electrode surface, no high voltage is applied to the analyte
spray. The spray droplets hit the surface of the thin film of
electrolyte solution, which is now thinner than 1 mm because
of the N2 nebulizing gas pressure, on the surface of the working
electrode. Tinier secondary microdroplets are directed into the
mass spectrometer28,29 and are analyzed with an LTQ Orbitrap
XL hybrid mass spectrometer (Thermo Fisher Scientific, San
Jose, CA) where the m/z ratio is determined utilizing the high
mass accuracy and high resolving power of the Orbitrap mass
analyzer.30 A potentiostat (WaveNow, Pine Research Instru-
mentation, Durham, NC) is used to apply a potential across the
three electrodes. The electrolyte solution in the bath is 1 mM
ammonium acetate unless specified otherwise.
The electrochemical oxidation of triphenylamine (TPA) is
well-understood,18,31,32 so TPA was chosen as a proof-of-
concept analyte. A 100 μM TPA solution was prepared in 1:1
MeCN/H2O with 1% acetic acid. When the analyte solution is
sprayed at an infusion rate of 5 μL/min at the surface of the
rotating waterwheel electrode rotating at 2.5 rev/s, a cation at
m/z 246.1266 is observed, which is ascribed to protonated TPA
(Figure 2a; theoretical m/z 246.1277, error −4.5 ppm).
been previously reported,18,31,32 corresponding to the radical
cation dimer at m/z 488.2241 (theoretical m/z 488.2247, error
−1.2 ppm) and the dicationic dimer at m/z 244.1122
(theoretical m/z 244.1121, error +0.3 ppm). The signal
intensity of each dimer peak markedly increases when an
oxidizing potential is applied to the working electrode relative
to when no potential is applied, indicating that these species are
also formed as a result of TPA electrooxidation (see the
Supporting Information). TPA electrochemical oxidation as a
function of potential at different electrode rotation rates has
also been studied, and the results are presented in the
Supporting Information.
With the success of TPA electrochemical oxidation, the next
goal was to investigate the feasibility of using this experimental
setup to observe transient electrochemical reaction intermedi-
ates. Uric acid and xanthine, two biologically important
molecules, were chosen for investigation. It is known that the
electrochemical oxidation and enzymatic oxidation of uric acid
and a number of its derivatives proceed by similar reaction
pathways.33−35 The proposed mechanism for the oxidation of
uric acid at physiological pH initiates with a two-electron, two-
proton (2e−, 2H+) oxidation, leading to the formation of an
unstable intermediate proposed to be a diimine species
(Scheme 1, 1) on the basis of cyclic voltammetry.36 This
Scheme 1. Initial Steps of Uric Acid Oxidation
diimine species, with a measured half-life in solution of 23 ms at
pH 8, readily reacts with H2O to yield the imine alcohol,
whereas at higher or lower pH the half-life of the diimine
species is even shorter.33 Previous EC-MS studies have not
reported the observation of the diimine species in situ, but the
formation of the imine alcohol (Scheme 1, 2) has been
observed.13,35 The proposed diimine intermediate was observed
with MS when the imine alcohol was fragmented via tandem
MS,35 but the diimine intermediate has not been observed
using online EC-MS techniques.
A 100 μM solution of uric acid in water with 1% acetic acid
was prepared. When the analyte solution is sprayed at an
injection flow rate of 15 μL/min onto the working electrode
rotating at 2.5 rev/s, the deprotonated uric acid anion is
observed at m/z 167.0209 (Figure 3a; theoretical m/z
167.0211, error −1.2 ppm). When an oxidation potential of
1.0 V is applied across the rotating working electrode, a peak at
m/z 165.0053 is observed, which is ascribed to the diimine
intermediate (Figure 3b; theoretical m/z 165.0054, error −0.9
ppm). The MS signal of this species increases greatly when an
oxidizing potential is applied to the working electrode, as is
observed in the EIC (Figure 3c). This observation indicates
that it originates from the electrolysis of uric acid. Interestingly,
the imine alcohol 2 (theoretical m/z 183.0160) was not
observed to have a correlation with the application of the
oxidation potential, emphasizing the fast transfer of electro-
chemically generated species from the electrode surface to the
mass spectrometer. Previous work utilizing this waterwheel
setup has been performed in which the imine alcohol has been
observed (see the Supporting Information). This is the first
Figure 2. (a, b) Positive-ion-mode mass spectra of triphenylamine
with (a) 0.0 V and (b) 1.0 V applied to the working electrode. (c) EIC
for the m/z 245.1189 peak as a function of the applied potential.
Applying an oxidation potential of 1.0 V to the rotating
working electrode results in the formation of an ion at m/z
245.1189, which is attributed to the TPA radical cation as a
result of electrochemical oxidation of TPA (Figure 2b;
theoretical m/z 245.1199, error −4.1 ppm). The signal intensity
of the radical cation increases greatly when an oxidizing
potential is applied to the rotating working electrode relative to
when no potential is applied to the rotating working electrode,
as is observed in the extracted ion chromatogram (EIC) of the
m/z 245.1189 peak (Figure 2c). The high mass accuracy and
the signal response of the m/z 245.1189 peak to an oxidation
potential provides strong evidence for the formation of the
radical cation by way of electrochemical oxidation of TPA.
Peaks associated with dimer species are observed that have
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Communication
Figure 4. (a, b) Negative-ion-mode mass spectra of xanthine with (a)
0.0 V and (b) 2.0 V applied to the working electrode. The peak
regions for m/z 149.0106, 165.0053, and 167.0210 have been
magnified 50 times for clarity. (c) EIC for the 149.0106 peak as a
function of the applied potential.
Figure 3. (a, b) Negative-ion-mode mass spectra of uric acid with (a)
0.0 V and (b) 1.0 V applied to the working electrode. (c) EIC for the
m/z 165.0053 peak as a function of the applied potential.
online EC-MS observation of the unstable diimine, which
provides strong evidence for the formation of the diimine
intermediate in the electrochemical oxidation of uric acid.
Xanthine, another purine compound, was also examined.
Xanthine is proposed to undergo a series of electrochemical
oxidations, the first giving uric acid and the subsequent ones
following the oxidation pathway of uric acid.33,36 Unlike uric
acid, a diimine species produced from the direct 2e−, 2H+
electrooxidation of xanthine has not been observed by EC-MS
or cyclic voltammetry, leading to a proposed electrooxidation
mechanism from xanthine directly to uric acid.33,36
Scheme 2. Initial Steps of Xanthine Oxidation
A 1 mM xanthine solution was prepared with 10 mM
ammonium acetate in water and 0.8% acetic acid. When the
analyte solution is sprayed at an injection flow rate of 20 μL/
min onto the working electrode rotating at 1.5 rev/s, the
deprotonated xanthine is observed in negative-ion mode at m/z
151.0261 (Figure 4a; theoretical m/z 151.0261, error −0.3
ppm). When an oxidation potential of 2.0 V is applied to the
rotating working electrode, a peak at m/z 149.0106 is observed
(Figure 4b), ascribed to the xanthine diimine (Scheme 2, 1,
theoretical m/z 149.0105, error +0.5 ppm); this peak is
observed only when an oxidation potential is applied to the
working electrode (Figure 4c). This is the first online EC-MS
evidence for the formation of a xanthine diimine intermediate
in the electrochemical oxidation of xanthine.
Upon the application of an oxidation potential to the
working electrode, peaks were also observed at m/z 167.0210,
attributed to the water addition product to form uric acid
(Figure 4b; theoretical m/z 167.0211, error −0.4 ppm), and m/
z 165.0054, attributed to species 3 of Scheme 2, the 2e−, 2H+
electrooxidation product of uric acid (Figure 4b; theoretical m/
z 165.0054, error −0.3 ppm). The MS signal intensity of each
of these species increases greatly with the applied potential,
indicating that they originate from the electrochemical
oxidation of xanthine (see the Supporting Information).
Comparison of Figures 3b and 4b shows that the same m/z
165 and 167 ions observed for the electrochemical oxidations of
both uric acid and xanthine. This supports the proposed
mechanism that xanthine oxidizes to uric acid before under-
going further oxidation along the uric acid oxidation pathway.36
This study has presented a new application of electro-
chemistry coupled with ambient ionization mass spectrometry
that is capable of detecting transient intermediates generated on
the electrode surface. The transient diiimine intermediates
resulting from electrochemical oxidations of uric acid and
xanthine were successfully captured and analyzed by MS.
Because of the sensitivity of MS, minute amounts of analyte are
needed for this experiment. The mass information offered by
MS gives this approach high specificity. The results show
promise for capturing active electrochemical intermediates on
an electrode surface by employing a rotating waterwheel
electrode cell with fast transfer of electrogenerated species from
the electrode surface to the gas phase for MS detection. This
ambient ionization mass spectrometric technique probes
electrochemical reactions on the time scale of milliseconds,
which can elucidate mechanistic information for many
oxidation−reduction reactions.
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(25) Fernandes, A. M. A. P.; Fernandes, G. D.; Barrera-Arellano, D.;
de Sa, G. F.; Lins, R. D.; Eberlin, M. N.; Alberici, R. M. J. Mass
́
Spectrom. 2014, 49, 335.
ASSOCIATED CONTENT
■
S
* Supporting Information
Mass spectra, text, and figures giving experimental details. The
Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/jacs.5b03862.
(26) Perry, R. H.; Splendore, M.; Chien, A.; Davis, N. K.; Zare, R. N.
Angew. Chem. 2011, 123, 264.
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AUTHOR INFORMATION
■
Corresponding Authors
*chenh2@ohio.edu
*zare@stanford.edu
Notes
(32) Chiu, K. Y.; Su, T. X.; Li, J. H.; Lin, T.-H.; Liou, G.-S.; Cheng,
The authors declare no competing financial interest.
S.-H. J. Electroanal. Chem. 2005, 575, 95.
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ACKNOWLEDGMENTS
■
T.A.B. thanks the Center for Molecular Analysis and Design
(CMAD) for a graduate fellowship. H.C. is grateful for support
through an NSF Career Award (CHE-1149367), an ASMS
Research Award, and a sabbatical fellowship from Ohio
University for his visit to the Zare laboratory and for helpful
discussions with Jared Butcher and Howard Dewald. This work
was supported by a subcontract with the University of Utah
(Agreement 10029173-S2) for which the Air Force Office of
Scientific Research (Grant FA9550-12-1-0481) is the prime
sponsor.
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