Multistage Reactive Transmission-Mode Desorption Electrospray Ionization Mass Spectrometry

Article abstract of DOI:10.1007/s13361-015-1171-5

Elucidating reaction mechanisms is important for advancing many areas of science such as catalyst development. It is often difficult to probe fast reactions at ambient conditions with high temporal resolution. In addition, systems involving reagents that

Full text of DOI:10.1007/s13361-015-1171-5

B American Society for Mass Spectrometry, 2015
J. Am. Soc. Mass Spectrom. (2015) 26:1494Y1501
DOI: 10.1007/s13361-015-1171-5
RESEARCH ARTICLE
Multistage Reactive Transmission-Mode Desorption
Electrospray Ionization Mass Spectrometry
Kevin C. Peters, Troy J. Comi, Richard H. Perry
Department of Chemistry, University of Illinois, Urbana, IL 61801, USA
Abstract. Elucidating reaction mechanisms is important for advancing many areas of
science such as catalyst development. It is often difficult to probe fast reactions at
ambient conditions with high temporal resolution. In addition, systems involving
reagents that cross-react require analytical methods that can minimize interaction
time and specify their order of introduction into the reacting system. Here, we explore
the utility of transmission mode desorption electrospray ionization (TM-DESI) for
reaction monitoring by directing a microdroplet spray towards a series of meshes
with micrometer-sized openings coated with reagents, an approach we call multi-
stage reactive TM-DESI (TMn-DESI, where n refers to the number of meshes; n=2 in
this report). Various stages of the reaction are initiated at each mesh surface,
generating intermediates and products in microdroplet reaction vessels traveling towards the mass spectrometer.
Using this method, we investigated the reactivity of iron porphyrin catalytic hydroxylation of propranolol and other
substrates. Our experimental results indicate that TMn-DESI provides the ability to spatially separate reagents
and control their order of introduction into the reacting system, thereby minimizing unwanted reactions that lead to
catalyst deactivation and degradation products. In addition, comparison with DESI-MS analyses (the Zare and
Latour laboratories published results suggesting accessible reaction times <1 ms) of the reduction of
dichlorophenolindophenol by L-ascorbic acid suggest that TM1-DESI can access reaction times less than
1 ms. Multiple meshes allow sequential stages of desorption/ionization per MS scan, increasing the number of
analytes and reactions that can be characterized in a single experiment.
Keywords: Ambient mass spectrometry, Transmission-mode desorption electrospray ionization, Reaction mon-
itoring, Mechanisms, Time scale, Catalysis, Iron porphyrin
Received: 25 February 2015/Revised: 16 April 2015/Accepted: 18 April 2015/Published Online: 20 June 2015
pot complex transformations that improve efficiency, selectiv-
ity, and enantiomeric purity. Advancing these areas of research
Introduction
haracterizing reaction mechanisms is important for devel-
oping new catalysts, drugs, and materials that address
demand analytical technologies that can capture fleeting inter-
mediates in real-time at ambient conditions, as well as provide
the ability to separate reagents and specify their order of intro-
duction to minimize unwanted side reactions between incom-
patible reagents during analysis and to facilitate step-wise
elucidation of reaction mechanisms.
Electrospray ionization mass spectrometry (ESI-MS)
methods are one of the primary approaches for obtaining real-
time information about solution-phase molecular species
formed in the course of a reaction with high sensitivity, speed,
and selectivity [7]. The recent introduction of ambient mass
spectrometric techniques [8–15] such as desorption
electrospray ionization (DESI)[16] developed by Cooks and
co-workers has revolutionized analytical chemistry in the past
decade, allowing chemical analyses with minimal sample prep-
aration. A recent advance developed by Zare and co-workers
C
scientific and socioeconomic problems[1]. However, it is often
difficult to obtain detailed molecular mechanistic information
under normal operating conditions because intermediates have
short lifetimes (typically less than ~1 s), exist in a complex and
dynamic matrix involving multiple reaction pathways, and
have low concentrations [2–5]. These difficulties are signifi-
cantly more pronounced for homogenous multi-catalytic sys-
tems [6], which show great promise for rapidly achieving one-
Electronic supplementary material The online version of this article
(doi:10.1007/s13361-015-1171-5) contains supplementary material, which is
available to authorized users.
Correspondence to: Richard Perry; e-mail: rhperry@illinois.edu

K. C. Peters et al.: Multistage Reactive Transmission-Mode DESI
1495
Mesh 1 (M1) Mesh 2 (M2)
Desorbed microdroplets
(a)
involves using DESI to capture short-lived solution-phase re-
action intermediates (<1 ms) [17–23] at ambient conditions,
while minimizing sample preparation times, carry over effects,
and experimental complexity compared with ESI config-
urations. This elegant discovery has opened the possibility for
the development of new types of ambient ionization
sources and applications for characterizing fast solution-phase
processes [4, 24–32].
mCPBA
Liquid:10-30µL/min
+
+
+
+ +
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
N2 =0.5 L/min
MS
Substrate
Fe Porphyrins
Transmission-mode DESI (TM-DESI)[33–38], developed
1 mm 2-4 mm
6-8 mm
by Brodbelt and co-workers, is an ambient MS method that
involves directing an electrostatically charged solvent spray at
a mesh having micrometre-sized open areas on which analytes
of interest are deposited. TM-DESI requires small sample
(b)
Mesh Holder
Sprayer Holder
Latch
Micromanipulators
Meshes
y
x
volumes and minimal source optimization prior to analysis,
making it easily amenable to high-throughput analyses[33,
z
36, 39, 40]. TM-DESI has been previously used for direct
MS
analysis of samples containing analytes such as peptides and
small organic molecules [33]. In addition, the mesh can be
functionalized for selective extraction of analytes from com-
plex matrices followed by TM-DESI-MS [35, 40]. Despite its
simplicity, the utility of TM-DESI for probing chemical reac-
tivity has not yet been explored.
Herein, we describe a TM-DESI-based ionization source
that employs two meshes in series (M1 and M2) (Figure 1),
referred to hereafter as multistage reactive transmission-mode
Lab Jack
N2 Line
Rotatable
Posts
Microdroplet
Emitter/Sprayer
Stage
Alignment Bar
Fused Silica Capillary (FS1)
Figure 1. (a) Two-dimensional representation of TM2-DESI-
MS. (b) Three-dimensional rendition showing the meshes in
front of the MS inlet (for clarity, the micromanipulators that
attach the sprayer holder to the stage and the alignment bar
are not shown; their locations are indicated with dashed lines).
Inset shows a close-up 3D view of the rotatable posts, mesh
holder, and alignment bar. These images are not drawn to scale
DESI (TMn-DESI, where n represents the number of desorp-
tion stages; conventional TM-DESI configuration has n=1) for
characterization of reaction mechanisms. Using TM2-DESI-
MS, we studied iron (Fe) porphyrin-catalyzed hydroxylations
such as Fe tetra(pentafluorophenyl)-porphyrin (Fe-TPFPP, 1)
hydroxylation of substrate propranolol 2 (proposed mechanism
shown in Scheme 1 [41]), previously analyzed by ESI Fourier
transform ion cyclotron resonance MS[42, 43]. Experimental
results show that TM2-DESI provides the ability to spatially
separate reagents and to specify their order of introduction into
microdroplet reaction vessels, reducing off-path processes such
as oxidant-mediated hydroxylation of 2 (Scheme 1). In addi-
tion, comparison of TM1-DESI-MS and DESI-MS analyses of
the chemical reduction of dichlorophenolindophenol (DCIP)
by L-ascorbic acid (L-AA; Figure 3; reaction previously ana-
lyzed by liquid DESI-MS[44]) showed that TM1-DESI can
access reaction times less than 1 ms. These capabilities,
coupled with the high-throughput features of TM-DESI, dem-
onstrate a unique ambient ionization approach for chemical
analyses at ambient conditions. Multiple meshes increase the
number of analytes and reaction steps that can be characterized
per MS scan by a factor of n, which has great potential for
reducing analysis time and increasing high-throughput analy-
ses at ambient conditions.
Fe porphyrin catalysts (Fe tetra(pentafluorophenyl)-
porphyrin, Fe-TPFPP, 1; Fe tetraphenylporphyrin, Fe-TPP;
Fe tetramesitylporphyrin, Fe-TMP; Fe tetra(2,6-
dichlorophenyl)porphyrin TDCPP, Fe-TDCPP; Fe tetra(2,6-
difluorophenyl)porphyrin, Fe-TDFPP), and propranolol 2,
which were purchased from Frontier Scientific, Inc. (Logan,
UT, USA). The polyether ether ketone (PEEK) meshes (71 μm
strand diameter, 56% open area) were purchased from Small
Parts, Inc. (Miramar, FL, USA). PEEK was chosen as the mesh
material because of its high chemical resistance to dichloro-
methane (CH2Cl2) compared with other polymers such as
polypropylene.
Design of the Multistage Reactive Transmission
Mode Desorption Electrospray Ionization Source
The microdroplet sprayer of the TMn-DESI source has similar
specifications to the home-built DESI source described by
Cooks and co-workers[45]. Briefly, the sprayer consists of a
Swagelok (Swagelok Company, Fremont, CA, USA) 1/16′′
stainless steel (SS) tee through which a fused silica (FS1)
capillary (75 μm internal diameter (i.d.); 190 μm outer diameter
(o.d.); Polymicro Technologies, Phoenix, AZ, USA) delivers
Experimental
Materials
All chemicals were purchased from Sigma-Aldrich (St. Louis,
MO, USA) and used without further purification, except for the

1496
K. C. Peters et al.: Multistage Reactive Transmission-Mode DESI
(a)
connected to x, y, and z micromanipulators (location shown by
dotted lines in Figure 1b) mounted on a modified PicoView
550 (New Objective, Inc., Woburn, MA, USA) stage. The
stage mounts on the frame of an LTQ-Orbitrap XL mass
spectrometer[46–51] via a pair of latches (Figure 1b).
Each mesh is held in place by a pair of rotatable posts (6.5
cm in height) that are secured to separate mesh holders via set
screws (Figure 1b). The rotatable posts provide control of mesh
position and tension, which are critical parameters for
obtaining reproducible measurements; a loose mesh has erratic
motion in the microdroplet plume, which causes large varia-
tions in signal intensity at a given concentration of analyte. The
rotatable posts are composed of two segments that can be
clamped together by a pair of screws (Figure 1b inset) to
securely hold the meshes in place. The mesh holders are
controlled by a set of x, y, and z micromanipulators mounted
on a lab jack adjacent to the stage (Figure 1b). The microma-
nipulators are used to position the sprayer and meshes such that
the sprayer is normal to the meshes and 0o relative to the MS
inlet. To aid in positioning, the lower mesh holder rests against
an aluminium alignment bar (Figure 1b inset) that is affixed to
the TMn-DESI stage with a pair of clamps (location shown by
dotted lines in Figure 1b). The alignment bar ensures
that the mesh-to-inlet angle and distance remains constant
when analyzing multiple samples located at different positions
on the same mesh (different samples are accessed by motion in
the y-z plane). The inter-mesh distance and M2-to-inlet dis-
tances are shown in Figure 1a.
(b)
Mass Spectrometry
In a typical TM2-DESI experiment, the meshes are first washed
Scheme 1. (a) Catalytic cycle for Fe-TPFPP oxidation of pro-
pranolol. Species 1a and 1c (red) could not be unambiguously
identified because they are isobaric with degradation products
having oxygen insertion into the porphyrin ring. (b) Degradation
pathway of Fe-TPP from reference [55]
with a 1:1:1 mixture of water (H2O), methanol (CH3OH), and
acetone (C3H6O) for 10 s and then air-dried. Each mesh is then
secured to a holder via the rotatable posts. Using a micropi-
pette, microliter volumes of catalyst and substrate are deposited
on M1 and M2, respectively, and allowed to air-dry. The mesh
holders are then attached to the micromanipulators while being
careful to avoid contact between the meshes or with other
surfaces. The mesh holders are adjusted to set the inter-mesh
distance in the range of 2 mm–4 mm. Shorter M1–M2 dis-
tances have higher desorption/ionization efficiency for 2 de-
posited on M2, with 2 mm typically used for experiments
(Supplementary Figure S1b). This is supported by COMSOL
Multiphysics (ver. 4.4.0.248; COMSOL, Inc., Burlington, MA,
USA) laminar flow simulations showing that N2 velocity de-
creases by ~20% after passing through M2 (Supplementary
Figure S1b inset).
After setting the meshes and sprayer in desired positions
relative to the MS inlet, N2 gas and liquid flow are initiated to
generate a microdroplet spray containing a reagent such as
oxidant mCPBA (Figure 1). Detection of [2+H]+ from M2
indicates a linear dynamic range of approximately one to two
orders of magnitude and signal intensity scales linearly with
liquid flow rate (Supplementary Figure S1c and d). We hy-
pothesize that impact of the primary microdroplets on M1
liquid from a Harvard Apparatus Standard Pump 22 (Holliston,
MA, USA) syringe pump. FS1 passes through a 1/16′′ fluori-
nated ethylene propylene tubing (i.d.: 200 μm; length: 5 cm ;
IDEX Health and Science, Oak Harbor, WA, USA) that holds
FS1 in place with a stainless steel (SS) nut and ferrule. The
other end of FS1 passes through a second FS capillary (FS2;
length=~1.5–2 cm; i.d. = 250 μm; o.d. 360 μm), which is held
in place with a graphite ferrule and SS nut. FS1 and FS2
protrude from the end of the SS nut by ~0.7 and 0.5 cm,
respectively. The FS1-to-MS inlet distance ranges from 5 mm
to 13 mm (Figure 1a). Sheath gas (nitrogen; N2) enters the
sprayer at the 90o opening of the tee (line pressure=~150–200
PSI) and exits FS2, generating the microdroplet reagent spray.
Measurements of [2+H]+ (m/z 260.164) intensity as a function
of N2 line pressure show that a threshold value of ~150 PSI is
necessary for efficient desorption/ionization from M2 in TM2-
DESI (Supplementary Figure S1a). The sprayer is connected to
a 1/16′′ Swagelok bulkhead union that is mounted in an alu-
minium bar (sprayer holder; Figure 1b). The sprayer holder is

K. C. Peters et al.: Multistage Reactive Transmission-Mode DESI
1497
100
initiate reactions in desorbed secondary microdroplets generat-
ing active catalytic species that interact with the substrate upon
impacting M2. Catalytic conversion of the substrate occurs in
desorbed tertiary microdroplets as they travel towards the mass
spectrometer. Unless specified otherwise, the Orbitrap MS is
260.1645
260
x100
Propranolol
276.159
0
276
(a)
100
x8
306.182
1027.977
Dapoxetine
100
x8
typically operated using the following parameters for full-scan
mass spectra: m/z range=250–1500, resolution=100,000 at m/z
400, mass accuracy=2–5 ppm, microscan=1, injection time=
500 ms; capillary voltage=275°C, tube lens voltage=110 V,
and spray voltage=0 kV. Settings that are different for tandem
MS (MS/MS) include: microscans=3, injection time=50, 500,
or 1000 ms.
322.177
0
310
320
1043.972
0
1030
1040
1050
(b)
(c)
812.091
100
828.086
Results and Discussion
0
810
820
830
TMn-DESI has the ability to monitor catalytic cycles (on- and
off-path processes) and capture highly reactive intermediates.
Iron porphyrins such as Fe-TPFPP chloride ([1+Cl]) are a class
of biomimetic catalysts capable of a wide variety of oxidation
reactions including C–H hydroxylation[41, 52, 53]. The mech-
anism of Fe-mediated hydroxylation involves activation of 1
by an oxidant such as meta-chloroperbenzoic acid (mCPBA) to
produce a Fe peroxo intermediate 1a, which undergoes homo-
lytic and heterolytic cleavage to yield high-valent iron oxo
catalytic intermediates 1b and 1c, respectively (Scheme 1a).
The active catalytic species (1b and 1c) can then hy-
droxylate organic compounds such as propranolol[54] (2,
Scheme 1a) on the aromatic ring (2b), regenerating 1 to com-
plete the catalytic cycle[41].
943.849
100
959.842
960
0
940
950
(d)
(e)
1337.957
1353.951
100
1198.962
1078.939
1217.935
1097.914
1233.929
0
1100
1200
1300
When [1+Cl] (5 μL of 5×10–5 M in CH2Cl2) is deposited
on M1 and activated by mCPBA (5×10–5 M in 4:1
CH2Cl2:CH3OH) in the reagent spray (no ionization voltage
applied; 200 PSI N2 pressure), desorbed secondary
microdroplets containing active catalyst species react with
[2+HCl] (5 μL of 5×10–5 M in CH2Cl2) deposited on M2.
Desorbed microdroplets from M2 were analyzed by Orbitrap
MS to identify species 1 (m/z 1027.977), 2 (m/z 260.164), and
2b (m/z 276.159), indicating that the complete catalytic trans-
formation of 2 can be observed on the TM2-DESI time scale
(Figure 2a). Fe-TPFPP can hydroxylate other substrates such as
dapoxetine by TM2-DESI-MS (Figure 2a inset), as well as
activate catalysts such as Fe-TDFPP (Figure 2b) and Fe-
TDCPP (Figure 2c), indicating that the technique is generaliz-
able for studying chemical reactivity. Analysis of the activation
of 1 by mCPBA using negative mode TM1-DESI (Figure 2d)
identifies the formation of 1b at m/z 1078.939 [1b+Cl]– and
m/z 1198.962 [1b+mCBA]–, corresponding to the homolytic
catalytic pathway. To our knowledge, these results represent
the first direct observation of the Fe-porphyrin homolytic cat-
alytic pathway by MS (previous FT-ICR ESI-MS studies ob-
served the radical cation iron oxo 1a) [42, 43].
684.161
100
668.166
595.121
600
700.154
750.112
0
640
680
720
m/z
(f)
10
Aliphatic
Aromatic
0.68
0
TPFPP
TDFPP
TDCPP
TMP
TPP
Increasing Fe Electron Density
Figure 2. Positive-mode TM2-DESI-MS of the hydroxylation of
2 by (a) 1, (b) Fe-TDFPP, and (c) Fe-TDCPP using oxidant mCPBA.
Inset of (a) shows the hydroxylated products of 2 and dapoxetine.
(d) Negative mode TM1-DESI-MS of the hydroxylation of 2 by Fe-
TPFPP using oxidant mCPBA. (e) Positive-mode TM1-DESI-MS
showing self-oxidation of Fe-TPP (Scheme 1b). (f) Reactivity of five
Fe porphyrin catalysts towards aromatic and aliphatic oxidation of
2 by TM2-DESI-MS (green dotted line represents aliphatic oxidation
by mCPBA). Structures: TPFPP (Scheme 1a), TDFPP (Figure 2b),
TDCPP (Figure 2c), TMP (Figure 2f), and TPP (Scheme 1b)
Further analysis of acquired Orbitrap mass spectra indicate
that the m/z values corresponding to 1a (Figure 2d) and 1c
(Figure 2a) are isobaric with ligand oxidation degradation prod-
ucts (Scheme 1b)[41]. Oxidative degradation of porphyrin

1498
K. C. Peters et al.: Multistage Reactive Transmission-Mode DESI
ligands can produce species having opened rings and containing
oxygen atoms in various functional forms, as exemplified by
reacting Fe-TPP (nominal Δm/z=360 compared with Fe-
TPFPP) 3 (deposited on M1) with mCPBA (reagent spray)
yielding structures 3a–3c (Scheme 1b). The structures of these
TPP degradation products are in agreement with previous studies
employing ultraviolet-visible spectroscopy and MS[55]. For Fe-
TPFPP, the [TPFPP+O-H]– (m/z 989.044; Supplementary
Figure S2) degradation product is observed when free TPFPP
ligand ([TPFPP-H]– at m/z 973.051; 5 μL of 5×10-4 M in
CH2Cl2) and [1 + Cl] (5 μL of 5 × 10-4 M in 4:1
CH2Cl2:CH3OH) are deposited at the same spot and then reacted
with mCPBA in TM1-DESI (there is a 10-fold increase in the
signal compared with the absence of [1+Cl]; Supplementary
Figure S2). These results clearly indicate that [1+Cl] undergoes
self-oxidation. In addition to being isobaric with ligand oxidation
products, species 1a is estimated to have a lifetime on the order of
tens of microseconds [56], [see Supporting Information (SI)], for
the calculation), which is significantly shorter than the estimated
reaction time of TMn-DESI (based on the N2 velocity and DCIP
estimations discussed below). As a result, ion signals at m/z
1233.929 and 1353.951 are unlikely to contain the Fe-peroxo
intermediate. However, observation of both 1b and 2b combined
with the higher reported reactivity of 1c towards hydroxylation of
2[41] suggest that the peak at m/z 1043.972 possibly also con-
tains the high-valent radical cation Fe-oxo 1c.
microdroplet spray. These capabilities are useful for step-wise
elucidation of reaction mechanisms and for minimizing off-
path processes. For example, mCPBA can oxidize 2 without
catalyst interaction to generate 2a (aliphatic chain oxidation;
green dotted line in Figure 2f), which is isobaric with catalytic
product 2b (aromatic oxidation)[41]. Species 2a and 2b were
distinguished using MS/MS experiments (see SI;
Supplementary Figure S3). When a premixed solution of [2+
HCl] (2.5×10–6 M) and mCPBA (2.5×10–5 M) in 4:1
CH2Cl2:CH3OH was sprayed towards the mass spectrometer
at 30 μL/min (no ionization voltage applied), a peak at m/z
276.160 ([2a+H]+) is observed at ~4% intensity relative to m/z
260.165 ([2+H]+), indicating aliphatic oxidation in the absence
of the catalyst (Supplementary Figure S3). However, when [2+
HCl] (5 μL of 5×10–5 M in CH2Cl2) is deposited on a mesh
and analyzed using a microdroplet spray containing mCPBA
(5×10–5 M), the relative intensity of [2a+H]+ is ~0.06%,
indicating a reduction in the off-path process by a factor of
~60. When a mesh bearing [1+Cl] (5 μL of 5×10–5 M in
CH2Cl2) was placed in front of the mesh bearing 2, the
peak at m/z 276.160 had relative intensity of ~0.8% com-
pared with [2+H]+. So, when TM2-DESI was used to
separate reagents and specify their order of introduction,
the off-path process was reduced and the ion population at
m/z 276.160 primarily represented on-path product 2b.
It is essential to determine the reactivity of ligands with
different chemical motifs in the discovery and development
of new catalysts. TMn-DESI allows rapid simultaneous
TM2-DESI provides a simple approach for separating re-
agents and controlling their order of introduction into the
(a)
267.993
100
270.008
270
0
268
(b)
267.993
100
270.008
270
0
268
(c)
1000x
100
270.008
267.994
0
268
270
m/z
Figure 3. (a) TM1-DESI-MS of the reaction between L-AA and DCIP. (b) DESI mass spectrum of DCIP reduction by ascorbic acid.
(c) Continuous-flow ESI-MS of of L-AA reacting with DCIP (reaction time=~7 s)

K. C. Peters et al.: Multistage Reactive Transmission-Mode DESI
1499
characterization of reaction mechanisms and reaction progress.
Depositing simultaneously five Fe catalysts with different por-
phyrin ligands on M1 and substrate 2 on M2, allowed rapid
comparison of the relative yields of aliphatic and aromatic
hydroxylation products in a single experiment (Figure 2f and
Supplementary Figure S3; ratio of aliphatic to aromatic oxida-
tion determined by MS/MS as described in the SI). The ob-
served reactivity trend agrees with previously published reports
showing that oxidizing power decreases with increasing elec-
tron density at the metal centre[41], suggesting that TM2-
DESI-MS also provides information relevant to bulk solution-
phase chemistry. Other spray-based ionization sources (e.g.,
continuous-flow ESI, stopped-flow ESI, and DESI) would
require additional tubing, pumps, microdroplet emitters, or
valves to achieve high-throughput analyses of complex reac-
tions similar to TMn-DESI-MS.
In addition to 2, dapoxetine (Figure 2a inset) and 1-
naphthyl-3-pyrrolidinyl (Supplementary Figure S4), which
share an oxynaphthyl moiety, as well as four other substrates
were successfully hydroxylated using Fe-TPFPP as the catalyst
(structures of 1-naphthyl-3-pyrrolidinyl, rhodamine B, rhoda-
mine 6G, fluorescein, and lauric acid shown in Supplementary
Figure S4). A spray voltage of –5 kV was applied (Bertan 205b
high voltage power supply, Spellman, Hauppauge, New York,
USA) for TM2-DESI-MS oxidation of fluoroscein and lauric
acid to improve sensitivity (the effect of spray voltage on signal
intensities is discussed later in this article). Product-to-substrate
ratios (P:S) calculated from the single-stage MS experiments
indicate that the substrates containing aromatic rings are more
easily oxidized by Fe-TPFPP compared with alkyl compounds.
The location of the hydroxyl group was determined using MS/
MS (Supplementary Figures S5–S9).
suggest that applying standard ionization voltages and temper-
atures in TMn-DESI does not significantly influence the ob-
served reaction pathways in acquired mass spectra for this
catalytic system, thereby demonstrating the ability of TM-
DESI to provide information relevant to bulk-phase chemistry.
TMn-DESI has the ability to probe reactions on time scales
less than 1 ms. When L-AA[58] (2×10–3 M in 1:1
CH3OH:H2O) was sprayed (5 μL/min; 200 psi nebulizing gas;
0.6 cm and 1.0 cm emitter-to-inlet distances; +3 kV) towards a
mesh bearing DCIP (4a; 5 μL of 5×10–3 M in CH3OH) in TM1-
DESI-MS, [4b+H]+ was observed at m/z 270.008 (Figure 3a)
with intensity of ~4% relative to [4a+H]+ at m/z
267.993. Assuming that the concentration of DCIP in
the secondary microdroplets is low compared with L-
AA, the observed reaction time in microdroplets (t) for
TM1-DESI (tTM) and DESI (tDESI) can be approximated
using pseudo-first-order kinetics [58] (see SI)
ꢀ
ꢁ
À
Á
I4a þ I4b
t ¼ ln
= kf ⋅½AAŠ
0
I4a
where kf is the rate constant, [AA]0 the initial L-AA
concentration, I4a the intensity of [4a+H]+, and I4b the
intensity of [4b+H]+. For DESI (Figure 3b) , L-AA (2×
10–3 M in 1:1 CH3OH: H2O) was sprayed (5 μL/min;
200 PSI nebulizing gas; 0.6 cm and 1.0 cm emitter-to-
inlet distances; +3 kV applied) towards a paper surface
containing DCIP (deposited 5 μL of 5×10–3 M in
CH3OH). The ratio [tDESI/tTM]≅2–3 (Supplementary
Table S1), which suggests that tTM and tDESI have the
same order of magnitude of less than 1 ms (tDESI was
experimentally estimated by Latour and coworkers[59,
60]). These results agree with previous studies proposing that
TM-DESI and DESI have relatively similar desorption/
TM2DESI characterization of the Fe-TPFPP reactions de-
scribed above were performed without applying voltage to the
spray solutions in order to closely mimic bulk-phase reaction
conditions, except for fluorescein and lauric acid, which re-
quired –5 kV for adequate sensitivity. Application of 5 kV
(positive and negative modes) in TM1-DESI-MS characteriza-
tion of Fe-TPFPP activation by mCPBA (deposited 5 μL of
10–4 M Fe-TPFPP in CH2Cl2; DESI microdroplet spray con-
tains 10–5 M mCPBA in 4:1 CH2Cl2:CH3OH) increased abso-
lute signal intensities without significantly altering relative
intensities (Supplementary Figure S10; viz., applying a voltage
increased sensitivity). In addition, ion signals suggesting for-
mation of reactive oxygen species[57] or other unwanted by-
products were not observed (Supplementary Figure S10).
Experiments carried out at different capillary inlet tempera-
tures, 225°C (Supplementary Figure S11a), 275°C (Figure
2d), and 325°C (Supplementary Figure S11b) in negative mode
(0 kV) show that the relative intensities change, indicating
effects on desolvation/ionization processes and/or reaction ki-
netics. Ion signals for [1+2Cl]– (m/z 1097.916), [1b+mCBA]–
(m/z 1198.963), and [1+Cl+mCBA]– (m/z 1217.938) in-
creased relative to [1+2mCBA]– (m/z 1337.959). Increasing
the capillary inlet temperature did not yield any new ion signals
indicative of unwanted side reactions. All these observations
ionization mechanisms[34], calculations showing that [tESI
/
tTM]>40 at various emitter-inlet distances and concentrations
(Supplementary Table S1), and calculations showing that
microdroplets of TM2-DESI travel at twice the velocity of
droplets generated with DESI; see Electronic Supplementary
Information for the calculations) [45].
In this report, we describe an ambient ionization source that
involves directing a microdroplet spray through a series of
micrometer-sized meshes bearing reagents, referred to as mul-
tistage reactive transmission mode desorption electrospray ion-
ization. This technique provides a simple approach for separat-
ing reagents and specifying their order of introduction into the
microdroplet stream, which facilitates the step-wise analysis of
complex catalytic reactions. In addition, the short travel time of
the microdroplet spray from the emitter to the mass spectrom-
eter inlet provides access to short reaction time scales
less than 1 ms.
Acknowledgments
The Perry Research Laboratory gratefully acknowledges finan-
cial support from the University of Illinois at Urbana-

1500
K. C. Peters et al.: Multistage Reactive Transmission-Mode DESI
activation of organometallic iridium complexes for catalytic water and C–
H Oxidation. Inorg. Chem. 53(1), 423–433 (2014)
Champaign (UIUC). T.J.C. thanks the National Science
Foundation Graduate Research Fellowship, National
Institutes of Health Chemistry-Biology Interface Training
Program (NIH T32 GM070421), and UIUC Springborn
Fellowship for financial support. The authors also acknowl-
edge the work of Asenath Francis (Louisiana State University;
funded by the NSF Research Experience for Undergraduates
program at UIUC) for characterizing the oxidation of various
substrates. Kevin Parker (UIUC undergraduate) was instru-
mental in early studies characterizing reactions and ion source
configurations. The authors acknowledge Jedidiah Veach
(UIUC graduate student) for help in designing the
continuous-flow ESI-MS figure located in the SI. We also
thank the staff of the UIUC School of Chemical Sciences
Machine Shop for their expertise in designing and machining
ion source components.
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