Electronic Supporting Information
Multistage Reactive Transmission-Mode Desorption Electrospray Ionization
Mass Spectrometry
Kevin C. Peters, Troy J. Comi, and Richard H. Perry*
TM2-DESI Characterization
Propranolol Intensity
1.3×105
c)
Parameters
Liquid Flow Rate = 10 mL/min
[Propranolol] = 5.0 ×10-5 M
1.0×103
0
N2 Threshold
100
2.5×104
Propranolol Intensity
a)
200
[Propranolol] (M)
5.0×10-6 M
1.0×10-5 M
5.0×10-5 M
1.0×10-4 M
5.0×10-4 M
0
300
[Propranolol] (M)
b)
d)
0
1
2
3
Inter-Mesh
Distance (mm)
7.0×104
Propranolol Intensity
Propranolol Intensity
100
Relative N2 Velocity
2.0×105
0
6.0×10-4
0
Nitrogen Line Pressure (PSI)
0
Parameters
N2 Flow Rate = 200 PSI
Liquid Flow Rate = 10 mL/min
y = 2336.2x – 8546.8
R² = 0.9828
1.0×103
0
4
Parameters
N2 Flow Rate = 200 PSI
[Propranolol] = 5.0×10-5 M
10
20
Liquid Flow Rate (µL/min)
30
Figure S1. The performance characteristics of TM2-DESI were evaluated by monitoring the intensity of [2+H]+ from M2 as a
function of various experiment parameters. (a) N2 line pressure. (b) Inter-mesh distance (200 psi N2 pressure; 15 μL/min flow
rate; 5 μL of 5 x 10-5 M 2 deposited). Inset shows COMSOL Multiphysics simulation of N2 flow through M1 and M2 with 1.0
mm separation. (c) Concentration of 2 deposited on M2 (solvent is 4:1 CH2Cl2:CH3OH for all trials). (d) Liquid flow rate.
TPFPP Ligand Oxidation by Fe-TPFPP
Relative Abundance
a)
100
973.051
x10
No Catalyst
989 : 973 (x1000) = 2.5 ± 0.2
989.044
0
974
976
978
980
982
984
986
988
Relative Abundance
b)
100
973.051
x10
+ Fe-TPFPP
989 : 973 (x1000) = 23 ± 5
989.044
0
974
976
978
980
982
m/z
984
986
988
Figure S2. (a) Negative mode TM1-DESI mass
spectrum of free TPFPP ligand (5 μL of 5 × 10-4 M)
with 5 × 10-4 M mCPBA in 4:1 CH2Cl2:CH3OH as spray
solvent (10 μL/min, 200 psi N2). (b) Same experiment
performed as in part (a), with 5 μL of 5 x 10-4 M FeTPFPP added to a new TPFPP sample spot; n = 3 for
each experiment.
Tandem MS Characterization of Propranolol Hydroxylation (Aromatic and Aliphatic) and
Screening of Various Fe-porphyrin Catalysts
TM2-DESI-MS screening of five catalysts’ reactivities towards hydroxylation of 2 was carried out by
depositing five spots of [2+HCl] (5 μL of 5 × 10-5 M in CH2Cl2) on M2, and each spot was outlined with
red permanent marker for reference. Then, M1 was added in front of M2, and 5 mL of each catalyst (5 ×
10-5 M in CH2Cl2) was deposited directly over each deposited spot of 2. The meshes were sufficiently
spaced to prevent M1-M2 contact. In the TM2-DESI experiment, the mesh holders were positioned on
each of the five pairs of spots to initiate reaction (triplicate measurements were acquired for each).
Aliphatic hydroxylation product 2a yielded characteristic fragments at m/z 114, 183, 198, and 232 (Fig.
S3c). Aromatic hydroxylation gives characteristic fragment ions at m/z 98, 116, 173, and 199 in TM2DESI-MS/MS spectra (Fig. S3d), which all have O-atom addition on the ring evidenced by a shift of 16
Da on fragments containing the naphthyl ring (Fig S3e). The aliphatic to aromatic oxidation ratio (Fig. 2a)
was estimated by multiplying the summed intensities of the aromatic or aliphatic hydroxylation fragment
ions in MS/MS spectra (viz. the fragment ions mentioned above; Fig. S3) by the 276/260 intensity ratio.
Relative Abundance
a)
260.165
100
276.160
260
262
264
266
268
b)
270
272
183.1
Relative Abundance
100
260.2
157.0
98.0
218.1
100
120
140
160
180
200
242.2
220
240
c)
Relative Abundance
276
116.0
80
100
114.1
132.1 157.1
120
140
160
200
116.0
100
280
300
276.2
198.1
180
260
258.2
183.1
d)
Relative Abundance
274
234.1
220
240
260
280
300
258.2
199.1
276.2
173.0
98.0
80
100
234.1
120
140
160
180
200
m/z
220
240
260
280
300
e)
m/z 98.1
m/z 116.0
m/z 242.2
m/z 157.0
m/z 218.1
m/z 183.1
Figure S3. (a) Mass spectrum from spraying a pre-mixed solution of 2.5 × 10-6 M 2 and 2.5 × 10-5 M
mCPBA in a sonic spray ionization (SSI) experiment (30 μL/min, 200 psi N 2). (b) MS/MS spectrum of
[2+H]+ (m/z 260) from (a). (c) Tandem MS of [2a+H]+ at m/z 276 from (a), which was generated in the
absence of a catalyst. (d) Tandem MS of [2b+H]+ generated by Fe-TDFPP in TM2-DESI experiment (5 μL of
10-5 M catalyst on first mesh; 5 μL of 5 × 10-5 M propranolol on second mesh; 5 × 10-5 M mCPBA sprayed at
30 μL/min with 200 psi N2, and 1.0 cm sprayer-to-inlet distance). (e) Possible structures for some of the
fragments observed in (b) – (d) (other isomers are possible).
Fe-TPFPP Hydroxylation of 1-Naphthyl-3-Pyrrolidinyl Ether by TM2-DESI-MS
18x
90x
214.121
Relative Abundance
100
230.120
0
220
230
m/z
Figure S4. Positive mode TM2-DESI-MS showing the oxidation of 1-naphthyl-3-pyrrolidinyl ether ([M+H]+
= m/z 214.12) by mCPBA (5 × 10-5 M in 4:1 CH2Cl2:MeOH used as the spray solvent; 10 μL/min and 200
PSI N2 line pressure) and Fe-TPFPP (5 μL of 5 × 10-4 M deposited on M1; 2 mm between meshes; Emitterto-inlet distance was 1.25 cm).
Other Substrates Investigated by TM2-DESI-MS
Figure S5. Structures for various substrates that were hydroxylated using Fe-TPFPP in TM2-DESI-MS
experiments. Product to substrate intensity ratios (P:S) are shown below each structure (all P:S ratios are ×
1000).
Tandem Mass Spectrometry of Rhodamine B
Tandem MS of Rhodamine B (m/z 443.2) shows loss of C2H4 and C3H8 (m/z 415.2 and m/z 399.2,
respectively) from the alkyl chains (Fig. S5a). MS/MS of oxidized Rhodamine B (m/z 459.2) also shows a
primary fragment at m/z 415.2, indicating that the molecule is oxidized on the alkyl chain that undergoes
dissociation (Fig. S5b). Further evidence for this assignment is found at m/z 441.2 (loss of H2O), as
dehydration from an aromatic ring is unlikely. Another peak is observed at m/z 430.3, corresponding to
C2H5 loss. All of these results indicate that Rhodamine is oxidized on the aliphatic substituents rather than
the aromatic ring.
a)
443.2
Relative Abundance
100
399.2
415.2
395
b)
415
435
455
415.2
Relative Abundance
100
441.2
459.2
Figure S6. (a) Positive mode TM1-DESI
MS/MS of Rhodamine B (m/z 443.2).
Structure shown represents the m/z 399.2
fragment ion. (b) Positive mode TM2-DESIMS/MS of in-situ-oxidized Rhodamine B
(m/z 459.2). Structure shown represents the
m/z 415.2 fragment ion. (Parameters:
collision energy = 30%; activation time =30
ms; Solvent = acetonitrile).
430.2
395
415
435
455
m/z
Tandem Mass Spectrometry of Rhodamine 6G
MS/MS of Rhodamine 6G (m/z 443.2) also shows a significant fragment at m/z 415.2, indicating C2H4
loss (Fig. S6a). Oxidized Rhodamine 6G (m/z 459.2) shows a prominent fragment ion at m/z 415.2 (Fig.
S6b), indicating alkyl chain oxidation. H2O loss is also observed (m/z 441.22), as well as combined [C2H4
+ C2H4O] loss (m/z 387.17), all of which indicate alkyl chain, rather than aromatic, hydroxylation. The
only peak that does not definitively suggest aliphatic hydroxylation is m/z 431.20, which contains the
ethyl substituent and an oxygen atom.
a)
443.2
Relative Abundance
100
443.2
415.2
385
405
425
445
459.2
415.2
100
Relative Abundance
b)
431.2
441.2
459.2
387.2
385
405
m/z 425
445
Figure S7. (a) Positive mode TM1-DESI
MS/MS of Rhodamine 6G (m/z 443.2).
Structure shown represents the m/z 415.2
fragment ion. (b) Positive mode TM2DESI MS/MS of in-situ-oxidized
Rhodamine 6G (m/z 459.2). Structures
shown represent m/z 387.2 (left) and m/z
431.2 (right) fragments respectively.
(Parameters: collision energy = 30%;
activation time =30 ms; Solvent =
acetonitrile).
Tandem MS of Fluorescein
Tandem MS of fluorescein (m/z 331.1) produce fragment ions at m/z 287.1 and m/z 286.1, which
corresponds to a CO2 and -CO2H loss, respectively (Fig. S7a). Upon oxidation, there is a 16 Da mass unit
shift (Fig. S7b), which confirms hydroxylation.
a)
Relative Abundance
100
287.1
286.1
285
331.1
295
305
b)
31
5
325
335
345
303.1
Relative Abundance
100
347.1
302.1
285
295
Figure S8. (a) Negative mode TM1-DESI
MS/MS of fluorescein (m/z 331.1).
Structure shown represents m/z 286.1
fragment. (b) Negative mode TM2-DESI
MS/MS of in-situ-oxidized fluorescein
(m/z 347.1). Structure shown represents
m/z 302.1 fragment. Reaction performed
in 2:1 dichloromethane:acetonitrile.
305
315 m/z
32
5
335
345
Tandem MS of Lauric Acid
Finally, tandem MS of lauric acid (m/z 199.17) shows a single fragment at m/z 181.2, which corresponds
to loss of H2O from the carboxylic acid group (Fig. S8a). Tandem MS of oxidized lauric acid (m/z 215.2,
Fig. S8b), however, shows a peak corresponding to loss of water and hydroxyethene at m/z 153.1. This
result suggests that Fe-catalyzed oxidation of lauric acid occurs primarily on one of the two carbon atoms
furthest from the carboxylic acid group.
100
181.2
155
100
Relative Abundance
b)
199.2
199.2
Relative Abundance
a)
165
175
185
195
215.2
a
197.1
153.1
155
165
175
m/z
185
195
Figure S9. (a) Negative mode TM1-DESI
MS/MS of lauric acid (m/z 199.17). (b)
Negative mode TM2-DESI MS/MS of insitu-oxidized lauric acid (m/z 215.17).
Reaction performed in acetonitrile.
Comparison of Signal Intensities at 0 kV and 5 kV (Positive and Negative Modes)
Relative Abundance
(a)
100
1027.980
+0 kV
NL: 8.9E4
1043.975
0
1025
1050
Relative Abundance
(b)
100
1027.976
+5 kV
NL: 1.9E5
1043.971
0
1025
1050
Relative Abundance
(c)
-0 kV
NL: 9.0E4
100
1337.957
1198.962
1217.936
1097.914
0
1100
1200
1300
(d)
-5 kV
NL: 1.2E5
100
1097.914
1217.935
1198.961
1100
1200
m/z
1337.956
0
1300
Figure S10. TM1-DESI-MS of Fe-TPFPP (deposited 5 mL of 10-4 M Fe-TPFPP in CH2Cl2; DESI micrdroplet spray contains 10-5
M mCPBA in 4:1 CH2Cl2:CH3OH) using 0 kV ((a) and (c)) and 5 kV ((b) and (d)) in positive and negative modes.
Comparison of Signal Intensities at Different Inlet Capillary Temperatures
(a)
Relative Abundance
-0 kV; 225 oC
NL: 5.6E4
1337.958
100
1198.963
1097.915
1217.936
0
1100
1200
1300
(b)
Relative Abundance
-0 kV; 325 oC
NL: 4.2E4
1337.959
100
1198.963
1097.916
1217.938
0
1100
1200
1300
m/z
Figure S11. TM1-DESI-MS of Fe-TPFPP (deposited 5 mL of 10-5 M Fe-TPFPP in CH2Cl2; DESI micrdroplet spray contains 10-5
M mCPBA in 4:1 CH2Cl2:CH3OH) using -0 kV spray voltage, as well as 225 oC (a) and 325 (b) oC capillary temperatures.
Calculation for Estimating the Mean Lifetime of the Iron Peroxo Intermediate
An Arrhenius plot was constructed using previously reported data [1] describing the temperature
dependence of Fe-TMP peroxo (Fe-OOR) O-O bond cleavage. This Arrhenius plot is described by
where k is the forward rate constant for this reaction and T is the temperature (in Kelvin). Using this
relationship, the estimated activation energy (Ea, obtained by multiplying the slope by -R) is about 17
kJ/mol, which agrees with the previously reported value of about 17 kJ/mol. Substituting room
temperature (298 K) for T gives an estimated rate constant (k298) of 5.4 × 104 s-1, which corresponds to a
mean lifetime (τ298) of approximately 19 μs (assuming first order kinetics with respect to Fe-OOR).
Comparison of Time Scales of Ambient Mass Spectrometric Methods
The timescales of ESI-MS, TM1-DESI-MS, and DESI-MS can be compared by deriving mathematical
expressions for the reaction time (t) as a function of ion intensities in acquired mass spectra. The
integrated rate law for the second order reaction A + B  P that applies to the ESI-MS experiment is:
where kf is the forward rate constant (an unknown value), [4a] and [4a]0 refer to the concentrations of
final and initial concentrations of reduced DCIP, respectively, and [AA] and [AA]0 refer to initial and
final concentrations of ascorbic acid, respectively. The variables [4a]0 and [AA]0 are known, but the final
concentrations of DCIP and ascorbic acid must be estimated from ion intensities corresponding to
oxidized and reduced DCIP (I4a and I4b, respectively). [4a] is relatively simple to calculate, since any 4a
consumed turns into 4b. So, by multiplying [4a]0 by the fraction of unreacted 4a observed in the mass
spectrum yields [4a]. Because 4a and 4b have nearly equal ionization efficiencies, the fraction of
unreacted 4a is simply given by the intensity of 4a divided by the total DCIP intensity (I4a + I4b). Thus, the
concentration of 4a can be found by:
The final concentration of ascorbic acid can be found by assuming that all ascorbic acid consumed in this
reaction is consumed by DCIP reduction. Thus, the final ascorbic acid concentration is simply the
difference between the initial concentration and the amount of reduced DCIP ([4b]), which can again be
measured directly from the mass spectrum. Analogous to the calculation of [4a], [4b] can be given as:
So, substituting this term into the calculation of ascorbic acid remaining at the end of the reaction, the
equation becomes:
Substitution of these terms for [AA] and [4a] and solving for t yields the equation given in the main text:
Calculation of the reaction time of TM1-DESI-MS is made difficult by the fact that the starting
concentration of DCIP is not known, since it is desorbed from a mesh by the DESI spray. To perform this
calculation, we can make the reasonable assumption of pseudo-first-order kinetics due to an excess of
ascorbic acid in the spray relative to the small amount of DCIP that is desorbed from the mesh. This
assumes that the concentration of ascorbic acid is not significantly affected by its reaction with the small
amount of DCIP. The integrated rate law is then given by:
The ratio in the logarithm can be calculated from the MS data by the following:
If it is assumed that [AA]0 is the initial concentration placed in the DESI spray solvent, then substitution
yields:
An identical analysis can be performed for reactive DESI as well. These “time“ values can then be
compared to compute factor differences in apparent timescale for the three MS techniques. The only
shortcoming of this model is the fact that in both TM1-DESI and DESI, rapid microdroplet evaporation
increases the actual concentrations of both reagents in the droplets as the reaction proceeds [2].
Comparison of TM1-DESI-MS to Other MS Techniques in the Reduction of DCIP
Four separate sets of conditions were used to explore the reduction of DCIP by each of the three MS
methods, generated by varying both ascorbic acid concentration and the distance from the solvent emitter
to the MS inlet. The equations outlined above were used to estimate the timescales for continuous-flow
ESI-MS, DESI-MS, and TM1-DESI-MS. Table S1 shows a summary of the results for all of these
experiments. All eight experiments show shorter effective reaction time for TM1-DESI compared to the
other techniques.
Table S1. Comparison of TM1-DESI to ESI and DESI-MS for the reduction of DCIP
under various experimental conditions. For TM1-DESI and DESI reactions between
DCIP and L-AA, 5 μL of 5 x 10-3 M DCIP in CH3OH was deposited. For all
continuous-flow ESI-MS experiments, the concentration of DCIP was decreased by
one order of magnitude to compensate for desorption of DCIP from the surface. In all
experiments, the calculated reaction yield, calculated as I4b/(I4a + I4b), was statistically
different (p ≤ 0.05) between the two techniques under study in a give experiment. tTM
refers to the calculated timescale of TM 1-DESI; Same for tESI and trDESI.
[L-AA]
(M)
Emitter-Inlet
Distance (cm)
tESI
tTM
trDESI
tTM
4 × 10-3
4 × 10-3
2 × 10-3
2 × 10-3
0.6
1.0
0.6
1.0
48
47
244
200
3.5
1.6
3.0
1.6
Relative Abundance
Reduction of Dichlorophenolindophenol by Reactive Desorption Electrospray Ionization
267.993
100
270.008
0
268.0
268.2
268.4
268.6
268.8
269.0
m/z
269.2
269.4
269.6
269.8
270.0
Figure S11. Reactive DESI mass spectrum of DCIP reduction by ascorbic acid. L-AA (2 × 10-3 M
in 1:1 CH3OH: H2O) was sprayed (5 μL/min; 200 PSI nebulizing gas; 0.6 cm emitter-to-inlet
distance; +3 kV applied) towards a paper surface bearing DCIP (5 μL of 5 x 10-3 M in CH3OH).
Schematic of Continuous-Flow Electrospray Ionization Mass Spectrometry
Figure S11 shows a schematic of DCIP reduction studied by continuous-flow ESI-MS. A short capillary
connects the mixing tee to the ESI emitter, resulting in a reaction time of only ~7 seconds at the liquid
flow rate studied.
L-AA
Mixing Tee
Mass Spectrometer
Inlet
N2 In
Electrospray
Emitter
Flow Direction
Reaction
Mixture Out:
Vol = 574 nL
Fused Silica
Capillaries
DCIP
Figure S12. Schematic of the continuous-flow ESI-MS experiment.
Estimation of Microdroplet Velocities
Previous studies by Cooks and co-workers demonstrated that primary droplets had velocities of 120 m/s
at 2 mm distance from the DESI emitter tip [3]. COMSOL simulations show that the N2 velocity in
TMn-DESI is 35% slower between M1 and M2 and 20% slower after M2, which corresponds to a velocity
of ~9 m/s and a maximum microdroplet flight time of ~450 ms before entering the mass spectrometer,
which further supports an estimated tTM range of ~350 ms – 500 ms. Since the velocity of the microdroplets
desorbed from M2 is approximately 2-3 times faster compared to DESI (4 m/s after leaving the surface),
we hypothesise that the droplet velocity is a significant factor leading to the ability to access shorter
times. However, As a result, the tTM range of ~350 ms – 500 ms represents the experimentally ideal case,
and that TMn-DESI has the potential to access reaction time scales on the order of hundreds of
microseconds.
References
1.
2.
3.
Groves, J. T. and Watanabe, Y.: Reactive Iron Porphyrin Derivatives Related to the Catalytic Cycles of
Cytochrome-P-450 and Peroxidase - Studies of the Mechanism of Oxygen Activation. J. Am. Chem. Soc.
110(8443-8452 (1988)
Girod, M., Moyano, E., Campbell, D. I. and Cooks, R. G.: Accelerated Bimolecular Reactions in
Microdroplets Studied by Desorption Electrospray Ionization Mass Spectrometry. Chem Sci. 2(3), 501-510
(2011)
Venter, A., Sojka, P. E. and Cooks, R. G.: Droplet Dynamics and Ionization Mechanisms in Desorption
Electrospray Ionization Mass Spectrometry. Anal. Chem. 78(24), 8549-8555 (2006)