Joining Microfluidics with Infrared Photodissociation: Online Monitoring of Isomeric Flow-Reaction Intermediates

Article abstract of DOI:10.1021/acs.analchem.8b05532

A cryogenic ion trap vibrational spectrometer is combined with a microfluidic chip reactor in a proof-of-principle experiment on the Hantzsch cyclization reaction forming 2-amino-4-phenyl thiazole from phenacyl bromide and thiourea. First, the composition of the reaction solution is characterized using electrospray-ionization mass spectrometry combined with two-color infrared photodissociation (IRPD) spectroscopy. The latter yields isomer-specific vibrational spectra of the reaction intermediates and products. A comparison to results from electronic structure calculations then allows for an unambiguous structural assignment and molecular-level insights into the reaction mechanism. Subsequently, we demonstrate that isomeric and isobaric ions can be selectively monitored online with low process time, i.e., using a single IRPD wavelength per isomer, as the chip reaction parameters are varied.

Full text of DOI:10.1021/acs.analchem.8b05532

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Letter
Joining microfluidics with infrared photodissociation:
online-monitoring of isomeric flow-reaction intermediates
Maik Pahl, Martin Mayer, Maximilian Schneider, Detlev Belder, and Knut R. Asmis
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05532 • Publication Date (Web): 09 Jan 2019
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Maik Pahl,¥,£ Martin Mayer,¢,£ Maximilian Schneider,¥ Detlev Belder¥,* and Knut R. Asmis¢,*
¥Institut für Analytische Chemie, University Leipzig, Linnéstraße 3, 04103 Leipzig, Germany
¢Wilhelm-Ostwald-Institut für Physikalische und Theoretische Chemie, Universität Leipzig, Linnéstraße 2, 04103
Leipzig, Germany
£These authors contributed equally
ABSTRACT: A cryogenic ion trap vibrational spectrometer is combined with a microfluidic chip reactor in a proof-of-principle
experiment on the Hantzsch cyclization reaction forming 2-amino-4-phenyl thiazole from phenacyl bromide and thiourea.
First, the composition of the reaction solution is characterized using electrospray-ionization mass spectrometry combined
with two-color infrared photodissociation (IRPD) spectroscopy. The latter yields isomer-specific vibrational spectra of the
reaction intermediates and products. A comparison to results from electronic structure calculations then allows for an unam-
biguous structural assignment and molecular-level insights into the reaction mechanism. Subsequently, we demonstrate that
isomeric and isobaric ions can be selectively monitored online with low process time, i.e. using a single IRPD wavelength per
isomer, as the chip reaction parameters are varied.
The structural identification of reaction intermediates in so-
lution is an essential aspect in unravelling reaction mecha-
nisms and pathways in synthetic chemistry. The short life-
time of these transient species typically circumvents their
proper analytical characterization. The advent of microflu-
idic technology in chemistry provided alternative ap-
proaches to shed new light on chemical processes at time
and length scales hardly reachable with conventional tech-
nology.1 When chemical conversions are performed in con-
tinuous flow microreactors2-19 rather than in laboratory
flasks, the reaction time coordinate is translated to the
length scale of the reaction channel. By using short reaction
channels and respective flow rates chemical conversions
can be studied at minimum residence times far-from equi-
librium. This opens up new routes to probe fast reactions in
solution with minimal reagent consumption.20-27
into the gas phase, in particular, when this technique is com-
bined with messenger-tagging44-46 in cryogenic ion traps.47-
It's potential for characterizing solution phase reaction
50
intermediates has recently been demonstrated.51-59 Fur-
thermore, IRPD spectroscopy combined with double–reso-
nance laser excitation schemes in the form of double-reso-
nance IR2MS2 spectroscopy60-61 allows separating the spec-
troscopic signatures of isomeric and isobaric species in a
general way (i.e. without the need of a UV/VIS chromophore
as in ion dip spectroscopy62). This approach is highly com-
plementary, for example, to recent combinations of IRPD
spectroscopy with ion mobility measurements.63-64
For in-situ monitoring of chemical processes in microfluidic
chips techniques such as fluorescence microscopy28-31 or
Raman microspectroscopy32-34 are current state of the art.
The coupling of microfluidic reactor chips with downstream
mass spectrometry enables to probe the chemical constitu-
tion of reactor effluents.35-37 Mass spectrometry, however,
has limitations whenever isomeric compounds are present
and there is currently a great demand for a technology for
fast online monitoring of stereoselective processes at the
microscale. For slower processes this can be achieved by
on-chip integration of separation functionalities like elec-
trophoresis or chromatography.38-42
Figure 1. Schematic drawing of the microfluidic chip/IRPD
spectrometer set-up. Reactants are mixed on chip. The reaction
solution is continuously sampled using electrospray ionization
in order to transfer ions into the gas phase, where they are
characterized using vibrational action spectroscopy.
An interesting candidate for on-the-fly structure-sensitive
analysis is vibrational action spectroscopy by way of infra-
red photodissociation (IRPD).43 IRPD allows for the isomer-
specific monitoring of ions after they have been transferred
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Fig. 2b). To this end, the reactant solutions, containing ei-
ther R1 or R2, are introduced into the first two inlets (see
Fig. S-1) with an adjustable flow rate. A MeCN/H2O solution
(80:20, v/v) is supplied through a third inlet, at the end of
the chip, to enhance the electrospray efficiency. The on-chip
residence time is then determined from these flow rates and
considering the chip geometry. A characteristic mass spec-
trum for a residence time of 1 min is show in Fig. 2a. Two
dominant signals with m/z ratios of 177 amu and 195 amu,
whose intensities change with varying residence time (see
Fig. 2b), are detected. The signal at 177 amu corresponds to
the protonated product [P+H]+. Its relative intensity in-
creases with longer residence times. The signal of the inter-
mediates is found at 195 amu, which expectedly decreases
with increasing residence and hence reaction time.
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spectra for unambiguous identification of gas-phase ions
the rather high process times for recording a full spectrum
have prevented its use for monitoring short-lived interme-
diates. In this contribution, we present a two-step approach
to solve this challenge by joining microfluidics with IRPD. In
this approach, reaction intermediates are first spectroscop-
ically identified and subsequently the IRPD spectrometer is
used to isomer-specifically monitor the chemical composi-
tion of a reactor effluent constantly spraying from a micro-
fluidic chip (see Fig. 1).
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Scheme 1: Mechanism of Hantzsch Cyclization Reaction
In a second step, we measured the vibrational spectrum of
the product ion [P+H]+. To this end, ions with
m/z = 177 amu are mass-selected and subsequently
trapped in a buffer-gas filled cryogenic ion trap held at 12 K.
Here, the ions are thermalized close to the ambient temper-
ature and messenger-tagged with D2, which ensures meas-
uring an IRPD spectrum in the linear absorption regime (see
experimental section). The obtained IRPD spectrum in the
spectral range from 1100 to 3800 cm-1 is shown in Fig. 3a.
The spectrum reveals five characteristic IR bands, labeled
a1-a5, a7 and a9 as well as some weaker signals. Comparison
to the simulated IR spectrum of protonated 2-amino-4-phe-
nylthiazole (see Fig. S-4 in the supporting information), ob-
tained from density functional theory (DFT) calculations,
which reproduces all the experimental spectrum satisfacto-
rily, unambiguously identifies the structure of the [P+H]+
ions and allows assigning these bands to individual vibra-
tional modes (see Table S-1 for band assignments). Briefly,
the intense absorptions above 3400 cm-1 (a1-a3) correspond
to the N-H stretching modes of the primary and secondary
amino groups. Band a7 is the NH2 bending mode and a9 re-
sults from more delocalized modes involving C=C stretching
and C-N-H bending motions.
In order to characterize the properties and demonstrate the
advantages of the chip-IRPD set-up, we apply the approach
in
a proof-of-concept study on the well-understood
Hantzsch reaction shown in Scheme 1. This reaction is well
suited as a litmus test for the approach in which the thiazole
derivate P is formed from the reactants phenacyl bromide
(R1) and thiuorea (R2) via three consecutively formed in-
termediates I1-I3. Note, all three intermediates I1-I3 are
isomers with the same m/z ratio of 195 amu. In order to
characterize the structure and monitor the abundance of
the reaction participants, in particular of the intermediates,
we developed a tailored microfluidic chip (channel dimen-
sions: 50 µm wide, 20 µm deep, 30 cm long, see supporting
information for details) for this continuous flow reaction.
The chip-device has an integrated electrospray emitter at its
end to efficiently transfer the reaction solution into the
high-vacuum chamber of the IRPD-triple mass spectrome-
ter (Fig. S-3).
Subsequently, we investigated the IR spectrum of the reac-
tion intermediates, i.e. the ions with m/z = 195 amu. The ob-
tained IRPD spectrum in the O-H and N-H stretching region
is shown in the bottom panel of Fig. 3b. Similar to the spec-
trum of the product ion [P+H]+, it can be assigned based on
a comparison to simulated IR spectra of the three possible
reaction intermediates I1 to I3 (see Fig. S-5 and Table S-2
for band assignments). The assignment of the experimental
spectrum (Fig. 3b) is straightforward. Contributions from
I3, which contains the water adduct and hence is character-
ized by two characteristic O-H stretching modes in the re-
gion above 3600 cm-1, can be excluded, since only a single
band is observed at 3618 cm-1 (b1) in the experimental spec-
trum. Moreover, intermediate I1 does not contain an O-H
group and so I2 must be present to account for band b1. In-
deed, the predicted IR spectrum of I2 (Fig. S-5b) shows a
satisfactory agreement with the experimental one. How-
ever, the experimental spectrum reveals additional bands,
in particular b6, which can only be rationalized assuming the
coexistence of I2 and I1. Assuming the presence of both of
these intermediates with a 1:1 ratio results in the best
agreement with the experimental spectrum (see Fig. S-5d).
Figure 2. (a) ESI-MS spectrum of the continuous flow reaction
using 2.5 mmolˑL-1 phenacyl bromide and 1.0 mmol L-1 thiuo-
rea in acetonitrile and flow rates corresponding to a chip resi-
dence time of 1 min. (b) Relative ion signal intensities of the
protonated reaction product ([P+H]+, m/z 177 amu, blue) and
the intermediates (I1 and I2, m/z 195 amu, orange and red) at
residence times from 0.21 to 6.0 min. Relative ion signals are
obtained by normalizing the mass spectrum intensities to the
total ion yield.
First, we monitored the reaction yield as a function of the
residence time of the reaction solution on the chip (see
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Figure 3. (a) IRPD spectrum of D2-tagged [P+H]+ ions (blue). (b) The experimental spectrum of the ions I1 (orange) and I2 (red)
corresponding to the signal at m/z = 195 amu in Fig. 2. P, I1 and I2 are formed in the chip reaction, transferred in their protonated
form by ESI to the high vacuum chamber and their IR spectra are subsequently measured using IRPD. The corresponding minimum
energy structure (H: white, C: grey, N: blue, S: yellow, O: red) is also shown. Simulated IR spectrum of D2-tagged ions (Fig. S-4, S-5)
and band assignments (Tab. S-1, S-2) are shown in supporting information. (c,d) Isomer-specific IR2MS2 spectra of I1 (c, orange),
and I2 (d, red) in the range between 2400 and 3800 cm-1, confirming the presence of two stable intermediates. The spectra of I1
and I2 were obtained by pumping their isomer-specific modes at 2636 cm-1 and 3618 cm-1, respectively.
In order to confirm the presence of both intermediates and
reveal their individual IR signatures, we performed isomer-
specific IR2MS2 measurements in the range between 2400
and 3800 cm-1 and these are shown in Figs. 3c-d. Briefly, this
double-resonance method allows measuring the IRPD spec-
tra of coexisting isomers, whenever the IR spectra of the
corresponding isomers differ significantly at least at one
wavelength. In the present case, we obtained the IR2MS2
spectrum of I1 (Fig. 3c) by pumping the isomer-specific NH
stretching mode b6 and that of I2 (Fig. 3d) by pumping the
isomer-specific O-H stretching mode b1. The two IR2MS2
spectra convincingly show that these two, and only these
two, constitutional isomers are responsible for the ion sig-
nal at m/z = 195 amu.
A major advantage of the microfluidic setup is that the con-
ditions on the chip can be changed quickly. In combination
with the IRPD detection scheme it is then possible to moni-
tor the influence of these changes online and obtain isomer-
resolved kinetic information. Note, this does not require to
measure complete IRPD spectra over broad spectral ranges,
which indeed would be very time consuming. Once the IR
spectra of the individual reaction participants have been
disentangled using the IR2MS2 scheme, isomer-specific ion
signals can be obtained, in the best case, by probing at a sin-
gle wavelength per isomer and the resulting ion signals can
then be converted to ion yields based on calibration tables.
Such a procedure, in principle, takes only 100 ms, given the
10 Hz repetition rate of the photodissociation laser. How-
ever, fluctuations of the laser pulse energy as well as of the
ion signal require to typically average over at least 10 to 20
laser shots in order to obtained a satisfactory signal-to-
noise ratio.
Figure 4. The ratio of I2/I1 as a function of the reactant solu-
tion flow ratio Q(R1)/Q(R2) measured by monitoring the ion
yield of the intermediates I1 and I2 spectroscopically at
3445 cm-1 and 3618 cm-1, respectively.
To demonstrate the analytical possibilities of this approach
we determined the optimal flow rate ratio of the reactant
solutions with respect to the highest product yield by mon-
itoring the relative intermediate ratio I2/I1 (see Fig. 4) us-
ing this technique. The fastest possible reaction is obtained
when this yield is maximized. The ion yields were deter-
mined by monitoring the ion signals of the intermediates I1
and I2 by probing at 3445 cm-1 (I1) and 3618 cm-1 (I2),
while systematically changing the flow rate of the individual
reactant solutions containing R1 and R2. The initial concen-
trations, 2.5 mmolˑL-1 phenacyl bromide and 1.0 mmol L-1
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thiuorea, of both reactants and the total flow rate
(1.2 µL min-1, residence time: 25 s) were kept unchanged.
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The data in Fig. 4 shows that a flow rate ratio of one, i.e.
equal flow rates for both solutions is far from optimal. A
maximum in the I2/I1 ratio, indicating the reaction that has
proceeded the farthest, is obtained for a flow rate ratio of
around 4.0. Even though the conversion of flow rates to lo-
cal concentrations is not straight forward, the present re-
sults suggest that the transformation of R1 is involved in the
rate-limiting step of the reaction and therefore needs to be
present in excess. This is in agreement assuming a SN1 type
reaction, which exhibits a first order dependence on the
concentration of the electrophile (R1) and a zeroth-order
dependence on the concentration of the nucleophile (R2).
Similar results were obtained, when the initial concentra-
tions of the reactants were changed.
The Supporting Information is available free of charge on the
ACS Publications website.
Detailed description of the microfluidic fabrication process;
picture of the experimental setup; mechanism of the Hantzsch
reaction; MS spectrum of the Chip-MS-IRPD measurement;
Chip-MS spectrum of the changes conditions (PDF).
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¢ Phone: +49 341 97 36421; Fax: +49 341 97 36399; E-mail:
knut.asmis@uni-leipzig.de
¥ Phone: +49 341 97 36091; Fax: +49 341 97 36115; E-mail:
belder@uni-leipzig.de
In summary, we have demonstrated that IRPD combined
with lab-on-a-chip technology allows to identify, character-
ize and monitor reactants, products and short-lived inter-
mediates of continuous flow reactions. Vibrational spectra
of the corresponding gas phase ions can be measured over
a broad spectral region, allowing for an unambiguous struc-
tural assignment based on a comparison to simulated spec-
tra from electronic structure calculations. After a character-
istic wavelength has been determined for each reaction par-
ticipant, their abundance can be monitored isomer-specifi-
cally on a time scale of seconds or less as the on-chip reac-
tion parameters are optimized. Hence, the present tech-
nique represents a powerful new addition to the lab-on-a-
chip tool kit for studying reaction kinetics and unraveling
reaction mechanisms of continuous flow reactions.
The authors declare no competing financial interest.
Financial support by the German Research Foundation (DFG,
FOR 2177) is gratefully acknowledged. K.R.A. acknowledges
instrumental support from the Fritz-Haber-Institute of the
Max-Planck Society.
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