Real-Time Reaction Monitoring of an Organic Multistep Reaction by Electrospray Ionization-Ion Mobility Spectrometry

Article abstract of DOI:10.1002/cplu.201700296

The capability of electrospray ionization (ESI)-ion mobility (IM) spectrometry for reaction monitoring is assessed both as a stand-alone real-time technique and in combination with HPLC. A three-step chemical reaction, consisting of a Williamson ether synthesis followed by a hydrogenation and an N-alkylation step, is chosen for demonstration. Intermediates and products are determined with a drift time to mass-per-charge correlation. Addition of an HPLC column to the setup increases the separation power and allows the determination of further species. Monitoring of the intensities of the various species over the reaction time allows the detection of the end of reaction, determination of the rate-limiting step, observation of the system response in discontinuous processes, and optimization of the mass ratios of the starting materials. However, charge competition in ESI influences the quantitative detection of substances in the reaction mixture. Therefore, two different methods are investigated, which allow the quantification and investigation of reaction kinetics. The first method is based on the pre-separation of the compounds on an HPLC column and their subsequent individual detection in the ESI-IM spectrometer. The second method involves an extended calibration procedure, which considers charge competition effects and facilitates nearly real-time quantification.

Full text of DOI:10.1002/cplu.201700296

DOI: 10.1002/cplu.201700296
Full Papers
Real-Time Reaction Monitoring of an Organic Multistep
Reaction by Electrospray Ionization-Ion Mobility
Spectrometry
[
a]
Martin Zühlke, Stephan Sass, Daniel Riebe, Toralf Beitz, and Hans-Gerd Lçhmannsrçben*
The capability of electrospray ionization (ESI)-ion mobility (IM)
spectrometry for reaction monitoring is assessed both as a
stand-alone real-time technique and in combination with
HPLC. A three-step chemical reaction, consisting of a William-
son ether synthesis followed by a hydrogenation and an N-al-
kylation step, is chosen for demonstration. Intermediates and
products are determined with a drift time to mass-per-charge
correlation. Addition of an HPLC column to the setup increases
the separation power and allows the determination of further
species. Monitoring of the intensities of the various species
over the reaction time allows the detection of the end of reac-
tion, determination of the rate-limiting step, observation of the
system response in discontinuous processes, and optimization
of the mass ratios of the starting materials. However, charge
competition in ESI influences the quantitative detection of sub-
stances in the reaction mixture. Therefore, two different meth-
ods are investigated, which allow the quantification and inves-
tigation of reaction kinetics. The first method is based on the
pre-separation of the compounds on an HPLC column and
their subsequent individual detection in the ESI-IM spectrome-
ter. The second method involves an extended calibration pro-
cedure, which considers charge competition effects and facili-
tates nearly real-time quantification.
Introduction
A deeper understanding of chemical reactions is an essential
requirement for optimizations of chemical synthesis in research
and industry. Fast online detection methods enable the deter-
mination of reactive intermediates and the rate-limiting step,
and consequently, allow the elucidation of the reaction kinetics
and the underlying reaction mechanism. Typical techniques for
monitoring reactions are spectroscopic methods such as near-
ever, expensive equipment and, in the case of atmospheric
pressure ionization (API) sources, charge competition influenc-
ing the ionization efficiency of the individual components are
disadvantageous.
A technique less often applied in reaction monitoring is ion
mobility (IM) spectrometry, which is less expensive than MS.
The fundamental principle of IM spectrometry is the separation
of ions according to their mobility (K) in an electric field (E),
usually at atmospheric pressure. The mobility K is a characteris-
tic physical constant for a molecular ion in a drift gas, and is
[
1]
[2]
[3]
infrared (NIR), ultraviolet (UV), Raman, or nuclear magnetic
[4]
resonance (NMR) spectroscopy, which often allow direct con-
tactless access to the reaction mixture, avoiding any disturb-
ance and providing real-time monitoring. A number of differ-
ent syntheses can be monitored spectroscopically. Neverthe-
less, various properties can hamper applications, such as low
sensitivity for dilute intermediates (Raman), high complexity of
the reaction mixture (UV, NIR, Raman), or difficult interpretation
of spectra or impurities (NMR). Other techniques such as mass
[
8]
described by the Mason equation, shown in Equation (1).
ꢀ
ꢁ
ꢀ
ꢁ
1
=2
1=2
l
3 q
1
m
1
M
2p
1
WD
K ¼
¼
þ
ð1Þ
EtD 16 N
k T
B
eff
[
5,6]
[7]
spectrometry (MS)
and chromatographic methods allow
Here, l is the drift path length, t is the drift time, q is the
D
the characterization of more complex reactions. Gas chroma-
tography (GC) and liquid chromatography (LC) are proven
techniques for synthesis control in the industry, but are often
time-consuming. In the case of MS, many publications demon-
strate the fast analysis of the compounds in the reaction. How-
charge, N is the number density of the drift gas, m is the mass
of the drift gas molecules, M is the mass of the ions, Teff is the
temperature in the drift tube, k is the Boltzmann constant,
B
and W is the collisional cross section. W is a measure of the
D
D
shape of an ion, and is the most important parameter in IM
spectrometry. In contrast to MS, in IM spectrometry, the colli-
sional cross section determines the separation, and thus, iso-
mers can be separated. The ionization methods most frequent-
ly applied in IM spectrometry are either based on the evapora-
tion of analytes with subsequent ionization in the gas phase
by chemical ionization at atmospheric pressure (APCI) through
[
a] M. Zühlke, S. Sass, D. Riebe, T. Beitz, Prof. Dr. H.-G. Lçhmannsrçben
University of Potsdam, Physical Chemistry
Karl-Liebknecht-Strasse 24–25
1
4476 Potsdam–Golm (Germany)
E-mail: loehm@uni-potsdam.de
Homepage: http://www.chem.uni-potsdam.de/groups/pc
63
electron- or proton-transfer reactions (e.g., with Ni or corona
discharge), or rely on the direct ionization of liquids by electro-
Supporting information for this article can be found under:
https://doi.org/10.1002/cplu.201700296.
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spray ionization (ESI) or matrix-assisted laser desorption/ioniza-
followed by a hydrogenation and an N-alkylation reaction was
chosen for the characterization of reaction monitoring by ESI-
IM spectrometry. These reaction steps are used frequently in
chemical syntheses. The ether synthesis and the alkylation are
representative of classical electrophilic or nucleophilic substitu-
tions, whereas the hydrogenation is an example of heteroge-
neous catalysis. However, the product is BAPTA-AM, which is a
precursor of the calcium sensor BAPTA, which is used for spec-
[
9]
tion (MALDI).
Only a few applications of APCI-IM spectrometry for reaction
monitoring have been reported. These include the online mon-
itoring of monomer concentrations (vinyl acetate) in (semi-)
[10]
batch emulsion polymerization reactors, and a kinetic study
of the acetylcholine hydrolysis catalyzed by acetylcholinester-
ase (AChE) and its inhibition by neostigmine and galantha-
[
11]
[18,19]
mine. In 2010, the company Excellims demonstrated reaction
monitoring of the Michael addition of cyclohexylethylamine to
troscopic in vivo measurements in biological applications.
[12]
dimethlitaconate using ESI-IM spectrometry. Several studies
have focused on the potential of ESI-IM in combination with
mass spectrometry (IM-MS) for reaction monitoring. In one
Results and Discussion
[
13]
study, the monitoring of the reaction products formed upon
deprotonation of 7-fluoro-6-hydroxy-2-methylindole by aque-
ous sodium hydroxide was investigated, demonstrating the ad-
vantages of the IM-MS method combination. Other studies
have shown the detection of short-lived intermediates and
quantitative investigations monitoring the reductive amination
reaction of nicotinaldehyde and 4-picolylamine to di-(2-picolyl)-
In this study, all three steps of the reaction sequence (Figure 1)
were monitored over the whole reaction time. Each reaction
step was performed separately in a flask. The reaction condi-
tions of each step were based on literature-known synthe-
[
20–23]
ses.
The detailed conditions can be found in the Support-
ing Information (S.1.2 Synthesis). Each step was followed by a
work-up, and the purified products were analyzed by standard
analytical methods before their utilization as the starting mate-
rials for the respective next step. At different reaction times,
samples were taken by syringe and introduced into the ESI-IM
spectrometer (see Experimental Section for details). This sec-
tion is divided into three parts, which are focused on the de-
termination of reaction intermediates and products, on differ-
ent tasks of reaction monitoring based on the pure intensities,
and on two methods allowing the quantification of all compo-
nents in the reaction mixture. In these sections, ESI-IM spec-
trometry is characterized as a stand-alone technique as well as
in combination with HPLC for pre-separation. In each section,
only one reaction step is discussed for demonstration pur-
poses.
[
14]
amine. In another study, the combination of stable isotope
labelling with direct infusion IM-MS was investigated for quali-
[
15]
tative and quantitative monitoring of biocatalytic reactions.
This approach was applied to lipase and monooxygenase en-
zymes and included multisubstrate screening.
However, for the less expensive, stand-alone ESI-IM spec-
trometry, a demonstration of the quantitative monitoring of all
the components of a reaction mixture is still lacking. Herein,
the capability of ESI-IM spectrometry was evaluated, both as a
stand-alone method and in combination with HPLC, for quali-
tative and quantitative reaction monitoring. The enhancement
of the separation power through the combination of HPLC
[
16,17]
with ESI-IM spectrometry is based on our previous work.
A
three-step reaction consisting of a Williamson ether synthesis
Figure 1. Reaction sequence starting from 1,2-dibromoethane and sodium 2-nitrophenoxide to 1,2-bis(aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid meth-
ylester (BAPTA-AM).
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Intermediate and product detection
ogous series showing good correlations between drift time (or
inverse ion mobility) and mass, facilitating simple mass assign-
Direct detection by IM spectrometry
[
24]
ments of unknown members of the series. Assuming singly
protonated molecules (M+1) for the five peaks between 9 and
15 ms, as are usually observed for small molecules in ESI, the
drift times of the corresponding ions can be plotted against
the mass-to-charge ratio (m/z) to verify the species. The high
correlation of the detected constituents shown in Figure 2b
ESI-IM spectrometry enables the very fast detection of inter-
mediates and products. For demonstration purposes, the N-al-
kylation of substance 3 to 4, which constitutes the most com-
plex reaction step and includes the highest number of inter-
mediates, was chosen. Figure 2a shows three IM spectra taken
after reaction times of 3, 25, and 390 min. In the IM spectra,
the discharge current (intensity in nA) of all ions arriving the
detector at the same drift time is plotted over their drift time
2
possesses a coefficient of determination of R =0.99 and allows
reliable assignment of the IM peaks to constituents of the reac-
tion mixture. The starting material has the lowest drift time. In-
creasing degrees of N-alkylation lead to a peak shift to higher
drift times. Thus, the peak with the highest drift time can be
assigned to the product, and the intermediates to the one-,
two-, and three-times substituted species of the starting mate-
rial 3. The assumed M+1 peak for the starting material and
product was verified through MS measurements (see Support-
ing Information). In contrast to HPLC separation (Figure 3),
which shows a less clear order of substance peaks, in IM spec-
trometry, the analytes are ordered by size.
(
drift time in ms). The peak group at 5–7 ms observed in all IM
spectra is attributed to solvent cluster ions. The five peaks at
–15 ms can be assigned to the starting material, different in-
9
termediates, and the product. They have a constant peak-to-
peak distance (Dt ) of approximately 1.13 ms, indicating a
D
structural relation as well as a constant mass difference be-
tween neighboring peaks. In the IM spectrometry literature,
there are several examples of substances belonging to homol-
There is an alternative, more elaborate method of peak as-
signment based on the quantum chemical calculation of sub-
stance structures and the subsequent determination of colli-
sion cross sections by programs calculating collision trajecto-
[
25–27]
ries.
Mobilities can be obtained from the cross sections by
the Mason equation [Eq. (1)]. The cost of calculation increases
in the case of flexible molecules, for which all energy-averaged
conformations must be considered by applying molecular dy-
namics simulations.
Two-dimensional detection by HPLC-IM spectrometry
The peak capacity n in IM spectrometry is often limited to n<
1
0. Thus, the combination of IM spectrometry with a separa-
tion method of high orthogonality, for example, HPLC, can fa-
cilitate the complete separation of complex reaction mixtures.
New information can appear owing to the complementary sep-
aration on the LC column and in the drift tube. Because of the
different time scales of HPLC (minutes) and IM spectrometry
(
milliseconds), both methods can be combined easily, yielding
2
D spectra consisting of the dimensions of retention and drift
[
16,17]
time.
A 2D spectrum of the consecutive N-alkylation from
3
to 4 is shown in Figure 3 at the reaction time corresponding
to the maximum intensity of the disubstituted species (5 min).
All constituents of the reaction mixture can be separated in
the plane of the 2D spectrum. The species can also be deter-
mined from the drift time versus m/z ratio, as in the 1D IM
spectrum. In comparison with the 1D IM spectrum, additional
compounds are found. In addition to the five species observed
in the IM spectra (Figure 2a), the disubstituted species peak is
split into two different peaks, which can be assigned to two
constitutional isomers. The N,N-disubstituted species has the
two alkyl chains on the same nitrogen atom. In the N,N’-disub-
stituted species, each nitrogen atom is connected to an alkyl
chain. The N,N-disubstituted species elutes first because of its
higher polarity. Computational calculations using density func-
tional theory (DFT) show that the charge density at the nitro-
Figure 2. Consecutive N-alkylation of subtance 3 to 4. a) IM spectra at differ-
ent reaction times (drift time 5–7 ms for the solvent cluster peak, and 9–
14 ms for the five different species of the N-alkylation). b) Direct determina-
tion of the five species of the N-alkylation from drift time versus mass-to-
charge ratio (m/z).
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Figure 3. Consecutive N-alkylation of subtance 3 to 4. HPLC ESI-IMS spectrum at 5 min reaction time (the colors of the words refer to Figure 2b for color
code of the subtances; drift time 5–7 ms for solvent cluster peak and 9–14 ms for the five different species of the N-alkylation).
gen atom decreases with increasing substitution owing to the
electron-withdrawing effect of the carbonyl group. This corre-
step and the deduction of the reaction mechanism. As IM
spectra can be measured in less than one second, IM spec-
trometry enables the measurement of a large number of sam-
ples in a short time, and thus, allows almost real-time monitor-
ing (sampling is the limiting factor). The reaction progress of
the N-alkylation was monitored over 24 h and is presented in
Figure 4. The normalized intensities of all five N-alkylation spe-
cies over the reaction time are displayed, enabling a conver-
sion control. The intensity maxima of all components are
passed sequentially. Furthermore, the time between subse-
quent intensity maxima increases, which indicates a decrease
in the rate constants of each additional alkylation step. The
rate-limiting step is the last alkylation step; one reason for this
could be the steric hindrance of the three alkyl chains, imped-
ing this final alkylation.
lates with approximations of the pK value, which decreases at
a
[
28]
higher substitution. The area of the first peak is only half
that of the second peak. This suggests that the N,N’-substitu-
tion is preferred. This experimental result is in agreement with
quantum chemical calculations on the reaction pathway. The
three-step calculation procedure starts with a systematic varia-
tion of the torsion angle in AMBER, followed by PM7 and
B3LYP/6-31G(d)+D3 optimizations (see Supporting Informa-
tion, S.3). The energy calculated for the ground-state confor-
À1
mation of the N,N’-substituted species is 33 kJmol below
that of the corresponding N,N-isomer. This difference could ex-
plain the preferred formation. The separation on the reversed-
phase LC column is poor. The effect of the increasing mass
gained with each additional N-alkylation step is cancelled by
the increasing polarity of the alkylated amine caused by the
carbonyl groups.
A well-known problem in ESI is charge competition between
analytes, which can lead to their partial or complete suppres-
sion. As already discussed, the negative charge density at the
nitrogen decreases with each alkylation step. This is reflected
in the pK values, which also decrease with each alkylation
a
Process monitoring
step. Hence, it is probable that the intensity curves are influ-
[
29]
In certain process monitoring tasks, exact knowledge of the
concentrations is not always required; tracking of relative in-
tensities can provide the necessary information. These tasks in-
clude the detection of the end of reaction, determination of
the rate-limiting step, observation of the system response in
discontinuous processes, and optimization of the mass ratio of
the starting materials.
enced by charge competition. Therefore, HPLC-IM spectrom-
etry measurements (not shown) were taken to address this
issue. Although the intermediates could not be separated com-
pletely in the LC dimension, the curves of the educt, intermedi-
ates, and product in Figure 4 could be reproduced qualitative-
ly, indicating that the influence of the charge competition
effect is not strong.
Direct monitoring by IM spectrometry
Monitoring by HPLC-IM spectrometry
After the qualitative determination of the components in the
reaction mixture, their temporal appearance profile is interest-
ing because it allows the determination of the rate-limiting
The focus of the previous subsection was the detection of the
end of reaction and the determination of the rate-limiting
step. In this part, a discontinuous process is simulated, allow-
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Figure 4. Consecutive N-alkylation of subtance 3 to 4 over the reaction time (refer to Figure 2b for the color code: starting material 3 (black), 1”substituted
species of 3 (cyan), 2”substituted species of 3 (red), 3”substituted species of 3 (purple), product 4 (dark blue); intensities were normalized separately).
ing observation of the system response and optimization of
the mass ratio of the starting materials. In comparison with the
N-alkylation, charge competition is much stronger among the
species involved in the Williamson ether synthesis. Therefore,
HPLC-IM spectrometry was used for reaction monitoring.
product concentration increases before reaching a plateau.
The temporal concentration profile of the intermediate is more
interesting. The reaction pathway shown at the top of Figure 5
indicates that the starting material 1 first reacts with 1,2-dibro-
moethane to form the intermediate. Then, the intermediate
reacts with 1 again to produce 2. Therefore, for the formation
of product 2, 1 mol of 1,2-dibromoethane and 2 mol of 1 are
needed. Because of the initial molar ratio (1:1), the intermedi-
ate is formed as the main product. After the second injection,
a strong increase in the concentration of the intermediate is
observed, caused by the reaction of 1 with the remaining 1,2-
dibromoethane. Subsequently, the intermediate reacts to form
The discontinuous process is simulated by stepwise injection
of the starting material 1, changing the molar ratio between
the starting materials from 1:1 to 3:1. The intensities of the
starting material 1 (black), the intermediate (green), and the
product 2 (blue) were monitored (Figure 5). After each injec-
tion, the concentration of the starting material rises immedi-
ately before it declines, and after a short initial period, the
Figure 5. Intensity over reaction time of the Williamson ether synthesis for starting material 1, intermediate, and product 2, with a stepwise injection of start-
ing material 1.
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the product because of the optimal molar ratio between the
starting materials. After the third injection, the intensity of the
intermediate does not increase further owing to the surplus of
starting material 1. Thus, the ratio of both starting materials, 1
and 1,2-dibromoethane, determines the reaction yield and the
concentration level of the intermediate.
Quantification
Charge competition influences the quantitative detection of all
compounds in the reaction mixture, so two different methods
were investigated, which consider this effect and allow the
quantitative determination of all substances. The first method
is based on the pre-separation of the compounds on an HPLC
column and their subsequent individual detection in the ESI-IM
spectrometer. The second method involves an extended cali-
bration procedure that takes charge competition effects into
account. The benefit of this method is the nearly real-time
quantification by direct IM spectrometry without the need for
a pre-separation step. For the illustration of both methods, the
hydrogenation was chosen as an example of a heterogeneous
reaction.
HPLC-IM spectrometry: quantification, kinetics, and conver-
sion control
The separate calibrations of the isolated starting material 2
and product 3 show a linear range of around two orders of
magnitude (Figure 6a). From this calibration and the pre-sepa-
ration of all constituents of the reaction mixture, concentra-
tion–time profiles over the complete reaction time were deter-
mined (Figure 6b). There are three notable features in these
profiles. The first is the fast drop of the concentration of 2
owing to adsorption on the activated carbon (catalyst), which
is not sampled. The second feature is the linear decrease in
the concentration of the starting material after this initial
period of adsorption, and the linear increase in the product
concentration with reaction time. After 90 min, 2 and 3 reach a
plateau, signifying the final conversion of the reaction. Fitting
of the linear segment yields good coefficients of determination
Figure 6. Reaction monitoring of the hydrogenation of 2 (black traces) yield-
ing 3 (blue traces) with HPLC ESI-IM spectrometry: a) calibration, and b) con-
centration over reaction time.
the fact that there are a limited number of charges in a droplet
[
30]
produced during the ESI process. If there is only one ioniza-
ble compound in the droplet, all charges are available to it. If
there is more than one ionizable compound, all compounds
compete for the same pool of charges. Assuming that the
overall number of ions produced by ESI is nearly constant, the
sum area of all IM peaks should be nearly constant as well (ne-
glecting diffusion processes). The following procedure for the
calculation of the correction factors was applied: IM spectra
were measured using a constant concentration of 2 and a
varying concentration of 3, and vice versa. In the absence of 3,
the peak intensity of 2 is at its maximum. Increasing concen-
trations of 3 lead to a decrease in the peak intensity of 2. The
maximum peak intensity of 2 divided by the peak intensity of
2
2
(
R =0.86 for 2, R =0.97 for 3). This allows the determination
of rate constants and indicates a reaction order of zero. The
zero-order rate constant obtained for the decrease in starting
À1 À1
material is k =(2.1Æ0.2) mmols L , and that for the forma-
2
À1 À1
tion of the product is k =(2.2Æ0.1) mmols L . These rate
3
constants have similar values, indicating that side reactions do
not occur.
IM spectrometry: direct quantification
2
at a defined concentration of 3 yields an individual correc-
The quantification without pre-separation on an HPLC column
requires an extended calibration procedure, which considers
charge competition effects. In addition to the normal calibra-
tion procedure of the individual substances, the mutual sup-
pression of starting material and product must be considered.
One possible method is the calculation of concentration-de-
pendent correction factors. The correction factors are based on
tion factor. The correction factor (CF) depends on the relative
intensities of both compounds. Hence, for every intensity ratio
of 2 and 3, there is a CF for each compound. The relationship
for 2 is displayed in Figure 7a, and can be described by an
asymptotic function. Nonlinear regression gives the following
x
2
function parameters: CF=0.89–1.07”0.98 and R =0.995.
Here, x describes the ratio of the intensities of starting material
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genation and an N-alkylation step were investigated. On the
one hand, IM spectrometry can be used as a stand-alone
method for qualitative and quantitative real-time monitoring
of reaction mixtures of low complexity. On the other hand, the
inclusion of a pre-separation step through HPLC enables the
investigation of more complex mixtures, albeit at the expense
of the real-time ability. IM spectrometry was shown to allow
the determination of all reaction participants including inter-
mediates, relative-intensity-based (without calibration) moni-
toring, and concentration-based (with calibration) monitoring
of the reaction process.
The determination of the various compounds is based on
their ion mobilities, which are characteristic physical constants
for any ion-drift gas molecule pair. A reliable assignment of
substances on the basis of their measured mobilities can re-
quire demanding DFT calculations. However, simple drift time–
mass correlations of the reactants are often sufficient, but IM
resolutions are often limited to 60 results in overlapping peaks
[
31]
in the IM spectrum. If the composition of the reaction mix-
ture is too complex for stand-alone IM spectrometry, 2D LC-IM
spectrometry can enable the separation of all substances. Such
separation benefits from the high orthogonality of the differ-
[
16]
ent separation principles.
Relative-intensity-based measurements can yield valuable in-
formation, but avoid extensive calibration. This could be
shown for the detection of the end of reaction, the determina-
tion of the rate-limiting step, the observation of the system re-
sponse in discontinuous processes, and the optimization of
the mass ratio of the starting material components. Charge
competition in the ESI process can influence the measured in-
tensities; complete suppression of some substances is possible.
Therefore, 2D HPLC-IM spectrometric separations should be
performed for evaluation of these charge competition effects.
If the charge competition effects cannot be neglected, LC-IM
spectrometric separation in combination with calibration yields
concentration information on the reaction process. An alterna-
tive, allowing almost real-time measurements, involves the ap-
plication of an extended calibration procedure. Although this
procedure usually requires a large number of measurements,
the simplified version applied in this study can give satisfactory
results.
Figure 7. Reaction monitoring of the hydrogenation with ESI-IM spectrome-
try: a) correction factor of 2 for charge competition as a function of the
ratio of intensities of 2 and 3, b) concentrations of 2 (black) and 3 (blue)
over reaction time in direct ESI-IM spectrometry (filled symbols) versus
HPLC-ESI-IM spectrometry (open symbols).
2
and product 3. The correction function was applied to the
peak intensities, yielding concentrations. The temporal concen-
tration profiles obtained by both methods, that is, HPLC-IM
spectrometry and direct IM spectrometry with the extended
calibration procedure, are compared in Figure 7b.
Experimental Section
Both methods yield similar results. However, this finding
might only apply to this specific case, as the range of concen-
trations investigated is relatively narrow. For a more general
approach, the correction factor vector used here should be ex-
tended to a correction matrix that takes into account addition-
al constant concentrations of 2 at varying concentrations of 3
All IM spectra were recorded with a home-built ESI-IM spectrome-
ter, which has been described in detail previously. The spectrom-
[17]
eter is divided into four sections. The first part is the cylindrical ion-
ization region (inner diameter, ID=30 mm) containing an ESI capil-
lary (Hamilton, gauge 32) inserted into a 1/16“ steel capillary ar-
ranged orthogonally to the axis of the IM spectrometer. This
region is followed by a desolvation (length=50 mm, outer diame-
ter, OD=47.5 mm, ID=25 mm, thickness=23 mm, repeating
unit=3.17 mm, T=1608C) and a drift region (length=100 mm,
OD=47,5 mm, ID=25 mm, thickness=2.54 mm, repeating unit=
(
and vice versa).
Conclusion
3
.17 mm, T=1408C) separated by an ion gate in the Bradbury–
This study demonstrates that ESI-IM spectrometry can be ap-
plied for reaction monitoring. A three-step reaction sequence
consisting of a Williamson ether synthesis followed by a hydro-
Nielsen design. The fourth part is the detection region consisting
9
of a Faraday plate with a diameter of 18 mm connected to a 10 V/
A amplifier (Physimetron A6203) and an oscilloscope (Handyscope
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HS3, Tiepie). Nitrogen 5.0 (Praxair) was used as drift gas (flow
rate=1.3 Lmin ) and the sheath gas (flow rate=1.8 Lmin ).
[5] L. S. Santos, Reactive Intermediates: MS Investigations in Solution, Wiley-
VCH, Weinheim, 2009.
À1
À1
[6] D. Schrçder, Acc. Chem. Res. 2012, 45, 1521–1532.
The HPLC system for the pre-separation of the reaction mixture
consisted of an LPG pump (Azura P 6.1L, KNAUER), an injection
valve (KNAUER), and a reversed-phase (RP) column (Hypersil Gold,
Thermo, 100”2.1 mm, 3 mm). The mobile phase of all reaction mix-
ture separations was composed of acetonitrile (solvent A) and
[7] W. A. Schafer, S. Hobbs, J. Rehm, D. A. Rakestraw, C. Orella, M. McLaugh-
lin, Z. Ge, C. J. Welch, Org. Process Res. Dev. 2007, 11, 870–876.
[8] E. A. Mason, E. W. McDaniel, Transport Properties of Ions in Gases, John
Wiley & Sons, Weinheim, FRG, 1988.
[9] H. Borsdorf, T. Mayer, M. Zarejousheghani, G. A. Eiceman, Appl. Spec-
trosc. Rev. 2011, 46, 472–521.
10] W. Vautz, W. Mauntz, S. Engell, J. I. Baumbach, Macromol. React. Eng.
0
3
.1% aqueous formic acid (solvent B), and the flow rate was
00 mLmin . Different solvent gradients were used for the follow-
[
À1
2009, 3, 85–90.
ing reaction mixtures:
[
[
11] S. Armenta, M. Blanco, Anal. Chim. Acta 2011, 685, 1–8.
12] C. Krueger, C. Hilton, M. Osgood, J. Wu, C. Wu, Appl. Note IMS-2010-
01A, 2010, 1–4.
1
2
3
. Consecutive alkylation: 0–3 min, 30% A; 3–4 min, 30–90% A;
–7 min, 90% A.
. Alkylation: 0–1 min, 30% A; 1–2 min, 30–90% A; 2–5 min,
0% A.
. Hydrogenation: 0–3 min, 30% A; 3–4 min, 30–90% A; 4–7 min,
0% A.
[
[
[
[
[
[
[
13] E. L. Harry, A. W. T. Bristow, I. D. Wilson, C. S. Creaser, Analyst 2011, 136,
4
1728–1732.
14] K. M. Roscioli, X. Zhang, S. X. Li, G. H. Goetz, G. Cheng, Z. Zhang, W. F.
Siems, H. H. Hill, Jr. , Int. J. Mass Spectrom. 2013, 336, 27–36.
15] C. Yan, J. W. Schmidberger, F. Parmeggiani, S. A. Hussain, N. J. Turner,
S. L. Flitsch, P. Barran, Analyst 2016, 141, 2351–2355.
16] M. Zühlke, D. Riebe, T. Beitz, H.-G. H.-G. Lçhmannsrçben, S. Andreotti, K.
Reinert, K. Zenichowski, M. Diener, J. Sep. Sci. 2016, 39, 4756–4764.
17] M. Zühlke, D. Riebe, T. Beitz, H.-G. Lçhmannsrçben, K. Zenichowski, M.
Diener, M. W. Linscheid, Eur. J. Mass Spectrom. 2015, 21, 391–402.
18] R. Pethig, M. Kuhn, R. Payne, E. Adler, T. H. Chen, L. F. Jaffe, Cell Calcium
9
9
Acknowledgements
1989, 10, 491–8.
We gratefully acknowledge the financial support for this research
received from the German Federal Ministry for Economic Affairs
and Energy (BMWi) through the AiF (Grant No. KF2167703NT2)
and Rainer Neumann for the quantum-chemical calculations. Ex-
perimental work was supported by Stefan Mies.
19] K. Jahn, C. Hille, PLoS One 2014, 9, e105334.
[20] S. Heilporn, F. Broeders, D. Daloze, J. C. Braekman, J. M. Boeynaems,
Bull. Soc. Chim. Belg. 1994, 103, 309–319.
21] R. Y. Tsien, Biochemistry 1980, 19, 2396–2404.
22] Y. M. A. Naguib, Molecules 2009, 14, 3600–3609.
23] G. Grynkiewicz, M. Poenie, R. Y. Tsien, J. Biol. Chem. 1985, 260, 3440–
[
[
[
3450.
[
[
24] H. Borsdorf, H. Schelhorn, J. Flachowsky, H.-R. Dçring, J. Stach, Anal.
Chim. Acta 2000, 403, 235–242.
25] M. F. Mesleh, J. M. Hunter, A. A. Shvartsburg, G. C. Schatz, M. F. Jarrold, J.
Phys. Chem. 1996, 100, 16082–16086.
26] A. A. Shvartsburg, M. F. Jarrold, Chem. Phys. Lett. 1996, 261, 86–91.
27] I. Campuzano, M. F. Bush, C. V. Robinson, C. Beaumont, K. Richardson, H.
Kim, H. I. Kim, Anal. Chem. 2012, 84, 1026–1033.
28] A. C. Lee, G. M. Crippen, J. Chem. Inf. Model. 2009, 49, 2013–2033.
29] L. W. Beegle, I. Kanik, L. Matz, H. H. Hill, Jr., Anal. Chem. 2001, 73, 3028–
Conflict of interest
The authors declare no conflict of interest.
[
[
Keywords: electrospray ionization · HPLC · ion mobility
spectrometry · reaction mechanisms · reaction monitoring
[
[
3
4.
[
1] N. Heigl, C. Petter, M. Rainer, M. Najam-ul-Haq, R. Vallant, R. Bakry, G.
Bonn, C. Huck, J. Near Infrared Spectrosc. 2007, 15, 269–282.
2] K. Grabow, U. Bentrup, ACS Catal. 2014, 4, 2153–2164.
3] K. A. Esmonde-White, M. Cuellar, C. Uerpmann, B. Lenain, I. R. Lewis,
Anal. Bioanal. Chem. 2017, 409, 637–649.
[
[
30] G. S. Jackson, C. G. Enke, Anal. Chem. 1999, 71, 3777–3784.
31] K. Cottingham, Anal. Chem. 2003, 75, 435A–439A.
[
[
[
4] F. Dalitz, M. Cudaj, M. Maiwald, G. Guthausen, Prog. Nucl. Magn. Reson.
Spectrosc. 2012, 60, 52–70.
Manuscript received: July 4, 2017
Revised manuscript received: August 18, 2017
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