International Journal of Mass Spectrometry 444 (2019) 116183

Contents lists available at ScienceDirect

International Journal of Mass Spectrometry

journal homepage: www.elsevier.com/locate/ijms

Teaching venturi electrospray mass spectrometry with amino acid

analysis
Marcos A.S. Ribeiro a, b, Ange
lica P.P. Tonin a, b, Camila B. Poliseli b, Beatriz B. Lima a,
Luiz E.N. Castro a, Valquíria M. Silva a, Marcelo Vale
rio a, Jaime C. Cedran c,
a, b, *
Eduardo C. Meurer
a
, 86900-000, Jandaia do Sul, PR, Brazil
Universidade Federal do Parana
b
Universidade Estadual de Maringa
, 87020-900, Maringa

, PR, Brazil
c
Universidade Tecnolo
, Campus Medianeira, 85884-000, Medianeira, PR, Brazil

gica Federal do Parana

a r t i c l e i n f o a b s t r a c t

Article history: There is an urgent need to update the ways in which undergraduates learn science technology. In this
Received 26 March 2019 laboratory experiment, students improved their knowledge of the chemistry used in the analysis of
Received in revised form amino acids by mass spectrometry with electrospray ionization using venturi pump (VESI-MS) sample
24 June 2019
introduction. Amino acid analysis using mass spectrometry is connected to a variety of chemical issues,
Accepted 10 July 2019
Available online 13 July 2019
from macroscopic concepts of solution preparation and the use of average molecular mass (g mol
1) to
microscopic concepts of chemistry, such as exact molecular mass (Da), organic functional groups, single
mass spectrometry (MS) to identify ions from molecules in solution, and tandem mass spectrometry
Keywords:
Mass spectrometry
(MS2) to access structural features of the ions formed. The experiment dealt with solution preparation,
Qualitative analysis physical chemistry concepts involved in VESI, and organic chemistry concepts in ion analysis by MS and
Practical chemistry class MS2 experiments in a triple quadrupole mass spectrometer.
Undergraduate education © 2019 Published by Elsevier B.V.
Venturi electrospray ionization

1. Introduction
Science education is crucial for social and economic develop-
ment, but teaching in STEM (Science, Technology, Engineering, and
Math) areas is sometimes evaluated as unsuccessful [1]. There is an
urgent need to update the ways in which undergraduates learn
science technology. Usually, experimental chemistry classes in
Brazil do not use modern equipment to access concepts in chem-
istry due to the non-availability of the equipment and lack of
preparation of those responsible. However, it is a powerful tool to
consolidate chemistry concepts [2]. Attempts to integrate the
knowledge of chemistry with the use of specific equipment, such as
infrared and nuclear magnetic resonance spectroscopies, have
already been reported in the literature [2e4]. This approach
showed that the interaction of chemistry knowledge and specific
equipment can develop promising results for basic knowledge,
student-centred learning, developing evidence, and demonstrating
* Corresponding author. Universidade Federal do Parana
, 86900-000, Jandaia do
Sul, PR, Brazil.
E-mail address: eduardo.meurer@gmail.com (E.C. Meurer).
how chemists think and use certain equipment to justify their hy-
potheses [2]. This is a crucial point in our educational proposal: not
to limit experimental practice to previous protocols and results, but
to place the student in the position of the protagonist in activities in
which his or her concepts, reflections, and hypotheses can be
presented and discussed [5].
Mass spectrometry (MS) exhibits great potential as a knowledge
development tool due to its interdisciplinary and rich chemistry
concepts. It is used to analyse semi-polar and polar molecules
[6e10] with electrospray ionization (ESI), which has been
employed in almost all areas of science [11]. Thus, its use of MS in
undergraduate classes are of great impact on scientific education
[12]. Understanding of the technique by chemistry students could
consolidate concepts previously seen only in the classroom
[13e17]. There are successful examples of ESI experiments in the
curricular structure of undergraduate institutions for molecular
identification of peptides, compounds with multiple charges, for-
mation of complexes in the gas phase, and identification of fatty
acids, phosphorylated lipids, and their oxidized products
[13e15,17,18].
Thus, use of the Venturi Electrospray Ionization (VESI) tech-
nique was fast and efficient at introducing the sample into ESI-MS.

https://doi.org/10.1016/j.ijms.2019.116183
1387-3806/© 2019 Published by Elsevier B.V.
2 M.A.S. Ribeiro et al. / International Journal of Mass Spectrometry 444 (2019) 116183
VESI employs the Venturi Effect, which occurs when a high velocity
gas (shelth gas or nebulization gas) flows coaxially in a tube with
the electrospray capillary tube inside, constricting the gas to help
pneumatically the formation of the sprayed droplets. The result is
the reduction in tube pressure causing a self-pumping effect
[19,20], which does not require the use of high performance liquid
chromatography or auxiliary pumps and can easily be used on any
commercial ESI just by changing the diameter of the capillary [20].
In a nutshell, the objective of our laboratory experiment was to
explore chemical concepts related to solution preparation, molec-
ular mass calculation, ionization capacity of organic molecules, and
chemical bond stability using the ESI-related technique named
Venturi Electrospray Ionization with mass spectrometry (VESI-MS)
[20] and tandem mass spectrometry (MS [2] or MS/MS) experi-
ments as educational tools for students of chemistry classes.
2. Experiment development
The first contact of the students with the lab dealt with calcu-
lations of chemical concentration, dilutions, and handling of basic
laboratory instruments, such as pipets, volumetric flasks, and vials.
The second step consisted of preparation of a solution with four
amino acids at equimolar levels. Due to the ability of the equipment
to identify a wide range of organic compounds that may appear on
the mass spectrum, washing of glassware must be performed
before the experiment by the students to learn the cleaning pro-
cess, avoiding ionic contamination that make analyses and inter-
pretation more complex. Usually, glassware is washed with ultra-
pure water, methanol, acetonitrile, trifluoroacetic acid, or ammo-
nium hydroxide, depending on the residues in the glass surface,
which may include detergents.
Amino acids were selected because their acidic (carboxylic acid),
basic (amine), and polar, apolar, acidic, and basic moiety side
groups possess some of the richest chemistry among all small
molecules (amino acids used: arginine, glutamic acid, leucine, and
cysteine). Amino acids in neutral solution (pH~7) possess zwitter-
ionic forms (Fig. 1); the carboxylic acid turns into carboxylate and
the basic groups became protonated. Amino acids are species that
might be deprotonated at basic pH values (anion) and protonated at
acidic pH values (cation), and the side groups add additional
acidebase equilibrium to the two pre-existing from carboxylic and
amine groups (eg., arginine pKR value of 12.48 of the guanidine
group, glutamate pKR value of 4.07 of the geCOOH group, and
cysteine pKR value of 8.37 of the sulfhydryl group) [21,22].
The experiment was carried out in an acidic medium with a pH
of 2. The working solution (100 mL) was placed in an Eppendorf tube
Fig. 1. Structures of the amino acids used. Blue, arginine; green, glutamic acid; pink, leucine; and red, cysteine in pH near 7.0. (For interpretation of the references to colour in this
figure legend, the reader is referred to the Web version of this article.)
with 900 mL of methanol with 0.1% formic acid to adjust to an acidic
pH value and obtaining the final concentration of 5.0 mmol L-1. This
condition led to protonation of the basic groups of the zwitterion,
with groups possessing pKa values around to 2 first forming acidic
molecules [MþH]þ with net charges of þ1 in solution, which were
sucked towards the VESI source [23].
Usually in solution chemistry the basicity rule lead us to define
that an organic molecule with the highest pKa has better proton-
ation efficiency, otherwise the experimental results of the ioniza-
tion efficiency on the electrospray source show us that this rule is
not the only factor; there are a variety of factors, such as solubility
and matrix influence [24].
The VESI-MS technique works by selecting positively or nega-
tively charged ions (cations, VESI (þ); anions, VESI (
)). Due to
better ionization and ionic abundance, the positive ion mode was
used. The VESI source receives the solution containing neutral
analytes, protonated analytes and solvent by Venturi Effect without
a delivery system (syringe pump or High Performance Liquid
Chromatography (HPLC) system). The signal strength (intensity) in
the spectrum of each species depends on the acidebase properties,
solubility, and the organic compounds in the matrix, that was
introduced as the sample in the VESI source. In the positive mode
with the application of a positive spray voltage, the VESI capillary,
which has a high oxidant potential, neutralizes (oxidize) a small
fraction of the anions (that come from the acid used to protonate
the amino acid), unbalancing the equivalence of cations and anions
from the solution. The resulting solution tends to have more cations
than anions. In negative mode with the application of a negative
spray voltage, a solution with more anions than cations are pro-
duced. The organic modifier used was formic acid, and the
acidebase reaction produced protonated amino acids and formiate
anions in solution. Using VESI (þ), a high positive voltage was
applied in the solution to oxidize the carboxylate anion to CO and
water, which were eliminated during spraying of the solution.
After this redox reaction inside the VESI source, a spray of the
oxidized solution (positive mode) was produced and mixed with
nitrogen gas (inert gas) at high pressure. The concept of reduction,
the gain of electrons by chemical species, and oxidation the loss of
electrons by chemical species have to be dealt with during the
experimental class. The droplets formed can contain more cations
(positive mode) or more anions (negative mode). As the solvent
started to dry, the droplets shrank and exploded due to the high
charge density in what is called a coulombic explosion. Then, cat-
ions (positive mode) or anions (negative mode) were obtained in
the gas phase; they were attracted by the difference in potential
and delivered to the mass analyser of the mass spectrometer
 M.A.S. Ribeiro et al. / International Journal of Mass Spectrometry 444 (2019) 116183
[25e27].
During ion transference to the gas phase, the analytes with high
affinity for the surface of ESI droplets (surface-active analytes) have
a higher VESI response. The difference in affinity of the analytes on
the surface (with residual charge) and the droplet interior with no
residual charge is important. Usually, ions with nonpolar moieties
tend to be on the surface and are better transferred to the gas phase
than highly polar ions. That is, highly polar ions tend to be attached
to the droplets and their offspring since the solvent is polar,
impacting the ion intensity on the mass spectrum [27].
The chemistry of VESI-MS, such as ESI-MS, involves solubility,
acidebase reactions in solution, redox reactions in a VESI source,
and coulombic explosion/ion evaporation, which transfers ions
from solution to the gas phase. All of these chemical processes were
adjusted to obtain more species of interest in the mass spectrom-
eter. These parameters are mostly tuned by varying solvent polar-
ity, pH, spray voltage, temperature, and desolvation gas. After the
VESI process, the cations or anions were delivered to the quadru-
pole mass analyser in sequence, called tandem. This part of the
mass spectrometer deals with the physics of the ions in the gas
phase, which is used to measure the m/z ratio of stable ions in an
electric field. The m is the sum of the exact atomic weights of the
elements (not the mean) that the ion contains, molecular weight is
related to the neutral version of the ion observed. The students
should be awarded that mass spectrometers do not analyse mean
weight, but detect natural isotopes distribution, usually the higher
intense ions come from the most stable isotopes and they have to
be considered to calculate m. Roughly, the quadrupoles (Q) use the
Fig. 2. Venturi Electrospray Ionization (VESI) experiment for the amino acids with an equimolar 5.0 mmol Le1 solution resulting in the mass spectrum.
3
ion stabilities of the trajectories explained by Mathieu differential
equations, dependent on continuum voltage and alternate voltage
applied to the quadrupoles, which results in a tri-dimensional
electromagnetic field [28]. Analysis of the ions results in a report
of intensity vs. m/z, which is called a mass spectrum. The mass
spectra of cysteine, leucine, glutamic acid, and arginine are shown
in Fig. 2.
Fig. 2 represents the mass spectrum obtained for the experi-
ment in which the amino acids arginine, leucine, cysteine, and
glutamic acid were at the same concentration. The reproducibility
of the mass spectrum is usually an issue since the use of different
equipment, solvents from different brands, different analysts, and
different pH values may alter the intensities of the ions in different
ways. All of the variables must be the same from the beginning to
the last mass spectrum acquisition to obtain reliable sample results
between injections. It was observed that all presented different ion
intensities, a behaviour which might be related to a complex in-
fluence of a variety of factors, such as pKa and the active-surface
analytes in the droplets [27,28]. Fig. 2 clearly highlight that peak
intensity depends on ionization efficiency, not concentration.
Amino acids in neutral pH are in their zwitterionic form. When
an acid is added, protonation takes place in order to first protonate
the moieties with higher pKa values and then the moieties with
lower pKa values. Often, more concentrated species in solution do
not form the most intense ions in the mass spectrum, a behaviour
that was exhibited. Because the intensity of signal in spectrum
dependent a higher capacity to be protonated and transferred to
the gas phase.
4 M.A.S. Ribeiro et al. / International Journal of Mass Spectrometry 444 (2019) 116183
To access connectivity of the ions observed, MS [2] experiments
in triple quadrupole mass spectrometers were conducted by
selecting the ion of interest (m/z 122 cysteine, m/z 132 leucine, m/z
148 glutamic acid, m/z 175 arginine), named the precursor ion and
adjusting the difference of potential (from 10e50 eV) with the
collision chamber filled with inert gas (argon) (1x10e3 Torr). Colli-
sions of the accelerated selected ions with the neutral gas resulted
in fragmentation. The data was collected from m/z 50 to m/z 190,
and the fragmentation occurred on the most polar and weaker
covalent bonds for heterolytic cleavage. It is good to note that el-
ements that attract the electrons more efficiently (electronegative
elements such as, fluor, oxygen, nitrogen) promote cleavages dur-
ing fragmentation experiments. The collision chamber focused the
precursor ion and the fragments formed by collision-induced
dissociation (CID) to the last quadrupole mass analyser resulting
on the CID mass spectra. Usually, the acquisition is carried out in a
voltage ramp in order to obtain the precursor ion and as many
fragments as possible. Fragmentation features are dependent on
the organic groups that might give very interesting clues about the
structure (supplementary information). The bond energies keep
the atoms together; they are related to the bond strength or the
bond enthalpy. They might be experimentally obtained through
cleavage evaluating the energy that is absorbed to break the bond.
CID experiment control the energy transferred (laboratory energy,
Lab energy) to the ionic species controlling the extent of frag-
mentation. The Lab energy used might reveal the weaker bonds of
the ions. Usually the bond energies might be calculated with ab
initio or hybrid method using quantum physics. Today, the major
use of CID experiment results is databank comparison for molecule
identification for metabolomics and proteomics analysis.
3. Experimental overview
3.1. Participants
This study included students enrolled in chemistry classes at the
Federal University of Parana in food engineering, agricultural en-
gineering, and exact sciences courses in 2016 and 2017. The number
of participants in this study was 100 individuals, divided into
groups of 10 participants in the laboratory. The number of in-
dividuals per group was associated with class time and the number
of students per room. The participants were subjected to a test at
the end of the course, which required a report with pre-defined
questions that discussed concepts such as measurement of
atomic weights and molecular weights, chemical and structural
analysis, redox reactions of the VESI source, acidebase concepts,
electronegativity, and bond energies. This experiment has been
incorporated into mass spectrometry and organic chemistry classes
and is part of the educational curriculum of the institution. The
performance evaluation and report were used to evaluate the stu-
dents. Students developed the experimental procedure in the lab-
oratory with the preparation of the amino acid solution, and the
professor performed introduction of the sample and optimization
of the equipment.
3.2. VESI source and solution preparation
The solutions were prepared by the class attendants and the
concepts of solution, concentration, equimolar solutions, handling
of micropipettes, and dilution were explored. The amino acid
analysis was performed in positive ion-mode VESI-MS on a Waters
PREMIER/XE® Quattro MicroTM API (triple quadrupole MS). The
parameters were set as follows: spray voltage, 2.00e3.5 kV; cone
voltage, 10e20 V; source temperature, 110 C; desolvation temper-
ature, 250e350 C; desolvation gas flow rate, 400 L h
1; cone gas
flow rate, 0.0 L h
1; and scanning range, from m/z 50e500. These
parameters were optimized in preliminary experiments to get the
highest abundance of the targeted protonated ions. N2 was used as
both a dry gas and nebulizer gas. The direct VESI-MS sample
sucking process took place to obtain the mass spectrum. All sam-
ples were analysed in the positive mode using formic acid as an
organic modifier. All amino acids were purchased from Sigma and
were stored at
20 C. All solutions were prepared in equimolar
concentrations for arginine, leucine, cysteine, and glutamic acid
(1 mmol L
1 diluted to 100 mmol L
1 and diluted again to
500 nmol L
1) (Fig. S4).
3.3. Full scan MS and collision-induced dissociation MS/MS
experiments
The experimental procedure was developed for one laboratory
period (5 h). The spectra were obtained using a mass spectrometer
from Waters Company, but the experiments could be conducted on
any mass spectrometer with VESI. The experiment may also be
conducted with an ESI source with a conventional syringe pump or
HPLC sample introduction system. Also, the equipment and its
functionality in several areas were explained superficially. Learning
science requires that the student not only listen, but speak, write,
and perform science in the process [29]. Therefore, the participants
were challenged to predict the masses of the amino acids presented
in the spectrum, and the students compared their hypothesis with
the result acquired with a single mass spectrum [30e32]. Thus,
each participant saw the differences between the suggested values
and the ones obtained using VESI, obtaining a mass spectrum for
1e3 min. At this point, the limitations related to a quadrupole
analyser were discussed, such as unitary resolution, which makes
analysis of molecules with a difference in mass lower than 1 Da
difficult. Following a discussion of the problematic areas, the pro-
fessor suggested using the experimental output in the technique of
MS to define the chemical identity of a molecule target using the
concept of fragmentation. To exemplify, the professor showed some
the fragmentation of the amino acids using MS [2]. The students
were instructed to apply previous chemical knowledge to justify
the data shown in the exemplification.
Fig. 3 presents a fragmentation experiment using CID (MS/MS
experiment in the Supplementary Information) to access the
structural features of each ion, where the major fragmentation
pathways were directly related to particular chemical characteris-
tics of the amino acids and ion stabilities. This was expected since
the chemical properties were different. The students might
compare the structure of the ions and propose the neutral loss on
the fragmentation. Even though the MS/MS spectra appear to
possess different chemical features, they can be grouped by frag-
mentation mechanisms. The mechanism proposed in Fig. 4 sug-
gests NH3 loss followed by CO loss, H2O loss followed by CO loss,
and formic acid loss, accessing the structure of the amino acids [33].
Then until this time the experiment for each group was con-
ducted in 35 min, for solution preparation and data acquisition in
the mass spectrometer. During this time, concepts about atomic
and molecular weight, chemical structure, proton affinities, elec-
tronegativity, bond energies, molar concentration, and redox re-
actions were discussed, increasing the class time by another
25 min. In the third and last step, the students collected the data
from the mass spectrometer and a report with pre-defined ques-
tions linking the chemical concepts used in MS analysis was sug-
gested (available at the supplementary information). For each
group of 10 students, a time of 60 min per class is required, with a
total of 5 h for five classes. The commercial mass spectrometers
have built-in safety inter-locks, so there is low risk associated with
the operation of the instrument.
 M.A.S. Ribeiro et al. / International Journal of Mass Spectrometry 444 (2019) 116183
Fig. 3. Double-stage (MS [2]) sequential collision-induced dissociation product ion mass spectra of (a) the cysteine ion with m/z 122, (b) the leucine ion with m/z 132, (c) the
glutamic acid ion with m/z 148, and (d) the arginine ion with m/z 175.
Fig. 4. Principal neutral loss observed in the CID MS [2] fragmentation experiment of
the amino acids.
5
4. Hazards
Since electrospray sources have a source block and heated
drying gas, care should be taken when cleaning the source region
since source components may be hot. Skin contact with the pep-
tides and solvents, especially acetonitrile, should be avoided by
wearing gloves [32], and the level of exposure has to be carefully
calculated by the professor or technical assistant. Methanol may
cause irritation to skin, eyes, and the respiratory tract, and it is a
flammable liquid.
5. Educational value
Learning about the analysis of amino acid samples by MS,
including VESI-MS, in experimental chemistry classes enables rapid
analysis without complex sample preparation, making the study of
chemical concepts straightforward. Students learn how to use these
techniques for qualitative identification of organic molecules. The
amount of data analysis and interpretation may be tailored to the
capabilities and backgrounds of the students. Students were led to
relate fundamental concepts of chemistry, such as bond strength,
ion stability, and protonation sites, with the new concepts dealt
with in the experiment, connecting their previous knowledge,
already anchored in its conceptual system [34], with the new in-
formation. Thus, it was expected that this approach would be
meaningful for students and that the technique used would not
simply be the interpretation of the data presented in the spectra,
but that they would understand the chemical concepts inherent in
6 M.A.S. Ribeiro et al. / International Journal of Mass Spectrometry 444 (2019) 116183
the analysis. Students were also introduced to fundamental
chemistry concepts. The experiment can be adapted to a large va-
riety of instruments possessing ESI sources operating in VESI mode.
The analysis may be conducted with simple quadrupoles or in-
struments capable of MSn or high resolution. By taking on MS as a
didactic resource, we also contributed to overcoming the mis-
conceptions about science that students may have. We not only
taught the scientific knowledge, but showed the development of
science and how it is made [35].
Acknowledgements
The authors are grateful to the Waters Company, Forquimica
Company, Jamel Company, UEM, CAPES, and UFPR for financial
support.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ijms.2019.116183.
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