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ESI-High Resolution TOF MS
Abbott Hall Rooms 110, 343
1
Introduction
Electrospray ionization (ESI) is a frequently used technique for the analysis of polar
species of various molecular weights.1, 2 ESI coupled with a high-resolution time of flight (TOF)
mass spectrometer allows for a relatively easy and accurate identification.3 However, there are
multiple factors, which may affect the accuracy and precision of the results obtained.
Among the major factors influencing the resulting mass spectrum are the analyte
concentration, chemical nature and concentration of the electrolytes (frequently referred to as
ionization agents) used, as well as the selection of solvents. Obviously, too low concentrations of
the analyte may lead to the loss of a signal whereas too high concentrations may cause various
spectral artifacts, e.g., dimerization of the analyte molecules in the ion source. The essential
requirement on solvent and electrolyte is their volatility (to prevent their precipitation in the
source) as well as nebulizing and ionization capability. The acquired mass spectrum may also be
affected by the introduction of contaminants, which may be observed as interfering ions or may
cause the suppression of ionization.4 For this reason, only LCMS grade solvents (including water)
and reagents should be used. Similarly, disposable glassware for sample dilutions is preferred.
The optimization of ionization and focusing voltages plays important role in quality of
the resulting mass spectrum. The optimized voltages include the electrospray voltage providing
ionization (i.e., capillary voltage), and voltage for further fragmentation [collision included
dissociation (CID)] and transfer of the analyte from source to MS (fragmentor voltage). In order
to perform the analysis with high mass accuracy, a mass calibration has to be performed
frequently (typically daily). Addition of an internal reference standard, which may correct for the
mass errors, is another way to improve mass accuracy. Although such reference may appear to
be preferred for all of the measurements, it is important to note that reference compounds may
also suppress the ionization of analytes or be suppressed themselves (e.g., have lower response,
or do not appear in the mass spectrum at all).
In addition to measurements, data processing plays a key role in the presentation of data.
The highest molecular weight ion observed in the mass spectrum does not have to be a
protonated ion (pseudomolecular ion in older terminology). Barring contamination, those ions
may be various adducts, including the dimmers of the analyte or adducts of molecular
ions/fragments and electrolytes/solvents.
2
Objectives
The aim of this laboratory assignment is to obtain practical experience in sample
preparation and ESI-HRMS analysis with respect to the main optimization parameters for
small molecular weight species. The lab will be focus on optimization using direct infusion
and one variable at time (OVAT) approach (training required for all ESI-MS users).
Within this lab assignment, a student will gain experience in 1) sample preparation for
ESI-MS analysis, 2) data acquisition and optimization on the ESI-TOF MS instrument, 3)
data processing of data obtained 4) data reporting in the form of a manuscript to a journal
with peer review process (i.e., J. Chromatography).
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Preparation for the lab
Preparation for the lab includes following items described below in detail:
a) Home to Take Lab Test
b) Schemes of chemical structures of tested analytes and calculation mass of their
ions.
c) Calculations for sample dilutions
d) Lab notebook
Prior to the lab, students are expected to complete the Home to Take Lab Test. The
results of this test have to be provided at the beginning of the lab and will be discussed and
reviewed with the instructor. Unsatisfactory preparation/knowledge will result in a
cancellation of a lab appointment and the lab will be deferred until new time slot is available.
The test has to be handwritten; the study materials include this lab manual and TOF user
guideline, which should be both read prior to the lab.
In addition, students must calculate a monoisotopic mass of the assigned analytes
(student may suggest for the study analyte of their interest upon request on the instructor) as
well as their protonated/deprotonated ions [A+H]+ and [A-H]- (tip: for the ion calculations do
not forget to add or subtract the mass of electron, the MS Excel calculator may be used), and
draw their structures in a chemical structure drawing software (e.g., Chem Draw or
Chemsketch).
Students should also precalculate preparation of the tested solutions from 100 mM
stock of electrolyte and 100 ppm stock of analyte. So they can easily prepare it during the lab.
Students will need to have a bounded lab notebook for records in the lab (it may be a
notebook used in previous analytical courses).
4
Lab assignment
The measurements will be performed in Abbott Hall room 110 under supervision of
an instructor or a TOF MS operator. Each student/pair of students will be responsible for the
data acquisition of at least two samples. The data processing must be accomplished on an
individual basis in room 343 using MassHunter software. Although students may discuss
(and this is encouraged) their data, the processing files cannot be duplicated even if students
worked on the same analytes. Submission of the same or nearly the same files will be
considered to be a plagiarism and students will be dismissed from the lab with zero score
and no further access to the MS lab.
Within the lab, students are expected to optimize ESI conditions and evaluate the
obtained mass spectra. The ESI optimizations will be performed for two analytes using one
variable at time (OVAT) screening analysis. For the OVAT, students will manually vary
electrolytes (in a lab slang terminology called ionization agents), solvents, and capillary and
fragmentor voltages for each analyte, and determine applicability of the positive and negative
ESI mode.
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Materials
Each student (student group) will receive 2 samples. The analyte samples (200 µL of
each) will be provided by the instructor in the concentration of 100 ppm (w/v) dissolved in 100%
methanol, acetonitrile, or water.
Solvent systems used for lab will be either 50% ACN or 50% MeOH in water. The
electrolytes available will be 100 mM acetic acid or ammonium acetate. The
solvents/electrolytes labeled in blue are for solutions making and solvents labeled in yellow are
for washing/cleaning steps (this labeling and correct use of solvents is essential to prevent
contamination).
For the OVAT experiment, students will prepare 1 mL of the following solutions:
a) 4 blanks (i.e., samples without the analyte) varying electrolytes (2.5 mM final
concentration) and two solvent systems.
b) 4 samples for each 1 ppm (w/v) 1 ppm varying electrolytes (2.5 mM final
concentration) and two solvent systems.
Students are expected to design in their lab notebooks labeling system, so they know
which samples is which.
Several files will be provided for students including the document wit ESI TOF MS
assignment, “Operation guideline with TOF.docx” Printouts of each of these two documents
should be brought to the lab. Additional files provided in the lab will include “Sharing data and
working with MassHunter.docx.”
5.1
Instrumentation and operation conditions
The mass spectrometric analyses will be performed using a high-resolution time of flight
G1969A MS equipped with ESI (Agilent, Santa Clara, CA, USA).
The lab involves a manual optimization, with the sample being introduced to the MS via
direct infusion with a 1 mL syringe at a flow rate of 5 µL/min (i.e., that is a mass spectrum is
acquired continuously). Nitrogen will be used as both the nebulizing (25 psi) and drying (12
L/min) gas at a temperature of 300 °C for all measurements. The measurements will be
performed in both positive and negative modes, with electrospray voltages being screened within
the range of 2500–5000 V and 2500–4500 V respectively. The fragmentor voltage will be
evaluated in the range between 125 and 250 V.
5.2
Measurements & data processing
Initial demonstration will be performed either by the instructor or the TOF operator. The
student will observe the direct infusion, data acquisition, and data processing. All these processes
are also described in the “Working guideline for TOF.” Further work on the OVAT will be
accomplished by student(s). Each student(s) must optimize ESI conditions for 2 samples. Even if
students work in the groups (pairs) each student within the particular group will individually
process the data in the computer lab (AH 343).
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5.2.1 Manual evaluation of ESI conditions using OVAT
For analysis students will be expected to follow the “Working guideline for TOF”
Manual. The instructor or the operator will provide advice only if the team of students will not be
able to resolve the problem on their own.
Student(s) will then evaluate ESI conditions for two analytes in following steps


Analysis of blanks
Analysis of samples
a) First task will be to select the mode (positive or negative) of ionization (note the
selection must be justified in the final paper)
b) Then, in selected polarity mode students will evaluate ionization in two different
electrolytes and solvent systems
Prior to the analysis of samples, students will acquire the mass spectra of the blanks (e.g.,
dilution solvent with ionization electrolyte) in both positive and negative modes (capillary
voltage 4000 V, fragmentor 150 V) to determine the contribution from the background species.
For each sample (solvent/electrolyte) system, students should experiment with capillary
and fragmentor voltages and acquire data at the voltages resulting in higher response of target
analytes, and if feasible the fragmentor voltage for which fragments are observed. Students
should record a) their analyses conditions b) the response of the target ions.
5.2.2 Data Processing & Interpretation
The processing will be performed using Mass Hunter software in AH room 343. The
guideline on the use of the software is provided in the additional document “Sharing data and
working with Masshunter.docx.” After the lab completion, students will transfer a copy of their
data onto the server and will be able to access their data from the computer lab (room 343).
The data processing and reporting in a form of manuscript should consist of three
parts:



Identification of major ions/fragments based on evaluation of mass spectra
Evaluation of ESI ionization conditions obtained with OVAT for two analytes
Discussion of the ESI data obtained for two analytes
1) The identification should be based on the evaluation of mass spectra and should
include figures consisting of mass spectra in a full and magnified (pertinent portion of
the mass spectrum), discussion of ions (adducts, fragments) found upon use of
different solvent system and electrolyte (if spectra differ) at different fragmentor
voltages. If the mass spectra obtained using different solvent systems are the same or
do not provide target ions, they do not need to be provided (however text should
explain this). The identification of observed ions/fragments should be reported, that is
determining whether the observed species are for example [M+H+] or [M+Na+] etc.
or specific fragments obtained by a loss of neutrals (e.g., [M-Cl+]). The tentative
identification provided (if possible) should be confirmed by calculating the mass
accuracy error (preferably in tabulated form) and isotopic ratios.
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2) The evaluation of ESI ionization results should be based on peak heights or areas
recorded in the laboratory notebook and reported as a table or a figure (chart) in the
manuscript. Students need to explain, which ions (i.e., protonated or adducts) were
used for optimization, and report the optimal conditions with regard to electrolyte use,
solvent system and voltages tested.
3) The obtained results (identification and optimal conditions) should be discussed with
respect to those found in the literature. Students are expected to do SCOPUS or
Scifinder literature search on the targeted analytes and ESI MS. If no such reports are
found, students should focus their discussion on comparison of behavior of two
targeted analytes.
5.3
Reporting the Results
The lab assignment for course should be reported as scientific investigation of optimal
ionization conditions following the J. Chromatography Authors'
guideline(http://www.elsevier.com/wps/find/journaldescription.cws_home/502688/authorinstruct
ions). In the case of the report for training purposes only data with explanation may be reported
the preferred format is the Power Point.
However, the following exceptions must be applied. The document has to be submitted
written in MS Word. The tables must be enclosed within the text including captions. Figures, and
figure legends may be provided in the text or MS Power Point file with the captions and properly
referenced in the text. No more than 6 figures should be included, however one figure may
consist of several charts. The text may not be longer than 3000 words excluding the references
and abstract. The references must be base on ACS style. The example of file labeling is
"AK_102511_MSlab_a" indicating your initials (AK), date when the lab was performed
(102511), and"MSlab_a or b" indicating 1st and 2nd submission, respectively. Similar labeling
needs to be provided for the MS Power Point file.
Timely delivery of the report by required deadline is required (5 pt); every day of delay
will result in the deduction of 1 point. After receiving their grade, student will have exactly 1week for resubmission of corrected report.
Grading will be based on the following items
a) Lab preparation, performance, notebook (5 pt).
b) Timely delivery (5 pt).
c) Proofreading and language expressions (5 pt).
d) Following the J. Chromatography guideline with respect to the nomenclature and
organization of the manuscript (5 pt).
e) Content of individual sections required in the guideline for J. Chromatography and
exceptions specified above (30 pt).
Note, the example of paper published by our lab is provided on blackboard.
Recording the acquisition conditions is critical to succeed in the lab, and
correctly present experimental section. The main sections should include:
Abstract (5 pt)
Introduction (3 pt) = paragraph explaining importance of optimization of ESI
ionization and also providing some background about the target analytes
Experimental (3 pt) = see any J. Chrom. Paper as example
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Results and Discussion (12 pt) requirements provided in section 5.3, this includes
also MS Excel file.
References in ACS format (2 pt) = should be used in the introduction and should
related to chemicals or method investigated
if needed, i.e., compare your results to other published work
Tables and/or Figures (5 pt) = should be provided at the end of report file. They
should follow guideline of J. Chromatography with clear labeling.
6 References
1.
Oss, M.; Kruve, A.; Herodes, K.; Leito, I., Electrospray ionization efficiency scale of
organic compound. Anal. Chem. 2010, 82, (7), 2865-2872.
2.
Cech, N. B.; Enke, C. G., Practical implications of some recent studies in electrospray
ionization fundamentals. Mass Spectrom. Rev. 2001, 20, (6), 362-387.
3.
Jiejun Wu, H. M., Exact mass measurement on an electrospray ionization time-of-flight
mass spectrometer: error distribution and selective averaging. J. Mass Spectrom. 2003, 38, (10),
1043-1053.
4.
Trufelli, H.; Palma, P.; Famiglini, G.; Cappiello, A., An overview of matrix effects in
liquid chromatography-mass spectrometry. Mass Spectrom. Rev. 2011, 30, (3), 491-509.
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