ESI-High Resolution TOF MS Abbott Hall Rooms 110, 343

Transcription

1 1 Introduction ESI-High Resolution TOF MS Abbott Hall Rooms 110, 343 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). ESI TOF MS Assignment_131102_OVAT.docx 1

2 3 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. ESI TOF MS Assignment_131102_OVAT.docx 2

3 5 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 V and 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). ESI TOF MS Assignment_131102_OVAT.docx 3

4 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 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). parts: The data processing and reporting in a form of manuscript should consist of three 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. ESI TOF MS Assignment_131102_OVAT.docx 4

5 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( 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 1- week 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 ESI TOF MS Assignment_131102_OVAT.docx 5

6 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), Cech, N. B.; Enke, C. G., Practical implications of some recent studies in electrospray ionization fundamentals. Mass Spectrom. Rev. 2001, 20, (6), 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), Trufelli, H.; Palma, P.; Famiglini, G.; Cappiello, A., An overview of matrix effects in liquid chromatography-mass spectrometry. Mass Spectrom. Rev. 2011, 30, (3), ESI TOF MS Assignment_131102_OVAT.docx 6