CH3F2 Mass Spectrometry Revision Lecture Slides 2023-2024 PDF

CH3F2 Advanced Analytical Chemistry Mass Spectrometry (principles): Revision lecture Dr. Mark P. Barrow Moodle https://moodle.warwick.ac.uk/course/view.php?id=57391 Books “Mass Spectrometry” James McCullagh and Neil Oldham Oxford Chemistry Primers ISBN: 9780198789048 “Mass Spectrometry: Principles a...

CH3F2 Advanced Analytical Chemistry Mass Spectrometry (principles): Revision lecture Dr. Mark P. Barrow Moodle https://moodle.warwick.ac.uk/course/view.php?id=57391 Books “Mass Spectrometry” James McCullagh and Neil Oldham Oxford Chemistry Primers ISBN: 9780198789048 “Mass Spectrometry: Principles and Applications” Edmond de Hoffmann and Vincent Stroobant Wiley ISBN: 978-0-470-03310-4 “Mass Spectrometry: A textbook” Jürgen Gross Springer ISBN: 978-3-319-54397-0 https://link.springer.com/book/10.1007/978-3-319-54398-7 Quizzes A note about past papers… https://warwick.ac.uk/services/exampapers Notice “2021” (year 2020-2021) is missing! For this year, search for CH3G5 instead of CH3F2 A note about past papers… Interpretation of EI (electron ionization) mass spectra was part of older course Not part of CH3F2 from 2019-2020 onwards (i.e. academic years 2019-2020, 2020-2021, 2021-2022, and 2022-2023) For mass spectrometry parts, exam papers for “2020,” “2021,” and “2022” are the most relevant https://webbook.nist.gov/cgi/cbook.cgi?ID=C112403&Mask=200 Exam CH3F2 exam: 13th March, 2024 Exam READ THE INSTRUCTIONS: front page and headings on later pages Style has not changed much, only number of questions 4 questions: all 3 from Section A, then a choice of 1 out of 3 from Section B Show your working: you can receive marks for it Remember your units Don’t only provide numeric values Work with SI units unless advized otherwise When calculating masses, use exact values (e.g. monoisotopic masses from a table); remember the “Basics” lecture Appendix in exam will include equations, exact masses, and amino acids Do I need to remember all the equations? What have we covered? Basics Ionization Analyzers Tandem mass spectrometry Data analysis Calculating masses Table of elements (including isotopes) from “Basics” lecture Remember to use exact masses in calculations; do not round to integer values! Concept of “mass defect” (i.e. not integer values; useful when measuring m/z accurately and assigning formulae) Calculating masses Need to know formula for neutral as a starting point Role of ionization method… ESI [M ± nH]n+/ [M+H]+ if singly-charged and in positive-ion mode (basic species) [M-H]- if singly-charged and in negative-ion mode (acidic species) APPI [M+H]+ for positive-ion mode (basic species) M+ for positive-ion mode too Example: naphthalene by APPI Neutral: C10H8 [M+H]+: C10H8 + H – (mass of electron) = C10H9 - mass of electron M+ : C10H8 – (mass of electron) Monoisotopic masses: C: 12.000000 H: 1.007825 N: 14.003074 O: 15.994915 Mass of electron: 0.000548 Charge on electron: 1.60 x 10-19 C 1 Da = 1.66 x 10-27 kg Calculating masses What’s wrong with this calculation? (Two big issues) Mass of of neutral for: C10H8 (10 x 12.011) + (8 x 1.008) = 128.74 Therefore, 128.74 amu Average masses used. Should be monoisotopic masses. “amu” is the wrong unit and uses the pre-1961 16O scale. Should be “Da” or “u.” Also note significant figures used. Started with 3 and ended up 2. Should have been ~5 (for monoisotopic masses) anyway. Mass defect and isotopes: carbon 12.000000 Da 98.93% 13.003355 Da 1.07% https://www.wavemetrics.com/products/igorpro/gallery/user_whittal Isotopologues (100%) C5 C5 12 (100%) C60 13C has a natural abundance of approximately 1.1% ~66% (60 x 1.1) 12C 13C 4 1 12 C3 13C2 ~5.5% (5 x 1.1) (100%) ~99% (90 x 1.1) C90 12C 13 119 C1 12C 120 (100%) ~132% (120 x 1.1) 12 C118 13C2 C120 Resolving power “Full width at half maximum” (FWHM) typically used Higher values (narrower peaks) are better Examples: ≈10,000 (FWHM) for TOF ≈500,000+ (FWHM) for FTICR When calculating what is needed to resolve two peaks, better to additionally quote ~1.5x minimum value Example from past paper: resolving power a) What was the charge state of the residue shown in Figure 2? Justify your answer. b) Using the FWHM definition of resolving power, calculate the resolving power for the monoisotopic peak shown in Figure 2. c) What types of mass analyser could have been used to acquire the data? Justify your answer. a) The m/z spacing between the isotopologues is approximately 0.33 and therefore the charge state is 3+. The residue shown is the [M+3H]3+ form. b) Using the FWHM definition of resolving power, the required equation is m/∆m (given in Appendix 1) and the resolving power is calculated to be approximately 50,000. c) This could be achieved by many time-of-flight mass spectrometers, but an Orbitrap or FT-ICR can easily achieve this or higher and so would also be acceptable answers. Ion traps and quadrupoles would NOT afford sufficiently high resolving power. Mass accuracy Figure of merit associated with measurement; measured in parts per million (ppm) Better mass accuracy allows user to have higher confidence in assignment Lower values are better Examples: TOF typically 3-5 ppm FTICR typically ≤ 1 ppm Calculating mass errors Example: naphthalene by APPI M+ : C10H8 – (mass of electron) Theoretical (“calculated”) m/z therefore: m/z 128.062052 Monoisotopic masses: C: 12.000000 H: 1.007825 N: 14.003074 O: 15.994915 Consider a mass spectrum where the peak is found (“observed”) at m/z 128.061668 Mass of electron: 0.000548 What is the mass error? Charge on electron: 1.60 x 10-19 C (10 × 12.000000) + (8 × 1.007825) – 0.000548 = 128.062052 Da (so m/z 128.062052 if singly-charged) ((mobserved-mtheoretical)/mtheoretical)) × 1,000,000 ((128.061668 - 128.062052) / (128.062052)) × 1,000,000 = -3 ppm Why is it negative? What if our formula assignment was wrong? 1 Da = 1.66 x 10-27 kg What have we covered? Ionization Electron ionization (EI) Matrix-assisted laser desorption/ionization (MALDI) MALDI imaging Electrospray ionization (ESI) Multiple charges (e.g. protein samples) Protonation (positive-ion mode) or deprotonation (negative-ion mode) Spacing between isotopologues determined by charge state Atmospheric pressure photoionization (APPI) Produces radical ions and protonated/deprotonated species Photoionization of analyte or dopant/solvent Suitable for many non-polar compounds Ionization methods “Mass Spectrometry: A textbook,” Jürgen Gross, Springer, ISBN: 978-3-319-54397-0 ESI: Ubiquitin (entire protein) Intens. x105 714.7 +MS, 0.1-1.3min #(7-79) 12+ [M + nH+]n+ 1.0 0.8 11+ 779.5 0.6 13+ 659.8 0.4 10+ 857.4 0.2 9+ 8+ 952.6 7+ 1071.5 0.0 500 600 700 800 900 1000 1100 1200 1300 1400 m/z Multiple charges (ESI) Measure m/z, not mass Spacing between isotopologues tells us about charge state Therefore useful to have resolving power to resolve isotopologues Adapted from "Mass Spectrometry," James McCullagh and Neil Oldham, Oxford Chemistry Primers, ISBN: 9780198789048 Determining charge state Consider two peaks at adjacent charge states, m/z values of “a” and “b” Mass of neutral molecule, “M” “n” charges Intens. x104 1147.5278 +MS, 0.0-0.3min #(3-16) a 6 956.4497 b 4 a = (M + nH+) / n b = (M + (n+1)H+) / (n+1) 2 n = (b – 1.007) / (a – b) 819.8634 1433.7548 0 600 800 1000 1200 1400 1600 1800 m/z Example from past paper: MALDI-TOF Explain why the combination of matrix-assisted laser desorption/ionization (MALDI) and a time-of-flight (TOF) mass analyser offer experimental advantages that make it well-suited to polymer analysis MALDI is suitable for ionization of polymers Also avoids formation of multiply-charged ions, which would lead to overlaps of polymer distributions and complicate data analysis TOF affords sufficient resolving power for the experiments TOF covers a wide m/z range, suitable for polymers TOF is also convenient due to speed of data acquisition Example from past paper: MALDI-TOF Briefly describe the design of a MALDI source and how ionization of the analyte occurs, including the role of the matrix. MALDI is a soft ionization technique, resulting in minimal fragmentation Organic matrix is chosen, according to the analyte used and the laser used Solution of the analyte and matrix is prepared (typically 1000s-to-1 ratio of matrix:analyte) Solution is deposited on to a stainless steel target and the solvent is allowed to evaporate UV laser is typically used with a nanosecond pulse length, can fire multiple times per second Plume of gas-phase analyte and matrix neutrals and ions Analyte is protonated (positive-ion mode) or deprotonated (negative-ion mode) through interaction with the matrix, resulting in the ionization of the analyte molecules Acceleration out of the ion source for m/z analysis ESI and APPI ESI APPI ESI and APPI ESI APPI Examples of APPI mechanisms S + h! ⟶ S+ + eS+ + M ⟶ S + M+ Charge transfer S+ + M ⟶ [S-H] + [M+H]+ where S is the solvent and M is the analyte Proton transfer APPI can produce both radical ions and protonated species Common observations and neutral losses Aliphatic compounds Cyclic structures Straight cleavages Rearrangements OH, C=O groups SO, CN groups Rearrangements Rearrangements Amines Loss of NH3 Hydroxyl groups Loss of H2O Acid groups Loss of CO2 and HCOOH Aromatic systems Difficult to fragment Peptides and proteins Roepstorff cleavage nomenclature UVPD Roepstorff, P.; Fohlman, J., Proposal for a Common Nomenclature for Sequence Ions in Mass Spectra of Peptides. Biomed. Mass Spectrom. 1984, 11, 601. ExD CID/IRMPD Liquid-liquid extraction Chrom Tech Solid-phase extraction Biotage web site https://www.hazardexonthenet.net Woods Hole Oceanographic Institution Good luck! Dr. Mark P. Barrow (principles) Prof. Peter O’Connor (applications) [email protected] [email protected]