Introduction to Walk-Up Mass Spectrometry Jonathan A. Karty, Ph.D. September 27 & 29, 2010

Topics Covered Molecular Weight and Isotope Distributions Accuracy and Resolution EI, ESI, and APCI ionization EI Fragmentation A Handful of MS Applications

Why Mass Spectrometry Information is composition-specific Very selective analytical technique Most other spectroscopies can describe functionalities, but not chemical formulae MS is VERY sensitive MSF personnel dilute NMR samples 1:500 Picomole sensitivity is common in the MSF Mass spectrometers have become MUCH easier to use in the last 15 years

Three Questions Did I make my compound? Did I make anything else? Molecular weight is an intrinsic property of a substance Did I make anything else? Mass spectrometry is readily coupled to chromatographic techniques How much of it did I make? Response in the mass spectrometer is proportional to analyte concentration (R = α[M]) Each compound has a unique response factor, α

Common MS Applications Reaction monitoring Crude reaction mixture MS Stable isotope labeling Stability studies Quick product identification (TLC spot) Confirmation of elemental composition Much more precise then EA Selective detector for GC/HPLC MS provides molecular weight information about each chromatographic peak

Important Concepts to Remember Mass spectrometers analyze gas-phase ions, not neutral molecules Neutrals don’t respond to electric and magnetic fields If a molecule cannot ionize, MS cannot help MS is not a “magic bullet” technique MS can describe atomic composition of an ion Connectivity of the atoms is much more challenging Although MS requires a vacuum, it cannot be performed in a vacuum of information Deriving useful information from MS data often requires some knowledge of the system under investigation

What is Resolution? Resolution is the ability to separate ions of nearly equal mass/charge e.g. C6H5Cl and C6H5OF @ 112 m/z C6H5Cl = 112.00798 amu (all 12C, 35Cl, 1H) C6H5OF = 112.03244 amu (all 12C, 16O, 1H, 19F) Resolving power >4700 required to resolve these two Two definitions Resolution = Δm/m (0.024/112.03 = 0.00022 or 2.2*10-4) Resolving power = m/Δm (112.03/0.024 = 4668)

Resolving Power Example RP= 3,000 RP= 5,000 RP= 7,000 C6H5Cl C6H5OF All resolving powers are FWHM

Mass Accuracy MSF reports mass accuracy as a relative value ppm = parts per million (1 ppm = 0.0001%) 5 ppm @ mass 300 = 300 * (5/106) = ±0.0015 Da High resolving power facilitates precise mass measurements Accurate mass spectrometry is used to confirm a molecular formula Walk-up instruments in the MSF should be treated as “nominal mass” accuracy +/- 0.15 Da mass accuracy

A Discussion of Molecular Ions

Molecular Weight Calculations Calculate molecular weights of expected components PRIOR to performing MS The molecular weight of a compound is computed by summing the masses of all atoms that comprise the compound. Morphine: C17H19NO3 = 12.011(17) +1.008(19)+ 14.007 + 15.999(3) = 285.34 Da Yet 285.136 is observed by EI-MS Molecular weight is calculated assuming a natural distribution of isotopes Molecular weights calculated with average masses for Br, Cl, and many metals will differ greatly from MS data

Monoisotopic vs. Average Mass Most elements have a variety of isotopes C  12C is 98.9% abundant, 13C is 1.1% abundant For C20, 80% chance 13C0, 18% chance 13C1, 2% chance 13C2 Sn has 7 naturally occurring isotopes @ >5% ab. F, P, Na, Al, Co, I, Au have only 1 natural isotope Mass spectrometers can resolve isotopic distributions Monoisotopic masses must be considered Monoisotopic masses are computed using the most abundant isotope of each element (12C, 35Cl, 79Br, 58Ni, 11B, etc.) For morphine, monoisotopic mass = 285.1365 (12.0000 * 17) + (1.0078 * 19) + 14.0031 + (15.9949 * 3)

Isotopic Envelopes Mass spectrometers measure ion populations 102 – 106 ions in MS peaks Any single ion only has 1 isotopic composition The observed mass spectrum represents the sum of all those different compositions “M+ peak” “M+1 peak” “M+2 peak”

C17H19NO3 Mass Spectrum 13C0, 15N0 285.36 avg. mass 13C1 or 15N1

Isotopic Envelope Applications Isotopic envelopes can be used to preclude some elements from ionic compositions Lack of intense M+2 peak precludes Cl or Br Many metals have unique isotopic signatures M+1/M+ ratio can be used to count carbons [(M+1)/M+]/0.011 ≈ # carbon atoms For morphine: (0.1901/1)/0.011 = 17.28  17 Isotope table can be found on NIST website Link from MSF “Useful Information” page

A few isotope patterns C12H27SnBr tributyltin bromide C2H3Cl3 trichloroethane C83H122N24O19 A 14-mer peptide

Last Comments on Molecular Ions Be aware of ionization mechanism EI, LDI, and CI generate radical cations M+• is an odd electron ion Nitrogen rule is normal Odd molecular ion mass implies odd # of N atoms M+• for morphine by EI is 285.136, odd # N (1) ESI, APCI, MALDI, and CI make cation adducts M+H and M+Na are even electron ions Nitrogen rule is inverted for these ions Even molecular ion mass implies odd # of N atoms M+Na for morphine by ESI is 308.126, odd # N (1) Metal atoms and pre-existing ions or radicals can override these rules

Some useful software tools The “exact mass” feature in ChemDraw will give you a monoisotopic mass Not always correct for complex isotope patterns Two freeware apps are available from MSF website “Links” page These can be used to predict the entire isotopic pattern as an exportable image MS-Search program on GC-MS computer can be used to retrieve mass spectra from NIST’02 library

Making ions: A Practical Primer

Mass Spectrometer Components Inlet Get samples into the instrument Source Ionize the molecules in a useful way Mass Analyzer Separates the ions by mass to charge (m/z) ratio Detector Converts ions into an electronic signal or photons Data system From photographic plates to computer clusters

Electrospray Ionization (ESI) Dilute solution of analyte (<1 mg/L) infused through a fine needle in a high electric field Very small, highly charged droplets are created Solvent evaporates, droplets split and/or ions ejected to lower charge/area ratio Warm nebulizing gas accelerates drying Free ions are directed into the vacuum chamber Ion source voltage depends on solvent Usually ±2500 – ±4500 V

Advantages of ESI Gentle ionization process High chance of observing molecular ion Very labile analytes can be ionized Molecule need not be volatile Proteins/peptides easily analyzed by ESI Salts can be analyzed by ESI Easily coupled with HPLC Both positive and negative ions can be generated by the same source

ESI Picture http://newobjective.com/images/electro/spraytip_bw.jpg

Characteristics of ESI Ions ESI is a thermal process (1 atm in source) Little fragmentation due to ionization (cf EI) Solution-phase ions are often preserved e.g. organometallic salts ESI ions are generated by ion transfer (M+H)+, (M+Na)+, or (M-H)-, rarely M+• or M-• ESI often generates multiply charged ions (M+2H)2+ or (M+10H)10+ Most ions are 500-1500 m/z ESI spectrum x-axis must be mass/charge (m/z or Th, not amu or Da)

ESI Disadvantages Analyte must have an acidic or basic site Hydrocarbons and steroids not readily ionized by ESI Analyte must be soluble in polar, volatile solvent ESI is less efficient than other sources Most ions don’t make it into the vacuum system ESI is very sensitive to contaminants Solvent clusters can dominate spectra Distribution of multiple charge states can make spectra of mixtures hard to interpret e.g. polymer mass spectra

ESI Example I (M+H)+ C26H18O4

ESI Example II 22% 78%

Atmospheric Pressure Chemical Ionization (APCI) APCI uses a corona discharge to generate acidic solvent cations from a vapor These solvent cations can protonate hydrophobic species not amenable to ESI APCI can be done from hexane or THF Often used to study lipids and steroids In MSF, completely protected macrocycles are routinely studied by APCI APCI is harsher than ESI Large # of variables in APCI make it less reproducible than ESI

APCI Diagram http://imaisd.usc.es/riaidt/masas/imagenes/apci1.jpg

APCI Example

Agilent 6130 Multi-mode Source http://www.chem.agilent.com/Library/Images1/MMS_schematic_300dpi_039393.jpg

Matrix-Assisted Laser Desorption/Ionization (MALDI) Analyte is mixed with UV-absorbing matrix ~10,000:1 matrix:analyte ratio Analyte does not need to absorb laser A drop of this liquid is dried on a target Analyte incorporated into matrix crystals Spot is irradiated by a laser pulse Irradiated region sublimes, taking analyte with it Matrix is often promoted to the excited state Charges exchange between matrix and analyte in the plume (very fast <100 nsec) Ions are accelerated toward the detector

MALDI Diagram Image from http://www.noble.org/Plantbio/MS/iontech.maldi.html

Some Common MALDI Matrices

MALDI Advantages Relatively gentle ionization technique Very high MW species can be ionized Molecule need not be volatile Very easy to get sub-picomole sensitivity Spectra are easy to interpret Positive or negative ions from same spot Wide array of matrices available

MALDI Disadvantages MALDI matrix cluster ions obscure low m/z (<600) range Analyte must have very low vapor pressure Pulsed nature of source limits compatibility with many mass analyzers Coupling MALDI with chromatography can be difficult Analytes that absorb the laser can be problematic Fluorescein-labeled peptides

MALDI Example (ACTH 7-38+H)+ (ACTH 18-37+H)+ (Ubiq+2H)2+ (Ubiq+H)+ (Ins+H)+

MALDI Example I Continued

Electron Ionization (EI) Gas phase molecules are irradiated by beam of energetic electrons Interaction between molecule and beam results in electron ejection M + e-  M+• + 2e- Radical species are generated initially EI is a very energetic process Molecules often fragment right after ionization

EI Diagram Image from http://www.noble.org/Plantbio/MS/iontech.ei.html

EI Mass Spectrum Figure from Mass Spectrometry Principles and Applications E. De Hoffmann, J. Charette, V. Strooband, eds., ©1996

Cocaine More EI Mass Spectra Vitamin B6 Androstenedione

Timescales for EI-MS

Basic Rules Electron is first removed from site with lowest ionization potential non-bonding electrons > pi bond electrons > sigma bond electrons NB > π > σ (think No Pizza from Sigma) Stevenson’s Rule: During a sigma bond dissociation, the charge will likely be retained on the fragment with the lowest ionization potential Odd electron species can fragment to give odd or even electron products Even electron species can only fragment to yield even electron products Only CHARGED species are detected

Four Basic Mechanisms to Learn Sigma Cleavage Alpha Cleavage Inductive Cleavage McLafferty Rearrangement

Sigma Bond Cleavage Removal of an electron from a sigma bond weakens it As bond breaks, one fragment gets the remaining electron, and is neutral (R•) The other fragment is a charged, even electron species (R+) Highly substituted carbocations are more stable (Stevenson’s Rule) Cleavage of the C1-C2 bond in long n-alkanes is not favored Lower IE fragments are favored Long n-alkane chains tend to make many fragments spaced by 14 from m/z 20-90

Sigma Cleavage Example: Hexane 57 43 8.0 eV 8.2eV 29 8.4 eV 86

43 71

Homolytic cleavage – Radical Site Driven Cleavage is caused when an electron from a bond to an atom adjacent to the charge site pairs up with the radical Weakened α-sigma bond breaks This mechanism is also called α-cleavage The charge does not move in this reaction Charged product is an even electron species α-cleavage directing atoms: N > S, O, π, R• > Cl, Br > H Loss of longer alkyl chains is often favored Energetics of both products (charged and neutral) are important

ΔHf = +117 kJ/mol 43 ΔHf = +145 kJ/mol 72 57

73 87 101

Heterolytic Cleavage: Charge Driven Charged site induces a pair of electrons to migrate from an adjacent bond or atom This breaks a sigma bond Also called inductive cleavage The charge migrates to the electron pair donor The electron pair neutralizes the original charge Even electron fragments can further dissociate by this mechanism Inductive cleavage directing atoms: Halogens > O, S, >> N, C

57 136

57 29 86

117 196 127 69

Benzylic Bond Cleavage The charge stabilizing ability of the aromatic group can dominate EI spectra Alkylbenzenes will often form intense ions at m/z 91 Tropylium ion 7-membered ring favored by >11 kJ/mol Tropylium ion can fragment by successive losses of acetylene 91  65  39 Phenyl ions (C6H5)+• decompose the same way (77  51)

91 120 65 39

74 91 120 165

3-Methyl-2-Pentanone 43 57 29 72 100

3-methyl-2-pentanone ions What about m/z 72?

McLafferty Rearrangement 72 Th fragment requires elimination of ethene A hydrogen on a carbon 4 atoms away from the carbonyl oxygen is transferred The “1,5 shift” in carbonyl-containing ions is called the McLafferty rearrangement Creates a distonic radical cation (charge and radical separate) 6-membered intermediate is sterically favorable Such rearrangements are common Once the rearrangement is complete, molecule can fragment by any previously described mechanism

EI Advantages Simplest source design of all EI mass spectrometers even go to other planets! Robust ionization mechanism Even noble gases are ionized by EI Fragmentation patterns can be used to identify molecules NIST ’08 library has over 220,000 spectra Structures of novel compounds can be deduced

EI Disadvantages Fragmentation makes intact molecular ion difficult to observe Samples must be in the gas phase Databases are very limited NIST’08 only has 190,000 unique compounds Interpreting EI spectra is an art

Huygens Probe (on Titan)

Problem Solving with MS

Problem Solving Examples Formula matching with accurate mass ESI-TOF data Discovery of a novel steroid (UCLA) Diagnosing a reaction with LC-MS and accurate mass LC-MS

Formula Matching Basics Atomic weights are not integers (except 12C) 14N = 14.0031 Da; 11B = 11.0093 Da; 1H = 1.0078 Da 16O = 15.9949 Da; 19F = 18.9984 Da; 127I = 126.9045 Da Difference from integer mass is called “mass defect” or “fractional mass” Related to nuclear binding energy Sum of the mass defects depends on composition H, N increase mass defect Hydrogen-rich molecules have high mass defects Eicosane (C20H42)= 282.3286 O, Cl, F, Na decrease it Hydrogen deficient species have low mass defects Morphine, (C17H19NO3) = 285.1365

More Formula Matching Accurate mass measurements narrow down possible formulas for a given molecular weight 534 entries in NIST’08 library @ mass 285 Only 3 formulas within 5 ppm of 285.1365 46 compounds with formula C17H19NO3 Mass spectrum and user info complete the picture Isotope distributions indicate/eliminate elements User-supplied info eliminates others (e.g. no F) Suggested formula has to make chemical sense 3 formulae are C17H19NO3 (46 entries), C16H23NSi2 (2 entries), and C15H21F2NS (1 entry)

Formula Matching Example Zoloft C17H18Cl2N Only 9 ways to combine up to 40 C, 50 H, 5 N, 5 O, and 2 Cl to get a mass within 20 ppm (0.0061 u) of 306.0820, only 3 have 2 Cl

Discovery of a Novel Steroid A researcher at UCLA was given an athlete’s used syringe that contained a suspected steroid GC-MS revealed a mass spectrum that matched no known steroid Compound was NOT detected by normal steroid screen The mass spectrum was similar to two other steroids Accurate mass spectrometry indicated a molecular formula of C21H28O2 (312.2080 Da) Rapid Communications in Mass Spectrometry vol. 18, page 1245 (2004)

Gestrinone mass spectrum Unknown mass spectrum New molecule dubbed THG or tetrahydrogestrinone is active ingredient in “The Clear” Gestrinone mass spectrum Used in Europe to treat endometriosis Trenbalone mass spectrum Trenbalone is used to aid growth in US beef cattle

Staudinger Rxn Gone Wrong? M+H for product is 582.19 M+Na is 604.18

LC-MS Chromatogram

Mass Spectra from Peaks

Mass Spectra from Peaks 2

Proposed Side Reaction

Accurate Mass Data M+H for deuterated amine is 583.1967 (-40 ppm)