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

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

3. 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

4. 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

5. 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

6. 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

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

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

9. Mass Accuracy MSF reports mass accuracy as a relative value
ppm = parts per million (1 ppm = %)
5 mass 300 = 300 * (5/106) = ± 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
+/ Da mass accuracy

10. A Discussion of Molecular Ions

11. 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 = (17) (19) (3) = Da
Yet 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

12. 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 >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 =
( * 17) + ( * 19) ( * 3)

13. 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”

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

15. 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
Isotope table can be found on NIST website
Link from MSF “Useful Information” page

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

17. 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 , 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 , odd # N (1)
Metal atoms and pre-existing ions or radicals can override these rules

18. 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

19. Making ions: A Practical Primer

20. 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

21. 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

22. 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

23. ESI Picture

24. 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 m/z
ESI spectrum x-axis must be mass/charge (m/z or Th, not amu or Da)

25. 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

26. ESI Example I
(M+H)+
C26H18O4

27. ESI Example II
22%
78%

28. 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

29. APCI Diagram

30. APCI Example

31. Agilent 6130 Multi-mode Source

32. 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

33. MALDI Diagram
Image from

34. Some Common MALDI Matrices

35. 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

36. 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

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

38. MALDI Example I Continued

39. 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

40. EI Diagram
Image from

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

42. Cocaine
More EI Mass Spectra
Vitamin B6
Androstenedione

43. Timescales for EI-MS

44. 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

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

46. 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

47. Sigma Cleavage Example: Hexane57 43
8.0 eV
8.2eV 29
8.4 eV 86

48. 43 71

49. 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

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

51. 73 87
101

52. 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

53. 57
136

54. 57 29 86

55. 117
196
127 69

56. 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)

57. 91
120 65 39

58. 74 91
120
165

59. 3-Methyl-2-Pentanone 43 57 29 72
100

60. 3-methyl-2-pentanone ions
What about m/z 72?

61. 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

63. 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

64. 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

65. Huygens Probe (on Titan)

66. Problem Solving with MS

67. 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

68. Formula Matching Basics
Atomic weights are not integers (except 12C)
14N = Da; 11B = Da; 1H = Da
16O = Da; 19F = Da; 127I = 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)=
O, Cl, F, Na decrease it
Hydrogen deficient species have low mass defects
Morphine, (C17H19NO3) =

69. More Formula Matching
Accurate mass measurements narrow down possible formulas for a given molecular weight
534 entries in NIST’08 mass 285
Only 3 formulas within 5 ppm of
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)

70. 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 ( u) of , only 3 have 2 Cl

71. 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 ( Da)
Rapid Communications in Mass Spectrometry vol. 18, page 1245 (2004)

72. 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

73. Staudinger Rxn Gone Wrong?
M+H for product is
M+Na is

74. LC-MS Chromatogram

75. Mass Spectra from Peaks

76. Mass Spectra from Peaks 2

77. Proposed Side Reaction

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