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
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



MS is not a “magic bullet” technique



Neutrals don’t respond to electric and magnetic fields
If a molecule cannot ionize, MS cannot help
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= 5,000
RP= 7,000
100
100
80
80
80
60
In ten sity (%)
100
In ten sity (%)
In ten sity (%)
RP= 3,000
60
40
40
20
20
20
0
111.95
112.00
Mass [amu]
112.05
112.10
C6H5OF
60
40
0
C6H5Cl
0
111.95
112.00
Mass [amu]
112.05
112.10
All resolving powers are FWHM
111.95
112.00
Mass [amu]
112.05
112.10
Mass Accuracy

MSF reports mass accuracy as a relative value

ppm = parts per million (1 ppm = 0.0001%)


High resolving power facilitates precise mass
measurements


5 ppm @ mass 300 = 300 * (5/106) = ±0.0015 Da
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”
100
80
Intensity (%)

60
40
“M+1 peak”
20
“M+2 peak”
0
285
286
287
M a s s [a m u]
288
289
2
C17H19NO3 Mass Spectrum
100
13C
0,
15N
0
80
Intensity (%)
285.36
avg. mass
60
40
13C
or
1
15N
20
1
13C
13C
1
2 or
+15N
1
0
285
286
287
M a s s [a m u]
288
2
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
100
100
C2H3Cl3
trichloroethane
80
Intensity (%)
60
40
20
20
100
C83H122N24O19
A 14-mer peptide
0
131
60
40
132
133
134
135
136
Mass [amu]
137
138
0
80
139
362
Intensity (%)
Intensity (%)
80
C12H27SnBr
tributyltin
bromide
60
40
20
0
1759
1760
1761
1762
Mass [amu]
1763
1764
1765
364
366
368
372
370
Mass [amu]
374
376
378
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



ESI, APCI, MALDI, and CI make cation adducts


M+H and M+Na are even electron ions
Nitrogen rule is inverted for these ions



Odd molecular ion mass implies odd # of N atoms
M+• for morphine by EI is 285.136, odd # N (1)
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


Two freeware apps are available from MSF
website “Links” page


Not always correct for complex isotope patterns
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


Source


Separates the ions by mass to charge (m/z) ratio
Detector


Ionize the molecules in a useful way
Mass Analyzer


Get samples into the instrument
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)


Solution-phase ions are often preserved


e.g. organometallic salts
ESI ions are generated by ion transfer


Little fragmentation due to ionization (cf EI)
(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



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


Hydrocarbons and steroids not readily ionized by ESI
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
js-29-1
LCT
js-29-1 54 (1.086) Cm (54:60)
395.1219
100
(M+H)+
%
C26H18O4
396.1333
304.0758
0
200
300
397.1367
400
500
600
700
ESI Example II
78%
22%
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



A drop of this liquid is dried on a target


Analyte incorporated into matrix crystals
Spot is irradiated by a laser pulse




~10,000:1 matrix:analyte ratio
Analyte does not need to absorb laser
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
(ACTH 7-38+H)+
(Ubiq+2H)2+
(ACTH 18-37+H)+
MALDI Example
(Ins+H)+
(Ubiq+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
82
100
Cocaine
More EI Mass Spectra
H
O
O
50
94
77
42
51
0
O
N
182
27
15
10 20 30 40
(mainlib) Cocaine
50
H O
105
303
122
59 68
60
70
80
140 152
198
166
272
90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310
151
100
94
Vitamin B6
106
N
169
HO
OH
50
122
OH
39
81
53
27 31
136
67
0
10
20
30
40
50
60
70
(mainlib) 3,4-Pyridinedimethanol, 5-hydroxy-6-methyl-
80
90
100
110
120
130
140
150
160
170
180
286
100
Androstenedione
124
O
244
148
50
109
O
79
41
0
18
55
91 97
67
29
10 20 30 40 50 60 70
(mainlib) Androst-4-ene-3,17-dione
86
80
131
201
162
173
187
216
229
258
271
90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300
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
39
65
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


Robust ionization mechanism


EI mass spectrometers even go to other planets!
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


Difference from integer mass is called “mass
defect” or “fractional mass”


= 14.0031 Da; 11B = 11.0093 Da; 1H = 1.0078 Da
16O = 15.9949 Da; 19F = 18.9984 Da; 127I = 126.9045 Da
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
Formula Matching Example
Elemental Composition Report
Tolerance = 20.0 PPM / DBE: min = -1.5, max = 50.0
Selected filters: None
Monoisotopic Mass, Even Electron Ions
370 formulas evaluated with 9 results within limits
Elements Used:
C: 0-40 H: 0-50 N: 0-5 O: 0-5 Cl: 0-2
Error
20 ppm
Zoloft C17H18Cl2N
error in:
Mass
intensity Calc. Mass mDa
PPM
i-FIT
Formula
306.082
100 306.0816
0.4
1.3
39.7 C17 H18 N Cl2
306.0776
4.4
14.4
376 C12 H18 N3 O2 Cl2
306.0875
-5.5
-18
701.7 C10 H22 N O5 Cl2
306.0798
2.2
7.2
1945.8 C18 H13 N3 Cl
306.0857
-3.7
-12.1
2205.2 C11 H17 N3 O5 Cl
306.0766
5.4
17.6
9102.8 C18 H12 N O4
306.078
4
13.1
9195.6 C19 H8 N5
306.0879
-5.9
-19.3
9289.5 C17 H12 N3 O3
306.0838
-1.8
-5.9
9543.2 C12 H12 N5 O5
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)
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
sdc2-271-001
sdc2-271-001
1: TOF MS ES+
BPI
4.25e3
10.20
100
%
3.23
5.82
9.97
8.15
15.32
5.62
4.08
18.73
0
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
Time
20.00
Mass Spectra from Peaks
sdc2-271-001
sdc2-271-001 489 (8.153) Cm (481:499)
1: TOF MS ES+
1.91e4
583.144
100
%
8.15 min peak
1 Th off from product (M+H)+
142.119
539.151
237.565
335.097
348.092
584.177
585.166
0
sdc2-271-001 350 (5.835) Cm (350:359)
1: TOF MS ES+
1.06e4
278.094
100
%
5.82 minute peak
unknown contaminant
142.119
229.140
279.110
453.347
0
sdc2-271-001 194 (3.234) Cm (188:205)
278.094
100
%
3.2 minute peak
unknown, non-retained compound
1: TOF MS ES+
3.36e4
279.110
0
100
200
300
400
500
600
700
800
900
m/z
1000
Mass Spectra from Peaks 2
sdc2-271-001
sdc2-271-001 922 (15.373) Cm (916:926)
1: TOF MS ES+
9.21e3
295.061
100
%
15.32 minute peak
unknown hydrophobic compound
296.075
185.168
229.147
420.977
475.337
651.071
697.035
0
sdc2-271-001 612 (10.204) Cm (610:618)
1: TOF MS ES+
3.59e4
279.056
100
%
10.20 minute peak
triphenyl phosphine oxide
(M+H)+, (2M+H)+, (2M+Na)+
280.089
342.100
343.115
142.125
557.179
579.145
580.174
0
sdc2-271-001 596 (9.937) Cm (595:600)
1: TOF MS ES+
1.03e4
608.141
100
%
9.97 minute peak
starting material M+H
609.184
142.125
610.194
397.338
0
100
200
300
400
500
600
700
800
900
m/z
1000
Proposed Side Reaction
Accurate Mass Data
sdc2-271-003
sdc2-271-003 (0.035) Is (1.00,1.00) C29H30N2O9SH 1: TOF MS ES+
583.1750
6.67e12
100
%
Theoretical M+H for
C29H30N2O9S
584.1782
585.1772
0
sdc2-271-003 417 (8.707) AM (Cen,8, 80.00, Ar,6000.0,622.57,0.70,LS 4); Sm (Mn, 2x4.00)
583.1733
1.40e3
100
%
Acc. Mass LC-MS data
584.1797
475.3204
585.1721
623.4202
539.1458
667.4045
0
475
500
525
550
575
600
625
650
675
m/z
700
M+H for deuterated amine is 583.1967 (-40 ppm)