Why Mass Spectrometry: An
Introduction to the IU MSF
Jonathan A. Karty, Ph.D.
jkarty@indiana.edu
http://msf.chem.indiana.edu
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
– mg/L to ng/L sensitivity possible
– 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?
– 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
• Quick product identification (TLC spot)
• Confirmation of elemental composition
– Much more precise then EA
• Selective detector for GC/HPLC
– MS provides retention time AND molecular weight
information about each analyte
• Reaction monitoring
– Crude reaction mixture MS
– Stable isotope labeling
– Stability studies
Important Concepts to Remember
• Mass spectrometers analyze gas-phase ions, not
neutral molecules
– Neutral molecules don’t respond to electromagnetic fields
– If you cannot make a stable ion, MS is impossible
• MS is not a “magic bullet” technique
– MS can tell you composition of an ion (CxHyOz)
– Connectivity of the atoms in that ion is much more
challenging
• Units
– 1 Da = 1 u = 1.6605*10-27 kg (1/12 of a 12C atom)
– 1 Th = 1 Da/e = 1.0364*10-8 kg/C
Molecular Weight Calculations
• The molecular weight is computed by summing the
masses of all atoms in the compound/ion.
– Erythromycin (M+H)+: C37H68N1O13+ = 12.011*37 + 1.008*67 +
14.007 + 15.999*13 = 734.93 Da
• Yet 734.5 is observed by ESI-MS
Isotopic Distributions
• Isotopes: same number of protons, different numbers
of neutrons
Unregistered
C mass spectrum
– 12100
C has 6 of each, 1340C has 6 protons and 7 neutrons
90
– Periodic
table assumes a natural distribution of stable
isotopes
(weighted average)
80
70
60
• Carbon
isotopes
– C 50

12C
is 98.9% abundant,
13C
is 1.1% abundant
• 40(0.989 * 12.0000) + (0.011 * 13.0034) = 12.011
– For30 C40: 64.2% 13C0, 28.6% 13C1, 6.2% 13C2
• 20Spectrum looks like 100% @ 480, 44.5% @ 481, 9.6% @ 482
10
• Many0479elements
of483isotopes
480 have
481 a variety
482
484
– Sn has 7 naturally occurring isotopes
– F, P, Na, I, Co, Au have only 1 natural isotope
485
Monoisotopic Masses
• Monoisotopic masses are considered for mass
spectrometry
– Monoisotopic masses are computed using the most
abundant isotope of each element (12C, 35Cl, 14N, 16O,
79Br, 11B, 120Sn etc)
• For erythromycin, monoisotopic mass = 734.468
– 12C37 1H68 14N1 16O13
– 12.000 * 37 + 1.0078 * 68 + 14.0031 + 15.9949 * 13
• Remember to include any ionizing reagent
– Electron loss, proton addition, etc.
C37H68NO13+ Mass Spectrum
13C , 2H , 18O
0
0
0
13C , 2H , 18O
1
0
0
13C , 2H , 18O
2
0
0
13C , 2H , 18O
0
0
1
13C , 2H , 18O
1
1
0
Average mass = 734.93 u
Observed isotope pattern is the convolution of
isotope patterns for all atoms
Isotopic Envelopes
• Isotopic distributions can indicate/preclude the
presence of an element
– Cl has a unique 3:1 pattern (M:M+2)
– B has a unique 1:4 pattern (M-1:M)
• M+1 / M+ ratio can be used to count carbon
atoms in a molecule
– [(M+1) / M+] / 0.011 ≈ # carbon atoms
– For morphine: (0.1901 / 1) / 0.011 = 17.28  17
• Significant deviation from expected isotope pattern
should be a warning sign
– Multiple compounds with similar masses in sample
– Partial isotopic enrichment (e.g. deuterated solvents used)
– Multiple ionization mechanisms at work
Two Complex Isotope Patterns
100
100
C2H3Cl3
trichloroethane
80
Monoisotopic
mass: 131.93
Average mass:
133.43
60
Intensity (%)
Intensity (%)
80
C12H27SnBr
tributyltin
bromide
60
40
40
20
20
0
0
131
Monoisotopic
mass: 370.03
Average
mass: 369.96
132
133
134
135
136
Mass [amu]
137
138
139
362
364
366
368
370
372
Mass [amu]
374
376
378
Instrumentation in the MSF
• One of 3 mass spectrometry facilities in the
department
• MSF is in Chemistry A411 and A454
• 1 GC-EI-Q-MS (A454)
• 1 LC-ESI/APCI-Q-MS (A454)
• 2 LC-ESI-TOF-MS (A411)
• 1 MALDI-TOF-MS (A411)
• 1 EI/CI-BE-MS (A411, staff only)
• Self-run experiments are $7-$10 per sample
• Staff-run experiments are $16-$30
• Training for Walk-Up MS will start after 11/7/10
Agilent 6890/5973 GC-MS
• 6890 GC
– 30 m long DB-5 (nonpolar) column installed
• Helium mobile phase
– Split/splitless injector
with autosampler
• 5973 MS
– Electron ionization
– Quadrupole MS (10-800)
– NIST 02 library installed
Agilent 1200/6130 LC-MS
• 1200 HPLC
– Binary pump
• 0.05-2 mL/min
– Autosampler with 6position column selector
– Diode array UV-VIS
detector
• 6130 MS
– Dual mode
electrospray/APCI source
• Can perform all 4 modes of
ionization in 1 experiment
– 50-3,000 m/z quadrupole
• Easy Access Software
Waters CapLC-LCT LC-MS
• CapLC
– Flow rates 1-40 uL/min
– C18, C8, and C4 columns
– Single wavelength UV-VIS
detector
• LCT
– Electrospray ionization
– Time-of-flight MS (TOF)
• 100-6,000 m/z
– Capable of accurate mass
spectrometry
• 5 ppm error for formula
confirmation
Bruker Autoflex III
• MALDI-TOF mass
spectrometer
– 200-150,000 m/z
• Can make both positive
and negative ions from
same spot
• Easy to interpret mass
spectra for large
polymers
• Matrices available for
wide array of analytes
Thermo MAT-95XP
• Magnetic sector mass
spectrometer
• Accurate mass spectrometry
is its main function
• Electron ionization and
Chemical ionization sources
• Trace GC available for low
MW species or mixture
analysis
Accurate Mass Spectrometry
• Accurate mass spectrometry can unambiguously
confirm chemical composition
• Mass accuracy is often reported as a relative value
– ppm = parts per million, 1 ppm = 0.0001%
• 5 ppm error is used by many journals as a standard
to confirm a chemical formula
– Instrumentation in the IU MSF routinely achieves this
level of accuracy
– 5 ppm at mass 300: 300 * (5/106) = ±0.0015 Da
Formula Matching Basics
• Atomic weights are not integers (except
12C)
– 14N = 14.0031 Da; 1H = 1.0078 Da
– 16O = 15.9949 Da; 127I = 126.9045 Da
– Table of isotopes link on MSF website
• Difference from integer mass is called “mass defect”
– Related to nuclear binding energy (E = mc2)
• Sum of the mass defects depends on formula
– H, N increase mass defect
• Eicosane (C20H42) = 282.3286
– O, Cl, F, Na decrease it
• 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
Accurate MS Notes
• Accurate MS is possible with the MAT-95 and
LCT mass spectrometers
– Bruker MALDI-TOF can do 20 ppm mass accuracy
• Only MSF staff can perform accurate mass
measurements
• All accurate MS submissions MUST include a
nominal (low res) mass spectrum to
demonstrate purity PRIOR to accurate MS
Sample LCT Accurate Mass Report
Sample MAT-95 Accurate Mass Report
Upcoming Lectures
•
•
•
•
All in C033 from 5:30-6:15
Small molecule EI and GC-MS (10/14)
Small molecule ESI/APCI and LC-MS (10/18)
Biomolecule/polymer analysis by ESI and MALDI
(10/20)
• Possible special interest seminars in November
– Quantitation and other advanced topics for small
molecule MS
– Intro to Bioinformatics
– These will only occur if sufficient interest is expressed
• For a more in-depth treatment of all topics covered,
take C613 in the spring!