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Ionization Methodsin Mass Spectrometryat the SCS Mass Spectrometry Laboratory
Steven L. Mullen, Ph.D.Associate Director
School of Chemical Sciences, University of Illinois
SCS Mass Spectrometry Laboratory
Contact Information31 Noyes Laboratory (8:00-5:00 M-F)333-2545www.scs.uiuc.edu/~mswebslm@uiuc.edu
School of Chemical Sciences, University of Illinois
SCS Mass Spectrometry Laboratory
StaffVera Mainz, Ph.D. (Acting Director)Steven L. Mullen, Ph.D. (Associate Director)Furong SunRachel TaylorRichard Milberg, Ph.D.
School of Chemical Sciences, University of Illinois
SCS Mass Spectrometry Laboratory
InstrumentationMicromass 70-VSE (3)Micromass 70-SE-4FMicromass QuattroMicromass Q-Tof UltimaApplied Biosystems Voyager DE-STRFinnigan LCQ Deca XP
School of Chemical Sciences, University of Illinois
SCS Mass Spectrometry Laboratory
70-VSE (A) 70-VSE (B)
70-VSE (C) 70-SE-4F
School of Chemical Sciences, University of Illinois
SCS Mass Spectrometry Laboratory
Quattro Q-Tof
Voyager LCQ Deca
School of Chemical Sciences, University of Illinois
Information from Mass Spectrometry
1. Molecular WeightFrom molecular ion
M+ or (M+H)+ [also (M+Na)+ or (M+K)+]
2. StructureFrom fragments
Hard ionizationMS/MS
3. Elemental CompositionFrom exact mass
School of Chemical Sciences, University of Illinois
Parts of a Mass Spectrometer
Inlet Source Analyzer Detector
EICIFI
FDFAB
MALDIESI
ProbeLeak Valve
SyringePlateGCLC
SectorQuadrupole
FTTOF
Ion Trap
Electron MultiplierPhotomultiplierArray Detector
School of Chemical Sciences, University of Illinois
Parts of a Mass Spectrometer
Inlet
Source
Analyzer
Detector
School of Chemical Sciences, University of Illinois
How a Mass Spectrometer Works
Source Analyzer Detector
School of Chemical Sciences, University of Illinois
Interpreting a Mass Spectrum
15
43
58
Acetone
CH3CH3
O 1543
MW=58
School of Chemical Sciences, University of Illinois
Isotopic Peaks
C6H6
C6H5Cl
C6H5Br
35Cl
37Cl
79Br 81Br
12C
13C
School of Chemical Sciences, University of Illinois
Resolution
R=1000
R=500
R=100
555.3
555.3
555.5
Leu-enkephalin Monoisotopic Mass = 555.3 Average Mass = 555.6
School of Chemical Sciences, University of Illinois
ChargeLeu-enkephalin Monoisotopic Mass = 555.3
555.3
556.3
557.3
277.6
278.6
278.1
+1 ionm/z = 555.3
+2 ionm/z = 277.6
School of Chemical Sciences, University of Illinois
HR Exact Mass Measurement
OH
HO
OCH3
O
OH
HO
OH
C21H22O4Nominal Mass: 338
Exact Mass: 338.1518Found Mass: 338.1518 (Error = 0.1 ppm)
C22H26O3Nominal Mass: 338
Exact Mass: 338.1882Found Mass: 338.1879 (Error = 1.0 ppm)
School of Chemical Sciences, University of Illinois
Ionization Techniques
Short name Full name How it works
EI Electron Ionization Impact of 70 eV electrons
CI Chemical Ionization Transfer of H+ from CH5+
FI Field Ionization High Field (12kV)
FAB Fast Atom Bombardment Beam of Xenon atoms
FD Field Desorption High Field (12kV)
MALDI Matrix-Assisted Laser Desorption Ionization Laser hits sample in UV-absorbing matrix
ESI Electrospray Ionization Spray and desolvation of preformed ions
School of Chemical Sciences, University of Illinois
Ionization Technique Summary
Ionization Volatile Thermal Size Amount Examples
EI Yes Stable Small 1-2 mg Organics
CI Yes Stable Small 1-2 mg Organics
FI Yes Stable Small 1-2 mg Organics
FAB No Labile Medium 50μg-1 mg Polar/ionic organics, peptides, biomolecules, organometallic
FD No Labile Medium 1-2 mgNon-polar organics, organometallics, porphyrins, PAHs
MALDI No Labile Large 250fmol-500pmol
Peptides, proteins, DNA/RNA, polymers, dendrimers, fullerenes
ESI No Labile Large 1-300pmol/μLPolar/ionic organics, peptides, proteins, biomolecules, organometallics, polymers
School of Chemical Sciences, University of Illinois
Electron Ionization (EI)
Ionization Volatile Thermal Size Amount Examples
EI Yes Stable Small 1-2 mg Organics
Short name Full name How it works
EI Electron Ionization Impact of 70 eV electrons
M - e- M+
Small stable organicsExtensive fragmentation, 70eV libraries availableHR available
School of Chemical Sciences, University of Illinois
EI Sample
OH
O
Chemical Formula: C11H12O2Exact Mass: 176.0837
Molecular Weight: 176.2118
School of Chemical Sciences, University of Illinois
EI Sample
O
O
Chemical Formula: C12H12O2Exact Mass: 188.0837
Molecular Weight: 188.2225(Typical EI sample)
School of Chemical Sciences, University of Illinois
EI Sample
N N
O O
Chemical Formula: C35H30N2O2Exact Mass: 510.2307
Molecular Weight: 510.6249
School of Chemical Sciences, University of Illinois
EI Sample
N
NH
Chemical Formula: C24H28N2Exact Mass: 344.2252
Molecular Weight: 344.4925
School of Chemical Sciences, University of Illinois
EI Sample
Br
Chemical Formula: C13H17BrExact Mass: 252.0514
Molecular Weight: 253.1781
(Typical EI sample)
School of Chemical Sciences, University of Illinois
Chemical Ionization (CI)
Ionization Volatile Thermal Size Amount Examples
CI Yes Stable Small 1-2 mg Organics
Short name Full name How it works
CI Chemical Ionization Transfer of H+ from CH5+
M + CH5+ (M+H)+
Small organics that fall apart in EIExtensive fragmentationHR available
School of Chemical Sciences, University of Illinois
CI Sample
O O
O
OH
Chemical Formula: C10H14O4Exact Mass: 198.0892
Molecular Weight: 198.2158
School of Chemical Sciences, University of Illinois
CI Sample
N
OH O
O
Chemical Formula: C18H25NO3Exact Mass: 303.1834
Molecular Weight: 303.396
School of Chemical Sciences, University of Illinois
CI Sample
OHO
O
O
Si(C2H5)3
Si(C2H5)3
Chemical Formula: C18H38O4Si2Exact Mass: 374.2309
Molecular Weight: 374.6629
School of Chemical Sciences, University of Illinois
CI Sample
H2N
NH2
Chemical Formula: C19H34N2Exact Mass: 290.2722
Molecular Weight: 290.4867
School of Chemical Sciences, University of Illinois
CI Sample
O
O
OH
Chemical Formula: C17H32O3Exact Mass: 284.2351
Molecular Weight: 284.4342
(Typical CI sample)
School of Chemical Sciences, University of Illinois
Field Ionization (FI)
Ionization Volatile Thermal Size Amount Examples
FI Yes Stable Small 1-2 mg Organics
Short name Full name How it works
FI Field Ionization High Field (12kV)
M + kV M+
Small organics that fall apart in EI, Isotopic enrichmentLittle fragmentationHR not available
School of Chemical Sciences, University of Illinois
Field Desorption (FD)
Ionization Volatile Thermal Size Amount Examples
FD No Labile Medium 1-2 mgNon-polar organics, organometallics, porphyrins, PAHs
Short name Full name How it works
FD Field Desorption High Field (12kV)
M + kV M+
Non-polar compoundsNo fragmentationHR not available
School of Chemical Sciences, University of Illinois
FD Sample
NN
OO
O O
HN
Chemical Formula: C48H59N3O4Exact Mass: 741.4506
Molecular Weight: 741.9998
(Typical FD sample)
School of Chemical Sciences, University of Illinois
FD Sample
NN
O
O
NN
Chemical Formula: C37H40N4O2Exact Mass: 572.3151
Molecular Weight: 572.7391
School of Chemical Sciences, University of Illinois
FD Sample
N
N
N
O
OO
Chemical Formula: C12H9N3O3Exact Mass: 243.0644
Molecular Weight: 243.2182
School of Chemical Sciences, University of Illinois
FD Sample
(CO)4Os
(OC)3Os Os(CO)3H
H
Chemical Formula: C10H2O10Os3Exact Mass: 857.8492
Molecular Weight: 852.8069
(Typical FD sample)
School of Chemical Sciences, University of Illinois
FD Sample
Chemical Formula: C42H24Exact Mass: 528.1878
Molecular Weight: 528.64
School of Chemical Sciences, University of Illinois
Fast-Atom Bombardment (FAB)
Ionization Volatile Thermal Size Amount Examples
FAB No Labile Medium 50μg-1 mg Polar/ionic organics, peptides, biomolecules, organometallic
Short name Full name How it works
FAB Fast Atom Bombardment Beam of Xenon atoms
M + H+ (M+H)+ M+ M+
Polar/ionic compounds in liquid matrix (DTT/DTE or 3-nba)Some fragmentationHR availableRarely used, superseded by ESI
School of Chemical Sciences, University of Illinois
FAB Sample
HNN
NH N
O
OO
Chemical Formula: C53H76N4O3Exact Mass: 816.5917
Molecular Weight: 817.1955
School of Chemical Sciences, University of Illinois
Electrospray Ionization (ESI)
Ionization Volatile Thermal Size Amount Examples
ESI No Labile Large 1-300pmol/μLPolar/ionic organics, peptides, proteins, biomolecules, organometallics, polymers
Short name Full name How it works
ESI Electrospray Ionization Spray and desolvation of preformed ions
M + H+ (M+H)+ M+ M+ M - H+ (M-H)- M- M-
Polar/ionic compounds, very little sample neededLittle fragmentationHR available
School of Chemical Sciences, University of Illinois
ESI SampleNH2
CH
C
H2C
O
CHO
O
NH
CH
C
H2C
O
H2C
H2C
HNCH2N
NH
NH
CH
C
CH
OCH3
H3C
HN
CH
CH2C
O
HO
HN CH C
CH
O
CH3
CH2
CH3
HN CH C
CH2
O
N
NH
N
C
O
NH
CH
C
CH2
O NH
CH
C
H2C
O
N
NH
NH
CH
C
H2C
OH
O
CH
CH3
CH3
Chemical Formula: C62H89N17O14Exact Mass: 1295.6775
Molecular Weight: 1296.4756
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu
Angiotensin
(Typical ESI sample)
School of Chemical Sciences, University of Illinois
ESI SampleO
HO
O
O
O
O
O
Chemical Formula: C21H32O7Exact Mass: 396.2148
Molecular Weight: 396.4746
School of Chemical Sciences, University of Illinois
ESI Sample
O O
OO
O
NO2
OO
O
Chemical Formula: C32H25NO10Exact Mass: 583.1478
Molecular Weight: 583.5416
School of Chemical Sciences, University of Illinois
ESI Sample
N
Ir
N
S
HH
OO
PF6
Chemical Formula: C31H37F6IrN2O2PSExact Mass: 839.1847
Molecular Weight: 838.8839
(Typical ESI sample)
School of Chemical Sciences, University of Illinois
ESI Sample
N
HN
N
O
O
Chemical Formula: C12H13N3O2Exact Mass: 231.1008
Molecular Weight: 231.2505
School of Chemical Sciences, University of Illinois
Matrix-Assisted Laser Desorption Ionization (MALDI)
Ionization Volatile Thermal Size Amount Examples
MALDI No Labile Large 250fmol-500pmol
Peptides, proteins, DNA/RNA, polymers, dendrimers, fullerenes
Short name Full name How it works
MALDI Matrix-Assisted Laser Desorption Ionization Laser hits sample in UV-absorbing matrix
M + laser (M+H)+
Large compounds, very little sample neededLittle fragmentationHR not available
School of Chemical Sciences, University of Illinois
MALDI SampleOTg
OTg
OTg
OTg
TgO
TgO
MW=1907
School of Chemical Sciences, University of Illinois
MALDI Sample
CO2Tg
TIPSTIPS
12
MW=3822
School of Chemical Sciences, University of Illinois
MALDI Sample
N
NN
N
NH2
O
HO
HH
HH
PO
O-
HO
O-
n
MW=34,000
School of Chemical Sciences, University of Illinois
MALDI Sample
H2N-UNPSTFPGHSALMICGTLTLK-OH
MW=1852
School of Chemical Sciences, University of Illinois
MALDI Sample
OBr
=
O
O
HO
OHO
OH
*
*
7
MW = 1270
School of Chemical Sciences, University of Illinois
MS Experiment Summary
Experiment Ionization Comments
LR All Mass Spectrum
HR EI, CI, FAB, ESI Exact Mass – Elemental Composition
MS/MS ESI, FAB, MALDI Structure, e.g. Peptide Sequence
Isotope Ratio FI Isotopic Enrichment
GC/MS EI, CI, FI
LC/MS ESI HPLC or Capillary HPLC
School of Chemical Sciences, University of IllinoisGraphic by Dorothy Loudermilk
School of Chemical Sciences, University of Illinois
Further InformationMass Spectrometry: A Textbook
Jürgen H. Gross2004, Springer-Verlag, ISBN 3-540-40739-1
Interpretation of Mass Spectra, 4th editionF. W. McLafferty1993, University Science Books, ISBN 0-93-570225-3
Mass Spectrometry for BiotechnologyGary Siuzdak1996, Academic Press, ISBN 0-12-647471-0
School of Chemical Sciences, University of Illinois
Use of this fileThis slide show was prepared by
Steven L. Mullen, Ph.D.Mass Spectrometry LaboratorySchool of Chemical SciencesUniversity of Illinois(217) 333-2545
It may be used for non-commercial educational purposes, withappropriate credit and no alterations.
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