FOS 6355
Summer 2005
Russell Rouseff
What is Mass Spectroscopy
Analytical Chemistry Technique
Used to identify and quantify unknown compounds
Can also elucidate structure and chemical
properties
Sensitive technique
As little as 10 -12 g can be analyzed
Identification based on molecular fragmentation
patterns
fragments are separated by their m/z ratio
1885
1905
1925
1945
1965
1996 - First proton transfer MS
1996 - MS of a virus
1993 - Protein Mass Mapping/Fingerprinting
1990 - Protein Structure
1980 - Inductively Coupled Argon Plasma
1978 - GC-C-IRMS
1968 - Atmospheric Pressure Ionization
1966 - Peptide Sequencing
1966 - Chemical Ionization
1956 - Identifying Organic Compounds
1956 - GC/MS
1953 - Quadrupole MS
1947 - Preparative Mass Spectrometry
1946 - Time of Flight MS
1943 - Double focusing analyzer
1899 - Early Mass Spectrometry
MS Time lines
1985
2005
Mass Spectroscopy Applications
Detect and identify the use of steroids in athletes
Monitor the breath of patients by anesthesiologists during
surgery
Determine the composition of molecular species found in
space
Determine whether honey is adulterated with corn syrup
Monitor fermentation processes for the biotechnology
industry
Detect dioxins in contaminated fish
Establish the elemental composition of semiconductor
materials
Perform forensic analysis – arson identification
Objectives
How did mass spectrometry originate?
What is a mass spectrometer?
What are the characteristics of a mass spectrum?
How is the sample introduced into the mass spectrometer?
How are the molecules and fragment ions produced in the
ion source?
How does the analyzer work?
How does the detector work?
What are the roles of computers in mass spectrometry?
Objectives
How can mass spectrometric data be used for structure
analysis?
What other techniques are used to produce ions?
How large a molecule can be analyzed?
How is mass spectrometry used with GC,LC, and other
separation techniques?
How is mass spectrometry used for quantitative analysis?
What is mass spectrometry/mass spectrometry (MS/MS)?
What is isotope ratio mass spectrometry?
Vacuum pumps
Ion Formation
source
Sample
Introduction
Ion Sorting
analyzer
Data Handling
Ion Detection
ion
detection
data
system
Data Output
Mass spectrum
Sample Introduction
Neutral molecules in gaseous state
Can be either liquid, solid or gas under STP
must volatilize under vacuum
must be pure
often coupled with GC, HPLC or CE
but can use solids probe if pure
Ionization Techniques
Electron Impact, EI
Chemical Ionization, CI
Fast Atom Bombardment, FAB
Atmospheric Pressure Chemical Ionization, APCI
Proton Transfer Reaction
Ion Formation (EI)
Electron collector
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Sample
entrance
repeller
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Ion focusing lenses
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filament
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44
100
CO2+
Molecular ion
Base ion
O
C
O
50
C+
12
O+
CO+
28
16
22
45
0
10
12
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16
(mainlib) Carbon dioxide
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Contains C, H, O
Sample Ionization
Electron Impact, EI, most common
70 eV standard ionization energy
M + e- => M+ + 2eBut sometime 70 eV too powerful
EI may overfragment
Molecular Weight =165.24
Exact Mass =165
Molecular Formula =C10H15NO
Chemical Ionization
A softer ionization technique
Use ionized molecules to transfer protons (+)
M + CH5+ => (M+H)+ + CH4
Chemical Ionization gases
methane
isobutane
Chemical Ionization
Produces spectra with little fragmentation
However, no standard spectral libraries
Spectra tends to be instrument specific
reagent gas pressures, ionization
efficiencies
Must generate own standards
Large Molecule Ionization
Can routinely analyze molecules of 10,000 Da or more
Upper limit used to be 2,000 Da
APCI
A form of chemical ionization
Used for LC/MS interfaces
Allows for removal of solvent before
vacuum
http://www.chm.bris.ac.uk/ms/theory/apci-ionisation.html
Proton Transfer Mass Spectrometery
glow
discharge
H30+ + M => (M + H) = H2O
M+
+
H30
M+
M
H30+
Europhysics News (2004) Vol. 35 No. 6
Proton Transfer Mass Spectrometery
Produces spectra with little ionization, quantify using M+
Detects aroma compounds at ppb levels
Europhysics News (2004) Vol. 35 No. 6
Mass Analyzer Types
Magnetic Sector
Quadrupole mass analyzers
Ion trap
Time of flight
Magnetic Sector Mass Spectrometers
eMolecular
Source
Ionization
chamber
H0
Magnetic Field
Exit slit
detector
Magnetic Sector Disadvantages
Not well-suited for pulsed ionization methods
Very High cost
Large size
High maintence costs
quadrupole mass analyzer
detector
resonant ion
nonresonant ion
quadrupole rods
source
focusing lens
Mass filters
Quadrupole Advantages
Classical mass spectra
Good reproducibility
Relatively small/ compact
Relatively low-cost systems
Can improve sensitivity 100x with SIM
Quadrupole limitations
Limited resolution
Peak heights variable as a function of mass
(mass discrimination).
Peak height vs. mass response must be
'tuned'.
Not well suited for pulsed ionization
methods
Ion Trap Advantages
High sensitivity
Multi-stage mass spectrometry, MSn
Compact mass analyzer
Ion Trap Limitations
Requires careful quantitation
Limited dynamic range
Subject to space charge effects and ion
molecule reactions
No sensitivity gain using SIM
Time of Flight MS
MS/MS
General Mass Spectrometry Sites
ASMS- American Society for Mass Spectrometry
http://www.asms.org/whatisms/edu_resources.html
Excellent educational MS resource with many MS related links.
Cambridge University WWW Mass Spectrometry Server
http://www-methods.ch.cam.ac.uk/meth/ms/theory/index.html
Comprehensive site with information on ionization processes, mass
analyzers, and other mass spectrometry techniques.
Organic Chemistry Online
http://chipo.chem.uic.edu/web1/ocol/spec/MS.htm
Introductory information about analysis of organic compounds by electron
impact mass spectrometry, tables of common fragment losses.
University of Leeds: Introduction to Mass Spectrometry
http://www.astbury.leeds.ac.uk/Facil/MStut/mstutorial.htm
Introductory site focusing on the analysis of biochemical samples.
Information on Specific Techniques
Misc. Mass Spectrometry Sites
Sheffield ChemPuter: Isotope Patterns Calculator
http://www.shef.ac.uk/chemistry/chemputer/isotopes.html
Site predicts isotope pattern based on molecular formula.
UCSF Protein Prospector
http://prospector.ucsf.edu/
Site includes useful programs for analyzing the mass spectra of proteins
and peptides.
Proteomics tools for mining sequence databases in conjunction with Mass
Spectrometry experiments