Mass Spectrometry

What is Mass Spectrometry?

How do we measure the mass
of a substance ?
1.66x10-24g/molecule

Methamphetamine?
C10H15N (M.W. 149.24 g/mol)
 Mass Spectrometry (MS)
• An analytical technique that analyze substances according to the mass-to-
charge (m/z) ratio of constituent atoms, groups of atoms or molecules
present
• Probably the most generally applicable analytical tool that provides both
qualitative and quantitative information about both atomic and molecular
compositions of both organic and inorganic materials
MS Applications
MS General Applications
Organic Chemistry
Molecular composition, Structure identification, Determination of molecular
weight, Functional group analysis, Medical application, Qualitative&Quantitative
analysis of organic compounds, Illegal drug analysis, Drug
metabolism&pharmacokinetics, etc.
Macromolecular Chemistry
Protein sequencing, Det.of M.W. of polymer and other macromolecules, Amino
acids
Inorganic Chemistry
Elemental composition, Isotope ratio measurement, Trace element analysis, etc.
Physical Chemistry
Investigation of gas-phase ion chemistry, Reaction mechanism, Metastable ions,
Bond energy, Dissociation energy, Ionization energy, etc.
Others: Space mission, Environment, Petrochem, Food&Drug, Forensic scince, etc.
Historical Perspectives
1886 E.Glodstein discovers positive ions using discharge tube
1912 J.J.Thomson analyzes these ions and obtains mass spectra of O2, N2, CO and CO2,
Also observes negative and multiply charged ions and discovers isotopes 20 and
22 of Ne. Nobel Prize
Magnet
pole DC electrical
Fluorescence screen pieces discharge
or photoplate
(-) (+)

Gas in

Pump
Pump
J. J. Thomson’s first mass spectrum (20Ne : 22Ne ~ 10:1)
1919 F.W.Aston develops first mass spectrometer with velocity focusing (Nobel Prize)
1942 First commercial MS for organic analysis
1948 Cameron discovers the measurement of ions by time of flight (TOF) analysis
1953 Paul describes quadrupole and ion trap mass analyzers (Nobel Prize)
1957 Kratos introduces the first MS with double focusing (high resolution) analyzer
1958 First spectrometer coupled with GC
1966 Munson discovers chemical ionization (CI) and Tandem MS
1973 McLafferty describes LC-MS
1974 Plasma desorption MS, FT-ICR-MS
1975 Mass spectrometers were on board spaceship Viking for the Mars mission
1980 Houk et al. describes ICP-MS for inorganic analysis (elemental composition)
1981 Barber describes fast atom bombardment (FAB) ionization source
1985 Hillenkamp discovers the matrix assisted laser desorption ionization (MALDI)
1988 Fenn developed the electrospray ionization and MS interface, first spectra of protein
above 20,000 Da obtained (Nobel Prize)
1988-present: Variety of new techniques and hybrid instruments and a lot more developments
Mass Spectrometers
 Overview of MS Technique *VACUUM*
(<10-5 Torr)

Sample Inlet System Ion Source Ion Focusing

Ion Separation
(Mass Analysis)

Data Recording Ion Detection

• Vacuum maintained in mass spectrometer to increase mean free path(= distance

travelled without collision) and reduce collision&loss of ions
• 1 Atm = 760 Torr
• 1 Torr ~ 1 mm Hg = 1.333 mbar = 0.01934 psi
Formation of ions and mass spectra
Consider an organic molecule (ABC) with E.I.(Electron Impact) source

Energy
(ABC+)* excited ion

ABC+ molecular ion, ground state

(2)
Ionization Energy
IE organic molecule (1)
~ 8-15 eV ABC* excited molecule

ABC neutral molecule, ground state

(1) ABC + Energy ABC+ + e-
Energy = high K.E. electron, hv (Laser), heat, electricity, etc.

e.g. Electron impact ionization: ABC + e-fast ABC+ + 2e-slow

I
ABC+

m/z

K.E. of e- ~ I.E.(ABC)
→ Molecular ions formed
(2) If energy in excess K.E. of e- >> I.E.(ABC) ==> Further fragmentation
ABC + e-fast (ABC+)* + 2e-slow
Parent ion
(Molecular ion)

A + BC+ AB+ + C
→ Daughter ions (Fragment ions) formed

I ABC+
AB+
BC+

m/z

Fragmentation pattern Structure I.D. (interpretation vs. spectrum

mapping/library search)
Structure Identificatiion by interpretation of fragmentation pattern
e.g. mass spectrum of alkanes
Mass spectrum
Peak position
Qualitative info.

Peak height or area

Quantitative info.
m/z ratio
M+-CH3

C6H5+
#
 How is mass defined?
• Assigning numerical value to the intrinsic property of “mass” is
based on using carbon-12, 12C, as a reference point.

• One unit of mass is defined as a Dalton (Da)

• One Dalton is defined as 1/12 the mass of a single carbon-12 atom

➔ one 12C atom has a mass of 12.0000 Da

 Isotopes
-Isotopes are atoms with the same number of protons but different
numbers of neutrons => same atomic number but different mass
- e.g. most carbon atoms have a mass of 12 Da, but in nature, 1.1% of
C atoms have an extra neutron, making their mass 13 Da.
- Why do we care?
Mass spectrometers can “see” isotope peaks if their resolution is high enough
Stable isotopes of selected elements
 Mass spectrum of peptide with 94 C-atoms
(19 amino acid residues)

“Monoisotopic mass”
No 13C atoms (all 12C)
1981.84

1982.84 One 13C atom

1983.84
Two 13C atoms
↓
 Average mass
Average mass corresponds
to the centroid of the
unresolved peak cluster

When the isotopes are not resolved, the centroid of the envelope
corresponds to the weighted average of all the the isotope peaks in
the cluster, which is the same as the average or chemical mass.
(most abundant isotope)
 MS Terminology
◼ Analyte
 a chemical substance or chemical constituent that is the
subject of chemical analysis (atoms, molecules, ions, etc.)
◼ Interference / Interferent
 the presence of species or substances in the sample
which affects the value of analytical signal determined
◼ Selectivity / Specificity ความสามาร !ในกา ารแบก interference

 relative freedom from interference effects

◼ Dynamic range / Linearity วงวเ ต
 concentration range over which the analytical curve is
linear or the calibration slope is constant
◼ Detection limit (DL) / Limit of detection (LOD)
 smallest concentration that can be reported as being
present in a sample with confidence
ช่
Mass Resolution (R)
• Ability of a mass spectrometer to separate two peaks of adjacent masses
• R = m/m
• m = mass difference between adjacent peaks OR peak width at specified
peak height, normally reported at 10% valley of peak height of overlapped
peaks or at 50% (FWHM) in case of single peak
how I m อ
peats m
resolutions ด
ห้
“High resolution” MS
• Separate chemically different ions with the same nominal masses
e.g. 87Sr+(86.908890) vs. 87Rb+(86.909180)
Rrequired = m/m ~ 87/2.9x10-4 ~ 300,000 (FT-ICR-MS needed)
• Assign elemental formula
C20H9+
C19H7N+
C13H19N3O2+ C20H9+ C19H7N+ C13H19N3O2+

Low resolution 3 different compounds

Nominal mass detected 3 different exact masses
at 249 Da High resolution
3 possible compounds

249 249.0070 249.0580 249.1479

Mass measurement accuracy depends on resolution

High resolution means better mass accuracy

Resolution =18100
8000

6000
Resolution = 14200
Counts

4000

Resolution = 4500
2000

2840 2845 2850 2855

Mass (m/z)
 What if the resolution is not so good?
At lower resolution, the mass measured is the average mass

Better
Poorer
resolution
resolution

6130 6140 6150 6160 6170

m/z
Mass Accuracy
• Ability of a mass analyzer to assign the mass of an ion close to its true
value (exact mass)
• maccuracy = mtrue – mmeasured
• High mass accuracy (exact mass measurement) is usually associated to
high resolution analyzers
• Exact mass helps to define atomic composition of a totally unknown cpd.

maccuracy
Mass Accuracy vs Resolution
 Sensitivity detect ความแตก
ไ แ
• Ability of the mass spectrometer to respond to a given amount of
sample analyte at a given mass to charge ratio
• The sensitivity of an MS system is directly related to the efficiencies of
all the processes of the entire system :
- Sample introduction เ องคอ น ดเ

- Interface I molecule

- Ionization efficiency
- Ion transmission
- Detection
ดี
นื
ด้
วั
ต่
ข่
ต่
 Sample Introduction (Inlet System)
Mission: Sample at ATM pressure Ion source/MS in vacuum
นอน
แน
น
เมา
(reduce P, minimize gas load)
5 amson + Worsolution
* iX
ของ:12
WS

I. Batch sample introduction

- Direct introduction of sample into ion source
- e.g. Direct insertion probe, desorption probe, MALDI, etc.
II. Coupled chromatographic sample introduction
- Online sample introduction through MS interface
- e.g. GC, LC, CE
 Sample Introduction System

GAS PHASE LIQUID PHASE SOLID PHASE

GC / EA HPLC / CE Laser Ablation
EI CI FI API ICP MALDI ICP

Sample Introduction
Gas Samples
• Septum inlet

– Gas or volatile compounds at room temp. stored in sample reservoir

with pressure maintained below 10-2-10-3 Torr (to avoid gas overload
in high vacuum MS chamber) by auxiliary pump
– Gas allowed to leak freely through septum (pin hole or glass sinter)
into ion source
– Samples occasionally heated to volatile
• GC Inlet
– Capillary GC: Low carrier gas flow rate ---> direct coupling to ion
source with heated transfer line and good pumping system
GC Ion source/MS

Hot transfer line through

wall into ion source
– Packed (Column) GC: - Much higher flow rate, effluent overload
- Use splitter/separator to reduce gas load
To ion source/MS

pump
Solid Samples
• Direct insertion probe
– Sample = solid, volatile, thermally stable compound
– Deposited on probe, inserted through vacuum interlock
– Often heated to volatilize sample
 Vacuum Interlock
• https://www.youtube.com/watch?v=OYcTmiCtCv8
Liquid Samples
• Batch introduction e.g. MALDI, FDI
• Online sample e.g. effluent from LC, CE, FIA, etc.
– Most difficult: Pressure overload by solvent & vapor must be avoided
– Samples must be vaporized and stripped off solvent (removing solvent)
to obtain gas phase ionization
• Electrospray ionization
• Thermospray ionization
• etc.
Total inlet system
• commercial instruments often couple several inlets into an MS system eg.
GC/septum/probe/ESI
 Pumps and Vacuum Technologies
Rotary Vane Pump (Mechanical Pump or Rough Pump)
- pumping speed ~ 20-150 L/sec
- used mainly in ionization source to obtain immediate pressure
(~ 10-4 up to 1 Torr) and also as a backup unit for turbo pump
Turbomolecular Pump (Turbo Pump)
- rotor-stator blades spinning at high speed of roughly 20,000-
90,000 rpm
- pumping speed ~ 200-5,000 L/sec
- used to obtain high vacuum (<10-5 Torr) for mass analyzers
- normally backed up by mechanical pump to facilitate the exhaust
20-200 L/sec
• Currently no single pump can handle such drastic pressure
reduction in a mass spectrometer
• In order to achieve high vacuum from higher pressure source,
differential pumping system is required
↳ อย ตลาด แต่ละ
Square
Quadrupole
Ion Transfer Inter-multipole
Tube
Lens 1 Split Gate Lens 3x10-3
Ion Transfer Detector Torr He
Electrospray Tube 1
Source Skimmer

Center
Section
Square
Tube Lens Quadrupole Octopole
Front Back
Tube Lens Lens Detector Lens
2
Front Back
rotary <- turbo < Section Section

8.3 L/sec 25 L/sec 300 L/sec 400 L/sec

ค่