1. Mass Spectrometry A.) Introduction:
Mass Spectrometry (MS) measures the atomic or molecular weight of a ion from the separation based on its mass to charge ratio (m/z)
- elemental composition of matter
- structures of inorganic, organic and biological molecules
- qualitative and quantitative composition of complex mixtures
- isotopic ratios of atoms in the sample
One of the MOST Routinely used Analytical Techniques

2. Mass Spectrometry Nobel Laureates: Joseph John Thomson
Physics 1906
first mass spectrometer
Francis William Aston
Chemistry 1922
mass spectrometry of isotopes
John B. Fenn
Chemistry 2002
electrospray ionization of
biomolecules
Wolfgang Paul
Physics 1989
quadrupole and
quadrupole ion trap MS
Koichi Tanaka
Chemistry 2002
Matrix-assisted laser
Desoprtion/ionization (MALDI)
A Long and Continuing History of Achievements

3. Mass Spectrometry Mass Spectrometry Data Quantitative Information
Qualitative Analysis
Molecular Weight Determination
Structure Determination
Quantitative Analysis
Biotechnology
analysis of proteins & peptides
analysis of oligonucleotides
Pharmaceutical
drug discovery, combinatorial chemistry
pharmokinetics, drug metabolism
Clinical
neonatal screening, hemoglobin analysis
drug testing
Environmental
water, food, air quality (PCDs etcs)
Geological
oil composition
Toxicology
Forensics
Abundance
Qualitative Information
Quantitative Information

4. Mass Spectrometry Disadvantages
Advantages Over Atomic Optical Spectrometric
Detection limits three orders of magnitude better
Remarkably simple spectra that are unique and easily interpreted
Ability to measure isotopic ratios
Disadvantages
Instrument costs are two to three times higher
Instrument drift that can be as high as 5- 10% per hour
Interference effects

5. Discriminates among the masses of isotopes
Atomic Weights in MS
Discriminates among the masses of isotopes
differs from other analytical techniques
Atomic mass units (amu) or daltons (Da)
Relative scale:  exactly 12 amu
amu or Da equals 1/12 mass of
x10-24 g/atom
All measured masses relative to
has a mass times
Da x = Da  atomic weight = g/mol
Exact Mass (m)
Sum of specific set of isotopes within compounds
12C1H4: m = x x 4 = Da
13C1H4: m = x x 4 = Da
12C1H32H1: m = x x x 1 = Da
3-4 significant figures to right of decimal
Nominal Mass (m)
Whole number precision in mass measurement
12C1H4: m = 12 x x 4 = 16 Da
13C1H4: m = 13 x x 4 = 17 Da
12C1H32H1: m = 12 x x x 1 = 17 Da

6. Chemical atomic weight or average atomic weight
Atomic Weights in MS
Chemical atomic weight or average atomic weight
A1, A2, An :atomic masses in Da
p1, p2, pn :fractional abundance of each isotope
n :number of isotopes
Weight of interest for most purposes
Sum of chemical atomic weights for the atoms in the compound formula
CH4 (m) = x = Da
typical atomic masses in periodic table
Boron: 10B 23%
11B 100%
B (m) = (23x x11)/123
= 10.81
Zirconium: 90Zr 51.5% 91Zr 11.2%
92Zr 17.1 % 94Zr 17.4% 96Zr 2.8%
Zr (m) = (51.5x x x x96)/100 =

8. Molecular Formulas from Exact Molecular Weights
Requires identification of molecular ion peak
Exact mass needs to be determined
High resolution instruments
detect mass differences of a few thousands of amu
Purine, C5H4N4 (m = )
Benzamidine, C7H8N2 (m = )
Ethyltolune, C9H12 (m = )
Acetophenone, C8H8O (m = )
Molecular ion peak is ±  only C7H8N2 is possible formula
Precision of a few parts per million is routinely possible

9. Molecular Formulas from Isotopic Ratios
Low-resolution instrument  only differentiate whole number masses
Requires sufficiently intense molecular ion peak
Requires accurate heights for (M+1)+ and (M+2)+
C6H4N2O4
13C
6 x = 6.48 2H
4 x = 0.060%
15N
2 x = 0.74%
17O
4 x = 0.16%
(M+1)+/M+ = 7.44%
C12H24
13C
12 x = 12.96% 2H
24 x = %
(M+1)+/M+ = 13.32%

10. differentiate between masses R = m/Dm or
Resolution in MS (R)
differentiate between masses
R = m/Dm or
Higher the number the better the resolution
500,000 is better than 500
Resolution of 4000 would resolve peaks occurring at m/z values of:
400.0 and 400.1
40.00 and 40.01

11. Example 22: Calculate the resolution to differentiate (a) C2H4+ (m = ) and CH2N+ (m = ) and (b) N2+ (m = ) and CO+ (m = ).

12. Identification of odd electron ions – The Nitrogen Rule
Based on an anomaly in the relationship between the atomic weights and valences of the common elements
An organic molecule containing the elements C, H, O, S, P or halogen
odd nominal mass if it contains an odd number of nitrogen atoms
even nominal mass if it contains an even number of nitrogen atoms (including 0)
Element
At Wt
Valence H 1 C 12 4 N 14 3 O 16 2 F 19 S 32 Cl
35/37
Compound MW
CO2 44 CO 28
CH4 16
HCO2H 46
HCl
36/38
HCN 27 N2
NH3 17
Even nominal mass  even valence
Odd nominal mass  odd valence
Nitrogen is the exception

13. Double Bond Equivalent (D)
number of rings or double bonds that an ion contains
Calculated from the elemental formula as follows:
where:
D is unsaturation
imax is the total number of different elements
Ni the number of atoms of element i and
Vi the valence of atom i
valence of 1 (H, F, Cl), valence of 2 (O, S).
valence of 3 (N, P), valence of 4 (C, Si).
Hexane: C6H14
Mass of molecular ion: 86
D = 1+1/2((6x(4-2) + 14x(1-2)) = 0
Hexene: C6H12
Mass of molecular ion: 84
D = 1+1/2((6x(4-2) + 12x(1-2)) = 1

14. Basic MS Instrument Design
Atomic Mass Spectrometry involves the following steps:
atomization
conversion of atoms to ions
most ions are single charge
separation of ions based on mass-to-charge ratio (m/z)
Counting the number of ions of each type or measuring the ion current
Principal Components
Vacuum system  maintain low pressure(10-5 to 10-8 torr)
Inlet: introduce m-amount of sample into ion source
Ion source: sample converted into gaseous ion by bombardment with:
Electrons
Photons
Ions
Molecules
Thermal/electric energy
Positive/negative ions accelerated
into mass analyzer
Mass analyzer: disperse ions (m/z)
Transducer: convert beam of ions to
electrical signal

15. Types of Atomic and Molecular MS
Thermal ionization & Spark source  first MS
Inductively coupled plasma (ICP)  current common approach
Differ by types ion sources and mass analyzer

16. F = e(E+ v x B) (Lorentz force law) where:
MS Theory:
1.) Mass analyzers use electric and magnetic fields to apply a force on charged particles
F = ma (Newton's second law)
F = e(E+ v x B) (Lorentz force law)
where:
F - force applied to the ion m - mass of the ion
a - acceleration e - ionic charge
v x B - vector cross product E - electric field
of the ion velocity and the applied
magnetic field
2.) Force is therefore dependent on both mass and charge
- spectrometers separate ions according to their mass-to-charge ratio (m/z)
- not by mass alone

17. Inlet Systems:
Introduce sample into ion source with minimum loss of vacuum
Spectrometer equipped with multiple inlets for different sample types
Batch Inlet
Direct Probe Inlet
Gas Chromatography
Liquid Chromatography

18. Inlet Systems: External (Batch) Inlet Systems: Simplest
Gas & liquid samples (bp < 500oC)
Sample heated (<400 °C) in small external oven
Sample pressure 10-4 to 10-5 torr
Vapor admitted to ionizer through valve
Gas stream added to analyte

19. Direct probe inlet system for solids
Inlet Systems:
Direct probe inlet system for solids
Probe moves through various lock system stages permits for a step-wise increase in the vacuum
The direct probe is only ¼" in diameter.
Direct Probe
Solids and non-volatile samples
Less sample is required & wasted (few ng)
sample held on the surface of glass or aluminum capillary tube, fine wire or small cup
Sample vial inserted through air-lock into ionizer chamber
Lock system minimizes amount of air that must be pumped from system
Vial heated to vaporize sample
Vial can be reduced to capillary or surface plate for small quantities

20. Inlet Systems: LC & GC coupled to mass spectrometer
Permits separation and determination of components for complex mixtures
Requires specialized inlet systems
Major interface problem – carrier gas dilution
Jet separator (separates analyte from carrier gas)
Lighter carrier gas deflected by volume
Heavier sample travels in straight line

21. Ion Sources: Formation of gaseous analyte ions
Mass spectrometric methods are dictated by ionization techniques
Appearance of spectrum highly dependant on ionization technique
Gas-phase
Sample first vaporized then ionized
Thermally stable compounds boiling points < 500oC
MW < 100 amu
Desorption
Solid or liquid directly converted to gaseous ion
MW as large as 105 daltons
Type
Name and Acronym
Ionizing Process
Gas Phase
Electron Impact (EI)
Exposure to electron stream
Chemical Ionization (CI)
Reagent gaseous ions
Field Ionization (FI)
High potential electrode
Desorption
Field Desorption (FD)
Electrospray Ionization (ESI)
High electric field
Matrix-assisted desorption ionization (MALDI)
Laser beam
Plasma Desorption (PD)
Fission fragments from 252Cf
Fast Atom Bombardment (FAB)
Energetic atomic beam
Secondary Ion Mass Spectrometry (SIMS)
Energetic beam of ions
Thermospray Ionization (TS)

22. Ion Sources: Hard sources Hard Ionization
Sufficient energy so analyte are in highly excited energy state
Relaxation involves rupture of bonds
Produces fragment ions with m/z < molecular ion
Kinds of functional groups  structural information
Hard
Ionization

23. Ion Sources: Soft Ionization Soft sources Cause little fragmentation
Mass spectrum consists of molecular ion and only few, if any, other peaks
Accurate mass
Soft
Ionization

24. M + e-  M●+ + 2e- Ion Sources: Electron-Impact Source (EI)
Sample heated to produce molecular vapor
Bombard with a beam of electrons
Electrons emitted from heated tungsten or rhenium filament
Electrons accelerated by a potential of 70V
Path of electrons and molecular ion at right angles
Form positive ions  electron beam expels electron due to electrostatic repulsion
M + e-  M●+ + 2e-
Not very efficient  one molecule in a million ionized
Positive ions attracted to first slit by small potential 5V
High potential applied at accelerator plates 103 to 104 V
Generates molecular ion velocity

25. Ion Sources: Electron-Impact Source (EI)
Hard source 50V higher energy than chemical bond
Highly excited vibrational and rotational state
Electron beam does not increase translational energy
Relaxation results in extensive fragmentation
Large number of positive ions of various masses
Typically less mass than molecular ion
Lower mass ions called daughter ions
Sometimes molecular ion not present

26. Ion Sources: Electron-Impact Source (EI) Base peak  most intense peak
Usually a daughter ion or fragment ion
Peaks at MW greater than molecular ion
Same chemical formula but different isotope composition
Size of peak depends on relative natural abundance of isotopes
Collision Product peak (M+1)+
Collision transfers a hydrogen atom to the ion to generate a protonated molecule
Second order reaction  depends on concentration
Increases with increase in pressure

27. Ion Sources: Electron-Impact Source (EI) Advantages Disadvantages
Good sensitivity
Fragmentation  unambiguous identification of analytes
Disadvantages
Need to volatize sample  thermal decomposition before ionization
Fragmentation  disappearance of molecular ion peak
MW not determined

28. Ion Sources: Chemical Ionization Source (CI)
Electron Impact and Chemical Ionization are Interchangeable in a Spectrometer
Chemical Ionization is the second most common procedure for generating ions
Gaseous atoms from the sample are:
Heated from a probe
Collide with ions produced reagent gas bombarded by electrons
Usually positive ions are used
Need to modify electron beam
Add vacuum pump capacity
Reduce width of slit for mass analyzer
Allow a reagent pressure of 1 torr in ionization area
Keep pressure below 10-5 torr in analyzer
Concentration ratio of reagent to sample is 103 to 104
Electron beam preferentially interacts with reagent instead of sample

29. Ion Sources: Hard EI Ionization Soft CI Ionization
Chemical Ionization Source (CI)
Soft Source
Methane is common reagent
Also use propane, isobutane and ammonia
Reacts with high-energy electron beam to generate several ions
CH4+, CH3+ (~90% of product) and CH2+
React with other methane molecules
CH4+ + CH4  CH5+ + CH3
CH3+ + CH4  C2H5+ + H2
Collisions Between sample molecule and CH5+ & C2H5+ are highly reactive
Involve proton or hydride transfer
CH5+ + MH  MH2+ + CH4 proton transfer
C2H5+ + MH  MH2+ + C2H4 proton transfer
C2H5+ + MH  M+ +C2H6 hydride transfer
Proton transfer  (M + 1)+
Hydride transfer  (M – 1)+
C2H5+ transfer (M + 29)+ CI
Soft
Ionization EI
Hard
Ionization

30. Ion Sources: Matrix-Assisted Laser Desorption/Ionization (MALDI)
Accurate MW for polar biopolymers
DNA, RNA, Proteins
Few thousands to several hundred thousand Da
Sample is mixed with large excess of radiation-absorbing matrix material
Solution is evaporated onto solid surface
Sample exposed to pulsed laser beam
Sublimation of analyte ions
MS spectra recorded between laser beam pulses

31. Ion Sources: Simulation of MALDI
Matrix-Assisted Laser Desorption/Ionization (MALDI)
Low background noise
At high MW, matrix causes significant background at low MW
Absence of fragmentation
Multiple charged ions (+2, +3)
Observe dimers trimers
Mechanism is not completely understood
Matrix compound must absorb the laser
radiation
Soluble enough in sample solvent to be
present in large excess
Analyte should not absorb laser radiation
Fragmentation will occur
Simulation of MALDI

32. Ion Sources: Matrix-Assisted Laser Desorption/Ionization (MALDI)
MALDI spectra are greatly influenced by type of matrix, solvent and additive
At high MW, matrix causes significant background at low MW
Dariusz Janecki et al. (2002) ASMS

33. Ion Sources: Electrospray Ionization (ESI)
One of the most important techniques for analyzing biomolecules
Polypeptides, proteins and oligonucleotides
Inorganic species synthetic polymers
MW >100,000 Da
Uses atmospheric pressure and temperature
Sample pumped through a stainless steel capillary
Rate of a few mls per minute
Needle at several KVs potential
Creates charged spray of fine droplets

34. Ion Sources: Electrospray Ionization (ESI)
Passes through desolvating capillary
Evaporation of solvent
Attachment of charge to analyte molecule
Molecules become smaller  charge density becomes greater  desorption of ions into ambient gas

35. Ion Sources: Electrospray Ionization (ESI)
Little fragmentation of thermally fragile biomolecules
Ions are multiply charged
m/z values are small
Detectable with quadrupole with mass range of 1500 or less
Average charge state increases ~linearly with MW
MW determined from peak distribution

36. Ion Sources: Fast Atom Bombardment Sources (FAB)
Major role for MS studies of polar high molecular-weight species
Soft Ionization technique
MW > 10,000
Structural information for MW ~3,000
Samples are in a condensed state
Glycerol solution matrix
Liquid matrix helps reduce lattice energy
Ionized by bombardment with energetic (several keV) xenon or argon atoms
Very rapid sample heating
Reduces sample fragmentation
Positive & negative analyte ions are sputtered from the surface
Desorption process
Must overcome lattice energy to desorb an ion and condense a phase
“healing” the damage induced by bombardment

37. Ion Sources: Fast Atom Bombardment Sources (FAB)
Beam of fast energetic atoms are generated by:
Passing accelerated argon or xenon ions from an ion source through a chamber
Chamber contains argon or xenon atoms at 10-5 torr
High-velocity ions undergo a resonant electron-exchange reaction without substantial loss of translational energy
Focusing
Extraction plate
Analyte ion beam
(secondary ions)
Probe tip
Analyte metrix
Atom beam
Production of “fast atoms”
Ar+* Ar > Ar Ar0*
Charge transfer
Accelerated argon ion from “ion gun”
Ground state argon atom
“slow ion”
“fast atom”

38. Example 23: Identify the ions responsible for the peaks in the following mass spectrum for 1,1,1,2-tetrachloroethane (C2H2Cl4):
MW =

39. Double-Focusing analyzer
Mass analyzers:
Double-Focusing analyzer
Two devices for focusing an ion beam
Electrostatic analyzer
Magnetic sector analyzer
Ions accelerated through slit into curved electrostatic field
Focus beam of ions with narrow band of kinetic energies into slit
Ions enter curved magnetic field
Lighter ions deflected more than heavier ions

40. Mass analyzers: Double-Focusing analyzer
Curved magnetic field of 180, 90 or 60 degrees
Vary magnetic field strength or accelerating potential to select for ions of different mass
Kinetic energy of accelerated ion (before magnetic field)
where:
V = voltage v = ion velocity e = electronic charge (1.60x10-19C)
z = ion charge
Path through magnet depends on the balance of two forces:
Magnetic force (FM) and centripetal force (Fc)
B = magnetic field strength r = radius of curvature of magnetic sector
Substitute velocity into kinetic energy equation
Select masses by varying B by changing current in magnet

41. Mass analyzers:
Double-Focusing analyzer

42. Quadrupole mass analyzer
Mass analyzers:
Quadrupole mass analyzer
More compact, less expensive, rugged
High scan rate  spectrum in < 100ms
Four parallel cylindrical rods serve as electrodes
Opposite rods are connected electrically
One pair attached to positive side of variable dc source
One pair attached to negative side of variable dc source
Variable radio-frequency ac potential (180o out of phase) applied to each pair of rods
Ions accelerated through space between rods
Potential of 5 to 10 V
ac and dc voltages increased simultaneously with ratio being constant
All ions without specific m/z strike rods and become neutral
only ions having a limited range of m/z reach transducer (detector)

43. Mass analyzers: Quadrupole mass analyzer Positive rods
Alternating ac causes ions to converge during positive arc and diverge during negative arc
If ions strike rod during negative arc  neutralized and removed by vacuum
Striking a rod depends on:
rate of movement through rod
Mass to charge ratio
Frequency and magnitude of the ac signal
dc current effects momentum of ions
Momentum directly related to square-root of mass
More difficult to deflect heavy ions than lighter ions
Prevents heavier atoms from striking rods
High-pass mass filter

44. Mass analyzers: Quadrupole mass analyzer Negative rods
In the absence of ac, all positive ions drawn to rods  annihilated
Offset for lighter ions by ac
Low-pass mass filter
For ions to pass through the rods (band of ions):
Significantly heavy to pass positive rods
Significantly light to pass negative rods
One pair attached to positive side of variable dc source
One pair attached to negative side of variable dc source
Adjusting ac & dc moves the center of band of ions which pass the rods

45. Mass analyzers: Time of Flight (TOF) Mass Analyzers
Ions generated by bombardment of the sample with a brief pulse of:
Electrons, secondary ions, laser-generated photons
Frequency of pulse 10 to 50 kHz, duration of pulse 0.25 ms
Ions accelerated by electric field pulse 103 to 104 V
Same frequency of ionization pulse, but lags behind
Accelerated particle enter field-free drift tube
Ions enter tube with same kinetic energy
Ion velocity vary inversely with mass
Lighter particles arrive at detector before heavier particles
Flight times are 1 to 30 ms
Requires fast electronics
Peak broadening due to variability in ion energies and initial position
Limits resolution compared to magnets and quadrupole
Less widely used than quadrupole
Advantages: unlimited mass range, rapid data acquisition, simplicity, ruggedness, ease of access to ion source

46. Transducers (detectors) for Mass Spectrometry:
Electron Multipliers
Detects positive ions
Similar to photomultiplier used in UV/vis
Each dynode held at successfully higher voltage
Dynodes have Cu/Be surfaces
Burst of electrons emitted when struck with energetic ions or electrons
Electron multiplies contain upwards of 20 dynodes
Typical current gain of 107
Continuous-Dynode electric multiplier
Glass that is heavily doped with lead
Potential of 1.8 to 2 V across length of transducer
Trumpet shaped
Ions strike surface near entrance that ejects electrons
Electrons then skip along surface ejecting more electrons with each impact
Typical current gain of 105 to 108
Rugged, reliable with high current gains
Positioned directly at the exit slit of magnet
Ions have enough energy to eject electrons
Requires accelerator with quadrupole MS

47. Transducers (detectors) for Mass Spectrometry: Faraday Cup
hollow collector, open at one end and closed at the other, used to collect beams of ions
incident ion strikes the dynode surface which emits electrons
induces a current which is amplified and recorded
Surrounded by cage
Prevents the escape of reflected ions and ejected secondary electrons
Inclined with respect to ion beam
Particles striking or leaving the electrode are reflected away from entrance
Connected to ground potential through a large resistor
Ions striking plate are neutralized by flow of electrons from resistor
Causes a potential drop across resistor
Independent of the energy, mass or chemical nature of ion
Inexpensive and simple mechanical and electronic device
Disadvantages:
Need for a high-impedance amplifier
Limits speed at which spectrum can be scanned
Less sensitive than electron multipliers

48. Atomic Mass Spectra and Interferences: Spectroscopic interference
An ionic species in the plasma has the same m/z values as an analyte
Isobaric interference
Two elements that have isotopes with nearly the same mass
Differ by less than 1 amu
113In+ overlaps with 113Cd+ and 115In+ overlaps with 115Sn+
Isobaric interference occurs with the most abundant and most sensitive isotope
40Ar+ overlaps with 40Ca+ (97%) need to use 44Ca+ (2.1%)
58Ni+ overlaps with 56Fe+ need to use 56Fe+
Exactly predictable from abundance tables
Polyatomic ion interference
More serious than isobaric
Polyatomic species form from interactions with species in plasma, matrix or atmosphere
Several molecular ions can form and interfere
Typically observed for m/z < 82 amu
Potential interference: 40Ar2+, 40ArH+, 16O2+,
H216O,16OH+, 14N+
Serious interference: 14N2+ with 28Si+,
NOH+ with 31P+,
16O2+ with 32S+,
40ArO+ with 56Fe+,
40Ar2+ with 80Se+
Correct with blank

49. Atomic Mass Spectra and Interferences: Spectroscopic interference
Oxide and Hydroxide species interference
Most serious
Oxides an hydroxides of analyte, matrix solvent
plasma gases
MO+ and MOH+ ions
Formation depends on injector flow rate, RF power,
sampler skimmer spacing, sample orifice size,
plasma gas composition, oxygen elimination
solvent removal efficiencies
Matrix Effects
Occur at concentrations > 500 to 1000 mg/ml
Reduction in analyte signal, sometimes signal enhancement
General effect
Minimized by using more dilute solutions, altering sample introduction procedure or by separating out offending species
Use internal standard to correct effect
Affect of pesticide response to matrix effects
Add protectant to remove matrix effect
Analytical Sciences 2005, 21, 1291

50. Fourier Transform (FT) MS: Improves signal-to-noise ratios
Greater Speed
Higher sensitivity and resolution
Requires an Ion Trap
Ions are circulated in well-defined orbits for extended periods
Ion cyclotron resonance (ICR)
Ions in a magnetic field circulate in a plane perpendicular to the direction of the field
Angular frequency of this motion  cyclotron frequency (wc)
Ion trapped in a magnetic field can absorb energy from an ac electric field
Frequency of field must match cyclotron frequency
Absorbed energy increases velocity of ion and radius without affecting wc

51. Fourier Transform (FT) MS: Detection of ICR
Ions circulating between plates induces current between plates
Image current
Non-destructive
Decays over a few seconds through collision
Decay over time of the image current after applying an RF pulse is transformed from the time domain into a frequency domain
Ions of two different m/q ratios excited on resonance for the same amount of time with the same excitation voltage. Ion [A] has the lower m/q ratio and thus has a higher cyclotron frequency. Ion [B] has the higher m/q ratio and thus a lower cyclotron frequency.