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22.1
Ch. 22 Mass Spectrometry (MS)
MS measures mass of atoms, molecules, or fragments of molecules
22-1. What is MS ?
Gaseous atom
from
condensed phase
ionized
Accelerated & separated
By mass to charge ratio
m/z
M=200: + 1 ion Æ m/z=200
+2 ion Æ m/z=100
• mass spectrum : detector response vs. m/z
Peak area ~ abundance of isotope
norminal mass: usual mass
Anal. Chem. by Prof. Myeong Hee Moon
22.2
22-1. What is MS ?
Magnetic Sector MS
• Uses magnetic field
: gas entering & ionization
at + ion mode
Æ acceleration by E. field
Æ expelled into analyzer tube
: high vacuum to reduce
collision with background gas
Æ magnet deflects ion toward D.
depending on B
Æ at electron multiplier detector
(105/electron)
Anal. Chem. by Prof. Myeong Hee Moon
22.3
How ions of different masses are separated by B ?
• Under electrical field
mass: m, charge: +ze
acceleration voltage: V
Kinetic E = potential E
1
mv 2 = zeV
2
v=
2zeV
m
• Under magnetic field
ion ze experiences a magnetic force Æ zevB
B deflects ion through a radius r
Centripetal force = magnetic force
2
mv
= zevB
r
zeBr
v=
m
zeBr
=
m
2zeV
m
m eB 2r 2
=
z
2V
If r fixed, then adjust B or V (~3kV)
Anal. Chem. by Prof. Myeong Hee Moon
22.4
22-1. What is MS ?
1. Electron Ionization (EI)
e- from hot filament. Accelerated through 70V.
M +
e- Æ
70eV
M+ ·
+
e-
+
e-
molecular ~55eV
0.1eV
ion
(1eV=96.5kJ/mol)
enough residual internal
energy (~1 eV) to break into
fragments
• for HCHO
70 eV is so large.
Remaining is used to break molecules
M+ (m/z 226) very rare, m/z 197,
Anal. Chem. by Prof. Myeong Hee Moon
22.5
22-1. What is MS ?
For HCHO, lower the energy Æ less fragmentation
Base peak: most intense peak
Anal. Chem. by Prof. Myeong Hee Moon
22.6
22-1. What is MS ?
2. Chemical Ionization (CI)
: less fragmentation than EI
: use reagent gas for ionization
– CH4, isobutane, ammonia, ~ 1 mbar
e- (100-200eV) convert CH4 Æ
CH4 + e − → CH4+• + 2e −
CH4+• + CH4 → CH5+ + • CH3
CH+4• → CH3+ + H•
CH3+ + CH4 → C2H5+ + H2
Proton donor ro analyte molecule
M + C2H5+ → MH+ + CH4
Anal. Chem. by Prof. Myeong Hee Moon
CH5+
22.7
22-1. What is MS ?
3. Resolving Power
R.P.=
m
m
or
Δm
m1/ 2
Higher, the better to separate two peaks
with similar mass
Anal. Chem. by Prof. Myeong Hee Moon
22.8
22-2 Oh, Mass Spectrum, Speak to Me
Each mass spectrum has a story to tell.
Nitrogen rule
: a compound has an odd # of N ÆM+ •
even #
1. Molecular ion & Isotope pattern
EI for aromatic compound
Æ M+ • usually Æ base peak
M+1 provides information on
elemental composition
: follow the natural abundance
of isotopes
Æ Table 22-1
Anal. Chem. by Prof. Myeong Hee Moon
odd nominal mass
even
22.9
Anal. Chem. by Prof. Myeong Hee Moon
22.10
22-2 Oh, Mass Spectrum, Speak to Me
• Intensity of M+1 : n x 1.08% + m x 0.012%
for CnHm
from 13C
from 2H
• For benzene: C6H6
M+• : m/z 78
m/z 79 : 6x1.08 + 6x0.012=6.55%
Check with Fig 22-6
• Cl, Br: nearly equal peaks at m/z 136, 138
Anal. Chem. by Prof. Myeong Hee Moon
22.11
22-2 Oh, Mass Spectrum, Speak to Me
2. High-Resolution mass spectrometry
C5H8O+ = 84.056 96 Da
C6H12+ = 84.093 35Da
With double focusing MS
Or accurate TOF MS
Resolve 0.001 at
m/z 100
m/Δm=4000
All with nominal
Mass=31
Anal. Chem. by Prof. Myeong Hee Moon
22.12
22-2 Oh, Mass Spectrum, Speak to Me
3. Identifying the molecular ion peak
How do we know m/z 100 Æ molecular ion ?
1)
M+• : highest m/z value but
5~20% intensity of the base peak
2) M+1, M+2 ratio
3) Heaviest fragment should not
correspond to improbable mass loss
from M+ •
loss of 3-14 or 21-25Da ? No way
CH3: 15, OH, NH3: 17
H2O: 18, C2H5: 29
OCH3:31, CH3CO:43
m/z 100, next highest m/z 85 (-15: CH3)
Anal. Chem. by Prof. Myeong Hee Moon
22.13
22-2 Oh, Mass Spectrum, Speak to Me
5. Interpreting fragmentation patterns
How many fragment ions from 2-hexanone ?
Anal. Chem. by Prof. Myeong Hee Moon
22.14
22-3 Types of Mass Spectrometers
1. Magnetic Sector MS -- Figure 22-2
Resolving power = ~0~1000, 0.1 at m/z 100
~ 105, 0.001 at m/z 100 (double focusing)
Double focusing: ion passes magnetic sector & electrostatic sector
Anal. Chem. by Prof. Myeong Hee Moon
22.15
22-3 Types of Mass Spectrometers
2. Transmission Quadrupole Mass Spectrometer
GC-Q: most common cheap analyzer,
vac ~10-9 bar by turbo molecular pump, Ion acceleration : 5-15V
E field deflects ion when
migration.
ÆAllows only one m/z
ion to reach detector
by applying constant V
& RF oscillating voltage
• Up to 4000m/z in 0.3 m/z
• constant Resolving power
: 100-101, 500-501
Anal. Chem. by Prof. Myeong Hee Moon
22.16
22-3 Types of Mass Spectrometers
3. Time of Flight MS (TOF-MS)
3000-20,000 times/s, 5kV to accelerate ions
kinetic energy of ions
KE =
1
mv 2
2
Same KE but diff.mass
Æ Different flight time
REFLECTRON SYSTEM : improved resolution
Anal. Chem. by Prof. Myeong Hee Moon
22.17
22-3 Types of Mass Spectrometers
• Reflectron system
: ions of different mass may have slight difference in KE
Æ may have same flight time. No good in resolution
at hollow rings with increasing positive potential,
- the last with more positive than accelerating potential : Repelling
ÆIons slow down, stopped and reflected back to left.
Æ all ions of same m/z – same flight time
: Rs = 1000~25,000 (accuracy ~0.001)
: but low pressure 10-12 bar
: high acquisition rate - 102~104 spectra/s to m/z 106
Anal. Chem. by Prof. Myeong Hee Moon
22.18
22-3 Types of Mass Spectrometers
4. Quadrupole Ion-Trap MS (TOF-MS)
: most widely used MS,
: interfaced with chromatography column
• constant DC-V, RF voltage to central ring electrode
Æ causes ion circulation
• increase amplitude of RF-voltage expels ions
of a particular m/x (unstable trajectory)
passing through exit holes Æ detection
• scanning m/z 10~650 can be 8 times/s
Resolving power = 1000~4000, m/z 0.1 acc.
max m/z ~ 20,000
• 10 times higher sensitivity than T-Quadrupole
Anal. Chem. by Prof. Myeong Hee Moon
22.19
22-4 Chromatography-Mass Spectrometry
GC: capillary GC column directly to MS
EI or CI used
LC : volatile mobile phase additives needed
spraying device by pneumatic or atmospheric pressure chemical ionization
1. Electrospray ionization (ESI) : for liquid sample
• ESI: pneumatically assisted electrospray
Anal. Chem. by Prof. Myeong Hee Moon
22.20
22-4 Chromatography-Mass Spectrometry
Electrospray ionization (ESI)
Nebulizer: 0V
Spray chamber: -3.5kV
: positive ion mode
Æ fine aerosol Æ vaporize
Æ ionization
MH+ (M+1)
M(NH4+) (M+18)
M(Na+) (M+23)
Anal. Chem. by Prof. Myeong Hee Moon
22.21
22-4 Chromatography-Mass Spectrometry
• fine droplets : ~1μm by solvent evaporization
repulsive force = cohesive force of surface tension.
• in ESI, LITTLE fragmentation of analyte Æ simple spectra
• for fragmentation ? Å collision induced dissociation (CID) between
capillary and skimmer cone
Anal. Chem. by Prof. Myeong Hee Moon
22.22
22-4 Chromatography-Mass Spectrometry
2. Atmospheric Pressure Chemical Ionization : APCI
: heat & coaxial flow N2 convert eluate into a fine mist
like CI, gas phase reaction creates new ions from gas phase reaction
between ions and molecules
: under High voltage to a metal needle,
Æ Corona (plasma containing charged particles) around needle.
Æ inject e- into aerosols Æ ionization Æ protonation
Anal. Chem. by Prof. Myeong Hee Moon
22.23
22-4 Chromatography-Mass Spectrometry
N2 + e − → N2+• + 2e −
M + e − → M− •
N2+• + 2N2 → N+4• + N2
N+4• + H2O → H2O +• + 2N2
H2O +• + H2O → H3O + + • OH
X − Y + e − → X• + Y −
Or
H3O + + nH2O → H3O + (H2O)n
+
+
H3O (H2O)n + M → MH + (n + 1)H2O
Y − + AH → A − + HY
Adv:
: Handle variety of analyte
: Upto ~ 2 mL/min
: but only singly charged ion
Disadv:
: Not good for macromolecules.
: Little fragmentation
Anal. Chem. by Prof. Myeong Hee Moon
22.24
22-4 Chromatography-Mass Spectrometry
3. Selected Ion Monitoring (SIM) & Selected Reaction Monitoring (SRM)
ÆTo increase selectivity & sensitivity of MS
by decreasing the response to everything else
Chromatogram (UV) of
Herbicide spiked in river water
Anal. Chem. by Prof. Myeong Hee Moon
TIC
22.25
22-4 Chromatography-Mass Spectrometry
In SIM, only a specific m/z is monitored.
S/N is greater
Anal. Chem. by Prof. Myeong Hee Moon
22.26
22-4 Chromatography-Mass Spectrometry
In SRM, triple quadrupole MS
Q1: select one ion
Q2: collision cell, N2 or Ar : ~10-8 ~ 10-6 bar
Q3: select only specific product ions to detector
Anal. Chem. by Prof. Myeong Hee Moon
22.27
22-4 Chromatography-Mass Spectrometry
ESI-tandem MS of pure estrogen EE2
By triple quadrupole MS
EE2: (17a-ethinylestradiol) : contraceptive
Ppt level Æ reproductive disturbance in fish
SRM chromatrogram showing the elution of
3.6 ngL of Human estrogens
(hormones in ovarian cycles) extracted from
in sewage water.
Signal : the sum of m/z 159 + 145 from Q3.
Anal. Chem. by Prof. Myeong Hee Moon
22.28
22-4 Chromatography-Mass Spectrometry
4. ESI of proteins : ESI well suited for charged macromolecules
• To find charge, m/z=mn
mn =
mass
M + n(1.008 ) M
=
= + 1.008
ch arg e
n
n
•The next peak at lower m/z
M + (n + 1)(1.008 )
M
m
=
=
+ 1.008
n
+
1
should have (n+1) protons
n +1
n +1
mm − 1.008
M/n
n +1
=
=
mn+1 − 1.008 M /(n + 1)
n
Charge:
n=
mm+1 − 1.008
mn − mm+1
• mass of neutral molecule
M = n × (mm+1 − 1.008 )
ESI-MS of cytochrome C
Anal. Chem. by Prof. Myeong Hee Moon
• Mass can be measured.
• Instrument calibration with
known mass proteins
22.29
22-4 Chromatography-Mass Spectrometry
Anal. Chem. by Prof. Myeong Hee Moon