20.1
Ch.20 Molecular Mass Spectrometry
MS
elemental composition
molecular structure (inorganic, organic, biological)
qualitative-quantitative composition
structure & comp. of solid surface
isotopic ratio of atoms
1940s 1st Molecular MS
1950s commercialized
1980s big change in MS
ions from nonvolatile & labile molecules
applicable to biological molecuels
1990s explosive growth into Bio-MS
polypeptides
proteins
high MW biopolymers
by Prof. Myeong Hee Moon
20.2
20A. Molecular Mass Spectra
Largest
ion peak
EI of ethylbenzene
Molecular ion
Radical ion
(same MW)
C6H5CH2CH3 + eC6H5CH2CH3·++ 2eElectron bombardment
After excitation -- relaxation & produce fragmentation ion
C6H5CH2CH3·+
by Prof. Myeong Hee Moon
C6H5CH2+ + CH3·+
20.3
20B. Ion Sources
: gaseous analyte ions to be formed
• Ionization sources
Gas-phase sources: vaporization then ionization
: for thermally stable sample (bp < 500oC), MW<103 Da
Desorption sources: sample in solid or liquid state
--- directly converted to gaseous ions
: applicable to nonvolitile & thermally labile sample
by Prof. Myeong Hee Moon
20.4
• Classification of ion sources
1-decanol
Hard sources
: energy imparted to molecules
relaxation --- fragment ions
: info about functional group
Soft sources
: little fragmentation.
: Info about MW
by Prof. Myeong Hee Moon
20.5
20B-1. The Electron-Impact (EI) sources
Vaporization
(high T)
electron bombardment (~70V)
~5V
103~104V
M + e-
M·+ + 2e- : 1/106 ionization (low eff.)
Kinetic energy
1
KE  qV  zeV  mv 2
2
e: 1.6x10-19C
z=1
by Prof. Myeong Hee Moon
20.6
20B-1. The Electron-Impact (EI) sources
• EI spectra
Excitation/relaxation causes fragment ions (daughter ions)
by Prof. Myeong Hee Moon
20.7
20B-1. The Electron-Impact (EI) sources
EI of Methylene chloride (MC)
Base peak: - Cl
Base m/z=44
-CH2CHOH
1-pentanol
Isotope peaks
12C1H 35Cl (m=84)
2
2
13C1H 35Cl (m=85)
2
2
12C1H 35Cl37Cl (m=86)
2
13C1H 35Cl37Cl (m=87)
2
Collision product peaks
protonated molecular ion peak
: (M+1)+
2nd order reaction
Amount of (M+1)+ conc.
(partial pressure)
MW peak
: not always present
but very important
by Prof. Myeong Hee Moon
20.8
20B-1. The Electron-Impact (EI) sources
•
Advantage & disadvantages
Advantages
convenient
high ion currents
good sensitivities
extensive fragmentation
– unambiguous identification
Disadvantages
low molecular ion peak
volatilization of sample needed
- causes thermal degradation before ionization
remedy: location of heated probe close to entrance slit
low T volatilization using lower pressure
MW < 1000 Da
by Prof. Myeong Hee Moon
20.9
20B-2. Chemical Ionization (CI) sources
: 2nd most common
EI & CI : interchangeably operated in most instruments
gaseous atoms
positive or negative ions
collision with
(rare)
reagent gas ions
(from electron bombardment) CH4, propane, isobutane etc.
: modify EI area with adding vacuum pump (~1 torr) &
by reducing slit width to mass analyzer
ionization area (~ 1 torr)
analyzer (<10-5 torr)
: reagent gas reduced (103~104 higher than sample source)
CH4
CH4+, CH3+ (90%), few CH2+,
CH4+ + CH4
CH3+ + CH4
CH5+ + CH3
C2H5+ + CH3
by Prof. Myeong Hee Moon
20.10
20B-2. Chemical Ionization (CI) sources
•
In collision with sample MH
CH5+ + MH
C2H5+ + MH
C2H5+ + MH
MH2+ + CH4
(M+1)+
MH2+ + C2H4
M+ + C2H6
(M-1)+
H+ transfer
H- transfer
EI : rapid & extensive fragmentation
Fig. 20-2a
CI : CI spectra provides
(M+1)+ or (M-1)+ peaks
by addition or subtraction of H under reagent ion
by Prof. Myeong Hee Moon
20.11
20B-3 Field Ionization Sources and Spectra
•
Ion formed under a large E field (108 V/cm)
10~20 kV total. applied to emitters having fine tips (d<1mm)
or carbon microtips
carbon dendrites at surface of W wires
by pyrolysis of benzonitrile
Ionization occurs via a
quantum mechanical tunneling mechanism
in which e- from analyte are
extracted by microtips at the anode
Limitation : sensitivity (one order less)
by Prof. Myeong Hee Moon
20.12
20B-4. Field Desorption
•
•
EI & CI: based on ionizing agents acting on gaseous sample
but for nonvolatile or thermally unstable samples (bio)--?
Desorption ionization methods (recently 1980s)
: volatilization then ionization
simple in MS spectrum .. molecular ion or
protonated molecular ion
• Field desorption sources
similar to field ionization
- probe coated with a solution of sample
- heat apply to emitter
(thermal degradation)
- but simpler than field ionization (see Fig 20-6)
by Prof. Myeong Hee Moon
20.13
20B-4. Field Desorption
• Matrix-Assisted Laser Desorption/Ionization (MALDI)
MALDI - good for accurate
MW of polar biopolymers
1988 by two groups
(German & Jap)
In German group.
sample (in aq. alcohol) mixed
with matrix (Table 20-4)
- evaporated on the surface of
metallic probe
- laser pulse causes
sublimation of analyte into
ions & introduces sample ions
to TOF
by Prof. Myeong Hee Moon
20.14
20B-4. Field Desorption
Matrix : nicotinic acid absorbs at 266nm (from laser)
Spectrum : multiply charged ions
low background noise
complete absence of fragmentation
• Mechanism of MALDI is not completely clear
But requires
1. matrix compd must absorb laser strongly
2. “
“
- soluble in solvent
3. analyte should not absorb laser radiation (fragmentation)
by Prof. Myeong Hee Moon
20.15
20B-4. Field Desorption
MALDI spectrum from a nicotinic acid matrix irradiated with
A 266-nm laser beam, 1990
by Prof. Myeong Hee Moon
20.16
• Electrospray Ionization (ESI)
1984. ESI/MS. Most important for biomolecules
even inorganic & synthetic polymers
~kV
by Prof. Myeong Hee Moon
Advantages
• useful for thermally fragile
biomolecules
(little fragmentation)
• multiply charged ions.
m/z within 1500 or less at Q
• direct introduction of sample
from HPLC or CE columns
20.17
by Prof. Myeong Hee Moon
20.18
• Fast Atom Bombardment (FAB)
FAB had a major role in MS for polar high MW species
: sample in a condensed state (in a glycerol solution matrix)
are ionized by bombarding with Xe or Ar atoms
1. very rapid heating of sample (reduce fragmentation)
liquid matrix – healing effect
(reduce lattice energy)
: healing the damage by bombardment.
2. acceleration of Ar or Xe by ion gun
FAB of organic or biochemical compounds
produces significant amount of molecular ions
(over 10,000 Mw)
by Prof. Myeong Hee Moon
20.19
20C. Mass Spectrometers
volatilizing solid or
liq. Sample --convert to gaseous
just like grating in
optical ins.
Ionization
high vacuum
needed
Why ?
by Prof. Myeong Hee Moon
20.20
20C-2. Sample Inlet Systems
Devices to put sample into ion source with minimal loss of vacuum
batch, direct probe, chromatographic, CE
• Batch inlet systems
by syringe
10-4~10-5 torr
by Prof. Myeong Hee Moon
20.21
20C-2. Sample Inlet Systems
•
Direct probe inlet
solid or nonvolatile liquid by using sample holder or probe
inserted into vacuum lock
• Chromatographic & CE inlet
On-line coupling with MS
Sections 27D-3, 28C-6, 30B-4
by Prof. Myeong Hee Moon
20.22
20C-3. Mass Analyzers
•
Ideal performance
: resolution – detect small difference in mass
: analyzer – should allow passage of a sufficient number of ions
to yield readily measurable ion currents
• Resolution of MS
R
m
m
m: mass difference between
two adjacent peaks
In case, R=4000
distinguish m/z =400.0 & 400.1
or m/z=40.00 & 40.01
Commercial instrument : 500~above 1,000,000
by Prof. Myeong Hee Moon
20.23
1) Magnetic Sector Analyzer (classic)
needs permanent magnet
or electromagnet
KE of ions
1
KE  ZeV  mv 2
2
V: voltage between A & B
e: 1.60x10-19C
All ions leaving the slit at app. same KE
Heavier ions travel at lower velocity
by Prof. Myeong Hee Moon
20.24
1) Magnetic Sector Analyzer (classic)
Magnetic force, FM
Centripetal force, FC
FM  BzeV
mv 2
FC 
r
B: magnetic field strength
r: radius of curvature
In order for an ion to traverse the circular path to the collector
FM=FC
m B2r 2e

z
2V
Vary one of B, V, r while holding two others.
: Modern MS - ion sorting
by holding V & r, vary B (by varying current in magnet)
: In case of photographic recording
by holding B & V, vary r.
by Prof. Myeong Hee Moon
20.25
2) Quadrupole MS
- less expensive, more rugged than magnetic sector
- compact, bench top
- low scan times (<100ms) : good in case of chromatographic
- most common (see section 11B-2)
by Prof. Myeong Hee Moon
20.26
3) Time of Flight (TOF) MS
See section 11B-3
In TOF-MS, ion acceleration into
field-free drift tube by
E pulse of 103~104V.
•
Advantages of TOF
Simplicity, Ruggedness
Ease of accessibility of ion source
Virtually unlimited mass range but limited resolution & sensitivity
by Prof. Myeong Hee Moon
20.27
4) Ion Trap analyzers (or Ion trap MS)
•
Ion trap : a device in which gaseous anions or cations can be
confined for extended periods by electric and/or magnetic fields
Conventional type
Ion cyclotron resonance trap
Principle: ions of certain m/z
circulate in a stable orbit within
the trap
When V increased, orbits of
heavier ions become stable
(lighter, unstable ions hit wall of
ring electrode– leave trap through
radio frequency
openings in the lower end cap)
voltage
by Prof. Myeong Hee Moon
20.28
Advantages of Ion trap
: rugged, compact, less expensive
500~1000 Da mass range
--- improved with ICRMS
20C-4. Fourier Transform (FT) Instruments
FTMS -- 1980s, it provides improved S/N
greater speed
higher sensitivity & resolution
FT-ICR MS
by Prof. Myeong Hee Moon
20.29
20C-4. Fourier Transform (FT) Instruments
by Prof. Myeong Hee Moon
20.30
20C-4. Fourier Transform (FT) Instruments
•
ICR phenomenon
: when gaseous ion drifts into a strong magnetic field
motions become circular but perpendicular to the field direction
c: angular frequency or cyclotron frequency

v zeB

r
m

velocity increase
increase in rotation radius of ions
1
m/z
Frequency in radians/s
If frequency of Electric field matches with c, trapped ions absorb energy
from AC electric field. Absorbed E increases the velocity & r
without disturbing c.
When AC field terminates, radius becomes constant.
----- Then, coherent motion of ensemble of ions of
same m/z at a given AC field.
(other m/z ions are not affected)
by Prof. Myeong Hee Moon
20.31
20C-4. Fourier Transform (FT) Instruments
•
Measurement of ICR signal
: coherent circular motion of resonant ions create image current
observed after termination of freq.sweep signal
(current decays with time)
Frequency of current
m/z

Decay pattern creates
time domain FT signal
• FT Spectrometers
Ions trapped in cell
Apply short Rf pulse
Image current amplification
digitization
by Prof. Myeong Hee Moon
20.32
20C-4. Fourier Transform (FT) Instruments
Time domain signal
Frequency domain
Expensive
(superconducting magnet)
Resolution in FTMS > 106
precision of
frequency measurements

Mass domain
by Prof. Myeong Hee Moon
20.33
20D. Applications of Molecular MS
by Prof. Myeong Hee Moon
20.34
20D-1. Identification of Pure Compounds
•
MW from MS
identification of molecular ion peaks
or (M+1)+, or (M-1)+
(except EI)
•
Molecular formula from Exact MW
ex) purine C5H4N4 (m=120.044)
benzamidine C7H8N2 (m=120.069)
acetophenone C8H9O (m=120.058)
In case, measured mass of 120.070 (+0.005)
only C7H8N2 is close.
• Molecular formulas from isotope ratios
ratio of (M+1)+ & (M+2)+
by Prof. Myeong Hee Moon
20.35
20D-1. Identification of Pure Compounds
• Structural information from fragmentation pattern
fragmentation
pattern
fragmentation mechanism
general rule to interpret spectra
14 m/z – CH2 -- paraffin
water -- (M-18)+
alcohol – (M-CH2OH)+
by Prof. Myeong Hee Moon
20.36
20D-1. Identification of Pure Compounds
•
Compound identification from comparison of spectra
: check with possible suspect molecules
and compare mass fragmentation
Modern Technique --- Library search
largest : John Wiley & Sons (>150,000 spectra)
use PC (PBM-STIRS)
small libraries – small number but similar group
: pesticides, drugs, forensics.
by Prof. Myeong Hee Moon
20D-2. Analysis of Mixtures
by Hyphenated MS methods
20.37
coupling with separation devices
• Chromatography/MS
GC/MS – most powerful
elution of gaseous sample --- sect.27D-3
LC/MS – for nonvolatile
--- sect.28C-6
CE/MS – for biopolymers
--- sect 30B-4
• Tandem Mass Spectrometry (or MSMS)
Coupling of one MS with second MS
First MS --- isolate the molecular ions from mixture
Second MS – fragmentation
in a chamber, He is filled (10-3 or 10-4 torr)
collisions bet. Fast moving parent ions and He
fragmentation scanned by second spectrometer
by Prof. Myeong Hee Moon
20D-2. Analysis of Mixtures
by Hyphenated MS methods
by Prof. Myeong Hee Moon
20.38
20D-2. Analysis of Mixtures
by Hyphenated MS methods
20.39
• most common Instruments– triple quadrupole MS (QQQ)
by Prof. Myeong Hee Moon
20D-2. Analysis of Mixtures
by Hyphenated MS methods
• Applications of MS/MS
Tandem MS is more sensitive
because chemical noise is smaller but expensive
Drugs
Hormones
Pheromones
Alkaloids DNA -- genomics
Peptides proteins -- proteomics
by Prof. Myeong Hee Moon
20.40
20.41
Protein Identification Scheme in Shotgun Approach (NanoLC-MSMS)
Sample : Rat Liver Cell Lysates
1: TOF MS ES+
nanoLC
BPI
2.87e3
chromatogram
~1.0g injection
NP3_P1_1_lab MS
Lv_1_0422_T08
48.49
100
83.07
49.56
88.89
67.43 70.72
110.16
104.77
59.01
%
75.96
36.97 39.76
36.54
100.03
114.86 126.55
143.36
127.19
140.98
33.56
0
40.00
60.00
80.00
100.00
120.00
1st MS
140.00
NP3_P1_1_lab MS
NP3_P1_1_lab MS
1: TOF
nd MS ES+
217
2
Lv_1_0422_T08 1082 (72.953)
766.88
768.38
723.34
100
MS
Lv_1_0422_T08 402 (73.217)
100
%
326.22
213.13
988.55
1063.57
%
1064.57
543.29
0
2: TOF MSMS 766.87ES+
GILAADESVGTMGNR
49
Fructose-bisphosphate aldolase B
397.26
635.34
635.28
547.31
821.41 950.45 1136.57
1321.75
m/z
200
Time
160.00
MSMS spectrum of m/z=766.88
Mass Spectrum at 72.92min
189.13
TIME (min)
145.67
400
600
800
1000
1200
1400
1600
m/z
0
m/z
250
500
750
1000
1250
1500
1750
m/z
by Prof. Myeong Hee Moon
Direct Interface between Nanoflow HPLC
and ESI-MS & On-line Sample Clean-up
20.42
on-off valve
for vent
Fritless pulled tip column
C18-5m-100A, 75m x 15cm
200nL/min.
Sample
trapping
column
C18-5m-200A
75m x 1.5cm
Pt lead
for electrical contact (2.0~2.5kV)
Mass Spectrometer
: Minimizes post-column band broadening
: Improves electrospray efficiency by using a low flow rate <250nL/min.
: On-line sample clean-up & minimization of dead volume between sample trap and anal. column (20nL)
by Prof. Myeong Hee Moon
20.43
Nanoflow LC/MS interface for Ion Trap MS
by Prof. Myeong Hee Moon
20.44
Shotgun Proteomics
: Shotgun Identification of Proteins in Mixture
Protein mixture
Peptide mixture
Digestion
HPLC
protease
Collision Cell
(tandem MS)
MS/MS
Database
Search
Tandem MS
P1
MS-1
P2
P3
P4
He
gas
MS-2
P5
F1 F2 F3 F4 F5
HPLC
ES Source
Select for a
particular ion
(peptide)
Input: peptides from
enzymatic digest
by Prof. Myeong Hee Moon
Detector
Output: fragments
from daughter ions
CID (Collision Induced Dissociation) Pattern
of a Tryptic Peptide
b1
b2
b3
b4
b5
b6
b series
114.1 261.2 348.2 476.3 575.3 632.3
ions
L F S Q V G K
[LFSQVGK+H]+
=778.4 Da
665.4 518.3 431.3 303.2 204.1 147.1
y6
K G
y5
y4
V
CID
spectrum
y3
y2
y1
y series ions
S
Q
b2
F
y5
y6
b4
y1
y2
by Prof. Myeong Hee Moon
y3
b3
b5
y4
b6
m/z
20.45