ch.20 molecular mass spectrometrychem.yonsei.ac.kr/~mhmoon/pdf/insanal/ch20.pdf · 20.1 by prof....
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![Page 1: Ch.20 Molecular Mass Spectrometrychem.yonsei.ac.kr/~mhmoon/pdf/InsAnal/Ch20.pdf · 20.1 by Prof. Myeong Hee Moon Ch.20 Molecular Mass Spectrometry elemental composition molecular](https://reader038.vdocuments.net/reader038/viewer/2022110112/5aeb28667f8b9ae5318d5f92/html5/thumbnails/1.jpg)
20.1
by Prof. Myeong Hee Moon
Ch.20 Molecular Mass Spectrometry
elemental compositionmolecular structure (inorganic, organic, biological)
MS qualitative-quantitative compositionstructure & comp. of solid surfaceisotopic ratio of atoms
1940s 1st Molecular MS1950s commercialized1980s big change in MS
ions from nonvolatile & labile moleculesapplicable to biological molecuels
1990s explosive growth into Bio-MSpolypeptidesproteinshigh MW biopolymers
20.2
by Prof. Myeong Hee Moon
20A. Molecular Mass Spectra
C6H5CH2CH3 + e- C6H5CH2CH3·++ 2e-Electron bombardment
Molecular ionRadical ion(same MW)
After excitation -- relaxation & produce fragmentation ion
C6H5CH2CH3·+ C6H5CH2+ + CH3·+
Largest ion peak
EI of ethylbenzene
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20.3
by Prof. Myeong Hee Moon
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
20.4
by Prof. Myeong Hee Moon
• Classification of ion sources
Hard sources: energy imparted to molecules
relaxation --- fragment ions : info about functional group
Soft sources: little fragmentation. : Info about MW
1-decanol
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20.5
by Prof. Myeong Hee Moon
20B-1. The Electron-Impact (EI) sources
Vaporization electron bombardment (~70V)
(high T)
M + e- M·+ + 2e- : 1/106 ionization (low eff.)
~5V
103~104V
Kinetic energy
2mv2
1 zeV qV KE
e: 1.6x10-19Cz=1
20.6
by Prof. Myeong Hee Moon
• EI spectra
Excitation/relaxation causes fragment ions (daughter ions)
20B-1. The Electron-Impact (EI) sources
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20.7
by Prof. Myeong Hee Moon
EI of Methylene chloride (MC)Base peak: - Cl
1-pentanol
MW peak : not always present
but very important
Base m/z=44 -CH2CHOH
Isotope peaks12C1H2
35Cl2 (m=84)13C1H2
35Cl2 (m=85)12C1H2
35Cl37Cl (m=86)13C1H2
35Cl37Cl (m=87)
Collision product peaksprotonated molecular ion peak: (M+1)+
2nd order reactionAmount of (M+1)+ conc.
(partial pressure)
20B-1. The Electron-Impact (EI) sources
20.8
by Prof. Myeong Hee Moon
• Advantage & disadvantages
convenienthigh ion currentsgood sensitivitiesextensive fragmentation
– unambiguous identificationDisadvantages
low molecular ion peakvolatilization of sample needed- causes thermal degradation before ionization
remedy: location of heated probe close to entrance slitlow T volatilization using lower pressure
MW < 1000 Da
Advantages
20B-1. The Electron-Impact (EI) sources
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20.9
by Prof. Myeong Hee Moon
20B-2. Chemical Ionization (CI) sources
EI & CI : interchangeably operated in most instruments
gaseous atoms positive or negative ionscollision 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 analyzerionization area (~ 1 torr)analyzer (<10-5 torr)
: reagent gas reduced (103~104 higher than sample source)
CH4 CH4+, CH3
+ (90%), few CH2+,
CH4+ + CH4 CH5
+ + CH3
CH3+ + CH4 C2H5
+ + CH3
: 2nd most common
20.10
by Prof. Myeong Hee Moon
• In collision with sample MH
CH5+ + MH MH2
+ + CH4
(M+1)+
C2H5+ + MH MH2
+ + C2H4
C2H5+ + MH M+ + C2H6
(M-1)+
H+ transfer
H- transfer
EI : rapid & extensive fragmentationFig. 20-2a
CI : CI spectra provides(M+1)+ or (M-1)+ peaksby addition or subtraction of H under reagent ion
20B-2. Chemical Ionization (CI) sources
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20.11
by Prof. Myeong Hee Moon
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 wiresby pyrolysis of benzonitrile
Ionization occurs via a quantum mechanical tunneling mechanismin which e- from analyte areextracted by microtips at the anode
Limitation : sensitivity (one order less)
20.12
by Prof. Myeong Hee Moon
20B-4. Field Desorption
• EI & CI: based on ionizing agents acting on gaseous samplebut for nonvolatile or thermally unstable samples (bio)--?
• Desorption ionization methods (recently 1980s): volatilization then ionization
simple in MS spectrum .. molecular ion orprotonated molecular ion
• Field desorption sourcessimilar 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)
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20.13
by Prof. Myeong Hee Moon
• Matrix-Assisted Laser Desorption/Ionization (MALDI)MALDI - good for accurateMW 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
20B-4. Field Desorption
20.14
by Prof. Myeong Hee Moon
20B-4. Field Desorption
Matrix : nicotinic acid absorbs at 266nm (from laser)
Spectrum : multiply charged ionslow background noisecomplete 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)
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20.15
by Prof. Myeong Hee Moon
MALDI spectrum from a nicotinic acid matrix irradiated with A 266-nm laser beam, 1990
20B-4. Field Desorption
20.16
by Prof. Myeong Hee Moon
• Electrospray Ionization (ESI)
1984. ESI/MS. Most important for biomolecules
even inorganic & synthetic polymers
~kVAdvantages• useful for thermally fragile
biomolecules(little fragmentation)
• multiply charged ions.m/z within 1500 or less at Q
• direct introduction of samplefrom HPLC or CE columns
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20.17
by Prof. Myeong Hee Moon
20.18
by Prof. Myeong Hee Moon
• 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 compoundsproduces significant amount of molecular ions
(over 10,000 Mw)
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20.19
by Prof. Myeong Hee Moon
20C. Mass Spectrometers
volatilizing solid or liq. Sample ---
convert to gaseous Ionization
just like grating in optical ins.
high vacuumneededWhy ?
20.20
by Prof. Myeong Hee Moon
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
10-4~10-5 torr
by syringe
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20.21
by Prof. Myeong Hee Moon
• Chromatographic & CE inlet
On-line coupling with MSSections 27D-3, 28C-6, 30B-4
• Direct probe inlet
solid or nonvolatile liquid by using sample holder or probe
inserted into vacuum lock
20C-2. Sample Inlet Systems
20.22
by Prof. Myeong Hee Moon
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
m
mR
m: mass difference between
two adjacent peaks
In case, R=4000 distinguish m/z =400.0 & 400.1or m/z=40.00 & 40.01
Commercial instrument : 500~above 1,000,000
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20.23
by Prof. Myeong Hee Moon
1) Magnetic Sector Analyzer (classic)
KE of ions
2
2
1mvZeVKE
All ions leaving the slit at app. same KEHeavier ions travel at lower velocity
needs permanent magnetor electromagnet
V: voltage between A & Be: 1.60x10-19C
20.24
by Prof. Myeong Hee Moon
Magnetic force, FM BzeVFM B: magnetic field strength
Centripetal force, FC
r
mvFC
2 r: radius of curvature
FM=FC
In order for an ion to traverse the circular path to the collector
V
erB
z
m
2
22
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 recordingby holding B & V, vary r.
1) Magnetic Sector Analyzer (classic)
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20.25
by Prof. Myeong Hee Moon
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)
20.26
by Prof. Myeong Hee Moon
• Advantages of TOF
Simplicity, RuggednessEase of accessibility of ion sourceVirtually unlimited mass range but limited resolution & sensitivity
3) Time of Flight (TOF) MS
See section 11B-3
In TOF-MS, ion acceleration intofield-free drift tube byE pulse of 103~104V.
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20.27
by Prof. Myeong Hee Moon
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 typeIon cyclotron resonance trap
radio frequencyvoltage
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 ofring electrode– leave trap throughopenings in the lower end cap)
20.28
by Prof. Myeong Hee Moon
Advantages of Ion trap
: rugged, compact, less expensive500~1000 Da mass range
--- improved with ICRMS
20C-4. Fourier Transform (FT) Instruments
FTMS -- 1980s, it provides improved S/Ngreater speedhigher sensitivity & resolution
FT-ICR MS
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20.29
by Prof. Myeong Hee Moon
20C-4. Fourier Transform (FT) Instruments
20.30
by Prof. Myeong Hee Moon
• ICR phenomenon
: when gaseous ion drifts into a strong magnetic field
motions become circular but perpendicular to the field directionc: angular frequency or cyclotron frequency
m/z
1
m
zeB
r
v Frequency in radians/s
velocity increase increase in rotation radius of ions
If frequency of Electric field matches with c, trapped ions absorb energy from AC electric field. Absorbed E increases the velocity & rwithout 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)
20C-4. Fourier Transform (FT) Instruments
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20.31
by Prof. Myeong Hee Moon
• Measurement of ICR signal
Decay pattern createstime domain FT signal
• FT Spectrometers
Ions trapped in cell
Apply short Rf pulse
Image current amplificationdigitization
: coherent circular motion of resonant ions create image currentobserved after termination of freq.sweep signal(current decays with time)
Frequency of current m/z
20C-4. Fourier Transform (FT) Instruments
20.32
by Prof. Myeong Hee Moon
Time domain signal
Frequency domain
Mass domain
Expensive(superconducting magnet)
Resolution in FTMS > 106
precision of frequency measurements
20C-4. Fourier Transform (FT) Instruments
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20.33
by Prof. Myeong Hee Moon
20D. Applications of Molecular MS
20.34
by Prof. Myeong Hee Moon
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 ratiosratio of (M+1)+ & (M+2)+
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20.35
by Prof. Myeong Hee Moon
• Structural information from fragmentation pattern
fragmentationpattern
fragmentation mechanismgeneral rule to interpret spectra
14 m/z – CH2 -- paraffinwater -- (M-18)+
alcohol – (M-CH2OH)+
20D-1. Identification of Pure Compounds
20.36
by Prof. Myeong Hee Moon
• Compound identification from comparison of spectra
: check with possible suspect moleculesand 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.
20D-1. Identification of Pure Compounds
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20.37
by Prof. Myeong Hee Moon
20D-2. Analysis of Mixtures by Hyphenated MS methods
coupling with separation devices
• Chromatography/MS
GC/MS – most powerfulelution of gaseous sample --- sect.27D-3
LC/MS – for nonvolatile --- sect.28C-6CE/MS – for biopolymers --- sect 30B-4
• Tandem Mass Spectrometry (or MSMS)
Coupling of one MS with second MSFirst MS --- isolate the molecular ions from mixtureSecond MS – fragmentation
in a chamber, He is filled (10-3 or 10-4 torr)collisions bet. Fast moving parent ions and Hefragmentation scanned by second spectrometer
20.38
by Prof. Myeong Hee Moon
20D-2. Analysis of Mixtures by Hyphenated MS methods
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20.39
by Prof. Myeong Hee Moon
• most common Instruments– triple quadrupole MS (QQQ)
20D-2. Analysis of Mixtures by Hyphenated MS methods
20.40
by Prof. Myeong Hee Moon
• Applications of MS/MSTandem MS is more sensitivebecause chemical noise is smaller but expensive
DrugsHormonesPheromonesAlkaloids DNA -- genomicsPeptides proteins -- proteomics
20D-2. Analysis of Mixtures by Hyphenated MS methods
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20.41
by Prof. Myeong Hee Moon
NP3_P1_1_lab MS
40.00 60.00 80.00 100.00 120.00 140.00 160.00Time0
100
%
Lv_1_0422_T08 1: TOF MS ES+ BPI
2.87e348.49
39.7636.97
36.54
33.56
83.0749.56
67.43
59.01
70.72
75.96
88.89
110.16
104.77
100.03
126.55114.86143.36127.19
140.98145.67 TIME (min)
nanoLC chromatogram
1st MS
Protein Identification Scheme in Shotgun Approach (NanoLC-MSMS)
~1.0g injection
NP3_P1_1_lab MS
250 500 750 1000 1250 1500 1750m/z0
100
%
Lv_1_0422_T08 402 (73.217) 2: TOF MSMS 766.87ES+ 49326.22
213.13397.26
635.34
635.28821.41 950.45 1136.57
1321.75
Mass Spectrum at 72.92minNP3_P1_1_lab MS
200 400 600 800 1000 1200 1400 1600m/z0
100
%
Lv_1_0422_T08 1082 (72.953) 1: TOF MS ES+ 217766.88
723.34
547.31
189.13 543.29
768.38
988.55
1063.57
1064.57
MSMS spectrum of m/z=766.88
2nd MS
m/z m/z
GILAADESVGTMGNRFructose-bisphosphate aldolase B
Sample : Rat Liver Cell Lysates
20.42
by Prof. Myeong Hee Moon
: 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)
Fritless pulled tip columnC18-5m-100A, 75m x 15cm
Sampletrappingcolumn
C18-5m-200A75m x 1.5cm
Pt leadfor electrical contact (2.0~2.5kV)
on-off valve for vent
Mass Spectrometer
Direct Interface between Nanoflow HPLC and ESI-MS & On-line Sample Clean-up
200nL/min.
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20.43
by Prof. Myeong Hee Moon
Nanoflow LC/MS interface for Ion Trap MS
20.44
by Prof. Myeong Hee Moon
Shotgun Proteomics
: Shotgun Identification of Proteins in Mixture
Digestion
protease
Protein mixture Peptide mixture
HPLC MS/MSDatabaseSearch
(tandem MS)
ES Source
MS-1 MS-2
Collision Cell
DetectorInput: peptides from enzymatic digest
Select for a particular ion
(peptide)
Hegas
F1 F5F4F3F2
Output: fragmentsfrom daughter ions
P1
P2
P3
P4
P5
HPLC
Tandem MS
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20.45
by Prof. Myeong Hee Moon
CID (Collision Induced Dissociation) Patternof a Tryptic Peptide
L F S Q V G Kb series ions
y series ions
b1
114.1b2
261.2b3
348.2b4
476.3b5
575.3b6
632.3
665.4y6
518.3y5
431.3y4
303.2y3
204.1y2
147.1y1
m/z
[LFSQVGK+H]+
=778.4 Da
b2
b3
b4b5
b6
y6
y5
y4y3y2
y1
K G V Q S FCIDspectrum