ch.20 molecular mass spectrometrychem.yonsei.ac.kr/~mhmoon/pdf/insanal/ch20.pdf · 20.1 by prof....
TRANSCRIPT
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
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
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
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
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
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)
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)
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
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)
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
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
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)
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.
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
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
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
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)+
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
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
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
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.
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
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