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

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


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


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


• 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


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


• EI spectra

Excitation/relaxation causes fragment ions (daughter ions)


20.7


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)


20.8


• 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


20.9


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


• 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


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


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


• 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


20.14



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


MALDI spectrum from a nicotinic acid matrix irradiated with A 266-nm laser beam, 1990


20.16


• 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


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 compoundsproduces significant amount of molecular ions

(over 10,000 Mw)

20.19


20C. Mass Spectrometers

volatilizing solid or liq. Sample ---

convert to gaseous Ionization

just like grating in optical ins.

high vacuumneededWhy ?

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

10-4~10-5 torr

by syringe

20.21


• 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


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


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


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


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


• 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


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


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



20.30


• 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)


20.31


• 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


20.32


Time domain signal

Frequency domain

Mass domain

Expensive(superconducting magnet)

Resolution in FTMS > 106

precision of frequency measurements


20.33


20D. Applications of Molecular MS

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 ratiosratio of (M+1)+ & (M+2)+

20.35


• Structural information from fragmentation pattern

fragmentationpattern

fragmentation mechanismgeneral rule to interpret spectra

14 m/z – CH2 -- paraffinwater -- (M-18)+

alcohol – (M-CH2OH)+


20.36


• 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.


20.37


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



20.39


• most common Instruments– triple quadrupole MS (QQQ)


20.40


• Applications of MS/MSTandem MS is more sensitivebecause chemical noise is smaller but expensive

DrugsHormonesPheromonesAlkaloids DNA -- genomicsPeptides proteins -- proteomics


20.41


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


: 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


Nanoflow LC/MS interface for Ion Trap MS

20.44


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


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

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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