Mass Spectrometry for Chemists and · PDF fileMass Spectrometry for Chemists and Biochemists...

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Instrumental Analysis - Module 2 -Lecture 1

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Mass Spectrometry for Chemists and Biochemists

Spiros A. PergantisAssistant Professor of Analytical ChemistryDepartment of ChemistryUniversity of Crete

[email protected]. +30 2810 545084

Office Number: A206

Erasmus Intensive Program SYNAPS

Univ. of Crete - Summer 2007


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Module OutlineWhat is Mass Spectrometry (MS) and what is it used for ?

Ionization Sources

Analyzers – Detectors

Modes of fragmentation in MS

Applications: Identification of organic compounds, proteomics, environmental analysis.

Hyphenated techniques (LC-MS and GC-MS)

Quantitation with MS

Problem Set

Mon

Tue

Wed

Thu

Fri

Take home

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Course Material

Lecture slides (pdf format) will be available on SYNAPS website and hardcopies will be handed out before each lecture

Chapter 9.4 Analytical Mass Spectrometry from the textbook: Analytical Chemistry, Edited by R. Kellner, J.-M. Mermet, M. Otto, H.M. Widmer; Wiley-VCH, ISBN 3-527-28881-3


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What can we do with mass spectrometry?1. Measure mass better than any other technique.

2. Obtain information about chemical structures.

What are mass measurements good for?To identify and quantitate, also help characterize:

metabolites, recombinant proteins, proteins isolated from natural sources, oligonucleotides, drug candidates and their metabolites, peptides, synthetic organic chemicals, polymers, natural products, organometallics

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Pharmaceutical analysisBioavailability studiesDrug metabolism studies, pharmacokineticsCharacterization of potential drugsDrug degradation product analysisScreening of drug candidatesIdentifying drug targets

Biomolecule characterizationProteins and peptidesOligonucleotides

Environmental analysisPesticides on foodsSoil and groundwater contamination

Forensic analysis/clinical

Some Applications of MS:


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m/zInte

nsity

Ionization Source

Mass Analyzer

Detector

Mass Spectrum

+m/z+

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Ionization SourcesElectrospray Ionization (ESI)

Nano Electrospray Ionization (NanoESI)Atmospheric Pressure Chemical Ionization (APCI)

Atmospheric Pressure Photo Ionization (APPI)Matrix-Assisted Laser Desoprtion/Ionization (MALDI)

Desorption/Ionization on Silicon (DIOS)Fast Atom/Ion Bombardment (FAB)

Electron Ionization (EI)Chemical Ionization (CI)

Mass AnalyzersQuadrupoles

Quadrupole Ion TrapLinear Ion Trap

Ion Trap LimitationsMagnetic Sector

Quadrupole Time-of-Flight Tandem MSThe MALDI with Time-of-Flight Analysis

Quadrupole Time-of-Flight MSFourier Transform Mass Spectrometry (FTMS)

DetectorsElectron Multiplier

Faraday CupPhotomultiplier Conversion Dynode

Array DetectorCharge(or Inductive) Detector

m/zInte

nsity

Ionization Source

Mass Analyzer

Detector

Mass Spectrum


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Electron Ionization Mass Spectrometry

http://www-chem.harvard.edu/mass/tutorials/eimovie.html

+

++

+

++

+

++

++

+

++

+

++

+Repeller (+V)

Collector (+V)

To mass analyzer

Extraction Plate (-V)

Filament70 V

10-6 Torr

N

S

5


9



10


6


11

Cl

Cl

NHO

N

CH3

CH3


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Advantages• Well-Established• Fragmentation Libraries*• No Suppression• Insoluble compounds• Interface to GC• Non-Polar Samples

Disadvantages• Molecular ion not always

present• Need Volatile Sample• Need Thermal Stability• No Interface to LC• Low Mass Compounds

(approx. 500 amu)

(low picomole)

* National Institute of Science and Technology (NIST) database (>100,000 compounds) available to compare fragmentation data


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Chemical Ionization Mass Spectrometry

+

++

++

+

++

+Repeller (+V)

Collector (+V)

To mass analyzer

Extraction Plate (-V)

Filament70 V

0.3 – 1 Torr

N

SReagent gas / analyte

1000 / 1

Chemical Ionization Mass Spectrometry

i) Acid – Base reactions

M + XH+ (reagent gas ions) → MH+ (protonated molecule) + X (reagent gas)

ii) Complex formation (electrophilic addition)

M + XH+ (reagent gas ions) → MXH+ (pseudomolecular ion)

iii) Charge exchange reaction

M + G+. (reagent gas radical ions) → M+. (pseudomolecular ion) + G (reagent gas)

Electron capture (highly selective)

M + e- → M-.

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Reagent gases commonly used for CI - MS: methane (CH4), ammonia (NH3), isobutane (i-C4H10)noble gases for the charge transfer reactions.

Methane as ionizing gas

CH4 + e- → 2 e- + CH4+.

CH3+ + H.

CH2.+ + H2

CH4 + CH4+. → CH5

+ + CH3.

CH4 + CH3+ → C2H5

+ + H2

CH4 + C2H3+ → C3H5

+ + H2

47%

41%

6%

CH4 + CH2.+ → C2H3

+ + H2 + H.


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M2-M1=28 amuM3-M1=40 amuM3-M2=12 amu

⇓M1-1=MW

CH5+ + M --> MH+ + CH4 [M+1]+

C2H5+ + M --> MH+ + C2H4

C2H5+ + M --> [M C2H5]+

C3H5+ + M --> [M C3H5]+

[M+1]+

[M+29]+

[M+41]+

Pseudomolecular ions in Methane CI

MW+1

MW+29

MW+41

0

5

10

15

20

25

m/z

rela

tive

abun

danc

e (%

)

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NH3 (EI ionization) NH3+•

NH3 + NH3+• NH4+ + NH2•

NH3 + NH4+ (NH 3)2H+

The reagent ions react with the molecules like Brønsted or like Lewis acids:

NH4+ + M MH+ + NH3 Mr+1

NH4+ + M --> [M NH4]+ Mr+18

Ammonia as ionizing gas

Isobutane chemical ionization

Negative chemical ionization

C4H9+ + M --> MH+ ([m/z]+1) or/and [M C4H9]+ ([m/z]+57)

C3H3+ + M --> MH+ ([m/z]+1) or/and [M C3H3]+ ([m/z]+39)

The reagent gases in negative CI

CH3O- + MH --> M- + CH3OH ([m/z]-1)

e- + R-Cl --> Cl- + R•

Cl- + R-Cl --> [RCl2]- ([m/z]+35)

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Chemical Ionization

Advantages• Parent Ion• Interface to GC• Insoluble Samples

Disadvantages• No Fragment Library• Need Volatile Sample• Need Thermal Stability• Quantitation Difficult• Low Mass Compounds

(<1000 amu)• Solids Probe Requires

Skilled Operator

(low picomole)


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The pentafluorobenzyl trimethyl silyl ether derivatives of steroids make them more amenable to high sensitivity measurements using negative chemical ionization.

Electron capture (highly selective)

M + e- → M-.

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+ 3-6 kV

Mass AnalyzerLiquid sample

2-10 µL/min

Electrospray Ionization Mass Spectrometry

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3-6 kV

Mass AnalyzerSolution

Pneumatically assisted Electrospray MS

2-1000 µL/min

Nebulization gas


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Introducing ions into the gas phase

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25


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14


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Basic groups [M+H]+(-NH2)

Acidic groups [M-H]-(-COOH, -OH)

60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230m/z0

100

%

0

100

%

217

195218

193

194Butyl Paraben

m.w. 194

Positive Ions

Negative Ions[M-H]-

[M+H]+

[M+Na]+

COOC4H9

OH


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Peptide: Substance P

300 400 500 600 700 800 900 1000 1100 1200 1300

Da/e0

100

%

674.7

666.1600.4462.8

685.7

693.6 1348.1

[M+2H]2 +

[M+H] +

Μr 1347

1347+22

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M E H F R W G KH

H H 4+H

N-terminal amine

H H H

H

HH

4+

HH

3+ 2+

H

1+

H

ES-MS

1+

2+

3+

4+

Rel. Inten.

m/z

CHH2N COOH

CH2

CH2

CH2

NH

CHN NH2

CHH2N COOH

CH2

NHN

CHH2N COOH

CH2

CH2

CH2

CH2

NH2


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Determination of Protein Relative Molecular MassUsing ESI MS

Παραδείγµατα

Ακρίβεια προσδιορισµού µάζας 0,01 %

16951,5

+15

+14

+13

+16+17+18

+19

+20

+21

+22

+15

+14

+13

+16

+17+18

+19

+20

17828

Determination of Protein Relative Molecular MassUsing ESI MS

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ESI Spectrum of Trypsinogen (MW 23983)

1599.8

1499.91714.1

1845.91411.9

1999.62181.6

M + 15 H+

M + 13 H+

M + 14 H+M + 16 H+

m/z Mass-to-charge ratio


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ESI - MSAdvantages

• Parent Ion• High Mass Compounds

(>100,000 amu)• Thermally Labile

Compounds (<0º C)• Easy to Operate• Interface to HPLC• Zeptomole sensitivity with

nanospray

Disadvantages• No Fragmentation• Need Polar Sample• Need Solubility in Polar

Solvent (MeOH, ACN, H2O, Acetone are best)

• Sensitive to Salts• Suppression

(low femtomole to zeptomole)

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Matrix Assisted Laser Desorption Ionization

(MALDI)Mass Spectrometry


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The mass spectrum shows the results

Rel

ativ

e A

bund

ance

Mass (m/z)

0

10000

20000

30000

40000

50000 100000 150000 200000

MH+

(M+2H)2+

(M+3H)3+

MALDI TOF spectrum of IgG


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Advantages• Parent Ion• High Mass Compounds

(>100,000 amu)• Thermally Labile

Compounds (R.T.)• Easy to Operate

Disadvantages• No Fragment Library• Wide variety of matrices• Quantitation Difficult

(low femtomole)

MALDI MS

Mass Spectrometry for Chemists and · PDF fileMass Spectrometry for Chemists and Biochemists...

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