Russell Rouseff

FOS 6355Summer 2005

What is Mass Spectroscopy Analytical Chemistry Technique

Used to identify and quantify unknown compounds

Can also elucidate structure and chemical properties

Sensitive technique

As little as 10 -12 g can be analyzed

Identification based on molecular fragmentation patterns

fragments are separated by their m/z ratio

1885 1905 1925 1945 1965 1985 2005

189

9 -

Ear

ly M

ass

Sp

ectr

om

etry

1943

- D

ou

ble

fo

cusi

ng

an

alyz

er

1946

- T

ime

of

Flig

ht

MS

1947

- P

rep

arat

ive

Mas

s S

pec

tro

met

ry

1953

- Q

uad

rup

ole

MS

1956

- G

C/M

S

1956

- Id

enti

fyin

g O

rgan

ic C

om

po

un

ds

1966

- C

hem

ical

Ion

izat

ion

196

6 -

Pep

tid

e S

equ

enci

ng

1968

- A

tmo

sph

eric

Pre

ssu

re Io

niz

atio

n

1978

- G

C-C

-IR

MS

1980

- In

du

ctiv

ely

Co

up

led

Arg

on

Pla

sma

1990

- P

rote

in S

tru

ctu

re

1993

- P

rote

in M

ass

Map

pin

g/F

ing

erp

rin

tin

g

1996

- M

S o

f a

viru

s

1996

- F

irst

pro

ton

tra

nsf

er M

S

MS Time lines

Mass Spectroscopy Applications

Detect and identify the use of steroids in athletes

Monitor the breath of patients by anesthesiologists during surgery

Determine the composition of molecular species found in space

Determine whether honey is adulterated with corn syrup

Monitor fermentation processes for the biotechnology industry

Detect dioxins in contaminated fish

Establish the elemental composition of semiconductor materials

Perform forensic analysis – arson identification

Determine exact atomic mass and isotope abundance

Objectives How did mass spectrometry originate?

What is a mass spectrometer?

What are the characteristics of a mass spectrum?

How is the sample introduced into the mass spectrometer?

How are the molecules and fragment ions produced in the ion source?

How does the analyzer work?

How does the detector work?

What are the roles of computers in mass spectrometry?

ObjectivesHow can mass spectrometric data be used for

structure analysis?

What other techniques are used to produce ions?

How large a molecule can be analyzed?

How is mass spectrometry used with GC,LC, and other separation techniques?

How is mass spectrometry used for quantitative analysis?

What is mass spectrometry/mass spectrometry (MS/MS)?

What is isotope ratio mass spectrometry?

source analyzerion

detection

datasystem

Vacuum pumps

SampleIntroduction

Ion Formation Ion Sorting Ion Detection

Data Handling

Data OutputMass spectrum

Sample Introduction

Neutral molecules in gaseous state

Can be either liquid, solid or gas under STP

must volatilize under vacuum

must be pure

often coupled with GC, HPLC or CE

but can use solids probe if pure

Ionization Techniques

Electron Impact, EI

Chemical Ionization, CI

Fast Atom Bombardment, FAB

Atmospheric Pressure Chemical Ionization, APCI

Proton Transfer Reaction

Sampleentrance

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filament

Electron collector

Ion focusing lenses

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+ + +

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repeller

Ion Formation (EI)

+++

(m a in lib ) C a rb o n d io xid e

10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 600

50

100

12 16

22

28

44

45

C

O

O

CO2+ Molecular ion

Base ion

CO+O+

C+

Contains C, H, O

Sample Ionization

Electron Impact, EI, most common

70 eV standard ionization energy

M + e- => M+ + 2e-

But sometime 70 eV too powerful

Molecular Weight =165.24Exact Mass =165Molecular Formula =C10H15NO

EI may overfragment

Chemical Ionization

A softer ionization technique

Use ionized molecules to transfer protons (+)

M + CH5+ => (M+H)+ + CH4

Chemical Ionization gases

methane

isobutane

Chemical Ionization

Produces spectra with little fragmentation

However, no standard spectral libraries

Spectra tends to be instrument specific

reagent gas pressures, ionization efficiencies

Must generate own standards

Large Molecule Ionization

Can routinely analyze molecules of 10,000 Da or moreUpper limit used to be 2,000 Da

APCI

A form of chemical ionization

Used for LC/MS interfaces

Allows for removal of solvent before vacuum

http://www.chm.bris.ac.uk/ms/theory/apci-ionisation.html

Proton Transfer Mass Spectrometery

Europhysics News (2004) Vol. 35 No. 6

glow discharge

H30+

H30+ + M => (M + H) = H2O

H30+

M+

M

M+

Europhysics News (2004) Vol. 35 No. 6

Proton Transfer Mass Spectrometery

Detects aroma compounds at ppb levels

Produces spectra with little ionization, quantify using M+

Mass Analyzer Types

Magnetic Sector

Quadrupole mass analyzers

Ion trap

Time of flight

H0

Magnetic Field

Magnetic Sector Mass Spectrometers

Exit slit

MolecularSource

Ionizationchamber

e-

detector

Magnetic Sector Disadvantages

Not well-suited for pulsed ionization methods

Very High cost

Large size

High maintence costs

resonant ion

nonresonant ion

detector

source

focusing lens

quadrupole rods

quadrupole mass analyzer

Mass filters

Quadrupole Advantages

Classical mass spectra

Good reproducibility

Relatively small/ compact

Relatively low-cost systems

Can improve sensitivity 100x with SIM

Quadrupole limitations

Limited resolution

Peak heights variable as a function of mass (mass discrimination).

Peak height vs. mass response must be 'tuned'.

Not well suited for pulsed ionization methods

Ion Trap Advantages

High sensitivity

Multi-stage mass spectrometry, MSn

Compact mass analyzer

Ion Trap Limitations

Requires careful quantitation

Limited dynamic range

Subject to space charge effects and ion molecule reactions

No sensitivity gain using SIM

Time of Flight MS

MS/MS

ASMS- American Society for Mass Spectrometryhttp://www.asms.org/whatisms/edu_resources.html Excellent educational MS resource with many MS related links.Cambridge University WWW Mass Spectrometry Server http://www-methods.ch.cam.ac.uk/meth/ms/theory/index.htmlComprehensive site with information on ionization processes, mass analyzers, and other mass spectrometry techniques. Organic Chemistry Online http://chipo.chem.uic.edu/web1/ocol/spec/MS.htmIntroductory information about analysis of organic compounds by electron impact mass spectrometry, tables of common fragment losses. University of Leeds:  Introduction to Mass Spectrometry http://www.astbury.leeds.ac.uk/Facil/MStut/mstutorial.htmIntroductory site focusing on the analysis of biochemical samples. Information on Specific Techniques

General Mass Spectrometry Sites

Sheffield ChemPuter: Isotope Patterns Calculator

http://www.shef.ac.uk/chemistry/chemputer/isotopes.html

Site predicts isotope pattern based on molecular formula.

UCSF Protein Prospector

http://prospector.ucsf.edu/

Site includes useful programs for analyzing the mass spectra of proteins and peptides.

Proteomics tools for mining sequence databases in conjunction with Mass Spectrometry experiments

Misc. Mass Spectrometry Sites