lecture date: february 20 th, 2012 mass spectrometry and related techniques 1

Lecture Date: February 20th, 2012

Mass Spectrometry and Related Techniques 1

Mass Spectrometry

Mass Spectrometry (a.k.a. MS or “mass spec”) – a method of separating and analyzing ions by their mass-to-charge ratio

MS does not involve a specific region of the electromagnetic spectrum (because it is not directly interested in the energies of emitted photons, electronic or vibrational transitions, nuclear spin transitions, etc…)

Ionabundance

Up to m/z = 100000!m/z

Ion

Ion Ion

History of Mass Spectrometry

J. J. Thomson at Cambridge reported the first MS experiment in 1913 and discovered isotopes.

F. W. Aston built the first MS in 1919 and studied isotopes, winning the 1922 Nobel Prize in Chemistry.

In the 1930’s, Ernest Lawrence invented the calutrons used in WW2 to separate 235U.

Nobel Prize in Physics (1989) to Wolfgang Paul for the ion trap.

Nobel Prize in Chemistry (2002) to John Fenn (electrospray ionization) and Koichi Tanaka (MALDI). Calutron at the Y-12 Plant at Oak Ridge,

Tennessee, used during the Manhattan Project

J. J. Thomson F, W, Aston

General Notes on Atomic and Molecular Mass

Helpful units and conversions:– 1 amu = 1 Da = 1/12 the mass of a neutral 12C atom.

– 1 kDa = 1000 amu

Atomic weights of other elements are defined by comparison.

Mass-to-charge ratio (m/z): the ratio of the mass of an ion (m) to its charge (z)

Molecular ion: an ion consisting of essentially the whole molecule

Mass Spectrometers

A block diagram of a generic mass spectrometer:

IonizationSource

MassAnalyzer

Detector

This lecture covers the ionization source – the method of making the ions for MS analysis.

Ionization Sources

Electron Ionization (EI)

Chemical Ionization (CI/APCI)

Photo-ionization (APPI)

Electrospray (ESI)

Matrix-assisted Laser Desorption (MALDI)

Field Desorption (FD)

Plasma Desorption (PD)

Fast atom bombardment (FAB)

High-temperature Plasma (ICP)

See also Table 20-1 in Skoog, et al.

Desorption

Gas Phase

IonizationSource

MassAnalyzer

Detector

EI: Electron Ionization/Electron Impact

The electron ionization (EI) source is designed to produce gaseous ions for analysis.

EI, which was one of the earliest sources in wide use for MS, usually operates on vapors (such as those eluting from a GC)

Heated IncandescentTungsten/Rhenium Filament

Accelerate!

e-

Vaporized Molecules

70 eV

Ions ToMass

Analyzer


How EI works:– Electrons are emitted from

a filament made of tungsten, rhenium, etc…

– They are accelerated by a potential of 70 V

– The electrons and molecules cross (usually at a right angle) and collide

– The ions are primarly singly-charged, positive ions, that are extracted by a small potential (5V) through a slit

Diagram from F. W. McLafferty, “Interpretation of Mass Spectra”, 3rd Ed., University Science Books, Mill Valley, CA (1980).


When electrons hit – the molecules undergo rovibrational excitation (the mass of electrons is too small to really “move” the molecules)

About one in a million molecules undergo the reaction:

M + e- M+ + 2e-


Advantages:

– Results in complex mass spectra with fragment ions, useful for structural identification

Disadvantages:

– Can produce too much fragmentation, leading to no molecular ion (makes structural identification difficult!)

CI: Chemical Ionization

Chemical ionization (CI) is a form of gas-phase chemistry that is “softer” (less energetic) than EI

– In CI, ionization occurs via proton transfer reactions

A gas (ex. methane, isobutane, ammonia) is introduced into the source at ~1 torr.

Example: CH4 reagent gas

CH4

EICH4

+

CH4+ + CH4 CH5

+ + CH3

AH + CH5+ AH2

+ + CH4

Strong acidSee B. Munson, Anal. Chem., 49, 772A (1977).

CI: Hard and Soft Sources

The energy difference between EI and CI is apparent from the spectra:

CI gases:– harshest (most

fragments): methane

– softest: ammonia

APCI: Atmospheric-Pressure Chemical Ionization

APCI – a form of chemical ionization using the liquid effluent in a spray chamber as the reagent

APCI is a form of API (atmospheric pressure ionization or ambient ionization) - these are a range of ionization techniques that operate at higher pressures, outside the vacuum MS regions, and sometimes at normal pressures and temperatures

Examples of ambient ionization methods to be discussed later in this lecture: DESI, MALDI

APCI: Atmospheric-Pressure Chemical Ionization

The APCI process:– The sample is in a flowing stream of a carrier liquid (or gas)

and is nebulized at moderate temperatures.

– This stream is flowed past an ionizer which ionizes the carrier gas/liquid.

63Ni beta-emitters Corona (electric) discharge needle at several kV

– The ionized stream (which can be an LC solvent) acts as the primary reactant ions, forming secondary ions with the analytes.

– The ions are formed at AP in this process, and are sent into the vaccuum

– In the vaccuum, a free-jet expansion occurs to form a Mach disk and strong adiabatic cooling occurs.

Cooling promotes the stability of analyte ions (soft ionization)

See A. P. Bruins, Mass Spec. Rev., 10, 53-77 (1991).

APCI: Chemical Ionization

An APCI source:

Diagram from Agilent Technologies

760 torr

10-6 torr

APCI: Chemical Ionization

An APCI mass spectrum:


Electrospray Ionization (ESI)

The ESI process:– Electrospray ionization (ESI) is accomplished by flowing a

solution through an electrically-conductive capillary held at high voltage (several keV DC).

– The capillary faces a grid/plate held at 0 VDC.

– The solution flows out of the capillary and feels the voltage – charges build up on nebulized droplets, which then begin to evaporate

– Coulombic explosions occur when the repulsion of the charges overcomes the surface tension of the solution (holding the drop together) – known as the Rayleigh limit.

– Depending on whose theory you believe the analyte ion is eventually the only ion left or…the analyte ion is evaporated from a small enough droplet

Electrospray Ionization (ESI) A picture of two ideas for the electrospray process:

El Aneed, et al. , Applied Spectroscopy Reviews, 44: 210–230, 2009.Jet image from http://www.newobjective.com/electrospray/electrospray.html

Note – ions which are surface-active will be preferentially ionized – this can lead to ion suppression!

The Taylor cone – the shape of the cone that shoots from the needle when surface tension is overcome by electrostatic forces, and forms a jet


An ESI source:



A selection of modern ESI and heated ESI designs:

Stanke et al., J. Mass. Spectrom. 2012, 47, 875–884.

Typical ESI Spectra

An ESI mass spectrum:


Typical ESI Spectra

An ESI mass spectrum of a 14.4 kDa enzyme:

Diagram from http://www.nd.edu/~masspec/ions.html

ESI and APCI

ESI and APCI are complementary techniques for solution-phase analytes:

Figure from Agilent Instruments

ESI and APCI

ESI and APCI –complementary techniques:

ESI APCI

Very “soft” ionization – can ionize thermally

labile samples

Some sample volatility needed (nebulizer)

Ions formed in solution Ions formed in gas phase

Singly- and multiply-charged ions [M+H]+

Singly-charged ions, [M+H]+ and [M-H]-

Atmospheric Phase Photo-ionization APPI ionizes using UV irradiation and (usually) a dopant:

D. A. Robb and M. W Blades, Anal. Chim. Acta, 2008, 627, 34-49.

Atmospheric Phase Photo-ionization APPI can ionize things that ESI and APCI can’t:

Comparison of Ionization Methods

How to choose an ionization technique:

Figure from Agilent Instruments

MALDI: Matrix-Assisted Laser Desorption/Ionization

A method for desorbing a sample with a laser, while preventing thermal degradation

A sample is mixed with a radiation-absorbing “matrix” used to help it ionize

MALDI is heavily used for large biomolecules and polymers.

Diagram from Koichi Tanaka (Nobel Lecture), 2002

MALDI: Matrix Effects

The role of the matrix– Must absorb strongly

at the laser wavelength

– The analyte should preferably not absorb at this wavelength

Common matrices include nicotinic acid and many other organic acids

Batoy et al., Applied Spectroscopy Reviews, 2008, 43, 485–550.

MALDI at Atmospheric Pressure

Advantages: fast, easy and sensitive

Disadvantages: no LC, matrix still needed

S. Moyer and R. Cotter, “Atmospheric Pressure MALDI”, Anal. Chem., 74, 468A-476A (2002)

FAB: Fast Atom Bombardment A soft ionization technique

– Often used for polar, higher-mwt, thermally labile molecules (masses up to 10 kDa) that are thermally labile.

Samples are atomized by bombardment with ~keV range Ar or Xe atoms.

– The atom beam is produced via an electron exchange process from an ion gun.

K. L. Rinehart, Jr., Science, 218, 254 (1982)K. Biemann, Anal. Chem., 58, 1288A, (1986).

Xee-

Xe+ + 2e-

Advantages:– Rapid sample heating – reduced fragmentation

– A glycerol solution matrix is often used to make it easier to vaporize ions

Xe+

accelXe+ (high KE)

Xe+ (high KE) + Xe Xe (high KE) + Xe+

SIMS: Secondary Ion MS

Focused Ion Beam – 3He+, 16O+, 40Ar+

– Beam energy 5 to 20 keV

– Beam diameter – 0.3 to 5 mm

Beam Hits Target – A small % of the target material is “sputtered” off and enters

the gas phase as ions (usually positive)

Advantages: – Imaging of ions (characteristic masses) on a surface or in

biological specimens

– Surface analysis using beam penetration depth/angle

– Can be used for both atomic and molecular analysis

– Sensitive to low levels, picogram, femtogram and lower

Will discuss more in surface analysis/microscopy talk…

Desorption Electrospray: DESI

Desorption-electrospray ionization (DESI) is an ambient ionization technique

A new technique for desorbing ions using supersonic jets of solvents (charged like in electrospray)

From Z. Takats et al., Science, 2004, vol 306, p471.

Inductively Coupled Plasma (ICP) as an MS Source

The inductively-coupled plasma serves as an atomization and ionization source (two-in-one!) for elemental studies.

Photo by Steve Kvech, http://www.cee.vt.edu/program_areas/environmental/teach/smprimer/icpms/icpms.htm#Argon%20Plasma/Sample%20Ionization

See optical electronic lecture for more details

Solution flow rates up to: 50-100 mL/min

Further Reading

Required (please skim):J. Cazes, Ed. Ewing’s Analytical Instrumentation Handbook, 3rd Ed., Marcel Dekker, 2005,

Chapter 7.

Optional:

http://www.spectroscopynow.com/raman/details/education/sepspec13199education/Introduction-to-Raman-Spectroscopy-from-HORIBA-Jobin-Yvon.html

D. A. Skoog, F. J. Holler and S. R. Crouch, Principles of Instrumental Analysis, 6th Edition, Brooks-Cole, 2006, Chapter 18.

D. A. Long, The Raman Effect, Wiley, 2002.

S. Hooker, C. Webb, Laser Physics, Oxford, 2010.

P. W. Atkins and R. S. Friedman, Molecular Quantum Mechanics, 3rd. Ed., Oxford, 1997.

http://www.rp-photonics.com/yag_lasers.html

lecture date: february 20 th, 2012 mass spectrometry and related techniques 1

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