lecture date: february 25 th, 2008 mass spectrometry and related techniques 1

Lecture Date: February 25th, 2008

Mass Spectrometry and Related Techniques 1

Ion and Particle Spectrometry 1 - Outline

Atomic and Molecular Mass Spectrometry– Skoog et al. Chapter 11 (atomic) and Chapter 20 (Molecular).

– Cazes Chapter 14

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

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

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

Accel!

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

See also Fig. 20-3, pg. 502 in Skoog, et al.

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

ions! (makes structural identification difficult!)

CI: Chemical Ionization

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

– Ionization 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

A form of API (atmospheric pressure ionization) – a range of ionization techniques that operate at higher pressures, outside the vaccuum MS regions.

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

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

APCI (diagram from Agilent)

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


A picture of two ideas for the electrospray process:

Diagram from John B. Fenn (Nobel Lecture), 2002Picture from http://www.newobjective.com/electrospray/electrospray.html

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

http://www.newobjective.com/electrospray/electrospray.html


An ESI source:


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 – complementary techniques:

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 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 mostly 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 – see Table 20-4 (pg. 509) in Skoog et al.

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 and DART

Desorption-electrospray ionization (DESI)

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)

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

Mass Analyzers - Outline

Sector Mass Analyzers (Magnetic and Electrostatic)

Quadrupole Analyzers

Ion Traps

Ion Cyclotron Resonance

Time-of-Flight

and many more….

IonizationSource

MassAnalyzer

Detector

Properties of Mass Analyzers

Resolution (R):

R = m/m

m = mass difference of two adjacent resolved peaks (typically

m = mass of first peak or average

Example: R = 500 (“low” resolution)

resolves m/z=50 and 50.1, and m/z=500 and 501

Example: R = 150000 (“high” resolution)

resolves m/z=50 and 50.0003, and m/z=500 and 500.0033

Sector Mass Analyzers

Basic Features– A sector: a geometrical construction that has

two arcs inside of one another.

– (Technically, a pie slice!)

Types:– Magnetic

– Electrostatic

– Combination (e.g. double-focusing)

Magnetic Sector Mass Analyzers

Ion kinetic energy:

V

erB

z

m

2

22

221 mvzeVT

BzeVFm

r

mvFc

2

mc FF

Forces:

Only ions with equal forces will pass:

Therefore:

Where:T is kinetic energyz is charge on ione is electron charge (1.60 x 10-19 C)B is magnetic field (T)v is velocity (m/s)V is the accelerating voltagem is the mass

Diagram from Strobel and Heineman, Chemical Instrumentation, A Systematic Approach, Wiley, 1989.

Electrostatic Sector Mass Analyzers

2

v

reV

z

m

Therefore:

Ion kinetic energy:2

21 mvzeVT

eVFm

r

mvFc

2

Mc FF

Forces:

Only ions with equal forces will pass:

V can be varied to bring ions of different KE (and different m/z ratio to the exit)


Double-Focusing Sector Mass Analyzers

If a batch of ions of equal m/z but with different kinetic energies enters a magnetic sector instrument, this will result in a spread-out beam

Soution: minimize directional and energy differences between ions of the same m/z.

Example of a double-focusing MS: the Nier-Johnson geometry


Double-Focusing Sector Mass Analyzers Another design, the Mattauch-Herzog geometry

This geometry is analogous to CCD-based optical electronic spectroscopy systems, while Nier-Johnson instruments are similar in nature to traditional scanning monochromator spectrometers.


Time-Of-Flight (TOF) Mass Analyzers

The principle of “Time-of-flight” mass analysis: – A batch of ions is introduced into a chamber by an

pulse of accelerating current.

– This chamber has no fields, and is a “drift tube”

– Since the ions have the same kinetic energy, their velocities vary inversely with their mass during their drift.

Notes:– Typical flight times are 1-30 us

– Lighter ions arrive at the detector first

221 mvT

M. Guilhaus; Journal of Mass Spectrometry, 30; 1995, p1519.

Time-Of-Flight (TOF) Mass Analyzers

Delayed extraction – anything you can do to tighten the KE spread will help a TOF instrument

M. Guilhaus; Journal of Mass Spectrometry, 30; 1995, p1519.

m/z is mass-to-charge ratio of the ion

E is the extraction pulse potential(V)

s is the length of flight tube over which E is appliedd is the length of field free drift zonet is the measured time-of-flight of the ion

zeEsmvT 221

2

2

v

eEs

z

m

2

2

d

teEs

z

m

Time-Of-Flight (TOF) Mass Analyzers The reflectron – a method of compensating for different ion KE’s

Figure from http://www.abrf.org/ABRFNews/1997/June1997/jun97lennon.html

Quadrupole Mass Analyzers

The quadrupole (named for its “electrical structure”) is one of the simplest and most effective mass spectrometers.

Diagrams from Skoog et al.


How a quadrupole works:– Most important points:

It is easier for an applied AC field to deflect a light ion than a heavier ion

Conversely, it is easier for an AC field to stabilize a light ion

– Using this knowledge – a combined AC/DC potential is applied to the rods. Via the DC, the ion is attracted to one set of rods and repelled by the other

– The DC serves to stabilize heavy ions in one direction (high pass filter). The AC serves to stabilize light ions in the other direction (low pass filter).

– The ion must pass through the quadrupole to make it to the detector

Diagrams from Skoog et al.


Another view – and the concept of the mass scan…

Images from http://www.jic.bbsrc.ac.uk/SERVICES/metabolomics/lcms/single1.htm

Light ion:(ex. m/z = 100)Dragged by AC

Heavy ion:(ex. m/z = 500)Dragged by DC

Just right:Dragged by both,But equally balanced

Ion Trap Mass Analyzers

Ion trap: a device for trapping ions and confining them for extended periods using EM fields

Used as mass analyzers because they can trap ions and eject them to a detector based on their mass.

Theory is based on Mattieu’s work on 2nd order linear differential equations (in the 1860’s), and on Wolfgang Paul’s Nobel Prize winning implementations

R. E. March and R. J. Hughes, Quadrupole Storage Mass Spectrometers, Wiley, 1989.See also Chem. Eng. News 1991; 69(12):26-30, 33-41

Figure from W. Paul Nobel Lecture, December 8, 1989.


The stability region of an ion trap – based on differential equations

220

8

mr

eUaz

220

4

mr

eVqz

)cos(0 tVU

Most ITMS systems don’t use DC (U), i.e. only qz is controlled

R. E. March and R. J. Hughes, Quadrupole Storage Mass Spectrometers, Wiley, 1989.


Layout of an ion trap mass analyzer:

Diagram courtesy of M. Olsen, GlaxoSmithKline

+

Main RF

Ring

Endcap

Lenses

Octopole

Optimized Asymptote Angle

End Cap

Shutter

Focus

Electron Multiplier

Conversion Dinode

Low Amplitude Dipole Field(1/3 frequency of main RF)

++

++ + +


The Bruker Esquire ESI ITMS - a typical ion-trap LC-MS system:

Photo courtesy of M. Olsen, GlaxoSmithKline


FT-ICR: a FT-based mass spectral method that offers higher S/N, better sensitivity and high resolution

Also contains a form of ion trap, but one in which “ion cyclotron resonance” occurs.

When an ion travels through a strong magnetic field, it starts circulating in a plane perpendicular to the field with an angular frequency c:

m

zeB

r

vc


How ICR works:– The ions are circulated in a field

– An RF field is applied to match the cyclotron frequency of the ions – this field brings them into phase coherence (forming ion “packets”)!

– The image current is produced as these little packets of ions get near the plates. The frequency of the image current is characteristic of the ion packet’s m/z ratio.

http://www-methods.ch.cam.ac.uk/meth/ms/theory/fticr.html

Ion Cyclotron Resonance and Magnetic Field

Parallels between NMR/EPR and ICR:

B

B= q B m=

B

Picture courtesy Prof. Alan Marshall, FSU/NHMFL

The OrbitrapTM: A “Hybrid” Trap – Between IT and ICR

The Orbitrap is a recently developed electrostatic ion trap with FT/MS read-out of image current, coupled with MS/MS

Advantages– Ease of use

– Resolving power (superior to TOF)

– Precision and accuracy

– Versatility, dynamic range

A lower-resolution, more economical ICR

LTQ Orbitrap schematic

API Ion source Linear Ion Trap C-Trap

Orbitrap

Finnigan LTQ™ Linear Ion Trap

Differential pumping

Differential pumping

Image/animation from Thermo Electron Inc. See A. Makarov et al., Anal. Chem. 2006, 78, 2113-2120.

LTQ Orbitrap Operation Principle

1. Ions are stored in the Linear Trap2. …. are axially ejected3. …. and trapped in the C-trap4. …. they are squeezed into a small cloud and injected into the Orbitrap5. …. where they are electrostatically trapped, while rotating around the central electrode and performing axial oscillation

The oscillating ions induce an image current into the two outer halves of the orbitrap, which can be detected using a differential amplifier

Ions of only one mass generate a sine wave signal


The axial oscillation frequency follows the formula Where = oscillation frequency

k = instrumental constant m/z = mass-to-charge ratio

zm

k

/

Frequencies and Masses

Many ions in the Orbitrap generate a complex signal whose frequencies are determined using a Fourier Transformation


Multiple-Stage MS: MS-MS, and MSn

Also known as Tandem MS or MSn

MassAnalyzer

MassAnalyzer

Multiple quadrupoles are very common (e.g. triple-quad or QQQ systems, EB for double-focusing, Q-TOF for quad time-of-flight…)

Why tandem MS? Because of the possibility of doing CID – collisionally induced dissociation. Ions are allowed to collide with a background gas (He) for several millliseconds, prior to analysis. Allows for MSn experiments in an ion trap.

…

Comparison of Mass Analyzers

A brief overview of the properties of common mass analyzers

Analyzer Cost Scan speed Resolution

Double-focusing High Slow High

Quadrupole Low Medium Low-medium

Trap Low Medium Medium

TOF Medium Medium Medium-high

ICR High Fast High

Detectors for Mass Spectrometry

Electron multipliers: like a photomultiplier tube. Ions strike a surface, cause electron emission. Each successive impact releases more electrons

Faraday Cups: Ions striking a cup cause charge to flow across a load. The potential across the load is monitored.

See pg 257 of Skoog et al. for more details.

IonizationSource

MassAnalyzer

Detector

Figure from D. W. Koppenaal, et al.; Anal. Chem., 77; 2005, 418A-427A.

Detectors: Electron Multipliers Electron multiplier (EM): most common design in current use

High gain (107), low noise, good dynamic range (104-106)

Several designs:


Detectors: Others

Super-conducting tunner junction – high mass range, used with MALDI

– Can detect fmol of 150 kDa proteins

– Can measure both energy and arrival time (2D MS – plots of m/z vs. kinetic energy)

Focal-plane array detectors/CCD– Like in electronic spectroscopy, much more challenging to design

for ion detection

– Would combine well with “mini-traps” or other small MS systems


MS-Chromatography Interfaces

GC-MS: gas eluent from a column is piped directly to the MS source

LC-MS: the ionization methods themselves serve as interfaces – techniques like ESI, APCI and APPI work on liquid phase samples. The methods are generally tolerant to RP LC solvents and some NP solvents. Some buffers can quench ionization of analytes though:

– Bad: Phosphate – leaves a solid upon evaporation. Also ionizes preferentially

– Bad: any other non-volatile additives are also bad

– Good: TFA, ammonium acetate, formic acid

– Good: lower concentrations, <50 mM

Homework

Choose one of these references to read:– R. E. March, "An Introduction to Quadrupole Ion Trap Mass

Spectrometry", J. Mass. Spec., 1997, 32, 351-369.

– D. H. Russell and R. D. Edmondson, "High-resolution Mass Spectrometry and Accurate Mass Measurements with Emphasis on the Characterization of Peptides and Proteins by Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry", J. Mass. Spec., 1997, 32, 263-276.

– R. Aebersold and D. R. Goodlett, "Mass Spectrometry in Proteomics", Chem. Rev., 2001, 101, 269-295.

– L. Sleno and D. A. Volmer, “Ion activation methods for tandem mass spectrometry”, J. Mass Spectrom. 2004; 39: 1091–1112

References

Note: see Mass Spectrometry and Related Techniques Part 2 for applications of MS, and theory/applications of Ion Mobility Spectrometry

R. M. Silverstein, et al., “Spectrometric Identification of Organic Compounds”, 6th Ed., Wiley, 1998.

R. E. March and R. J. Hughes, “Quadrupole Storage Mass Spectrometers”, Wiley, 1989.

F. W. McLafferty, “Interpretation of Mass Spectra”, 3rd Ed., University Science Books, 1980.

lecture date: february 25 th, 2008 mass spectrometry and related techniques 1

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