Lecture Date: February 27th, 2012

Mass Spectrometry and Related Techniques 2

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

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 is a geometrical construction that consists of two arcs

inside of one another.

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)

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

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

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

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.

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

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

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.

Quadrupole Mass Analyzers

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.

Quadrupole Mass Analyzers

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.

Ion Trap Mass Analyzers

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.

Ion Trap Mass Analyzers

Layout of a cylindrical 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)

++

++ + +

Ion Trap Mass Analyzers

The Thermo LTQ Velos Pro, a modern dual-pressure linear ion trap

For more about dual-pressure linear traps, see J. V. Olsen et al., A dual pressure linear ion trap Orbitrap instrument with very high sequencing speed, Molecular and Cellular Proteomics, 8, 2759-2769.

For more about linear traps, see D. J. Douglas et al., Linear ion traps in mass spectrometry, Mass Spectrometry Reviews, 2005, 24, 1–29

Ion Cyclotron Resonance

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

Ion Cyclotron Resonance

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= ze B m=

B

Picture courtesy Prof. Alan Marshall, FSU/NHMFL

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

The Orbitrap is an 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

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

The axial oscillation frequency follows the formula Where = oscillation frequency

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

zm

k

/

Frequencies and Masses

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

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

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 comparison of the properties of some 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:

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

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

References

Optional:• R. M. Silverstein, et al., “Spectrometric Identification of Organic Compounds”, 6 th

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.

• 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.

• Q. Hu, R. J. Noll, H. Li, A. Makarov, M. Hardman, and R. G. Cooks, “The Orbitrap, a new mass spectrometer”, J. Mass. Spectrom., 2005, 40, 430-443.

• 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.