Lecture Date: February 25 th, 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…) Ion abundance Up to m/z = !m/z Ion

General Notes on Atomic and Molecular Mass  Helpful units and conversions: –1 amu = 1 Da = 1/12 the mass of a neutral 12 C 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: Ionization Source Mass Analyzer 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 Ionization Source Mass Analyzer 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 Incandescent Tungsten/Rhenium Filament Accel! E- Vaporized Molecules 70 eV Ions To Mass Analyzer

EI: Electron Ionization/Electron Impact  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).

EI: Electron Ionization/Electron Impact  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 -

EI: Electron Ionization/Electron Impact  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: CH 4 reagent gas CH 4 EI CH 4 + CH CH 4 CH CH 3 AH + CH 5 + AH CH 4 Strong acid See 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.  63 Ni 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, (1991).

APCI: Chemical Ionization  APCI (diagram from Agilent) Diagram from Agilent Technologies 760 torr torr

APCI: Chemical Ionization  An APCI mass spectrum: Diagram from Agilent Technologies

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: Diagram from John B. Fenn (Nobel Lecture), 2002 Picture from Note – ions which are surface-active will be preferentially ionized – this can lead to ion suppression!

Electrospray Ionization (ESI)  An ESI source: Diagram from Agilent Technologies

Typical ESI Spectra  An ESI mass spectrum: Diagram from Agilent Technologies

Typical ESI Spectra  An ESI Mass Spectrum of a 14.4 kDa enzyme: Diagram from

ESI and APCI  ESI and APCI – complementary techniques: Figure from Agilent Instruments

ESI and APCI  ESI and APCI –complementary techniques: ESIAPCI Very “soft” ionization – can ionize thermally labile samples Some sample volatility needed (nebulizer) Ions formed in solutionIons 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:

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). Xe e-e- Xe  + + 2e -  Advantages: –Rapid sample heating – reduced fragmentation –A glycerol solution matrix is often used to make it easier to vaporize ions Xe  + accel Xe  + (high KE) Xe  + (high KE) + Xe Xe (high KE) + Xe  +

SIMS: Secondary Ion MS  Focused Ion Beam – 3 He +, 16 O +, 40 Ar + –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,  See optical electronic lecture for more details  Solution flow rates up to: mL/min

Mass Analyzers - Outline  Sector Mass Analyzers (Magnetic and Electrostatic)  Quadrupole Analyzers  Ion Traps  Ion Cyclotron Resonance  Time-of-Flight  and many more…. Ionization Source Mass Analyzer 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 = (“high” resolution) resolves m/z=50 and , and m/z=500 and

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: Forces: Only ions with equal forces will pass: Therefore: Where: T is kinetic energy z is charge on ion e is electron charge (1.60 x C) B is magnetic field (T) v is velocity (m/s) V is the accelerating voltage m is the mass Diagram from Strobel and Heineman, Chemical Instrumentation, A Systematic Approach, Wiley, 1989.

Electrostatic Sector Mass Analyzers Therefore: Ion kinetic energy: 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 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 applied d is the length of field free drift zone t is the measured time-of-flight of the ion

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

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

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 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 2 nd 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, See also Chem. Eng. News 1991; 69(12):26-30, Figure from W. Paul Nobel Lecture, December 8, 1989.

Ion Trap Mass Analyzers  The stability region of an ion trap – based on differential equations  Most ITMS systems don’t use DC (U), i.e. only q z is controlled R. E. March and R. J. Hughes, Quadrupole Storage Mass Spectrometers, Wiley, 1989.

Ion Trap Mass Analyzers  Layout of an ion trap mass analyzer: Diagram courtesy of M. Olsen, GlaxoSmithKline

Ion Trap Mass Analyzers  The Bruker Esquire ESI ITMS - a typical ion-trap LC-MS system: Photo courtesy of M. Olsen, GlaxoSmithKline

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 :

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.

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 Orbitrap TM: 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 Image/animation from Thermo Electron Inc. See A. Makarov et al., Anal. Chem. 2006, 78,

LTQ Orbitrap Operation Principle 1. Ions are stored in the Linear Trap 2. …. are axially ejected 3. …. and trapped in the C-trap 4. …. they are squeezed into a small cloud and injected into the Orbitrap 5. …. 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,

The axial oscillation frequency follows the formula Where  = oscillation frequency k = instrumental constant m/z = mass-to-charge ratio Frequencies and Masses Many 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,

Multiple-Stage MS: MS-MS, and MS n  Also known as Tandem MS or MS n Mass Analyzer Mass Analyzer  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 MS n experiments in an ion trap. …

Comparison of Mass Analyzers  A brief overview of the properties of common mass analyzers AnalyzerCostScan speedResolution Double-focusingHighSlowHigh QuadrupoleLowMediumLow-medium TrapLowMedium TOFMedium Medium-high ICRHighFastHigh

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. Ionization Source Mass Analyzer 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 (10 7 ), low noise, good dynamic range ( )  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 Figure from D. W. Koppenaal, et al.; Anal. Chem., 77; 2005, 418A-427A.

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, –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, –R. Aebersold and D. R. Goodlett, "Mass Spectrometry in Proteomics", Chem. Rev., 2001, 101, –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”, 6 th Ed., Wiley,  R. E. March and R. J. Hughes, “Quadrupole Storage Mass Spectrometers”, Wiley,  F. W. McLafferty, “Interpretation of Mass Spectra”, 3rd Ed., University Science Books, 1980.