Lecture Date: March 11th, 2013

Mass Spectrometry and Related Techniques 3

Applications of Mass Spectrometry

Interpretation of mass spectra is the key to most applications of the technique

Information contained in a mass spectrum:– Molecular weight (via exact or mono-isotopic mass). Usually

obtained though a suitably accurate measurement of: M+• (the molecular ion, an odd-electron species) [M+H]+ and [M-H]- (the protonated/de-protonated molecule, an even-

electron species) In some techniques, can be confirmed by [M+Na]+, [M+K]+,

[M+NH4]+, dimers, trimers, and other adducts, etc…

– Molecular formula

– Ionization energies

– Isotopic incorporation (ex. 13C, 14C, 2H, 3H…)

– Fragmentation and ion stability

Quasi-equilibrium Theory

Once we make an ion, what happens to it?

In EI, and similar techniques: the ionizing electron has little mass and high KE, so it barely moves the molecule that it hits but leaves it in a higher rotational/vibrational state.

Ionization energies can sometimes be determined from ion intensities.

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

Molecular Structural Analysis: Fragmentation

Fragmentation can also be used to determine structure – common fragmentation pathways and rearrangements can be predicted in many cases

General rules:

- More stable carbocations are more stable fragments (ex. tertiary carbocations are more stable than primary)

- Resonance can stabilize fragments, ex. allylic carbocations and benzyl/tropylium ions

- Loss of small, neutral, stable molecules is favored

Figure from R. M. Silverstein, Spectrometric Identification of Organic Compounds, 6 th Ed., Wiley, 1998.

O

p-chloro-benzophenoneCl

Molecular Structural Analysis: Fragmentation

A large number of gas-phase fragmentation reactions commonly observed in MS analysis. Some examples:

See R. M. Silverstein, Spectrometric Identification of Organic Compounds, 6 th Ed., Wiley, 1998.

Rearrangements also occur (the McLafferty rearrangement is shown):

limonene

OH

H

HH

OH2

H

H -H2O

H

H

Molecular Structural Analysis: Adducts

Typical adducts observed in MS:– [M+H]+

– [M+NH4]+

– [M+Na]+

– [M+K]+

– etc…

So for a molecular ion with mwt = 400– [M+H]+ = 401, [M + Na]+ = 423, etc…

Dimers and other molecular adducts are also observed, e.g.:– [2M+H]+ = 801

– [2M+Na]+ = 823

– etc…

See R. M. Silverstein, Spectrometric Identification of Organic Compounds, 6 th Ed., Wiley, 1998.

Molecular Structural Analysis: Isotope Patterns

Isotope patterns can be used to determine molecular structure

– Example: the well-known methods of calculating (M+1) and (M+2) intensities

– Especially useful for detecting chlorine, bromine, sulfur, silicon and many other elements with characteristic profiles

Isotope patterns can also be used to extract out “isotope incorporation profiles” for labeled compounds

– Examples: 13C, 14C, 2H, 3H-labeled molecules for metabolism studies

– Applications in isotope chemistry include the detection of stable and radioactive isotopes in synthetic products and in nuclear chemistry.

M(100%)

M+1(19.28%)

M+2(33.99%)

M+3(6.21%)

m/z215 220

O

p-chloro-benzophenoneCl

Molecular Structural Analysis: Accurate Mass

Nuclide masses are not integers. Example: Four things that weigh “28” amu:

– CO, 27.9949

– 14N2, 28.0062

– CH2N, 28.0187

– C2H4, 28.0312

m/z measurements to four decimal places or higher are needed

Accurate mass analysis is often used as a final confirmation of structure, or for unravelling complex fragmentation

Molecular Structural Analysis: Mass Defects

-0.04

-0.03

-0.02

-0.01

0

0.01

0.02

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

1H

2H13C 14N 15N

16O

31P32S

12C

AtomicMass Defects(All Different)

Mass Defect (Da)

Mass Defect =

Atom Mass – Nearest Integer

Every CcHhNnOoSs massis unique!

Mass (Dalton)

Picture courtesy Prof. Alan Marshall, FSU/NHMFL

Molecular Structural Analysis: MS-MS, and MSn

Step 1 – mass selection of an ion formed in the source

Step 2 – dissociation of the parent ion via collisions

Step 3 – mass analysis of the dissociated “daughter” ions

Step 4 – repeat…

++ +

+++

+ +

CollisionsMass

Analyzer 1Mass

Analyzer 2

++ +

+++

+ + Mass Analyzerand Collision

Chamber

More About MSn Systems

Tandem-in-space– Means that the mass selection and fragmentation occur in

different physical locations within the spectrometer.

– Examples: Triple-quad (QQQ), in which…

Tandem-in-time– Means that the mass selection and fragmentation occur in the

same part of the MS but at different times

– Example: ion traps

++ +

+++

+ +

CollisionsMass

Analyzer 1Mass

Analyzer 2

Dissociation and Controlled Fragmentation in MSn

Collisionally-Induced Dissociation (CID)– Also known as collisionally-activated dissociation (CAD), CID is the

principal ion-dissociation method for MSn. In CID, stable ions are fragmented by collisions with neutral gas atoms/molecules

CID uses low-pressure He or Ar gas– Ion traps typically use 10-3 torr of He

– Triple-quadrupole systems typically use 10-6 torr of Ar

– Also can use N2, Xe, etc…

Other methods:– Photo-induced dissociation

IRMPD (IR multiphoton dissociation) – via IR lasers BIRD (blackbody infrared radiative dissociation)

– Surface-induced dissociation (SID)

– Electron-capture dissociation (ECD)

– Electron-transfer dissociation (ETD) – uses a molecular anion to deliver charge

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

Collisionally-Induced Dissociation

Low-energy CID – ions traveling with typical KE of <100 eV.

– Ions excited to a higher vibrational state, ion-target complex has a lifetime

High-energy CID – ions travelling with typical KE > 1 keV– Ions excited to higher electronic states, no detectable ion-target

complex

CID occurs via a two-step mechanism:Step 1. An endothermic activation step to form an M+ ion that is

internally excited (usually to a higher vibrational state)

Step 2. An exothermic unimolecular decomposition to a fragment ion and a neutral.

For more information about CID, see:L. Sleno and D. A. Volmer, “Ion activation methods for tandem mass spectrometry”, J. Mass Spectrom., 2004, 39, 1091-1112.

K. R. Jennings, Int. J. Mass Spectrom. Ion Phys., 1968, 1, 227.F. W. McLafferty, et al., “Collisional Activation Spectra of Organic Ions”, J. Am. Chem. Soc., 1973, 95, 2120-2129.

K. Levsen and H. Schwarz, “Gas-phase Chemistry of Collisionally-activated Ions”, Mass Spectrom. Rev., 1983, 2, 77-148.S. A. McLuckey, “Principles of Collisional Activation in Analytical Mass Spectrometry”, J. Am. Soc. Mass Spectrom., 1992, 3, 599-614.

Molecular Structural Analysis with MSn

CID and MSn opens up a range of possiblities for MS Scan Modes

– Precursor ion scans: keep MS2 constant, scan MS1

– Product ion scans: keep MS1 constant, scan MS2

– Neutral loss scans: scan MS1 and MS2 “in sync”, offset by the difference (neutral) of interest (ex. set MS2 to follow MS1 by 32 Da).

– Selected reaction monitoring: hold MS1 and MS2 constant (observe a selected fragmentation)

CollisionsMass

Analyzer 1Mass

Analyzer 2

Applications of MSn Experiments

A short list of applications: MSn studies of drug metabolism, environmental samples,

Especially useful in drug metabolism because key “pieces” of drugs can be selected via their product (daughter) ions or their neutral loss characteristics

MSn is applicable to any analytical situation where complex, overlapping spectra are detected and need to be interpreted

For more information about MS applications in drug metabolism, see:R. J. Perchalski, R. A. Yost and B. J. Wilder, Anal. Chem., 1982, 54, 1466-1471.

M. S. Lee and R. A. Yost, Biomed. Environ. Mass Spectrom., 1988, 15, 193-204.

Applications of MSn Experiments

Example: Structural analysis of linear alkylbenzylsulfonates - a common anionic surfactant that can be a soil pollutant

Can be monitored in soil by LC-ESI-MSn

Samples extracted with methanol, concentrated with SPE

Bruker Esquire 3000 ITMS, negative ion mode (compounds are negatively charged) in this mobile phase:

water/methanol/tributylamine/NH4COOCH3

m/z = 183 obtained from CID MS-MS of all chain lengths as a characteristic ion

m/z = 119 obtained from CID MS-MS-MS of m/z = 183 by loss of SO2

V. Andreu and Y. Pico, Anal. Chem., 2004, 76, 2878-2885

Molecular MS Applications: Environmental Science

A compound was discovered in smoke derived from burning plant material that increases germination of a range of plant species that typically follow forest fires.

The compound is 3-methyl-2H-furo[2,3-c]pyran-2-one, and it was synthesized after being isolated and analyzed by MS and NMR

GC-MS was able to detect this butenolide at low levels in “smoke waters”

The compound is stable at higher temperatures, and is active at 1 ppm to 100 ppt levels. It is derived from the combustion of cellulose.

G. R. Flemmatti, Science., 305, 977 (2004)

GC-MS (EI) Data:

m/z = 150 (100%, M+)

m/z = 122 (25%, loss of CO)

m/z = 121 (71%)

m/z = 66 (14%)

m/z = 65 (16%)

OO

O

CH3

C8H6O3Exact Mass: 150.03Mol. Wt.: 150.13

m/e: 150.03 (100.0%), 151.04 (8.8%)C, 64.00; H, 4.03; O, 31.97

Molecular MS Applications: Proteomics

Proteome: The group of proteins related to a cell type (with a certain genome) under certain conditions (often forced on the cell)

Genome: The complete DNA sequence of a set of chromosomes.

Proteomics: The analysis of native and post-translationally modified proteins to characterize complex biological systems. There are at least three “types” of proteomics:

– Profiling Proteomics: Identify the proteins in a biological sample (or differences between proteins in multiple samples)

– Functional Proteomics: Determine protein functions by finding specific functional groups or interactions

– Structural Proteomics: Determine the tertiary structure of proteins and their complexes (e.g. using H/D exchange).

Sequencing: The analysis of primary structure of an oligomer (e.g. a protein) by bottom-up (chemical digestion, then MS) or top-down (do it all via selective fragmentation in the MS)

D. Figeys, Anal. Chem., 75, 2891-2905 (2003)

Molecular MS Applications: Proteomics

MS is historically used for profiling proteomics but has greatly increasing applications to other areas.

MS can be used in conjunction with gel electrophoresis techniques (2D GE, SDS-PAGE, etc…) or with separations (LC)

MS can be used to study post-translational modifications of proteins (e.g. phosphorylation, glycosylation)

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

Molecular MS Applications: Proteomics

“Peptide mass mapping”: used to ID proteins by comparison to a database (bottom-up proteomics).

Accurate mass methods (single MS stage) are usually used, following digestion by an enzyme (e.g. trypsin) that “chews up” the peptide into fragments.

The better the mass accuracy, the less chance of isobaric (same mass) interferences.

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

Molecular MS Applications: Proteomics Sequence-specific peptide MS:

usually done with MSn methods involving CID

Produce MS data that contains signals for each amino acid in the protein.

Results in complex spectra, which can be handled in different ways…

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

1. Searched against DB

2. Directly analyzed (top down)

3. Use chemical tools to ID fragments (bottom up)

1. Edman degradation

2. H2O trypsin proteolysis

3. Methyl esterification

MS Methods for Surface Analysis

Secondary-ion MS (SIMS) in surface analysis

– Secondary analyte ions are produced by impact from a primary ion

– Can depth-profile (sputtering and ionization)

– Typical analysis depths – 10-30 Å, with lateral resolution of < 1 um

TOF-SIMS – why is this combination so special?

– SIMS works well with delayed-extraction methods

– Pulsed ion guns (time-resolved pulses followed by drifts)

Ions: Ar+, Cs+, N2+, O2+5-20 keV

Sputtered Atoms

IonsTo

MassAnalyzer

Surface Analysis: TOF-SIMS

Micropatterning of biomolecules on a substrate: potential applications for biosensors

Example: a surface-derivatized polymer (PET, with COOH groups) is used to couple biological ligands:

– Biotin-Steptavidin

Figure from Z. Yang, et al. Langmuir 2000, 16, 7482-7492

Mass Spectrometers as GC/LC Detectors

MS is increasingly used as a routine chromatography detector (especially in GC and LC)

Two modes:

• Single-ion monitoring (SIM): observe 1-4 ions selectively – improved signal-to-noise for ions of interest

• Total ion current (TIC): sum of all ions – can be noisy but also captures potential unknown m/z ratios

In these cases, the basic MS system (usually simple quadrupoles with limited resolution and mass ranges) is known as a “mass-spectrometric detector” or MSD

D. Figeys, Anal. Chem., 75, 2891-2905 (2003)

Ion Suppression

Ion suppression (one ion preferentially ionizing to the detriment of another) can cause quantitative issues in LC-MS analyses when multiple compounds elute simultaneously.

Complex biological matrices (e.g. in clinical samples) often lead to ion suppression.

Can be avoiding by matching standards and matrices to the analyte

Niesson et al., Mass Spectrometry Reviews, 2006, 25, 881– 899

Elemental Analysis with ICP-MS

ICP-MS is similar to ICP-AES – the sample is vaporized and desolvated, and vaporized atoms are then ionized

Isobaric interferences from plasma or matrix components

Diagram from Agilent Instruments Promotional Literature.

Advantages of ICP-MS

Typical sensitivity: 0.1-1 ug/L (ng/mL) in solution

Many elements at once (~50 at a time)

Different interferences than ICP-OES

Can achieve ppt (ng/L) detection limits for rare earths and actinides

Metallic Ion Speciation using HPLC-ICPMS

• What is an metallic ion species? It is the valence state of a metal (or the organometallic form)

• Example:• chromium +3 (Cr+3) - essential nutrient• chromium +6 (Cr+6) – highly toxic (Cr+6 is the contaminant made famous by

Erin Brockovich in the groundwater of Hinckley, CA)

• HPLC/ICP-MS specifically detects Cr+6 with an LOD of 0.06 ng/mL

• Sample prep – addition of “harsh” chemicals can alter equilibrium, and alter the concentration of species. Example - Dissolution of Cr samples in hot acid converts Cr+6 to Cr+3

• Typical HPLC flow rates 0.1 – 0.5 mL/min – can extinguish plasma if too high.

AMS: Accelerator Mass Spectrometry

• We know that MS can determine isotope ratios. But what happens if we want to determine isotope ratios when the isotopes differ in quantity by a factor of 10-5 to 10-9 ?

• AMS offers isotope quantification at attomole (10-18 mole) sensitivity

• Numerous applications to “long-lived” radioisotopes, which are a challenge to detect by decay counting methods

• Features:• High-efficiency negative ion source (cesium sputter)• Tandem electrostatic acceleration• High energy ions detected by counting in a gas ionization detector (fast ion

causes gas to ionize itself, emit x-ray, which is detected.)

• The AMS design is essentially a sector system with an accelerator and a “stripper” (argon gas unit – to destroy molecular ions)

For reviews of AMS, see:K. W. Turteltaub and J. S. Vogel, “Bioanalytical Applications of Accelerator Mass Spectrometry for Pharmaceutical Research”,

Current Pharmaceutical Design, 2000, 6, 991-1007.J. S. Vogel, et al., “Accelerator Mass Spectrometry”, Anal. Chem., 1995, 353A-359A.

AMS: Basic Instrument Design and Operation

• The ions then pass into a high resolution double-focusing sector instrument allows e.g. separation of 14C and 14N

• Includes pre-selection of a narrow KE spread (velocity selector)

• The AMS system at the University of Arizona is shown

• Negative ions are created (usually from a solid sample)

• These ions are accelerated (MeV) by ever increasing positive potentials

• The ions are rammed into a carbon sheet, creating positive ions (i.e. the charge is “reversed”)

Velocity selector

University of Arizona

AMS: Radiocarbon Dating

• The 14C isotope:• Half-life (1/2): 5730 years• Abundance: 1 part per trillion• Produced in the atmosphere from cosmic rays, 14CO2

• All terrestrial life maintains a constant 14C level (although ocean life and “land” life differ)

• When a plant or animal dies, its uptake of 14C stops, and the equilibrated levels in its tissue begins to decay.

• If the remaining amount of 14C can be measured, the age of the plant or animal can be estimated.

• In AMS, the ratio of 13C to 14C is measured (sequentially, with two different detectors) and ages can be determined by comparison to calibrated references

• Prepared isotope ratios are used to calibrate the ratio• Samples of known age are used to calibrate the dating method

AMS: Other Applications• Other applications of AMS:

• Pharmaceuticals (ADME – absorption, distribution, metabolism, excretion) – using “microdoses” in humans even before tox studies!

• Biochemical pathways

Element IsotopeHalf life

(years)

Sensitivity

(parts per 1015)

Hydrogen 3H 12.3 0.1

Beryllium 10Be 1.6 x 106 5

Carbon 14C 5730 2

Aluminum 26Al 720,000 3

Chlorine 36Cl 300,000 5

Calcium 41Ca 105,000 2

Iodine 129I 16 x 106 10

K. W. Turteltaub and J. S. Vogel, “Bioanalytical Applications of AMS for Pharmaceutical Research”, Cur. Pharm. Design, 2000, 6, 991-1007.J. S. Vogel, et al., “Accelerator Mass Spectrometry”, Anal. Chem., 1995, 353A-359A.

See also C&E News, July 11, 2005, pg. 28.

IMS: Ion Mobility Spectrometry• In IMS:

• Sample vapor introduced by thermal desorption or other techniques• The vapors from the above are ionized using 63Ni (~10 mCi sample) to

produce molecular ions or clusters of molecular ions• An electronic shutter gates ions into a drift tube with a ~200 V/cm potential• Ions drift down the tube, colliding with neutral gas molecules (~760 torr)• Larger ions have longer drift times because of their larger cross-sections

Diagram from G. W. Eiceman and J. A. Stone, Anal. Chem., 76, 390A-397A (2004).G. W. Eiceman, Critical Rev. Anal. Chem., 22, 471-489 (1991).

D. C. Collins and M. L. Lee, “Developments in ion mobility spectrometry – mass spectrometry”, Anal. Bioanal. Chem., 372, 66-73 (2002).

• The ions strike a detector (can be a MS), and are identified by flight time• Typical drift times 3-50 ms, typical time resolution +/- 0.040 ms

Ion Source DetectorDrift Tube

IMS: Theory

• In IMS, larger ions have longer drift times because of their larger cross-sections

• The difference in drift time is proportional to the electric field strength and a mobility (kim):

G. W. Eiceman, Critical Rev. Anal. Chem., 22, 471-489 (1991).D. C. Collins and M. L. Lee, “Developments in ion mobility spectrometry – mass spectrometry”, Anal. Bioanal. Chem., 372, 66-73 (2002).

Where:vd is the average velocity of an ion (cm s-1)kim is the ion mobility constant (cm2 V-1 s-1)E is the applied electric field strength (V cm-1)

Dim kTN

zek

122/1

0163

z is the charge of the ion and e is the electron charge (1.602x10-19 C)k is Boltzmann’s constant and T is the temperature (K) is the reduced mass of the ion-drift gas pairD is the ion-neutral cross-section area (=d2 for rigid-sphere collisions where d is the sum of the ion and drift-gas radii)

)cm V 1000(for 1

E

Ekv imd

• The Mason-Schump equation predicts kim, which is a function of temperature and pressure as well as other factors:

IMS: Ion Mobility Spectrometer Design• Advantages

• No vacuum pumps needed• Can be operated at room temperature, with air as a drift gas• Small enclosures (handheld) are possible – drift tubes can be ~6

cm long and 1 cm in diameter

• Disadvantages• Flight times must not overlap and must be carefully calibrated• Low information content

Diagram from G. W. Eiceman and J. A. Stone, Anal. Chem., 76, 390A-397A (2004).

IMS: Applications

• Airport Security• IMS is used to detect explosives through a luggage checking system - more

than 10,000 units are in use at airports worldwide• When a piece of luggage is searched by hand, often after a suspicious X-

ray image is observed, swabs can be taken and run through an IMS spectrometer to detect many common explosives

• Example: IMS can easily detect RDX (a.k.a. hexogen, cyclonite). This explosive was used in several recent terrorist attacks in Russia (August 2004) - see C&E News, 6-Sep-2004, pg. 15

Picture and Data from G. W. Eiceman and J. A. Stone, Anal. Chem., 76, 390A-397A (2004).

• Military/Defense• IMS can be used to

detect common chemical weapons - more than 50,000 systems (many handheld) are deployed with military units worldwide, as of 2004

IMS: Applications

• Handheld units• Early units weighed ~1.6 kg,

were used extensively in the 1991 Gulf War to test for nerve and blister agents

• Newer units weigh less than 0.5 kg

• The radioactive source has been replaced with a corona discharge ion source – can run for up to 40 hours continuously on a single battery charge

Photo and Data from G. W. Eiceman and J. A. Stone, Anal. Chem., 76, 390A-397A (2004).

IMS: Dopants and Reactant Ions

• Proton affinity determines ionization (especially in 63Ni sources)

• Reactant ions are used to achieve selectivity

• The analyte ion actually forms a pair with whatever suitable reactant ion is in the drift gas

• Examples:• Water (in air) the hydrated proton [H2O]nH+

• Acetone (Ac) AcH+ and Ac2H+

• Ammonia [H2O]nNH4+

• Methylene chloride Cl- (by dissociative e- capture)

G. W. Eiceman and J. A. Stone, Anal. Chem., 76, 390A-397A (2004).

IMS Example

• DMMP - Dimethyl methylphosphonate (Used to simulate organophosphorus nerve agents like sarin, tabun, and soman safely)

• Using acetone as a reagent gas• The resulting mobility spectrum:

Figure from G. W. Eiceman and J. A. Stone, Anal. Chem., 76, 390A-397A (2004).

IMS: Pharma Applications

• IMS can be used to detect pharmaceuticals (small organics)

• Can outperform HPLC• Smith’s detection

IONSCAN

Figures from Y. Tan and R. DeBono, Today’s Chemist at Work, 15-16 (November 2004). www.tcawonline.org

• Disadvantage: the drug (or impurity) needs to be ionized – it can decompose during this process, leading to multiple ions

Hyphenated Ion Methods

Note – here we refer to ion methods only (i.e. no LC/GC)

MALDI-ion mobility-orthogonal TOF MS (MALDI-IM-oTOF)– Used to study biomolecular structure

– Detection limit approaches conventional MALDI-MS

A MALDI-IM-oTOF experiment can simultaneously give mass spectra and molecular “conformation” (size and overall shape) information on desorbed ions.

Applications: mixture analysis, proteomics, analysis of complex tissues and micro-organisms.

A. S. Woods, et al. Anal. Chem., 2004, 76, 2187-2195.

Hyphenated Ion Methods

Mobility differences for different biomolecule classes can differ by ~15%

2D resolution!

A. S. Woods, et al. Today’s Chemist at Work, May 2004, 32-36.

MALDI-IM-oTOF enabling technology: medium-pressure IM cells that do not lose ions in the differential pumping region

References

Atomic and Molecular Mass Spectrometry– Optional:

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

– H. A. Strobel and W. R. Heineman, “Chemical Instrumentation, A Systematic Approach”, Wiley, 1989.

– Skoog et al. Ch 11 and 20.

Ion Mobility Spectrometry– Optional:

G. W. Eiceman, Critical Rev. Anal. Chem., 1991, 22, 471-489. D. C. Collins and M. L. Lee, “Developments in ion mobility

spectrometry – mass spectrometry”, Anal. Bioanal. Chem., 2002, 372, 66-73.