mass spectrometry 2

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Lectu re Date: February 27th, 2008

Mass Spectrometry and RelatedTechniques 2

Ion and Particle Spectrometry 2 - Outline

Atomic and Molecular Mass Spectrometry

Skoog et al. Ch 11 and 20.

Please read this additional reference:

R. Aebersold and D. R. Goodlett, Mass Spectrometry in

Proteomics, Chem. Rev., 2001, 101, 269-295.

Ion Mobility Spectrometry

If interested, see: G. W. Eiceman, Critical Rev. Anal. Chem., 1991, 22, 471-489.

D. C. Collins and M. L. Lee, Developments in ion mobili ty

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


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Homework Problems

If you read Marchs paper on ion traps:

What is resonant excitation? Summarize how resonant excitation

is used in typical ion trap MS experiments.

If you read the Russell and Edmondson paper on MALDI-

TOF and accurate mass:

Summarize the advantages and disadvantages of MALDI-TOF

(with DE and reflection) versus FTICR (including ESI-FTICR),

especially in biochemical applications.

If you read the Aeberold and Goodlett proteomics paper:

Why is MS used so heavily in the study of post-translational

modifications? Briefly describe an application to phosphopeptide

sequence determinations.

If you read the Sleno and Volmer ion activation methodspaper:

Pick any two of the ion activation processes described in the

paper (e.g. in Table 1), describe how it works and the

approximate energies involved, and list one advantage

Appl ications 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


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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 energiescan 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 stablecarbocations are more

stable fragments (ex.tertiary carbocations are

more stable than

primary)

- Resonance can stabilizefragments, ex. allylic

carbocations andbenzyl/tropylium ions

- Loss of small, neutral,stable molecules is

favored

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

O

p-chloro-benzophenone

Cl


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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/z

215 220

O

p-chloro-benzophenone

Cl

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


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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

31

P 32S

12C

AtomicMass Defects

(All Different)

Mass Defect (Da)

Mass Defect =

Atom Mass Nearest Integer

Every Cc

Hh

Nn

Oo

Ss

mass

is 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 1

Mass

Analyzer 2

++

+

++

+

+ + Mass Analyzerand Collision

Chamber


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More About MSn Systems

Tandem-in-space

Means that the mass selection and fragmentation occur indifferent 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 1

Mass

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)

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


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Collisionally-Induced Dissociation

Low-energy CID ions traveling with typical KE of 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 v ibrational 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 Colli sional Activati on in Analytical Mass Spectromet ry, 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 1

Mass

Analyzer 2


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Appl ications of MSn Experiments

A short list of applications: MSn

studies of drugmetabolism, 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.

Appl icat ions 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


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Molecular MS Applications: Environmental Science

A compound was discovered in smoke derivedfrom 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%)

O

O

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 (ordifferences 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.

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


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MS is primarily used for profiling proteomics but hasapplications to other areas.

MS is often used in conjunction with gel electrophoresis

techniques (2D GE, SDS-PAGE, etc)

MS can be used to study post-translational modifications

of proteins

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


Peptide mass mapping:

used to ID proteins by

comparison to a database.

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 massaccuracy, the less chance of

isobaric (same mass)

interferences.



11/21


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 two major ways


1. Searched against DB

2. Used to ID new peptides (de novo

sequencing), using chemical tools to

ID fragments: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-

30A, with lateral resolution of < 1

um

TOF-SIMS why is thiscombination 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

Ato ms

Ions

To

Mass

Analyzer


12/21

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.Langmui r 2000, 16, 7482-7492

Mass Spectrometers as GC/LC Detectors

MS is increasingly finding use 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


13/21

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 earth

and actinides


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Metallic Ion Speciation using HPLC-ICPMS

What is an metallic ion species? It is the valence state of a metal (or theorganometallic 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 ifwe want to determine isotope ratios when the isotopes differ in quantityby 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 achallenge 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 astripper (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.


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AMS: Basic Instrument Design and Operation

The ions then pass into a highresolution double-focusing

sector instrument allows e.g.

separation of 14C and 14N Includes pre-selection of a

narrow KE spread (velocityselector)

The AMS system at theUniversity of Arizona is shown

Negative ions are created (usuallyfrom a solid sample)

These ions are accelerated (MeV)by ever increasing positive

potentials

The ions are rammed into a carbonsheet, creating positive ions (i.e. the

charge is reversed)

Velocity selector

University of Arizona

AMS: Radiocarbon Dating The 14C isotope:

Half-li fe (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 equilibratedlevels in its tissue begins to decay.

If the remaining amount of 14C can be measured, the age of the plant oranimal can be estimated.

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

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


16/21

AMS: Other Appl ications

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

Calcium41

Ca 105,000 2Iodine 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 Detector Drift Tube


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IMS: Theory

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

The difference in drift time is proportional to the electric fieldstrength 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:

vdis 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)

D

imkTN

zek

122/1

0

163

z is the charge of the ion ande is the electron charge (1.602x10-19 C)k is Boltzmanns constant andT is the temperature (K)

is the reduced mass of the ion-drift gas pair

D is the ion-neutral cross-section area (=d2 for rigid-sphere

collisions where d is the sum of the ion and drift-gas radii)

)cmV1000(for1

E

Ekv imd

The Mason-Schump equation predicts kim, which is afunction of temperature and pressure as well as otherfactors:

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. Ei ceman and J. A. Stone,Anal. Chem., 76, 390A-397A (2004).


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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 IMSspectrometer to detect many common explosives

Example: IMS can easily detect RDX (a.k.a. hexogen, cyclonite). Thisexplosive 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 commonchemical weapons -more than 50,000systems (many

handheld) are deployedwith mili tary unitsworldwide, 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. St one,Anal. Chem., 76, 390A-397A (2004).


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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. Sto ne,Anal. Chem., 76, 390A-397A (2004).

IMS Example

DMMP - Dimethyl methylphosphonate (Used to simulateorganophosphorus nerve agents like sarin, tabun, and

soman safely)

Using acetone as a reagent gas

The resulting mobility spectrum:

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


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IMS: Pharma Applications

IMS can be used to

detect pharmaceuticals(small organics)

Can outperform HPLC

Smiths detection

IONSCAN

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

Disadvantage: the

drug (or impurity)

needs to be ionized it

can decompose duringthis 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.


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Hyphenated Ion Methods

Mobility differences for

different biomoleculeclasses can differ by ~15%

2D resolution!

A. S. Woods, et al. Todays 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

Further Reading

Mass Spectrometry:

1. F. W. McLafferty, Interpretation of Mass Spectra, 3rd Ed., University

Science Books, Mill Valley, CA (1980).

2. H. A. Strobel and W. R. Heineman, Chemical Instrumentation, A

Systematic Approach, Wiley, 1989.

mass spectrometry 2

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