CH 415 Chapter 20: Molecular Mass Spectrometry

Study of +molecular ions" M+ e- -> M+ + 2e-

Purpose of Mass Spectroscopy

Use the difference in mass-to-charge ratio (m/z) of ionized atoms or molecules to separate them. Thus, allowing quantitation of atoms or molecules and providing structural info by the identification of distinctive fragmentation patterns.

General operation of mass spectrometer:

1. create gas-phase ions

2. separate the ions in space or time based on their m/z ratio

3. measure the quantity of ions of each m/z ratio

Instrumentation:

inlet → gaseous ion source (ionization) → mass analyzer (sorting of ions) → ion transducer (detection of ions) → signal processor → mass spectrum

Ionization Sources: EI, CI, FAB, MALDI, ESI

Analyzers: Quadrupoles, Time-of-Flight (TOF), magnetic sectors, Fourier transform, quadrupole ion traps

Detectors: electron multiplier, Faraday cup

Ion sources for molecular mass spectrometry

Gas-phase sources - sample is first vaporized then ionized (volatile analyses): EI (electron impact) and CI (chemical ionization)

Desorption sources - sample is converted directly into gaseous ions (non-volatile analyses): FAB (fast atom bombardment), MALDI (matrix-assisted laser desorption ionization), ESI (electrospray ionization)

Increasing Softness: (hardest) EI<CI<FAB<MALDI<ESI (softest)

Interpretation of Spectra

Ion Sources for Mass Spectrometers

Gas-Phase: EI (ionizing agent = energetic electrons) and CI (ionizing agent =reagent gaseous ions)

Desorption: FAB (ionizing agent = energetic atomic beam), MALDI (ionizing agent = laser beam), ESI (ionizing agent = high electrical field)

Energy driven process → hard ionization, soft ionization

MS with “Hard” and “Soft” Sources

A hard ionization source (EI) - shows fragments

A soft ionization source (CI) - shows parent

Electron Impact Source (EI)

• electrons from filament hit the sample, path of electrons and molecules are on a right angle

• this method is applicable to all volatile compounds (>10³ Da) and gives reproducible mass spectra with fragmentation to provide structural info.

Filament - tungsten or rhenium (our source of 70 eV e-s)

Target - anode used in association w/ the filament to produce e-s

Repeller - positively charged electrode used to “push” + ions out of the ionization source

Lens stack - series of increasingly more negative electrodes used to accelerate our ions to constant KE

• Why vacuum?

-to ensure filament does not burn out

-to help vaporize samples

-to reduce collision b/t formed ions and atmospheric gases

-to remove sample from instrument after analysis

M + e- (70 eV) → M+ + 2e-

Electron Ionization Process

M (IE)→ M+. (odd-electron ion) (excess energy) → EE+ (fragment ion)

Electron removed from orbital with lowest IE n < pi< sigma

Methane (CH4 IE = 12.6 ev)

Ethene H2C=CH2 IE = 10.5 eV)

Methyl amine H3C-NH2 IE = 10.3 eV

Typical Reactions during EI

Energy = 70 eV → 6700 kJ/mol

Typical bond energies → 200 to 600 kJ/mol → EXTENSIVE FRAGMENTATION

EI spectra

• different molecules behave differently

• Good molecular (parent) ion → little fragmentation

• No molecular (parent-far right side signal?) ion → extensive fragmentation

• isotopes are extremely important!

• Molecular ion isotopic cluster

84 = 12CH235Cl2

85 = 13CH235Cl2

86 = 12CH235Cl37Cl

87 = 13CH235Cl37Cl

88 = 12CH237Cl2

Advantages and Disadvantages of EI Ion Sources

Advantages:

• subpicomole to picomole detection limits

• availability of computer data bases of over 100,000 compounds

• use of fragmentation pattern as a fingerprint w/ databases to identify unknowns

• Structural info obtained from fragmentation pattern

Disadvantages:

• limited mass range (about 600 Da) due to thermal desorption (volatility) requirement; derivatization can extend range.

• possible decomposition prior to vaporization

• severe fragmentation; often resulting in no observable molecular ion

Chemical Ionization (CI)

• development from EI

• same compound classes as EI

• gives molecular weight

• Softer ionization technique

• Produces M+H+ ions or M-H- ions

• used to produce more abundant molecular ions when the molecule under investigation fragments using EI

CI MS Sources

• a soft ionization method

• gaseous molecules of the sample are ionized by collision rxns with ions produced from a reagent gas (methane, propane, isobutane, ammonia). They produce different spectra.

• It relies on our charge being transferred from a reagent molecule to our sample. Reagent ion + molecule → molecular ion + reagent ion

• This method gives molecular weight info and reduced fragmentation compared to EI

Positive CI Mass Spectrometry

Reagent gases are used and after several ion-molecule rxns, produce a species BH+ which have a variety of proton affinities

Efficiency of CI: Proton affinity of analyte > proton affinity of the reagent gas

• selective ionization method (ex. ammonia)

• softness fo ionization depends on differences in proton affinities b/t the analyte and BH+ - large differences in PA’s results in more fragmentation

Why EI and CI (gas-phase sources) not enough?

• sample must be in gas phase

• not for nonvolatile or thermally unstable compounds

→ Desorption Sources

Matrix-assisted laser desorption ionization (MALDI) - soft ionization

Analyte protonation occurs (M+H+ → MH+)

analyses: polymers, proteins, DNA, large fragile molecules

matrix: large excess over analyte, absorbs UV laser light, transfer energy to analyses (common matrix is strong UV chromophore (sinapinic acid (SA))

• matrix absorbs the laser light and transfer energy to analytes

• analyses are desorbed, ionized (by taking proton), and accelerated to mass analyzer

MALDI matrix must:

• be able to embed and isolate analyses (ex. by co-crystallization)

• be soluble in solvents compatible with analyte

• be vacuum stable

• Absorb the laser wavelength

• cause co-desorption of the analyte upon laser irradiation

• Promote analyte ionization

Essential Functions of the Matrix:

1. isolate and encase the analyte molecules (analogous to a solvent shell). (matrix encases the analyte)

2. Absorb the laser energy via electronic or vibrational coupling/excitation (photons from laser excite the matrix)

3. Facile desorption from the condensed phase WITH the analyte molecules but WITHOUT destructive heating of the analyte molecules (“softness”)

4. efficient ionization of analyte molecules

MALDI is:

• more tolerant of salts and complex mixture analysis than ESI

• Important for huge molecules: proteins, polymers

• Spectra often contain multiple charged ions

More MS Analyzers

Electrospray Ionization Source (ESI)

Excess ion charges accumulate at droplet surface evaporation → Rayleigh limit reached → Coulomb explosion (droplet fusion) → desolated ions

Rayleigh limit - electrostatic repulsion forces of ions at droplet surface equal the surface tension forces holding the droplet together

At the Rayleigh limit, surface tension can no longer support the charge leading to Coulombic explosion

Iribarne-Thompson Model

• charge density increases

• Rayleigh limit (Coulomb repulsion = surface tension)

• Coulomb explosion (daughter driblets)

• Evaporation of daughter droplets

• Desorption (desolation) of ions from the droplets into the ambient gas (IONS FORMED)

ESI mass spec of myoglobin - multiple intact ion charge states

mass = 16992 Da +20 ion

m/z = 19992 + 20/ 20 = 850.6

Protein molecular weight = M = 16,951.5 Da

add n protons → m/z = (M +1.0078n)/n

m/z (at z=20) = (16,951.5 +20)/20 = 848.6

• multiply charged ions result in lower m/z!

• lower mass range spectrometer can be used

Advantages of ESI

• allows for the direct coupling of liquid separations to mass spectrometer

• multiple-charging extends the mass range of an analyzer by a factor equal to z

• Soft(est) ionization technique which allows for the analysis of non-covalent complexes

• “No” matrix interference

• Practical mass range up to 100 kDa

• good detection limits (fentomole to attomole)

More MS Analyzers

Resolution? - the capability of a MS to differential b/t masses

R = m/deltam

deltam : mass difference b/t two adjacent peaks that are just resolved (height of the valley < 10% of the peak)

M: nominal mass of the first or mean of the two peaks

1. Estimate accuracy of measurement if resolution is known 

Ex. If R = 5000, at mass 500 range

R = m/delta m : 5000 = 500/delta m → deltam = 500/5000 = 0.10

2. Determine R required C2H4+ and CH2N+ have masses of 28.0313 and 28.0187

R = m/deltam = 28.025/0.0126 = 2220 (average of the two/distance b/t the two)

Sample Inlet Systems

1. Batch inlet systems

2. direct probe inlets

3. Chromatographic inlets

4. Capillary electrophoretic inlets

Inlet Systems

Gas/Liquid Inlet System

Solid/Matrix Inlet System

Mass Spectrometry Mass Analyzers

1. Magnetic Sector Mass analyzers

2. ion-trap analyzers

3. TOF mass analyzers

4. Quadrupole mass analyzers

How does an ion with a velocity vector (v) behave in a magnetic field (B)?

• magnetic field (B): F = v x B

• the ion undergoes a force perpendicular to velocity and magnetic field vectors, and is deflected through a circular path with radius r.

• Ions w/ different m/z will be deflected with different radii of curvature

Orbits of charged particles moving in magnetic field:

qvB > mv²/r (spiral)

qvB = mv²/r (circle)

qvB = mv²/r (unbound/escapes)

Magnetic Sector Physics

KE = zeV = ½ mv²

Magnetic force = FM = Bzev

centripetal force = Fc = mv²/r

FM = Fc

Bzev = mv²/r

v=Bzer/m

m/z = (B²r²e)/2V

r= radius of curvature

path of heavier and lighters ions (they will hit the outside)

Magnetic Sector Mass analyzer

The mass spectrometer is an instrument which can measure the masses and relative concentrations of atoms and molecules. It makes use of the basic magnetic force on a moving charged particle.

After ionization, acceleration (accelerating voltage applied), and selection of single velocity particles, the ions move into a mass spectrometer region where the radius of the path and thus the position of the detector is a function of the mass (r = mv/qB = mEs/qBBs)

Ion Trajectory:

- ions from the ion source are accelerated to high velocity through a magnetic sector, in which a magnetic field is applied perpendicular to the direction of ion motion.

-Ion velocity then becomes constant but in a circular path at angles of 180, 90, or 60 deg.

-Ions are sorted mass to charge ratio by holding V and r constant while varying B (m/z = (B²r²e)/2V

Advantages:

• high resolution, sensitivity, and dynamic range

• high-energy CID MS/MS spectra are very reproducible

Disadvantages:

• not well-suited for pulsed ionization methods (ex. MALDI)

• usually larger and higher cost than other mass analyzers

Single Focusing Magnetic Sector vs. Double Focusing Mass spectrometers

Single Focusing Magnetic Sector:

• ions at source with same m/z ratio

• ions with diverging directional substitution will be acted upon in the same way

• Brings ions with different directional orientations to focus

• limits the resolution

Double Focusing Mass spectrometers:

• passes ions through an electrostatic analyzer (ESA) which limits the range of the KE of ions reaching the magnetic sector

• only ions with the same average kinetic energy pass through the ESA slits into the magnetic sector

• Two focal planes at the ion collector (energy focal plane -takes place in electrostatic analyzer) and directional focal plane - occurs in the magnetic sector)

• Increases resolution

Ion Cyclotron Resonance Mass Analyzer

Ion Cyclotron Resonance:

r = mv/zeB

wc = zeB/m

FT-MS (Ion cyclotron):

• ions are arrested in cell inside a static magnetic field

• ions will move in a circular trajectory radius given by: r = mv/zeB

• the corresponding cyclotron frequency is: wc = zeB/m

First two are the ion cyclotron resonance phenomenon:

1. ions before excitation. They are in their natural cyclotron radius within the magnetic field.

2. Ions during excitation with a radio frequency. This excites the ions to a larger cyclotron radius

3. Ions after excitation. The cyclotron radius remains in its larger state

Detection of image current:

• independent of B0 strength

• increases linearly with r and with ion charge