Mass Spec

What are the key components of mass spec?

• sample introduction inlet

• sample ionization ion source

• ion sorting mass analyzer

• detector

• data processing

• data output and informatics

We analyze _____ in MS

ions (m/z)

MS is performed in a vacuum in order to prevent

ions from colliding w other molecules

For mass spec, what should be much larger than the distance from source to detector?

mean free path

What are the types of ion sources?

• electron ionization (EI)

• chemical ionization (CI)

• electrospray ionization (ESI)

• matrix-assisted laser/desorption ionization (MALDI)

What are the types of mass analyzers?

• time of flight

• triple quad

• ion trap

• FTIC

• orbitrap

Element

most fundamental chemical substance, cannot be broken down into simpler substances

Atom

basic unit of matter, 1 element

Atomic number

number of protons

Mass Number/Nominal mass

number of protons plus number of neutrons

Isotope

different forms of the same element that contain different numbers of neutrons; defined based on the isotopic mass number

Atomic mass

average mass of an atom (amu) in a mole

Molecule

group of atoms bonded together, forming the smallest unit of a chemical compound

Molecular weight

sum of the atomic masses of all atoms in a molecule (calculated using average mass)

Monoisotopic mass

mass if we were to take and consider only the most abundant of each isotope (342.297 amu for sucrose)

For sucrose, what are the other 2 peaks seen on MS?

next peak is if 1 C-13 atoms is present, next is if 2 C-13 atoms are present

Charge state or z =

1/deltaM where deltaM is spacing between isotope peaks, where deltaM = 1 if z = +/- 1, .5 if z = +/- 2, 1/3 is z = +/- 3

Resolution

defines our ability to separate two things

Resolving power

function of instrument performance, higher means peak width become much narrower; essentially peak width, allows us to separate things with similar masses

10% valley method

can measure instrument resolving power when you have 2 peaks of equal abundance separated by 10% valley (axis to dip in peak is 10%), Rp = m/deltaM where m is average mass, deltaM is difference between two masses

Single peak resolving power

much more common, take a single peak, Rp = m/deltaM where m is mass of single peak, deltaM is FWHM (width of peak at 50% height)

Having better resolving power will usually always (except for isomers) give better

resolution

Isobar

atoms of different elements with the same mass number (A) but different atomic numbers (Z)

Mass accuracy (ppm) =

(m_measure – m_theoretical)/m_theoretical x 1E6

Is better mass accuracy higher or lower number?

lower number

How does mass spec work?

ions move in an electric field

What is the simplest mass spec?

time of flight

In TOF, since all elements should have the same KE, velocity is

inversely proportional to mass

For time of flight, what molecules move the fastest?

the smallest

Is EI a gentle technique?

no

Advantages of EI

• lots of predictable fragmentation → structural info

• universal - ionization cross sections similar for all compounds at 70 eV

• large libraries of EI spectra

• simple, cheap, reproducible

Disadvantages of EI

• molecules must be in gas phase (volatile, not labile, typically MW < 500)

• sometimes no M_+ ion observed → no MW info

• No negative ions

Chemical Ionization

ionization by ion-molecule reactions between reagent ions and sample molecules

Positive Ion CI (PCI)

• basic EI source - tighter (smaller holes)

• Add large excess of reagent gas

• electrons impact almost exclusively on CH4

The reaction in PCI is more probable if M is more _____ than CH4

basic (PA_M > PA_CH4, where PA is proton affinity)

Choosing reagent gas changes

PA and fragmentation

As PA of reagent gas goes up, the deltaPA gets

smaller and gentler (less fragmentation)

Why is PCI more sensitive than EI?

• higher cross section for ion/molecule reactions than e-/molecule reactions

• Tighter CI source → longer M residence times

• Ion current concentrated in fewer ions

Charge exchange is probable if the recombinant energy of C6H6+ is

> ionization energy of M

As in PCI, in Ce, you can control the degree of fragmentation by selecting

reagent gas

If RE goes down in CE, fragmentation

goes down

What are the practical issues with CI?

• need tight source → P~1 torr

• if source P too low → “poor” CI

• to maintain low enough analyzer P, need differential pumping

• PCI spectra may be function of source T due to large number of collisions with walls

• In PCI, often set e- KE > 70 eV to increase e- penetration into source

• Ion traps can do PCI at low P

Some reagent gases can form _____ reagent ions

negative (NCI)

Why is NCI much less common than PCI?

thermal electron captures is much simpler for producing negative ions under CI conditions

ECNI sensitivity is much higher than EI for compounds with

high electron affinity (-COOH, NO2, X)

How does Atmospheric Pressure Chemical Ionization (APCI) work?

at high P complex series of ion molecule reactions typically occur w air reagent ions

What are the advantages/disadvantages of APCI?

• can be very sensitive

• can be very selective

• easily saturated

• subject to interferences, matrix effects

Field Desorption

field at sharp points; high enough to cause e- tunneling, leaving an ion which is repelled

Problems with Field Desorption

not routine; emitter prep not easy, emitter positioning critical, signal often transient

What are the possible desorption ionization techniques?

• laser desorption (laser beam)

• secondary ion mass spec (energetic ion)

• fast atom bombardment (energetic atom)

• plasma desorption (fission fragment)

Dynamic SIMS

• high primary ion flux

• elemental ions

Static SIMS

• low primary ion flux

• molecular ions formed

MALDI

pulsed laser beam absorbed by matrix that strongly absorbs laser, analytes dissolved in matrix become ionized and are analyzed by MS

Is MALDI a soft ionization method?

yes

Organic acids as matrix

• nicotinic acid

• 2,5-dihydroxybenzoic acid

• A-cyanohydroxycinnamic acid

With MALDI, the matrix is usually

an organic acid with strong molar absorptivity at 337 nm

In MALDI, the matrix-to-analyte molar ratio is usually

1,000 or 10,000 to 1

What are the goals of a matrix in MALDI?

• absorb UV/IR photons

• separate analyte molecules

• donate protons

Thermospray

ions produced by neutrals from spray being ionized or spray producing ions

Thermospray is ____ based CI

solvent

ESI

high voltage applied to needle with sample solvent flowing → formation of ions in solution → droplet evaporation to leave bare gas phase ions

What are the evaporation regimes for ESI?

ion evaporation model (small)

charged residue model (large, globular)

chain ejection model (large, linear)

What are the advantages of ESI?

• gentle, noncovalent complexes survive

• very sensitive

• LC, CE interfacing

• MS/MS

• Multiple charging reduces m/z reqs

MALDI Advantages

• straightforward, unsophisticated

• highest MW

• very sensitive

• tolerant of salts, buffers, detergents

• microprobe/imaging capabilities

• handles polar and nonpolar compounds

ESI Disadvantages

• Intolerant of salts, buffers, and detergents

• Not truly “quantitative” (ion suppression/matrix effects)

MALDI Disadvantages

• Not easily interfaced with LC, CE

• MS/MS not as easily implemented on TOFs

• m/z range <500 obscured by matrix

• Quantitation typically poor

Time of Flight

• Ions accelerated to constant KE

• ¨Accelerated ions travel through a field-free region (flight tube) and impact a detector

• An ion’s “time of flight” is measured (relative to the time of initial acceleration, t₀)

• Mass calibration plot (not linear) created based on known m/z ions to determine the m/z of unknowns

• High vacuum req (P < 10^-7 Torr) à long mfp required, due to 1+ m flight

• high resolution, high speed

• low quantification

Early TOF challenged by differences in

• Initial ion velocity (different KE)

• Initial ion position

• Ion formation (time)

How did early TOF resolve its challenges?

• Understanding of space focus/foci

• Orthogonal acceleration (or axis) = oaTOF

• Reflectron geometry

Mass-Selective Quadrupole

• Opposite rods with DC/RF voltage applied

• Mass-selective “stability scan”

• DC/RF voltages are ramped (at constant DC/RF)

• Each successive step allows for ions of higher m/z (1 amu/step) to have a stable trajectory to Reach detector

• Good speed; simple and robust; good sensitivity

• Most common MS in the world; common with GC-MS and LC-MS instruments

• Low resolution (unit)

Triple Quad

Q1 selects a fragment, Q2 = collision cell where m/z is fragmented again, Q3 select for another fragment

What is triple quad good for?

Single Reaction Monitoring (SRM)  Parent Scan/Daughter Scan

Targeted Fragmentation Spectrum   Neutral Loss Scan

The additional stages of triple quad make what better?

S:N

Orbitrap

• Ions injected into “trap” which confines ions in all 3-dimensions using quadrupolar (DC/RF fields)

• All ions initially trapped

• Mass-selective “instability scan”

• DC/RF voltages are ramped (at constant DC/RF)

• Each successive step allows for ions of higher m/z (1 amu/step) to have a unstable trajectory à Ejection from the trap à Reach detector

• Decent speed; simple and robust; good sensitivity; MSⁿ

• Low resolution (unit)

Tandem MS can be either

in space or in time

FTICR

• Ions injected into “trap” which confines ions using electric and magnetic fields

• Stable ion orbit measured by two receiver plates à Image Current detection

• Time-domain signal is Fourier transformed à Mass Spectrum

• Highest Resolution available (>>1M)

• Slow speed; extreme requirements (space, magnets, high vacuum)

Orbitrap

• Ions injected into “orbitrap” which confines ions using only electric fields

• Ions orbit radially around a central “spindle” electrode; Also oscillate axially

• Axial ion movement measured by two electrodes → Image Current detection

• Time-domain signal is Fourier transformed à Mass Spectrum

• Very high resolution (>100K); Benchtop availability

• Slow speed; high cost