Exam 3: MS

displays m/z ratio by

desorbing gaseous species from condensed phases, which get fragmented and ionized

• these fragments are accelerated by an E-field, which separates the fragments based on their m/z ratio

• the area of each peak is proportional to the abundance of each isotope

nominal mass

the whole number masses, based on the most abundant isotope of an element that we use to calculate m/z in MS

• 1 Da=1 amu

• so C=12 Da, H=1 Da, etc

main 3 parts of a MS instrument

• ionization source

• mass separator/selector

• detector

MS ionization sources

• EI

• CI

• ESI

• APCI

• ICP

• MALDI

EI (e- ionization/ e- impact ionization)

• MS ionization source

• uses an e- beam at +70eV to ionize the gaseous molecule, forming fragments

• uses hard ionization since it imparts a lot of enegry onto the molecule

  • dissociative (the parent molecule) and non-dissociative (ionized parent molecule, which is unstable) results are formed

EI instrumentation

• usually starts with GC to form gaseous molecules

• when the molecules ionize, they for radicals

not every collision event in EI is

equally likely to occur

• at 1eV, most molecules will ionize, so at 70 eV, almost anything will ionize and form a cation, as e- are ejected off

• so the molecule (M) gets hit with 70 eV e- beam and forms M^+. (the molecular ion) and an e-, which is ejected from the molecule

resonance capture

when you use a lower energy e- beam (less than 20 eV) to obtain anionic fragments

• resonance capture uses the lowest energy and forms M^-.

• dissociative capture uss a lower, but intermediate energy to break apart a bond in a molecule (XY) to form X + Y^-.

CI (chemical ionization) instrumentation

the products are protonated, so have higher mass than the original molecules

• uses some reactive gas like methane or ammonia

• soft ionization, imparts low energy onto molecule

in CI to form positive ions, you are trying to

generate a proton to transfer onto your molecule

• CH₅⁺ is a common proton donor used

• one molecule that has become ionized reacts with the next

CI to form negative ions

• ions in sln can produce neg ions

• methane and ammonia promote formation of M^-

• also sometime uses O₂^-, F-, SF₆

• either way, produces fragments that are anionic

EI characteristics in its MS spectra

• forms many small fragments

• no clear molecular ion formed

• lots of isotopic patterns

• uses hard ionization

CI characteristics in its MS spectra

• high abundance of molecular ion peak (MH⁺)

• not as much fragmentation

• uses soft ionization

hard vs soft ionization

hard: imparts high energy onto the molecule and leads to a high degree of fragmentation

soft: imparts lower energy onto the moelcule, so forms fewer fragments

EI and CI are often used

in a complimentary fashion

What are the only 2 MS ionization sources we have talked about? more exist but ignore that

EI and CI

How MS selector separates masses using a B field

• an ion with both mass and charge (duh) is accelerated through a potential difference given by the acceleration plates

• the ion acquires a KE equal to the electric potential difference given by the plates

• so an ion with some velocity perpendicular to that of the B field experiences a force (centripetal force), which is proportional to the mass:charge of a given ion

mass separator magnetic sector instrumentation

• ionization source

• magnet

electric sector instrumentation (MS mass separator)

• like magnetic sector, works to direct ions

• has lower LOD, good for nuclear materials

• helps isolate isotopes

Why is there both a magnetic and a electric sector in a MS mass separator?

• using both together helps improve resolution

Quadrupole (mass separator)

common detector/separator for small molecules

• called quadrupole bc has 4 rods in it (one set is pos charged and other 2 are neg)

• pos ions go away from the pos and towards the neg and vice versa, so that ions travel in waves through the Quadrupole

• constant DC and RF voltages oscillate between the rods

• + set filters for high m/z

• - set filters for lower m/z

• so for a given amplitude of DC and RF voltages, only certain m/z will resonate, and all other m/z will be deflected

• so only ions of the m/z you want are detected

because a Quadrupole is operated at constant resolution, the spacing between data points is constant as a function of

m/z

• whereas magentci sectors operate at constant revolving power, so that spacing between data points increases with m/z

mass accuracy of Quadrupole

0.1-0.2 Da

Quadrupoles can be used in 2 diff modes:

• scan-sweep DC/RF voltages to collect m/z spectra

• selected-ion monitoring to set to a specific m/z or DC/RF range to monitor what is eluted from the initial GC so that only your analyte goes through, used for trace molecules

What mass separators have we talked abt?

• Quadrupole

• electric sector

• magnetic sector

Only MS detector we are talking abt:

continuous-dynode electron multiplier

continuous-dynode electron multiplier (detector)

multiple e- are created evcerytime an ion collides with a side of the detector, so amplifies the detected signal

resolution

the separation between 2 distinguishable features

resolving power

to what m/z we can reliably separate peaks that are 1 Da apart

either quantified as m/∆m OR m/m_1/2

• must specify which form you are using since they yield diff answers

• m/∆m: x-value where peak is/distance between two peaks

• m/m_1/2 : x-value where peak is/FWHM

• m1/2 is FWHM

is resolving power of a spectrometer is say 14320 Da, then

it has good res at m/z values below 1420 Da, but not as you approach 1420 Da and higher

regarding isotope abundance factors, something with x+2 of 97.3n will have a much higher isotopic abundance than something with say, 3.35n

isotopologues

molecules with the same structure but diff isotopes (say a C12 is subbed for a C13)

• can verify a likely structure by looking at the relative abundance of isotopologues

• in benzene, C6H6, M⁺ is given at 78 Da and we know for M+1, C is 1.08n and H is 0.012n

• so 6×1.08 + 6×0.012 gives a value of 6.55%

• so the 6.55% represents the expected relative intensity (or abundance) of the M+1 isotope peak at m/z = 79 relative to the molecular ion base peak (M⁺) at m/z = 78

halogens have m/z +____

+2 isotopologues

• due to the high abundance of x+2 isotopes

• M-79 is M- a bromide

rings and double bonds formula (tells us the degree of unsaturation, aka double bond equivalency)

R + DB= c-h/2 + n/2 + 1

c is G14 atoms (things that make 4 bonds)

h is hydrogen and halogens (things that make 1 bond)

n is G15 atoms (things that make 3 bonds, so have a lp)

• allows us to propose a plausible structure

What is the highest peak on a MS spectra?

M^+.

• the peak from the heaviest fragment should be a mass loss from this M^+. peak

common loses

-15 (methyl group)

-17 (OH or NH3)

-18 (water)

common fragments

2 peaks can be similar bc they undergo similar fragmentation pathways

ToF (time of flight) mass selector/separator

uses the principle that all ion fragments have the same KE, so ions of diff masses will have different velocities and travel diff distances

• lighter ions travel faster and reach the detector first uses a Quadrupole to focus the ions

• is a cheap, but very large device

• good for biomolecules/macromolecules

• collects spectra fast

• very high resolving power

• has 2ppm=0.001m/z accuracy

ToF instrumentation

larger ions travel farther path so that all have equal KE

• reflectron reflects ions at some offset distance depending on energy of the particle, so particles travel a set distance based on their m/z

ion-trap MS

accumulates ions at same m/z, the ions then generate e-, which are detected

• good with LC-MS

• has high sensitivity, especially for low m/z

orbitrap MS

• highest mass accuracy and resolving power

• for high-precision and accuracy of macromolecules and biomolecules

FTICR MS (Fourier-transform ion cyclotron resonance)

• has highest resolution

• things make it thru if they are a certain m/z

all MS methods are

destructive

why is it hard to interface liquids with MS

• must be aerosolized

• have a bunch of solvents with them

• large pressure difference

interfacing with GC to do MS

good bc eluent is such a small volume

interfacing with LC

• solids can deposit and clog/foul/damage ionization sources

• must be mindful that what you are using is compatible with the ionization source

• mobile phase must be volatile

common ways to interface LC-MS

• ESI electrospray ionization

• APCI atmospheric pressure chemical ionization

ESI electrospray ionization

• good for large molecules, up to 100,000 Da

• little fragmentation occurs, ions are formed in sln

• target analytes are charged in eluent

• nebulizes liquid sln, focuses droplets, which become ionized

zoom in of ESI nebulizer

the neg voltage pulls cations in and the Taylor cone focuses ions in

APCI

• useful for smaller molecules that are also in sln

• mainly used for polar, thermally stable compounds

• takes liquid eluent from LC, heats it to gas, forms cations

• soft ionization

diagram of our ESI-MS, which can also do APCI-MS

has Quadrupole analyzer, ToF selector, LC pump inlet

MALDI matrix-assisted laser ionization

• useful for things that are hard to ionize (proteins, bio samples, solids)

• matrix is usually organic crystal like 2,5-dihydroxybenzoic acid

• can map a surface to do imaging

• the molecule absorbs UV-photon and ionizes to create a singly charged species, which is easier to analyze

how does MALDI work

• laser pulses sample

• another laser pulse (337 nm) hits the sample

• matrix absorbs UV radiation, becomes ionized

• some charge transfers to the molecule of interest

• supersonic expansion of matrix with molecule of interest occurs

when to use ESI-MS vs MALDI

• both are soft ionization techniques

ESI-MS: larger molecules that have higher valency charges of liquid samples

MALDI: rapid imaging, solid samples, singly charged molecules

we can tell there are multiple protonation combinations in a MS spectra by the

gaussian nature of the curve, which represents the probabalistic distribution of charge states

• compounds all being + shows that mobile phase from the LC was likely acidic, so protonated to form all these species

when n = the # acidic protons

MS has lower res at higher m/z, so number SFs decr

comparing ESI and MALDI

ESI gives way more peaks due to complex charge states (lots of multiply charged M)

MALDI has way fewer peaks bc mostly just singly charged molecules

how could you determine lower-order structures at the molecular subunnit level?

by doing MS-MS

ESI and MALDI both

dont make fragments, so are soft-ionization methods

other ionization methods that exist

• open-air sampling-low temp plasma

• DESI (open-air sampling desorption electrospray ionization)

• DART (direct analysis in real time) analyzes drugs, cookies

DART (direct analysis in real time)

closest thing to non-destructive MS

• uses He or N2 to form reactive water droplets

• used to analyze drugs