Mass Spectrophotometers: Ionization Methods

What type of molecules does Mass Spectrometry detect?

• mass spec detects a variety of ionized molecules including large biomolecules (proteins, peptides, DNA, carbohydrates), small organic compounds, drug metabolites, and inorganic elements

• measures their mass-to-charge ratio (m/z) to determine molecular weight, quantify compounds, and identify chemical structures

Goldilocks Molecules

• molecules that fall within the detectable m/z range of a mass spec

  • “just right” = not too small and not too large for the instrument

• lower limit: an e^- or smth smaller than an e^- (small ions); typically 1-10 Da and above)

• upper limit: ~5 mil Daltons (depends on the instrument)

  • the limits refer to the instrument’s detection limits; the smallest or largest m/z that the instrument can measure)

• Different mass spectrometers have different detectable m/z ranges, and therefore different “Goldilocks zones.”

JJ Thomson

• sealed glass container (vacuum sealed) to prevent collisions w/ air

  • electrode on 1 end to create charged particles (ions)

• looking at how charged atoms move in an electric field

  • measure deflection → determine m/z

• Charged particles are deflected by electric fields

  • if (-) charged, deflected from (-) plate and toward (+) plate

  • if (+) charged, deflected from (+) and toward (-) plate

• ionization method: used high energy to remove electrons to create ions

  • often caused fragmentation of molecules

  • works well for small atoms/molecules

John Fenn

• Developed Electrospray Ionization (ESI) for mass spectrometry; Solved the problem of analyzing large biomolecules (DNA, RNA, proteins)

• converts biomolecules from solution → gas phase gently, allowing intact mass spec analysis of large molecules

Key challenge:

• Molecules must be in the gas phase for mass spectrometry

• Traditional high-energy ionization would fragment large biomolecules

John Fenn: How ESI Works

• Sample is in liquid solution, creating an electrical connection between the sol. and mass spec

  • ions in sol. allow current to flow, effectively “closing the circuit”

• A high voltage is applied to a narrow capillary

• This produces a fine spray of charged droplets

• only very small charged droplets are emitted from the capillary

What happens next:

• Droplets evaporate solvent as they travel toward an oppositely charged plate (electrode)

• Droplets become smaller → charges become more concentrated

• Eventually, ions are released into the gas phase
  (large biomolecules remain intact)

ESI: Coulombic Repulsion & Fission

• like charges in a droplet repel each other (Coulombic repulsion

• when repulsion becomes too strong, the droplets becomes unstable and undergoes Coulomb fission → “explodes into smaller droplets”

• repeated evaporation + fission leads to formation of gas-phase ions that can enter the mass spec for detection

More ESI Figures

Tanaka & MALDI

• developed laser-based ionization (MALDI approach) for analyzing large biomolecules like proteins

• MALDI allows gentle ionization: proteins and other large biomolecules must be converted into the gas phase, where high-energy methods would normally fragment them

How MALDI works

• sample (protein) is mixed with a matrix (light absorbing chemical) and crystallized

• a laser pulse is fired at the sample surface, hitting the matrix which absorbs the laser energy and rapidly vaporizes

  • it carries embedded protein molecules into the gas phase, and in this process, proteins become ionized

MALDI vs ESI

• matrix-assisted laser desorption ionization

• electrospray ionization (more popular)

• MALDI: 1 charge, solid, smaller (better for smaller molecular weights)

• ESI: many charges, liquid (good for DNA/proteins, which are happier in solution)

Types of Mass Spectrometers

Why mass spectrometry?

• the most powerful of MS is its selectivity

  • very little ambiguity, however may struggle with isomers

  • measures mass very specifically; can fragment the molecules to figure out the original molecule

• Versus UV/visible spectrophotometry of a complex mixture, we may be unsure of what we’re looking at

World Records of Mass Spec

• Can distinguish very small mass differences (~0.01 Da or even smaller in high-resolution instruments)

  • allows detection of isotopes, small chemical modification, and tiny differences

• applying (-) or (+) voltage allows us to see diff things

The 1st Mass Spectrum by Iwan. W. Griffiths

• showed that mass spec can separate ions by m/z ratio

• demonstrated that ions form a pattern of peaks based on m/z

• helped establish that mass spec can be used to analyze and distinguish ions based on mass differences (an analytical method)

Mystery of Neon

• the mass spectrum of Ne showed two distinct peaks instead of one

  • at the time, this could not be explained by existing atomic theory

• Thomson thought he had discovered a new element

• actual explanation: the two peaks were isotopes of neon

Preparative Mass Spec: Calutron

• Calutron: large scale mass spec used for isotope separation

• purpose: Separate U²³⁵ and U²³⁸ isotopes (purifying uranium)

• mass spec is very sensitive

• U atoms are ionized into charged particles and accelerated thru an electric field

  • ions are separated based on m/z, where light ions (U²³⁵) deflect slightly differently than heavier (U²³⁸)

Ionization

• removal or addition of an electron

• Energy required depends on how tightly a nucleus binds its highest energy electrons

  • outer orbit e^-’s are easier to pull off

• biomolecules (larger molecules) tend to break apart

  • you risk breaking apart the molecule when trying to peel off just one e^-

• noble gases are most reluctant to give up e^-

Soft Ionization - MALDI

• usually N₂ laser (337 nm, 3-10 ns pulses)

  • very short, high energy pulses that rapidly excite the matrix

  • causes desorption and ionization in a very short burst

• soft method: minimizes fragmentation of biomolecules

• how ionization by MALDI occurs is poorly understood (what’s going on is occurring at the atomic/molecular level isn’t understood)

MALDI Matrices

• biological samples coated by a matrix

• must absorb light at wavelengths emitted by laser

• efficient at converting light energy to heat

• charge-transfer reactions

First ESI-MS of Proteins

ESI: Taylor Cone

• When high voltage is applied to a liquid at the capillary tip, the liquid does not spray straight out

• Instead, it forms a cone-shaped meniscus called the Taylor cone

• From the tip of this cone, a fine charged jet of liquid is emitted

The cone forms because…

• Strong electric field pulls liquid outward; Surface tension pulls inward

  • The balance of forces creates a stable cone shape rather than a flat droplet surface

Charge distribution in droplets

• Charges move toward the surface of the droplet due to Coulombic repulsion

• This makes the droplet surface highly charged and unstable

ESI: Jet Fission (Droplet Breakup)

• As solvent evaporates, droplets shrink and become unstable

• They undergo Coulombic fission (explosive breakup)

• During this process:

  • Small fragments of the droplet are ejected

  • Mass loss is relatively small (~2–5%)

  • Charge loss is proportionally larger (~20%) because charge is concentrated at the surface

ESI: Charged Residue Model

• Each droplet contains a single analyte molecule (e.g., a protein) as it gets smaller.

• A charged droplet is formed from the Taylor cone jet

• Solvent evaporates → droplet continuously shrinks

• Repeated Coulomb fission reduces droplet size

• Eventually, a droplet contains one analyte molecule + remaining charge

• Final solvent evaporates completely

• final outcome: The analyte is left as a charged, gas-phase ion; the charge originally on the droplet is transferred to the molecule