Ionization Techniques Mass Spectrometry Ionization Techniques

Mass Spectrometer All Instruments Have: Sample Inlet Ion Source Mass Analyzer Detector Data System http://www.asms.org

Ionization Techniques Gas-Phase Methods Electron Ionization (EI) Chemical Ionization (CI) Desorption Methods Matrix-Assisted Laser Desorption Ionization (MALDI) Fast Atom Bombardment (FAB) Secondary Ion MS (SIMS) Spray Methods Electrospray (ESI) Atmospheric Pressure Chemical Ionization (APCI)

Electron Ionization http://www.noble.org/PlantBio/MS/ion_tech_main.html

Electron Ionization Samples must be vaporized in the ion source Typically 1 of 1000 molecules entering the source is ionized 10-20 eV of energy is imparted to the molecule ~10eV is enough to ionize most molecules Up to 230 kcal/mol is left to cause fragmentation Using lower energies can reduce fragmentation, but since efficiency falls off, less molecular ion is generated under these conditions

Electron Ionization b-lactam: Effects of ionization energy Beta Lactam gives lower overall efficiency at 15 eV negating the effects of reduced fragmentation.

Electron Ionization (low picomole) Advantages Disadvantages Well-Established Fragmentation Libraries No Supression Insoluble Samples Interface to GC Non-Polar Samples Disadvantages Parent Identification Need Volatile Sample Need Thermal Stability No Interface to LC Low Mass Compounds (<1000 amu) Solids Probe Requires Skilled Operator

Chemical Ionization Reagent gas is introduced into the source at ~0.5 torr Reagent gas is preferentially ionized. Ions react mostly with neutral reagent gas Reactions occurring depend on the nature of the reagent gas Ions in the reagent gas plasma react with the analyte

Chemical Ionization http://www.noble.org/PlantBio/MS/ion_tech_main.html

Chemical Ionization: Methane Methane primarily forms CH4+• with CH2+• and CH3+ CH4+• + CH4 → CH5+ + CH3 (m/z 17) CH2+• + CH4 → C2H3+ + H2 + H• C2H3+ + CH4 → C3H5+ + H2 (m/z 41) CH3+ + CH4 → C2H5+ + H2 (m/z 29)

Chemical Ionization: Methane

Chemical Ionization: Methane Ions other than saturated hydrocarbons react via proton transfer CH5+ + M → MH+ + CH4 (or via C2H5+ or C3H5+) For saturated hydrocarbons, hydride abstractions is common CH5+ + RH → R+ + CH4 + H2 For polar molecules, adducts can form CH3+ + M → (M+CH3)+ MH+, R+, and adducts are pseudomolecular ions.

Chemical Ionization: Isobutane

Chemical Ionization: Isobutane

Chemical Ionization: Isobutane Reacts through Proton Transfer C4H9+ + M → MH+ + C4H8 For saturated hydrocarbons, no reaction For polar molecules, adducts can form C4H9+ + M → (M+C4H9)+ Lack of reaction with hydrocarbons can be used for selective detection of compounds in mixtures containing hydrocarbons Less fragmentation is observed with isobutane. (molecular species is more reliably formed)

EI vs. Methane vs. Isobutane Methane CI Isobutane CI

Chemical Ionization: Negative Ions Low energy electrons are present in the CI plasma These can attach to molecules with high electron affinities There are two principal pathways: N2O/CH4 reagent gas AB + e- → AB-• (associative resonance capture) AB + e- → A• + B- (dissociative resonance capture) Deprotonation can also occur if a basic ion is formed in the reagent gas plasma

Chemical Ionization (low picomole) Advantages Disadvantages Molecular Ion Interface to GC Insoluble Samples Disadvantages No Fragment Library Need Volatile Sample Need Thermal Stability Quantitation Difficult Low Mass Compounds (<1000 amu) Solids Probe Requires Skilled Operator

Ionization Sources - II EI and CI have limitations Both require a volatile sample Samples must be thermally stable Neither lends itself to LC/MS analysis Other techniques have been developed FAB (Fast Atom Bombardment) SIMS (Secondary Ion MS) MALDI (Matrix Assisted Laser Desorption) ESI (Electrospray)

FAB Sample is dissolved in a non-volatile liquid matrix Glycerol and m-Nitrobenzyl alcohol are common matrices A high energy (5kV) beam of neutral atoms (typically Ar or Xe) is focused onto the sample droplet Dissolved Ions and Molecules are ejected into the gas phase for analysis

FAB

FAB For Organic Molecules M+H and M+Na ions are typically observed M+H ions typically fragment more than M+Na ions Salts such as NaI can be added to the matrix to induce M+Na formation

FAB (nanomole) Advantages Disadvantages No Fragment Library Stable Molecular Ion High Mass Compounds (10,000 amu) Thermally Labile Compounds (R.T.) Disadvantages No Fragment Library Solubility in Matrix (MNBA, Glycerol) Quantitation Difficult Needs Highly Skilled Operator Not amenable to automation Relatively Low Sensitivity Can be used with HPLC (Continuous flow FAB) Difficult to implement Has been largely replaced by ESI/APCI/MALDI

SIMS Analysis of surfaces in situ A high energy (15-40 keV) beam of primary ions (In+, Ga+) or clusters (SF6, Au3, C60) is focused onto the sample droplet Surface and slightly sub-surface atoms or molecules are ejected and ionized Clusters increase secondary ion yield dramatically

SIMS Courtesy of Mike Kurczy, Winograd Group, Penn State University

SIMS Ga+ vs. C60+ J. Phys. Chem. B 2004, 108, 7831-7838 Videos

SIMS Advantages Disadvantages No Fragment Library Surface analyses Stable Molecular Ion Mass Limit ~ 10000 amu Small primary ion beam spots and beam rastering make imaging possible Disadvantages No Fragment Library Quantitation Difficult Needs Highly Skilled Operator Not amenable to automation Relatively Low Sensitivity Can be used with HPLC (Continuous flow FAB) Difficult to implement Has been largely replaced by ESI/APCI/MALDI

MALDI Matrix Assisted Laser Desorption Sample dissolved in a solid matrix Typically mixed in solution Small droplet applied to target and dried A wide variety of matrices exist Choose based on hydrophobic/hydrophilic character of sample Also based on laser absorbance (usually UV) An ionization agent is often added Agent must bind to the sample TFA and its Na+ Ag+ salts are common Acid for basic analytes such as peptides/proteins Na for good lewis bases. Ethers/esters Ag for pi systems (polystyrene, polyisoprene, polybutadiene

MALDI

MALDI Choice of matrix based on empirical evidence http://polymers.msel.nist.gov/maldirecipes/maldi.html Typically singly charged ions observed Some matrix adducts/cluster ions Difficult to analyze low MW compounds due to matrix background Typically used for MW 500-500,000

MALDI MALDI of a large protein (monoclonal antibody)

MALDI MALDI of polymethylmethacrylate

UV-MALDI Matrices Matrix Application Structure α-Cyano-4-hydroxycinnamic acid (CCA) peptides 3,5-Dimethoxy-4-hydroxycinnamic acid (sinapinic acid) proteins 2,5 Dihydroxybenzoic acid (DHB) peptides, proteins, polymers, sugars 3-Hydroxypicolinic acid (HPA) oligonucleotides Dithranol (anthralin) polymers

MALDI (low femtomole) Advantages Parent Ion High Mass Compounds (>100,000 amu) Thermally Labile Compounds (R.T.) Easy to Operate Easily Automated Disadvantages No Fragment Library Wide variety of matrices Quantitation Difficult Matrix Background

ESI Electrospray Ionization Sample dissolved in a polar solvent Solution flows into a strong electric field (3-6 kV potential) Electric field induces a spray of highly charged droplets (charges at surface) As droplets shrink, repulsion increases until they break into smaller droplets In small enough droplets, surface charges can be desorbed into the gas phase. Solvents: MeOH, H2O, ACN,

ESI

ESI Ions formed via charge-residue or ion- evaporation Molecules form M+H+ or M-H- ions Large molecules: 1 charge / 1000 amu Small molecules: Usually singly charged Molecules with no acid/base groups Can form adduct ions with Na+ K+ NH4+ Cl- OAc-, etc. Salts may be added or already present in sample. At higher concentrations, non-covalent dimers, trimers, etc can be observed.

ESI ESI ions formed at high pressure must be transferred into high vacuum Differential pumping is needed to move ions through small openings while maintaining low pressures Ions become super-cooled by expansion. Solvent can recondense Two methods to reduce cluster formation High temperature transfer tube Heated counter-current flow of N2

ESI

ESI-Multiply Charged Ions Large Molecules produce an envelope of charge states Deconvolution must be done to determine the charge states if isotopic resolution is not possible Typically, MS data systems use software to deconvolute automatically

ESI-Multiply Charged Ions Δm = 1 amu ; ∆(m/z) ≈ 0.055; z = 18 Δm = 1 amu ; Δ(m/z) ≈ 0.10 z = 10

ESI-Multiply Charged Ions Consider (M+zH)z+ z1m1 = M + z1mp (m1 = measured m/z) Consider a peak of m/z=m2 which is (j- 1) charge states away from peak m1 m2(z1-j) = M + (z1-j)mp z1 = j(m2-mp) (m2-m1) M = z1(m1-mp)

ESI-Multiply Charged Ions j=10 1303.8 1621.3 z1 = j(m2-mp) (m2-m1) M = z1(m1-mp) z1 = 10(1621.3-1.0073) (1621.3-1303.8) = 51.0 M = 51.0(1303.8-1.0073) M = 66485

ESI (low femtomole to zeptomole) Advantages Disadvantages Parent Ion High Mass Compounds (>100,000 amu) Thermally Labile Compounds (<0º C) Easy to Operate Interface to HPLC Zeptomole sensitivity with nanospray Disadvantages No Fragmentation Need Polar Sample Need Solubility in Polar Solvent (MeOH, ACN, H2O, Acetone are best) Sensitive to Salts