2. Lecture 3 Ionisation techniques Gas Phase Ionisation Techniques : Chemical Ionisation

3. At the end of this lecture you should be able: To explain how chemical ionisation works To instruct a MS operator about the type of chemical ionisation reagent gas required for your experiment

4. Ionisation Techniques: Overview Gas-Phase Methods Electron Impact (EI) Chemical Ionization (CI) Desorption Methods Secondary Ion MS (SIMS) and Liquid SIMS Fast Atom Bombardment (FAB) Laser Desorption/Ionization (LDI) Matrix-Assisted Laser Desorption/Ionization (MALDI) Spray Methods Atmospheric Pressure Chemical Ionization (APCI) Electrospray (ESI)

5. EI: electron ionisation: recap 1 st step: sample must be in gas phase 2 nd step: bombarded by electron beam Generates high-energy analyte ions, which can fragment Analyte ions are always odd-electron Advantages: Simple to use, provides library- searchable fingerprint data Disadvantages: –Applicable only to volatile (i.e. small) and thermally stable compounds –Extensive fragmentation, can be difficult to detect molecular ion

6. Chemical ionisation Introduced by Munson and Field 1966 Ion source similar to that for EI Suitable for small, volatile molecules Higher pressures: ca. 1 Torr for ionisation, 10 -4 Torr for injection into mass analyser Generates less energetic, more stable ions CI yields even-electron ions: more stable Mainly molecular ion Simple spectra – But: fragmentation not straightforward Good for mixtures and quantitation Routinely used in gas chromatography (GC-MS)

7. Chemical ionisation - details Step 1: “Reagent gas” R, present in large excess (10 to 100 fold higher partial pressure) over analyte, is ionised (leading to R +● ) at 0.1-1 Torr by electron beam of 200- 500 eV e.g.: CH 4 → CH 4 +● → CH 3 +, CH 2 +● Step 2: Stable reagent ions are generated via ion- molecule interaction e.g.: CH 4 +● + CH 4 → CH 5 + + CH 3 ● CH 3 + + CH 4 → C 2 H 5 + + H 2 CH 2 +● + CH 4 → C 2 H 3 + + H 2 + H ● C 2 H 3 + CH 4 → C 3 H 5 + + H 2 Step 3: Ion-molecule interactions generate [M+H] + of analyte (see next slide)

8. Mechanisms of chemical ionisation: Ion-molecule interactions between reagent gas and analyte Most important: Proton transfer –Reagent gases generate Brønsted acids, e.g. CH 5 +, C 2 H 5 +, H 3 + –Gas-phase acid-base reactions, e.g.: M + C 2 H 5 + → MH + + C 2 H 4 Other mechanisms: –Adduct formation: M + C 2 H 5 + → [M+C 2 H 5 ] + –Anion abstraction: M + C 2 H 5 + → [M-H] + + C 2 H 6

9. Selective fragmentation after proton transfer Parent ion, e.g. MH +, can fragment Extent of fragmentation is proportional to transferred energy during ion-molecule interaction Transferred energy depends on exothermicity of reaction Exothermicity is function of proton affinities (PA) of reagent gas (R) and analyte (M) R + H + → [R+H] + PA(R) = -  H (of this reaction) M + H + → [M+H] + PA(M) = -  H (of this reaction) M + [R+H] + → [M+H] + + R  H 0 = – [PA(M) – PA(R)] Exothermic (  H 0 PA(R)

10. Proton affinities of common reagent gases (kJ/mole) Methane, CH 4 423 Ammonia, NH 3 854 Iso-butane, (CH 3 ) 3 CH819 Ethane601 Water697 Methanol761 Hydrogen423 Acetone823 Methylamine882

11. Example: selective fragmentation 81 95 109 123 137 CH 4 100200 214 M+NH 4 + 197 137 NH 3 197 MH + 137 81 95 109 123 Iso-butane Lavanduyl acetate (MW 196) PA = 840 kJ/mole 137 O HO+ PA=423 kJ/mole PA=819 kJ/mole PA=854 kJ/mole

12. Other modes of CI Charge-Exchange Chemical Ionisation: with toluene, benzene, NO, CS 2, COS, Xe, CO 2, CO, N 2, Ar, He as reagent gas: –M + X +● → M +● + X (creates radical cations) –Can use mixtures to generate both kinds of ions (conventional CI and CE-CI) Negative CI: electron capture

13. Self-assessment questions Q1Describe chemical ionisation mass spectrometry. How does it work, what is the nature of the reagent gas, what function(s) does the gas serve, and what type of mass spectra are generated from the analyte species ? Q2 Compare and contrast EI and CI Q3 Explain why EI and CI are not applicable to large non-volatile samples. Q4Explain how the choice of reagent gas (eg NH 3 or CH 4 ) affects the appearance of the mass spectra in chemical ionisation with respect to ionisation by proton transfer.

14. Lecture 4 Condensed phase ionisation techniques (1): Desorption methods

15. At the end of this lecture you should be able to: describe the differences and similarities of SIMS, LSIMS and FAB explain how laser desorption works describe MALDI and preparation of samples

16. Condensed phase ionisation techniques (1): solid state samples Field desorption (FD) Plasma desorption (PD) Secondary-ion Mass Spectrometry (SIMS) Fast Atom Bombardment (FAB) Laser Desorption/Ionisation (LDI) MALDI

17. Field ionisation/field desorption Developed in 1969 by Beckey No primary beam to bombard sample Field ionisation: Volatile samples brought into gas phase e.g. by heating Field desorption: Non-volatile sample is applied to “whiskers” which are grown on thin metallic wire filament (“emitter”) FD: Suitable for non-volatile and thermally labile samples, e.g. peptides, sugars, polymers, organometallics, carbohydrates

18. Field ionisation/ field desorption Ionisation is induced by high electric field gradient (10 8 V/cm) Distorts electron cloud around atoms and facilitates electron tunnelling from sample molecules to emitter electrode Yields M +●, then [M+H] + Hardly any fragmentation + - 8 keV emitter cathode + + + + + + - + + + + + + - + + + + + + emitterto cathode M adsorbed on emitterelectron tunnelsM +● is desorbed

19. Secondary Ion Mass Spectrometry (SIMS) Mainly for surface analysis Beam of Ar + (or Xe + ) ions with energy of 5-15 keV bombards solid surface Secondary ions from surface are sputtered Used for: –Mass analysis –Chemical composition of material Drawback: Rapid damage to surface: rapid decrease in signal http://www.whoi.edu/science/GG/people/acohen/research/hurricanes_slide9.html

20. Variations of SIMS: Fast atom bombardment (FAB) and Liquid SIMS FAB: Developed in 1980 by Barber et al. Improved version of SIMS Sample is dissolved in inert liquid matrix Common Matrix: Glycerol (amongst others). Protects sample from destruction and helps ionisation and desorption FAB: Bombardment with high-energy ATOMS (e.g. Xe) LSIMS: Similar, but bombardment with IONS (e.g. Cs + at 25-40 keV) instead of ATOMS Mass limits: 7 kDa standard, 24 kDa possible Often used in conjunction with magnetic sector mass analysers

21. FAB schematic probe Atom gun Slow Xe 0 Fast Xe o Extraction and focusing Sample ion beam Sample 1. Ionisation  slow Xe + 2. Acceleration of Xe + ions 3. Neutralisation by collision and charge exchange with slow atoms: Xe + (fast) + Xe(slow)→ Xe(fast) + Xe + (slow) [M+H] + Primary beam

22. Laser Desorption/Ionisation (LDI) Solid sample Laser beam with UV, Vis, or IR wavelength Sample required to absorb at laser wavelength Applied in surface and cluster analysis Drawbacks: –Difficult to control –Thermal degradation –No or low molecular ion –Only useful for < 1kDa Laser beam Desorbed ions and neutral species

23. Matrix-assisted Laser Desorption/Ionisation (MALDI) Nobel Prize in 2002 Soft ionisation technique Generates low-energy ions Lasers: UV or IR Most frequently combined with TOF mass analyser Can work for up to 1 MDa Matrix molecules Analyte molecule/ion Laser beam

24. Analyte ionisation in MALDI Step 1: Laser beam generates reactive/ excited matrix ionic species Matrix ions can be protonated, deprotonated, sodiated, or radical cations Step 2: In-plume ion-molecule charge transfer reactions between matrix ions and neutral analyte molecules Reactions: Proton transfer, cation transfer, electron transfer, electron capture Plume: Ions and molecule in gas phase

25. MALDI – sample preparation Sample/matrix mix (1:10,000 molar excess) in volatile solvent Requires only pico- to femtomoles of analyte Matrices: Solid organic, liquid organic, ionic liquids, inorganic materials 80x magnification of dried sample/matrix drop on target Sample target Drying

26. Instrumentation Most common combination: MALDI-TOF Instrument: MALDI generates pulses of ions, TOF works with pulses of ions Insertion of target into instrument

27. Most common: Organic solids, e.g.: MALDI matrices 3,5-Dimethoxy-4- hydroxycinnamic acid (sinapinic acid; C 11 H 12 O 5 )  -Cyano-4-hydroxycinnamic acid (4-HCCA; C 10 H 7 O 3 N) 2,5-Dihydroxybenzoic acid (gentisic acid; C 7 H 6 O 4 )

28. MALDI matrix absorbs photon energy and transfers it to analyte minimises aggregation between analyte molecules Matrix must –Absorb strongly at Laser wavelength –Have low sublimation temperature –Have good mixing and solvent compatibility with analyte –Have ability to participate in photochemical reaction

29. Absorbance Wavelength (nm) matrix analyte Common lasers; N 2 (337 nm), ArF excimer (193), Nd-YAG frequency tripled (355 nm) and quadrupled (266 nm) Matrices and analytes: desired photochemical characteristics Laser 200500

30. Applications: Mass determination of intact proteins www.membrane.unsw.edu.au/alumni/robert.htm MALDI-TOF spectrum of a protein mixture Predominantly M + ions (singly charged)

31. Applications: Molecular weight distribution of polymers www.arkat-usa.org/?VIEW=MANUSCRIPT&MSID=869 poly(dimethyl)siloxane 2.25 kD

32. Summary - MALDI Disadvantages MALDI matrix cluster ions obscure low m/z (<600) range Analyte must have very low vapor pressure Pulsed nature of source limits compatibility with many mass analyzers Coupling MALDI with chromatography is very difficult Analytes that absorb laser light can be problematic Advantages Relatively gentle ionization technique Very high MW species can be ionized Molecule need not be volatile Very easy to get femtomole sensitivity Usually 1-3 charge states, even for very high MW species Positive or negative ions from same spot

33. Self-assessment questions Q1Describe SIMS, LSIMS and FAB Q2In FAB, how is the fast atom beam produced and why is a fast atom beam used instead of the ion beam for the production of the secondary ions? Q3How does Laser Desorption/Ionisation work? Q4Why is LDI not being used with high molecular weight molecules? Q5Describe MALDI and sample preparation for MALDI Q6Explain why time-of-flight is suitable for mass detection in MALDI. Given the choice between a sector instrument and a TOF instrument, which one would you use to detect MALDI produced ions of 100 kDa and why ?