How Mass Spectrometers Work

1. How Mass Spectrometers Work • Principles • New Technologies • Application areas for specific technologies

2. Name of this game is technology, physics and engineering design. J.J. Thompson Discoverer of electron 1912, showed 2 neon isotopes deflected differently by magnet His student Aston, (1918) designed more elaborate instruments.

3. Diverse Interfaces/Sources Let us Cope with a variety of Molecular Types Need to get the molecule into the vapor phase before it can be ionized…. Unfortunately many more molecules are non-volatile than are volatile. The Interface problem Then we have the problem that different structure types make ions with a variety of mechanisms and degrees of difficulty. The Source problem We have a range of solutions to the Interface problem. These generally fall into: Aerosolizing Blasting off a surface Heating

4. As liquid, in solution, flow from GC, eluent from LC, solid on probe or moving belt Charged plate collimator Interface Slit Ion Separator Detector Source To Vacuum To Vacuum To Vacuum A Generic Picture Sample is volatilized and ionized either with electrons or charged gas molecules. Fragments break off the starting molecule Different technologies to achieve this. E.g. Magnets, RF quadrupoles, “ion trap” (cyclotron) time-of-flight tube. All these, in different ways determine specific curves or paths through the separator based on molecular weight/charge. Some parameter e.g. Voltage is scanned, so that at some Vm only masses of m have proper curve and pass to detector Typically this is a photomultiplier tube

5. As liquid, in solution, flow from GC, eluent from LC, solid on probe or moving belt Interface Source To Vacuum Sources

6. Sources we will cover Electron Impact Chemical Ionization Fast Atom Bombardment Matrix Assisted Laser Desorption Ionization Electrospray Ionization Atmospheric Pressure Chemical Ionization APPCI Secondary Ionization (SIMS)

7. Different Classes of compounds may need different Ionization techniques Many compounds have adequate volatility on heated probe. Often careful tracking of ion current vs. time will reveal mixtures of compounds selectively “boiling off” a heated probe. Use by placing about a microgram in solution on probe tip, allow to dry, insert to source. An important variation on this is Desorptive heating (very fast, like a “thermal shock”

8. e- e- e- Electron Impact Sources To Vacuum, Maintains ca. 10-5 torr Sample inlet, heated probe, etc. M+ To mass spectrometer M+ M+ M M M M+ Incandescent Rhenium filament, electron source, 70ev, can be lowered

9. Chemical Ionization (CI)-Why? Variety of chemistry, tailor to the sample One gas doesn’t work, try another Dial in more fragmentation (hard, soft CI reagent gases) Can be universal or compound selective Positive or negative ion chemistry can be easily achieved Most organics with mass < 800 Daltons have sufficient volatility for CI.

10. Chemical Ionization Sources To Vacuum RG+ RG+ Sample inlet, heated probe, etc. RG+ To mass spectrometer RG+ RG+ RG+ M+ RG+ RG+ M+ RG+ M M M M+ RG RG RG RG+ RG Incandescent Rhenium filament, electron source Inlet, choice of gasses (RG), ca. 1torr. (maintained by valve, pumping system)

11. Ion Chemistry and Chemical Ionization Proton transfer (proton addition or abstraction) Charge transfer Electron capture Addition of Reagent ion Higher adduct Cluster formation Governed by the heat of formation of the various products vs the reactants

12. Example for CI Overdose case, gastric contents examined for drugs. Here, the soft ionization potential of Isobutane as CI reagent gas could be counted on to provide molecular ions of all the compounds. Milne, et al. Anal. Chem. 1970 (42) 1815-1820.

13. Different Reagent Gases give somewhat different Mass Specs We can see why CI data don’t make it into “fingerprint” databases R.G.Cooks, et al. Org Mass Spec. 1976, 11, 975-983

14. Better Molecular Ions for fragile compounds

15. The Ion-molecule chemistry can be diagnostic for stereoisomers CI in C6F6 You might say we rationalize the proclivities based on ease of H atom abstraction from para position Harrison and Lin, Org. Mass Spec. 1984, 19, 67-71, See also, Keogh, et.al. Anal Chem 1984, 56, 1849-1852.

16. Chemical Ionization-Diversity of Chemistry possible Different reagent gases Take advantage of chemical affinities to tailor the ionization “Hard Ionization” gasses (big-∆H) produce high energy MH+, leading to more fragmentation. Example, Hydrogen Energy scale in reagent gasses H2>>CH4>iC4H10>NH3>CH3-ONO>NF3>N2O(last three are proton abstraction reagent gases for negative ion MS. N2O is also pos. CI gas) Compare proton affinities (PA) of conjugate acid of reagent gas to that of substrate. e.g., C4H9+ is a strong enough acid to give a H+ to any nitrogenous base. Contrary example, NH4+is not acidic enough to protonate ethers

17. Other Reagent Gases Ar CS2 N2

18. Liquid Mobile phase from LC or direct injection of solution Capilliary Potential at 3-8 kVolts (M+nH)n+ (M+nH)n+ H+ M H+ M H+ M H+ (M+nH)n+ H+ H+ (M+nH)n+ H+ (M+nH)n+ (M+nH)n+ Electrospray Ionization (ESI) Lower vacuum To mass spec Coulombic explosion after desolvation in vacuum skimmers

19. Ion optics in a source after using phosphate buffer. Can work for a while but salts on highly charged surfaces can arc etc. Limitations on E-Spray Compound must be polar enough to spray in a substantially aqueous mobile phase Relies on electrostatic charging of aerosol droplets Non-volatile salts in mobile phase can foul the ion optics. (Buffers)

20. Atmospheric Pressure Ionization(API) Mass Spec Source works in near to atmospheric pressure Uses heated nebulizer Corona discharge uses solvent CI Useful in Normal Phase HPLC Logical choice for very non-polar molecules.

21. SH+ SH+ SH+ M M Liquid sample (LC) MH+ MH+ SH+ M M MH+ M MH+ Heater And fragments! High voltage needle “corona” discharge, ionizes, breaks down, the gas close to it Atmospheric Pressure Ionization Vacuum, to mass spec SH+ SolventH+ Sufficient concentration to act as Chemical Ionization Reagent gas High-velocity nebulizer gas

22. Atmospheric Pressure Photoionization Chemical Ionization (APPCI) Cutting edge source technology Uses UV photon flux to transit chromophores to excited states, able to ionize other molecules. When there is no chromophore, a dopant that has a chromophore, like acetone is used.

23. Atmospheric Pressure Ionization Atmospheric Pressure Photoionizaton (APPI) SH+ SH+ SH+ M M Liquid sample (LC) MH+ Vacuum, to mass spec MH+ SH+ SH+ M M MH+ M MH+ Heater High-velocity nebulizer gas And fragments! h h h h h h UV light source

24. Ion Separator To Vacuum Ion Separation

25. Various Ion Separation Technologies • Different for different applications • Big variable in cost • Different in resolving power • Differ in the accessible mass range • We will cover: • Magnetic sector • Quadrupole • Time-of-flight • Ion Trap cyclotron • Combinations, triple quad, Q-TOF, double sector

26. As the magnetic field is scanned, only one mass at a time has the right curvature to make it through the slit Ions from source Magnetic Sector Ion Separation magnet Curvature of pathway varies as function of mass/charge Detector

27. Quadrupole mass analysis Mass range 10–4000 daltons (amu) Resolution, typically 1000 Scan rate 5000 daltons/min Accuracy .1–.2 daltons Not for high resolution mass spec Great for most organic chemistry applications

28. Ions with wrong spiral, crash into sides and don’t get through Ions with proper spiral make it to the detector Accelerated beam of all the ions How do Quadrupoles separate ions of different m/z? pos Neg DC voltage And AC (Radio Frequency)

29. The Quadrupole Orbits A Constant A/Q ratio: steeper slope=lower resolution Where RF and DC are the dc and ac voltages m is the mass and e is the charge,  is the radiofrequency and r is the space between the quad rods A “Mathieu” diagram Q Regions of orbit stability Lifetimes are 50-100 sec

30. Ion Traps Use RF fields to bring the ions into orbits (like Quadrupole, but made into a ring. Scanned RF can sequentially make different masses have stable orbits Ion chamber is swept clean thousands of times per second, then the RF voltage changed for a new orbit. Ideal for MS-MS. In this technique we can keep a selected mass in orbit for a long time, then introduce a collision gas for secondary Chemical Ionization. Either get new fragment, or use to track the origin of smaller fragments (Did that fragment come from the molecular ion, or from fragmentation of a product ion?)

31. Ion Traps Ion Source End Cap Electrodes DC voltage Ring Electrode Ions held in orbits RF, frequency can be swept. Electron Multiplier

32. Ion Trap Equation of motion From the stability digram, qz gives instability at 0.98 V is the peak voltage between ring and endcap electrodes.  Is the RF frequency, ca. MHz for a 1 cm gap(radius)

33. C20H9+ C19H7N+ C13H19O2N3+ ??? All same nominal mass C19H7N+` C20H9+ C13H19O2N3+ 249.058 249.070 249.1479 249 Resolution and Mass Spec Done with TOF, FT mass spec or double sector magnetic instrument Exact mass calibration compounds used. 5ppm precision is sufficient

34. Exact Mass MS Curves back, focusses Highly refocussed, accuracy is is ca 10ppm 4-sector instruments;2 electric field, 2 magnet sectors

35. A second mass separation sector Collision cell, hexapole acceleration region without selectivity accd to mass detector Can pass single ion m/z for selective Ion Monitoring. We can exploit this as way to eliminate chemical noise from a dirty matrix Can have collision cell for further CI on a specific, selected ion or fragment ion. A normal quadrupole for mass separation Three sector Quad Mass SpecsMS-MS Ions from source

36. Time-of-Flight Mass Spectrometry Totally different concept. Have a fixed electric field down a “flight tube” A fixed distance from start to detector. Accelerate ions to the field, start timing. The time to the detector is inversely proportional to mass (smaller mass gets there first) Time therefore is calibrated to mass Great precision, accuracy--can do exact Mass determination

37. Time-of-Flight mass spectrometry Simple principle: Drift time over a fixed distance is related to momentum. “time” of transiting over this distance is proportional to Best technique for macromolecules. (have done over 500kDa). Has very high mass resolution, fortunately. Since biomolecules like this suffer from bad volatility issues, TOF is happily paired with MALDI ionization.

38. How Flight Tubes work Detector Ion path Accelerate ions (“pusher”) From source Electric field “reflectron” gives longer path for resolution

39. MatrixAssistedLaserDesorptiveIonization Or… What to Do, when your sample has no volatility whatsoever How do you get a protein to oblige you by vaporizing nicely and going down the mass spec? Molecular ions laser Exquisite sensitivity Great marriage with TOF for high mass range, resolution Dilute biomolecule in e.g. cinnamic acid for proteins, picolinic acids for nucleotides

40. MALDI-TOF A mixture of peptides ca. 10 amino acid in length Injected to mass spectrometer 20 femtomoles (10-15)each, of angiotensin I, angiotensin II, bradykinin, and fibrinopeptideA 1296.7 1060.6 1048.9 1537.6

41. Voltage to Detector Ions+ + + + + + + + h + e- h e- h h e- h e- e- Phosphor plate h e- Mass Spec Detectors Conversion Dynode PhotoMultiplier Tube

42. Specific Detectors Theimportant point is the 106-fold amplification by these stages (e- per ion) Cascade of Faraday Cups