1. FC-MS from Teledyne Isco CombiFlash ® a Name You Can Rely On

2. Overview of Mass Spectroscopy: Goals: Terminology Major parts of a mass spectrometer Ionization techniques; advantages & disadvantages Mass analyzers; advantages & disadvantages PurIon Mass Spectrometer overview Why use PurIon? FC-MS overview

3. Terminology ■ daltons (Da) ■ Unified atomic mass unit (u)- same as Da, based on 12 C ■ amu (atomic mass units)- archaic, technically based on 16 O, but people still use it… ■ m/z- charge-to-mass ratio, may see as m/q (older literature)

4. Summary of mass spectrometry ■ Analytes are converted to gas-phase ions (source). ■ The ions are separated by their mass-to-charge ratios (m/z, Analyzer) and are detected (Detector). ■ Relative ion current (signal) is plotted versus m/z to produce a mass spectrum.

5. More Terms that I chuck around casually ■ M: mass of a given molecule ■ [M+H] + : mass of a molecule with a proton; carries a positive charge ■ MeOH: methanol ■ MeCN, ACN: acetonitrile ■ EtOAc: ethyl acetate

6. Isotopes ■ Same element (determined by number of protons) ■ Different number of neutrons ■ Changes mass of atom (and molecules containing this atom) ■Hydrogen ( 1 H, 2 H deuterium, 3 H tritium) ■Carbon ( 12 C, 13 C, 14 C) ■Chlorine ( 35 Cl, 37 Cl; ~75:25) ■Bromine ( 79 Br, 81 Br; ~50:50)

7. Mass ■ Masses based on 12 C=12.0000 ■ Other elements/isotopes do not have integer masses ■ 1 H=1.0079 ■ 16 O=15.9949 ■ 14 N=14.00307 ■ 79 Br= 78.9183

8. Mass ■ Nominal mass= integer mass using most abundant isotope; the number we usually state in conversation ■ Monoisotopic mass: Sum of atomic masses using the most common isotope of each element in a molecule ■ Exact Mass: used by some chemistry software; sum of atomic masses of most common isotope when no isotopic species specified. ■ Average mass: sum of average atomic masses in a molecule

9. Mass Spectrometer block diagram Inlet Source Region Mass AnalyzerDetector Vacuum system

10. ■ Roughing pump ■ Turbo molecular pump ■ Vacuum needed: ■ To avoid further reactions (fragmentation, reactions, etc.) ■ Increase mean-free path (maintain ion energy)

11. Mass analyzers ■Single quadrupoles (MS) ■Triple quadrupoles (MS/MS) ■Ion traps ■Time-of-flight (TOF) ■Sector analyzers ■Hybrids (ex. QTOF) ■and more hybrids (magnetic sector TOF)

12. Types of Mass Analyzers: Sector ■ Separate ions by charge-to- mass ratio (m/z; m/q) ■ Generally have a electrical field sector (not shown) to focus ion energies ■ Classical mass spectrometer ■ Good resolution, dynamic range ■ Large, higher cost

13. Types of Mass Analyzers: Time-Of-Flight (TOF) ■ High mass range ■ Well suited for pulsed ionization methods (MALDI) ■ Requires pulsed ion injection or ion beam switching “Drift” area

14. Fourier Transform analyzers ■ Ions drift into area of constant magnetic field- ions move in circular motion ■ Use oscillating electric field to “excite” ions ■ Detect ions by their cyclotron radiation, use Fourier transform to obtain masses Highest mass resolution Expensive analyzer

15. Quadrupoles ■ Less expensive; compact design; low scan times, very common ■ Limited resolution, not suited for pulsed ionization methods ■ Scan the electric fields/ frequency to scan mass range The correct combination of AC and DC electric fields allow resolution of ions by their m/z ratio

16. Triple Quadrupoles (MS-MS) Select ion of interest Collision induced disassociation Fragment mass analyzer Q1Q2 Q3 Select ion of interest in Q1 Fragment this ion in Q2 Scan Q3 for fragment masses; fragmentation pattern used to deduce original ion structure

17. Ion traps ■ Trap ions for other purposes ■ Quadrupoles ■ Linear ion traps ■ Orbitraps ■ Some used for analyzers as well as traps

18. Hybridized techniques ■ Two or more m/z analyzers of different types ■ QTOF: Triple quadrupole, but the last quadrupole replaced by a TOF analyzer (Quadrupole TOF) ■ Provide different information on a molecule ■ Improve signal-to-noise

19. Ionization Techniques ■ Required to put charge on molecule ■ While ionizing, get molecule into gas phase ■ No ions- no mass spectrometry!!

20. Ionization Techniques ■ Atmospheric Pressure Ionization (API) ■ Electron and Chemical Ionization (EI/CI) ■ Photo-ionization ■ Matrix-assisted laser desorption (MALDI) ■ Fast atom bombardment (FAB)

21. Atmospheric Pressure Ionization ■ “Soft” techniques- reduced fragmentation, more molecular ions ■ Useful for liquid chromatography ■ ESI, APCI ■Ionize compounds ■Remove solvents ■Get compounds into analyzer

22. Electrospray ionization (ESI)- Used on PurIon

23. ESI Solvents & Additives ■ Solvents ■ Water ■ Acetonitrile ■ Methanol ■ Ethanol ■ Propanol ■ 2-propanol ■Additives ■ Acetic Acid ■ Formic Acid ■ Ammonium hydroxide ■ Ammonium formate * ■ Ammonium acetate * * <= 10 mM What happened here?!

24. What’s with the solvent additives? ■ Help charge the analyte ■ Acids add protons (positive charge) ■ Bases remove protons (negative charge)

25. ESI Solvents- Use with care ■ Trifluoroacetic acid ■ Strong ion pair causes neutral molecule? ■ Still, commonly used for LC-MS, some TFA runs seem Ok ■ Triethylamine- may suppress less basic compounds

26. Atmospheric Pressure Chemical Ionization (APCI)- Used on PurIon ■ Heated probe evaporates solvent ■ Corona discharge places charge on molecules ■ Tetrahydrofuran- very flammable

27. Compound Ionization Technique Map

28. Other Ionization- APPI ■ Atmospheric Pressure Photo-Ionization ■ Light “kicks off” an electron, charging molecule

29. Other ionization- Electron Ionization ■ Formerly known as “electron impact” ■ Molecule charged in vacuum ■ Not compatible with LC ■ Fragments molecules

30. Other ionization- Chemical Ionization ■ Primary ionization: ■ CH 4 + e -  CH 4 + + 2e - ■ Secondary reagent ion ■ CH 4 + CH 3 +  CH 5 + + CH 3 ■ Product Ion Formation ■ M + CH 5 +  CH 4 + [M+H] + (protonation) ■ M + CH 3 +  CH 4 + [M-H] + (proton abstraction) ■ M + CH 4 +  CH 4 + [M] + (charge exchange)

31. Other ionization- MALDI ■ Matrix Assisted Laser Desorption/Ionization ■ UV LASER ablates matrix & compound ■ Matrix transfer proton(s) to molecule ■ Commonly used with TOF (pulsed LASER) ■ Used with macromolecules, proteins, bacteria, viruses

32. Other ionization-DESI, DART ■ Desorption ESI ■ Direct Analysis in Real Time ■ Useful for QC, forensic analysis

33. Detectors ■ Faraday cup ■ Ions hits cup ■ Enough ions generate a measurable charge ■Electron multiplier ■Used in PurIon ■Amplifies signal by generating electrons

34. PurIon system

35. Get Sample into Mass Spectrometer ■ Split/dilute sample ■ Solvent good for ionization (generally methanol) ■ Consistent delivery ■Tubing with restrictions ■“MRA” valve (used in PurIon) ■Both use “make-up” or “carrier solvent” pump

36. Fluid Interface

37. Why use FC-MS? Traditional open-access LC/MS workflow Save steps Save time Move right into the next step

38. Advantages for the chemist ■ Collect only the desired compound(s) ■ Verification the correct compound is being collected ■Ignore previously known compounds (natural products, reverse engineering)

39. Other uses for PurIon- Flow Injection Analysis Use “Method Development Screen No flow from CombiFlash Useful for reaction monitoring 1 mg/20 mL or less

40. Chemistry ■ Isotopes ■ Nitrogen Rule ■ Fragmentations/ rearangements ■ Adducts ■ Multiply charged ions ■Note: Many of the rules written for electron ionization Parent peak is M + ■ESI parent is usually [M+1] ■What does this mean?!

41. Nitrogen rule ■ [M+1] even: odd # of nitrogens ■ [M+1] odd: 0 or even # of nitrogen ■ Applies only to the parent ion!! What is the [M+1] ? Is it even or odd?

42. Nitrogen rule ■ Synthesized compound has 2 nitrogens ■ See peak at m/z=168 ■ Is this a fragment or a parent ion? ■ Why?

43. Mass Spectroscopy Chemistry ■ Ions- what we produce ■ Rearrangements- move the charge someplace else ■ Adducts- “share the charge” ■ Fragments- make both charged and uncharged stable pieces ■Lower the energy of the molecule!!!

44. Ions ■ Multiply charged species ■ What m/z would they appear at? ■ Probably not common on small molecules 93, 94

45. Fragmentations, rearrangements ■ Generally, fragments occur near heteroatoms (N, O, S) ■ Also can occur with “good” leaving groups, stable ions

46. Fragmentation case study ■ [M+1] + = 185 ■ Base peak = 168 ■ What drives this reaction?

47. Rearrangement case Study ■ λ max 200-220 nm ■ [M+1] = 141 Da expected ■ Solvent system Hexane/EtOAc ■ User advised to use a range of masses, would get weak peak @ m/z 123

48. Rearrangement case Study Not charged- not seen. Major product (loss of another hydrogen between methyls Very minor product- charged, m/z=122 or 123 (depending on loss of H) H + binds to non- bonding electrons on oxygen

49. Good leaving groups ■ -NH 2 (leaves as ammonia) ■ -OH (leaves as water) ■ COOH (leaves as CO 2 ) ■ Look for increase in conjugation ■ Look for easily formed, stable molecules

50. Adducts ■ Bond to the molecule- usually detected as [M+H+Adduct] ■ May be more intense than [M+1] ■ May occasionally see dimers [M+H+M] +

51. Adduct List

52. Adduct example & sources ■Solvent ■Glassware ■Syringe

53. Adducts- potential confusion ■ Sample dissolved in methanol- use method development, [M+MeOH+H] observed. Purification in hexane/ethyl acetate- will you see adduct? ■ Sample run on LC-MS mobile phase water/MeCN, [M+MeCN+H] observed. Purification in hexane/ethyl acetate- will you see adduct? ■ Use a range that covers [M] through [M+adduct]

54. Another Adduct Example ■ Solvent system Hexane/EtOAc ■ m/z=141 expected ■ Carrier = MeOH/0.1% formic acid

55. Key Markets