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Mass Directed Purification from Teledyne Isco

Mass Directed Purification from Teledyne Isco. Combi Flash ® a Name You Can Rely On. Fundamentals of Mass Spectroscopy:. Goals: Terminology Major components of a mass spectrometer Ionization techniques Mass analyzers; PurIon Mass Spectrometer overview Why use PurIon ?

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Mass Directed Purification from Teledyne Isco

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  1. Mass Directed PurificationfromTeledyne Isco CombiFlash®a Name You Can Rely On

  2. Fundamentals of Mass Spectroscopy: • Goals: • Terminology • Major components of a mass spectrometer • Ionization techniques • Mass analyzers; • PurIon Mass Spectrometer overview • Why use PurIon? • Mass directed purification overview

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

  4. Mass Spectrometry Simplified • Analytesare 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. Some Common Terms • 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 • Base peak: Tallest peak in a mass spectrum

  6. Ion Current • XIC- Extracted Ion Current • Ion current for a limited mass range (often m/z range of 1 or 2) • Used to monitor characterized compounds • TIC - Total Ion Current • Uses the entire spectral range • Useful for natural products, unknown compounds • Useful for molecules that generate weak molecular ion peaks

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

  8. Isotopic Patterns Isotopic pattern for compound with single bromine Isotopic pattern for typical organic compound

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

  10. 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

  11. Mass Spectrometer block diagram Source Region Mass Analyzer Detector Inlet Vacuum system

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

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

  14. Quadrupoles (PurIon) • 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

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

  16. Ionization Techniques • Required to put charge on molecule • While ionizing, get molecule into gas phase • No ions = no mass spectrometry!!

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

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

  19. Electrospray ionization (ESI)- Used on PurIon

  20. 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?!

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

  22. 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

  23. Atmospheric Pressure Chemical Ionization (APCI)- Used on PurIon as an Option • Heated probe evaporates solvent • Corona discharge places charge on molecules • Tetrahydrofuran- very flammable when used for APCI (spark)

  24. Compound Ionization Technique Map

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

  26. PurIon system Good place to mention that Since the turbo is at 60,000 Rpm unit needs to be in Shutdown to prevent pump destruction

  27. 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

  28. Fluid Interface

  29. Why use Mass Directed Purification? Traditional open-access LC/MS workflow • Save steps • Save time • Move right into the next step

  30. 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)

  31. Fluid Interface Flow Diagram Pulse damper Pressure Transducer To Mass Spec Splitter Valve Prime valve Back pressure 40 psi To Rf port D From Rf port C Pump head Teledyne Isco Confidential

  32. Other uses for PurIon- Flow Injection Analysis Not running Not switching for FIA

  33. Flow Injection or Reaction Monitoring • Use “Method Development Screen • No flow from CombiFlash • Useful for reaction monitoring • 1 mg/20 mL or less 200 µL/min to ESI/APCI Mass Spectrometer Isocratic make up Pump 200 µL/min Methanol with 0.1% Formic Acid Teledyne Isco Confidential

  34. Chemistry • Isotopes • Nitrogen Rule • Fragmentations/ rearrangements • 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?!

  35. What are ions? • Charged molecule • Shown as M+ • Na+ • Cl-

  36. 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?

  37. Nitrogen rule • Synthesized compound has 2 nitrogens • See peak at m/z=168 using ESI+ • Is this a fragment or a parent ion? • Why?

  38. 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!!!

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

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

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

  42. Rearrangement case Study • λmax 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

  43. 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

  44. Good leaving groups • -NH2 (leaves as ammonia) • -OH (leaves as water) • COOH (leaves as CO2) • Look for increase in conjugation • Look for easily formed, stable molecules

  45. 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]+

  46. Adduct List

  47. Most common adducts seen: • Methanol • Acetonitrile • Sodium • Potassium

  48. Adduct example & sources • Solvent • Glassware • Syringe

  49. 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]

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

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