1. Mass Directed Purification from Teledyne 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
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. 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 TIC - Total 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=
Other elements/isotopes do not have integer masses
1H=1.0079
16O=
14N=
79Br=

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

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

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

33. Teledyne Isco Confidential
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. [M+1] even: odd # of nitrogens [M+1] odd: 0 or even # of nitrogen
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. Multiply charged species What m/z would they appear at?
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
Base peak = 168
What drives this reaction?

42. Rearrangement case Study
λmax nm
[M+1] = 141 Da expected
Solvent system Hexane/EtOAc
User advised to use a range of masses, would get weak 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

51. Key Markets Pharmaceutical Drug Discovery Agricultural Chemistry
Petrochemicals
Natural Products
Foods and Flavors

52. 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

53. 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

54. 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

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

56. 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

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

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

59. Other ionization- Chemical Ionization
Primary ionization:
CH4 + e-  CH4+ + 2e-
Secondary reagent ion
CH4 + CH3+  CH5+ + CH3
Product Ion Formation
M + CH5+  CH4 + [M+H]+ (protonation)
M + CH3+  CH4 + [M-H]+ (proton abstraction)
M + CH4+  CH4 + [M]+ (charge exchange)

60. 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

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