Presentation on theme: "FC-MS from Teledyne Isco CombiFlash ® a Name You Can Rely On."— Presentation transcript:
FC-MS from Teledyne Isco CombiFlash ® a Name You Can Rely On
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
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)
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.
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
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)
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
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
Mass Spectrometer block diagram Inlet Source Region Mass AnalyzerDetector Vacuum system
■ Roughing pump ■ Turbo molecular pump ■ Vacuum needed: ■ To avoid further reactions (fragmentation, reactions, etc.) ■ Increase mean-free path (maintain ion energy)
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
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
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
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
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
Ion traps ■ Trap ions for other purposes ■ Quadrupoles ■ Linear ion traps ■ Orbitraps ■ Some used for analyzers as well as traps
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
Ionization Techniques ■ Required to put charge on molecule ■ While ionizing, get molecule into gas phase ■ No ions- no mass spectrometry!!
Ionization Techniques ■ Atmospheric Pressure Ionization (API) ■ Electron and Chemical Ionization (EI/CI) ■ Photo-ionization ■ Matrix-assisted laser desorption (MALDI) ■ Fast atom bombardment (FAB)
Atmospheric Pressure Ionization ■ “Soft” techniques- reduced fragmentation, more molecular ions ■ Useful for liquid chromatography ■ ESI, APCI ■Ionize compounds ■Remove solvents ■Get compounds into analyzer
What’s with the solvent additives? ■ Help charge the analyte ■ Acids add protons (positive charge) ■ Bases remove protons (negative charge)
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
Atmospheric Pressure Chemical Ionization (APCI)- Used on PurIon ■ Heated probe evaporates solvent ■ Corona discharge places charge on molecules ■ Tetrahydrofuran- very flammable
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
Why use FC-MS? Traditional open-access LC/MS workflow Save steps Save time Move right into the next step
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)
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
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?!
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?
Nitrogen rule ■ Synthesized compound has 2 nitrogens ■ See peak at m/z=168 ■ Is this a fragment or a parent ion? ■ Why?
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!!!
Ions ■ Multiply charged species ■ What m/z would they appear at? ■ Probably not common on small molecules 93, 94
Fragmentations, rearrangements ■ Generally, fragments occur near heteroatoms (N, O, S) ■ Also can occur with “good” leaving groups, stable ions
Fragmentation case study ■ [M+1] + = 185 ■ Base peak = 168 ■ What drives this reaction?
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
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
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
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] +
Adduct example & sources ■Solvent ■Glassware ■Syringe
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]
Another Adduct Example ■ Solvent system Hexane/EtOAc ■ m/z=141 expected ■ Carrier = MeOH/0.1% formic acid