why mass spectrometry an introduction to the iu msf n.
Download
Skip this Video
Download Presentation
Why Mass Spectrometry: An Introduction to the IU MSF

Loading in 2 Seconds...

play fullscreen
1 / 25

Why Mass Spectrometry: An Introduction to the IU MSF - PowerPoint PPT Presentation


  • 113 Views
  • Uploaded on

Why Mass Spectrometry: An Introduction to the IU MSF. Jonathan A. Karty, Ph.D. jkarty@indiana.edu http://msf.chem.indiana.edu. Why Mass Spectrometry. Information is composition-specific Very selective analytical technique

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Why Mass Spectrometry: An Introduction to the IU MSF' - brent-workman


Download Now An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
why mass spectrometry an introduction to the iu msf

Why Mass Spectrometry: An Introduction to the IU MSF

Jonathan A. Karty, Ph.D.

jkarty@indiana.edu

http://msf.chem.indiana.edu

why mass spectrometry
Why Mass Spectrometry
  • Information is composition-specific
    • Very selective analytical technique
    • Most other spectroscopies can describe functionalities, but not chemical formulae
  • MS is VERY sensitive
    • mg/L to ng/L sensitivity possible
    • Picomole sensitivity is common in the MSF
  • Mass spectrometers have become MUCH easier to use in the last 15 years
three questions
Three Questions
  • Did I make my compound?
    • Molecular weight is an intrinsic property of a substance
  • Did I make anything else?
    • Mass spectrometry is readily coupled to chromatographic techniques
  • How much of it did I make?
    • Response in the mass spectrometer is proportional to analyte concentration (R = α[M])
      • Each compound has a unique response factor, α
common ms applications
Common MS Applications
  • Quick product identification (TLC spot)
  • Confirmation of elemental composition
    • Much more precise then EA
  • Selective detector for GC/HPLC
    • MS provides retention time AND molecular weight information about each analyte
  • Reaction monitoring
    • Crude reaction mixture MS
    • Stable isotope labeling
    • Stability studies
important concepts to remember
Important Concepts to Remember
  • Mass spectrometers analyze gas-phase ions, not neutral molecules
    • Neutral molecules don’t respond to electromagnetic fields
    • If you cannot make a stable ion, MS is impossible
  • MS is not a “magic bullet” technique
    • MS can tell you composition of an ion (CxHyOz)
    • Connectivity of the atoms in that ion is much more challenging
  • Units
    • 1 Da = 1 u = 1.6605*10-27 kg (1/12 of a 12C atom)
    • 1 Th = 1 Da/e = 1.0364*10-8 kg/C
molecular weight calculations
Molecular Weight Calculations
  • The molecular weight is computed by summing the masses of all atoms in the compound/ion.
    • Erythromycin (M+H)+: C37H68N1O13+ = 12.011*37 + 1.008*67 + 14.007 + 15.999*13 = 734.93 Da
  • Yet 734.5 is observed by ESI-MS
isotopic distributions
Isotopic Distributions

C40 mass spectrum

  • Isotopes: same number of protons, different numbers of neutrons
    • 12C has 6 of each, 13C has 6 protons and 7 neutrons
    • Periodic table assumes a natural distribution of stable isotopes (weighted average)
  • Carbon isotopes
    • C  12C is 98.9% abundant, 13C is 1.1% abundant
      • (0.989 * 12.0000) + (0.011 * 13.0034) = 12.011
    • For C40: 64.2% 13C0, 28.6% 13C1, 6.2% 13C2
      • Spectrum looks like 100% @ 480, 44.5% @ 481, 9.6% @ 482
  • Many elements have a variety of isotopes
    • Sn has 7 naturally occurring isotopes
    • F, P, Na, I, Co, Au have only 1 natural isotope
monoisotopic masses
Monoisotopic Masses
  • Monoisotopic masses are considered for mass spectrometry
    • Monoisotopic masses are computed using the most abundant isotope of each element (12C, 35Cl, 14N, 16O, 79Br, 11B, 120Sn etc)
  • For erythromycin, monoisotopic mass = 734.468
    • 12C37 1H68 14N1 16O13
    • 12.000 * 37 + 1.0078 * 68 + 14.0031 + 15.9949 * 13
  • Remember to include any ionizing reagent
    • Electron loss, proton addition, etc.
c 37 h 68 no 13 mass spectrum
C37H68NO13+ Mass Spectrum

13C0, 2H0, 18O0

13C1, 2H0, 18O0

13C2, 2H0, 18O0

13C0, 2H0, 18O1

13C1, 2H1, 18O0

Average mass = 734.93 u

Observed isotope pattern is the convolution of isotope patterns for all atoms

isotopic envelopes
Isotopic Envelopes
  • Isotopic distributions can indicate/preclude the presence of an element
    • Cl has a unique 3:1 pattern (M:M+2)
    • B has a unique 1:4 pattern (M-1:M)
  • M+1 / M+ ratio can be used to count carbon atoms in a molecule
    • [(M+1) / M+] / 0.011 ≈ # carbon atoms
    • For morphine: (0.1901 / 1) / 0.011 = 17.28  17
  • Significant deviation from expected isotope pattern should be a warning sign
    • Multiple compounds with similar masses in sample
    • Partial isotopic enrichment (e.g. deuterated solvents used)
    • Multiple ionization mechanisms at work
two complex isotope patterns
Two Complex Isotope Patterns

Monoisotopic mass: 370.03

Average mass: 369.96

C12H27SnBr

tributyltin bromide

C2H3Cl3

trichloroethane

Monoisotopic mass: 131.93

Average mass: 133.43

instrumentation in the msf
Instrumentation in the MSF
  • One of 3 mass spectrometry facilities in the department
  • MSF is in Chemistry A411 and A454
  • 1 GC-EI-Q-MS (A454)
  • 1 LC-ESI/APCI-Q-MS (A454)
  • 2 LC-ESI-TOF-MS (A411)
  • 1 MALDI-TOF-MS (A411)
  • 1 EI/CI-BE-MS (A411, staff only)
  • Self-run experiments are $7-$10 per sample
  • Staff-run experiments are $16-$30
  • Training for Walk-Up MS will start after 11/7/10
agilent 6890 5973 gc ms
Agilent 6890/5973 GC-MS
  • 6890 GC
    • 30 m long DB-5 (non-polar) column installed
      • Helium mobile phase
    • Split/splitless injector with autosampler
  • 5973 MS
    • Electron ionization
    • Quadrupole MS (10-800)
    • NIST 02 library installed
agilent 1200 6130 lc ms
Agilent 1200/6130 LC-MS
  • 1200 HPLC
    • Binary pump
      • 0.05-2 mL/min
    • Autosampler with 6-position column selector
    • Diode array UV-VIS detector
  • 6130 MS
    • Dual mode electrospray/APCI source
      • Can perform all 4 modes of ionization in 1 experiment
    • 50-3,000 m/z quadrupole
  • Easy Access Software
waters caplc lct lc ms
Waters CapLC-LCT LC-MS
  • CapLC
    • Flow rates 1-40 uL/min
    • C18, C8, and C4 columns
    • Single wavelength UV-VIS detector
  • LCT
    • Electrospray ionization
    • Time-of-flight MS (TOF)
      • 100-6,000 m/z
    • Capable of accurate mass spectrometry
      • 5 ppm error for formula confirmation
bruker autoflex iii
BrukerAutoflex III
  • MALDI-TOF mass spectrometer
    • 200-150,000 m/z
  • Can make both positive and negative ions from same spot
  • Easy to interpret mass spectra for large polymers
  • Matrices available for wide array of analytes
thermo mat 95xp
Thermo MAT-95XP
  • Magnetic sector mass spectrometer
  • Accurate mass spectrometry is its main function
  • Electron ionization and Chemical ionization sources
  • Trace GC available for low MW species or mixture analysis
accurate mass spectrometry
Accurate Mass Spectrometry
  • Accurate mass spectrometry can unambiguously confirm chemical composition
  • Mass accuracy is often reported as a relative value
    • ppm = parts per million, 1 ppm = 0.0001%
  • 5 ppm error is used by many journals as a standard to confirm a chemical formula
    • Instrumentation in the IU MSF routinely achieves this level of accuracy
    • 5 ppm at mass 300: 300 * (5/106) = ±0.0015 Da
formula matching basics
Formula Matching Basics
  • Atomic weights are not integers (except 12C)
    • 14N = 14.0031 Da; 1H = 1.0078 Da
    • 16O = 15.9949 Da; 127I = 126.9045 Da
    • Table of isotopes link on MSF website
  • Difference from integer mass is called “mass defect”
    • Related to nuclear binding energy (E = mc2)
  • Sum of the mass defects depends on formula
    • H, N increase mass defect
      • Eicosane (C20H42) = 282.3286
    • O, Cl, F, Na decrease it
      • Morphine (C17H19NO3) = 285.1365
more formula matching
More Formula Matching
  • Accurate mass measurements narrow down possible formulas for a given molecular weight
    • 534 entries in NIST’08 library @ mass 285
    • Only 3 formulas within 5 ppm of 285.1365
      • 46 compounds with formula C17H19NO3
  • Mass spectrum and user info complete the picture
    • Isotope distributions indicate / eliminate elements
    • User - supplied info eliminates others (e.g. no F)
    • Suggested formula has to make chemical sense
formula matching example
Formula Matching Example

Zoloft C17H18Cl2N

Only 9 ways to combine up to 40 C, 50 H, 5 N, 5 O, and 2 Cl to get a mass within 20 ppm (0.0061 u) of 306.0820, only 3 have 2 Cl

accurate ms notes
Accurate MS Notes
  • Accurate MS is possible with the MAT-95 and LCT mass spectrometers
    • Bruker MALDI-TOF can do 20 ppm mass accuracy
  • Only MSF staff can perform accurate mass measurements
  • All accurate MS submissions MUST include a nominal (low res) mass spectrum to demonstrate purity PRIOR to accurate MS
upcoming lectures
Upcoming Lectures
  • All in C033 from 5:30-6:15
  • Small molecule EI and GC-MS (10/14)
  • Small molecule ESI/APCI and LC-MS (10/18)
  • Biomolecule/polymer analysis by ESI and MALDI (10/20)
  • Possible special interest seminars in November
    • Quantitation and other advanced topics for small molecule MS
    • Intro to Bioinformatics
    • These will only occur if sufficient interest is expressed
  • For a more in-depth treatment of all topics covered, take C613 in the spring!