Why Mass Spectrometry: An Introduction to the IU MSF Jonathan A. Karty, Ph.D. [email protected] .
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Transcript of Why Mass Spectrometry: An Introduction to the IU MSF Jonathan A. Karty, Ph.D. [email protected] .
![Page 1: Why Mass Spectrometry: An Introduction to the IU MSF Jonathan A. Karty, Ph.D. jkarty@indiana.edu .](https://reader034.fdocuments.net/reader034/viewer/2022042814/551aa0ab5503466b3a8b56ba/html5/thumbnails/1.jpg)
Why Mass Spectrometry: An Introduction to the IU MSF
Jonathan A. Karty, [email protected]
http://msf.chem.indiana.edu
![Page 2: Why Mass Spectrometry: An Introduction to the IU MSF Jonathan A. Karty, Ph.D. jkarty@indiana.edu .](https://reader034.fdocuments.net/reader034/viewer/2022042814/551aa0ab5503466b3a8b56ba/html5/thumbnails/2.jpg)
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
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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
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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
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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
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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
O
O
O
O
O
OH
O
NH+HOO
OH
OHHO
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Isotopic Distributions• 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
Unregistered
485484483482481480479
100
90
80
70
60
50
40
30
20
10
0
C40 mass spectrum
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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.
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C37H68NO13+ Mass Spectrum
Average mass = 734.93 u
13C0, 2H0, 18O0
13C1, 2H0, 18O013C2, 2H0, 18O0
13C0, 2H0, 18O1
13C1, 2H1, 18O0
Observed isotope pattern is the convolution of isotope patterns for all atoms
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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
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Inte
ns
ity
(%
)
0
20
40
60
80
100
Mass [amu]362 364 366 368 370 372 374 376 378
Inte
ns
ity
(%
)
0
20
40
60
80
100
Mass [amu]131 132 133 134 135 136 137 138 139
Two Complex Isotope Patterns
C2H3Cl3
trichloroethane
C12H27SnBrtributyltin bromide
Monoisotopic mass: 131.93Average mass: 133.43
Monoisotopic mass: 370.03Average mass: 369.96
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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
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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
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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
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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
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Bruker Autoflex 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
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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
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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
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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
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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
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Formula Matching Example
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
Elemental Composition ReportTolerance = 20.0 PPM / DBE: min = -1.5, max = 50.0Selected filters: NoneMonoisotopic Mass, Even Electron Ions370 formulas evaluated with 9 results within limitsElements Used:C: 0-40 H: 0-50 N: 0-5 O: 0-5 Cl: 0-2 Error 20 ppm
Mass intensity Calc. Mass mDa PPM i-FIT
306.082 100 306.0816 0.4 1.3 39.7 C17 H18 N Cl2 306.0776 4.4 14.4 376 C12 H18 N3 O2 Cl2 306.0875 -5.5 -18 701.7 C10 H22 N O5 Cl2 306.0798 2.2 7.2 1945.8 C18 H13 N3 Cl 306.0857 -3.7 -12.1 2205.2 C11 H17 N3 O5 Cl 306.0766 5.4 17.6 9102.8 C18 H12 N O4 306.078 4 13.1 9195.6 C19 H8 N5 306.0879 -5.9 -19.3 9289.5 C17 H12 N3 O3 306.0838 -1.8 -5.9 9543.2 C12 H12 N5 O5
error in:Formula
Zoloft C17H18Cl2N
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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
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Sample LCT Accurate Mass Report
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Sample MAT-95 Accurate Mass Report
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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!