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Why Mass Spectrometry: An Introduction to the IU MSF Jonathan A. Karty, Ph.D.

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Presentation on theme: "Why Mass Spectrometry: An Introduction to the IU MSF Jonathan A. Karty, Ph.D."— Presentation transcript:

1 Why Mass Spectrometry: An Introduction to the IU MSF Jonathan A. Karty, Ph.D. jkarty@indiana.edu http://msf.chem.indiana.edu

2 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

3 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

4 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

5 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 (C x H y O z ) – 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 12 C atom) – 1 Th = 1 Da/e = 1.0364*10 -8 kg/C

6 Molecular Weight Calculations The molecular weight is computed by summing the masses of all atoms in the compound/ion. – Erythromycin (M+H) + : C 37 H 68 N 1 O 13 + = 12.011*37 + 1.008*67 + 14.007 + 15.999*13 = 734.93 Da Yet 734.5 is observed by ESI-MS

7 Isotopic Distributions Isotopes: same number of protons, different numbers of neutrons – 12 C has 6 of each, 13 C has 6 protons and 7 neutrons – Periodic table assumes a natural distribution of stable isotopes (weighted average) Carbon isotopes – C  12 C is 98.9% abundant, 13 C is 1.1% abundant (0.989 * 12.0000) + (0.011 * 13.0034) = 12.011 – For C 40 : 64.2% 13 C 0, 28.6% 13 C 1, 6.2% 13 C 2 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 C 40 mass spectrum

8 Monoisotopic Masses Monoisotopic masses are considered for mass spectrometry – Monoisotopic masses are computed using the most abundant isotope of each element ( 12 C, 35 Cl, 14 N, 16 O, 79 Br, 11 B, 120 Sn etc) For erythromycin, monoisotopic mass = 734.468 – 12 C 37 1 H 68 14 N 1 16 O 13 – 12.000 * 37 + 1.0078 * 68 + 14.0031 + 15.9949 * 13 Remember to include any ionizing reagent – Electron loss, proton addition, etc.

9 C 37 H 68 NO 13 + Mass Spectrum Average mass = 734.93 u 13 C 0, 2 H 0, 18 O 0 13 C 1, 2 H 0, 18 O 0 13 C 2, 2 H 0, 18 O 0 13 C 0, 2 H 0, 18 O 1 13 C 1, 2 H 1, 18 O 0 Observed isotope pattern is the convolution of isotope patterns for all atoms

10 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

11 Two Complex Isotope Patterns C 2 H 3 Cl 3 trichloroethane C 12 H 27 SnBr tributyltin bromide Monoisotopic mass: 131.93 Average mass: 133.43 Monoisotopic mass: 370.03 Average mass: 369.96

12 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

13 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

14 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

15 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

16 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

17 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

18 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 * (5/10 6 ) = ±0.0015 Da

19 Formula Matching Basics Atomic weights are not integers (except 12 C) – 14 N = 14.0031 Da; 1 H = 1.0078 Da – 16 O = 15.9949 Da; 127 I = 126.9045 Da – Table of isotopes link on MSF website Difference from integer mass is called “mass defect” – Related to nuclear binding energy (E = mc 2 ) Sum of the mass defects depends on formula – H, N increase mass defect Eicosane (C 20 H 42 ) = 282.3286 – O, Cl, F, Na decrease it Morphine (C 17 H 19 NO 3 ) = 285.1365

20 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 C 17 H 19 NO 3 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

21 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 Zoloft C 17 H 18 Cl 2 N

22 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

23 Sample LCT Accurate Mass Report

24 Sample MAT-95 Accurate Mass Report

25 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!


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