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Why Mass Spectrometry: An Introduction to the IU MSF

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Presentation on theme: "Why Mass Spectrometry: An Introduction to the IU MSF"— Presentation transcript:

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

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? Did I make anything else?
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 (CxHyOz) Connectivity of the atoms in that ion is much more challenging Units 1 Da = 1 u = *10-27 kg (1/12 of a 12C atom) 1 Th = 1 Da/e = *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)+: C37H68N1O13+ = * * *13 = Da Yet is observed by ESI-MS

7 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 * ) + (0.011 * ) = For C40: 64.2% 13C0, 28.6% 13C1, 6.2% 13C2 Spectrum looks like 480, 481, 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 C40 mass spectrum

8 Monoisotopic Masses Remember to include any ionizing reagent
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 = 12C37 1H68 14N1 16O13 * * * 13 Remember to include any ionizing reagent Electron loss, proton addition, etc.

9 C37H68NO13+ Mass Spectrum Average mass = 734.93 u
13C0, 2H0, 18O0 13C1, 2H0, 18O0 13C2, 2H0, 18O0 13C0, 2H0, 18O1 13C1, 2H1, 18O0 Average mass = u 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+] / ≈ # carbon atoms For morphine: ( / 1) / =  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
Monoisotopic mass: Average mass: C12H27SnBr tributyltin bromide C2H3Cl3 trichloroethane Monoisotopic mass: Average mass:

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 6130 MS Easy Access Software
Binary pump 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
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
,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 = % 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) = ± Da

19 Formula Matching Basics
Atomic weights are not integers (except 12C) 14N = Da; 1H = Da 16O = Da; 127I = 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) = O, Cl, F, Na decrease it Morphine (C17H19NO3) =

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 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

21 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 ( u) of , only 3 have 2 Cl

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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