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 techniqueMost other spectroscopies can describe functionalities, but not chemical formulaeMS is VERY sensitivemg/L to ng/L sensitivity possiblePicomole sensitivity is common in the MSFMass 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 substanceDid I make anything else?Mass spectrometry is readily coupled to chromatographic techniquesHow 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 compositionMuch more precise then EASelective detector for GC/HPLCMS provides retention time AND molecular weight information about each analyteReaction monitoringCrude reaction mixture MSStable isotope labelingStability studies
5 Important Concepts to Remember Mass spectrometers analyze gas-phase ions, not neutral moleculesNeutral molecules don’t respond to electromagnetic fieldsIf you cannot make a stable ion, MS is impossibleMS is not a “magic bullet” techniqueMS can tell you composition of an ion (CxHyOz)Connectivity of the atoms in that ion is much more challengingUnits1 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 = DaYet is observed by ESI-MS
7 Isotopic Distributions Isotopes: same number of protons, different numbers of neutrons12C has 6 of each, 13C has 6 protons and 7 neutronsPeriodic table assumes a natural distribution of stable isotopes (weighted average)Carbon isotopesC 12C is 98.9% abundant, 13C is 1.1% abundant(0.989 * ) + (0.011 * ) =For C40: 64.2% 13C0, 28.6% 13C1, 6.2% 13C2Spectrum looks like 480, 481, 482Many elements have a variety of isotopesSn has 7 naturally occurring isotopesF, P, Na, I, Co, Au have only 1 natural isotopeC40 mass spectrum
8 Monoisotopic Masses Remember to include any ionizing reagent Monoisotopic masses are considered for mass spectrometryMonoisotopic 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* * * 13Remember to include any ionizing reagentElectron loss, proton addition, etc.
9 C37H68NO13+ Mass Spectrum Average mass = 734.93 u 13C0, 2H0, 18O013C1, 2H0, 18O013C2, 2H0, 18O013C0, 2H0, 18O113C1, 2H1, 18O0Average mass = uObserved isotope pattern is the convolution of isotope patterns for all atoms
10 Isotopic EnvelopesIsotopic distributions can indicate/preclude the presence of an elementCl 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 atomsFor morphine: ( / 1) / = 17Significant deviation from expected isotope pattern should be a warning signMultiple compounds with similar masses in samplePartial isotopic enrichment (e.g. deuterated solvents used)Multiple ionization mechanisms at work
12 Instrumentation in the MSF One of 3 mass spectrometry facilities in the departmentMSF is in Chemistry A411 and A4541 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 sampleStaff-run experiments are $16-$30Training for Walk-Up MS will start after 11/7/10
13 Agilent 6890/5973 GC-MS6890 GC30 m long DB-5 (non-polar) column installedHelium mobile phaseSplit/splitless injector with autosampler5973 MSElectron ionizationQuadrupole MS (10-800)NIST 02 library installed
14 Agilent 1200/6130 LC-MS 1200 HPLC 6130 MS Easy Access Software Binary pumpmL/minAutosampler with 6-position column selectorDiode array UV-VIS detector6130 MSDual mode electrospray/APCI sourceCan perform all 4 modes of ionization in 1 experiment50-3,000 m/z quadrupoleEasy Access Software
15 Waters CapLC-LCT LC-MS Flow rates 1-40 uL/minC18, C8, and C4 columnsSingle wavelength UV-VIS detectorLCTElectrospray ionizationTime-of-flight MS (TOF)100-6,000 m/zCapable of accurate mass spectrometry5 ppm error for formula confirmation
16 Bruker Autoflex III MALDI-TOF mass spectrometer ,000 m/zCan make both positive and negative ions from same spotEasy to interpret mass spectra for large polymersMatrices available for wide array of analytes
17 Thermo MAT-95XP Magnetic sector mass spectrometer Accurate mass spectrometry is its main functionElectron ionization and Chemical ionization sourcesTrace GC available for low MW species or mixture analysis
18 Accurate Mass Spectrometry Accurate mass spectrometry can unambiguously confirm chemical compositionMass accuracy is often reported as a relative valueppm = parts per million, 1 ppm = %5 ppm error is used by many journals as a standard to confirm a chemical formulaInstrumentation in the IU MSF routinely achieves this level of accuracy5 ppm at mass 300: 300 * (5/106) = ± Da
19 Formula Matching Basics Atomic weights are not integers (except 12C)14N = Da; 1H = Da16O = Da; 127I = DaTable of isotopes link on MSF websiteDifference from integer mass is called “mass defect”Related to nuclear binding energy (E = mc2)Sum of the mass defects depends on formulaH, N increase mass defectEicosane (C20H42) =O, Cl, F, Na decrease itMorphine (C17H19NO3) =
20 More Formula MatchingAccurate mass measurements narrow down possible formulas for a given molecular weight534 entries in NIST’08 library @ mass 285Only 3 formulas within 5 ppm of46 compounds with formula C17H19NO3Mass spectrum and user info complete the pictureIsotope distributions indicate / eliminate elementsUser - supplied info eliminates others (e.g. no F)Suggested formula has to make chemical sense
21 Formula Matching Example Zoloft C17H18Cl2NOnly 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 NotesAccurate MS is possible with the MAT-95 and LCT mass spectrometersBruker MALDI-TOF can do 20 ppm mass accuracyOnly MSF staff can perform accurate mass measurementsAll accurate MS submissions MUST include a nominal (low res) mass spectrum to demonstrate purity PRIOR to accurate MS
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 NovemberQuantitation and other advanced topics for small molecule MSIntro to BioinformaticsThese will only occur if sufficient interest is expressedFor a more in-depth treatment of all topics covered, take C613 in the spring!