9Lecture I-1:MS Interpretation-1CU- Boulder CHEM 5181Mass Spectrometry & ChromatographyProf. Jose-Luis JimenezFall 2007Lecture based on an earlier version of notes from Dr. Dan Czizco11Introduction to InterpretationSample InletIon SourceMassAnalyzerDetector RecorderAerodynamicInletExcimer LaserTime ofFlightMicrochannelPlateDigitizer toComputerPALMS instrumentin nose of WB-57stratospheric aircraft.Murphy, Czizco et al.From Dr. Dan Czizco12...Now What? - ISample InletIon SourceMassAnalyzerDetector Recorder0.300.250.200.150.100.050.00 Signal (ion current)100806040200 Ion Mass / ChargeC+Mg+Na+NO+Ca+Fe+SA Frag.SA Frag.SA Frag.m/z 24?SO2+HSO3+PALMS single particle spectrum (stratosphere)From Dr. Dan Czizco13Clicker Question• What is the identify of the ion at m/z24 in the previous spectrum?–C2+–Mg+–SO2++–Li3H3+– Any of the above14...Now What? - II• Most mass spectra are not trivial to decipher due to:– interferences (Mg+or C2+on last page) – complexities of fragmentation – (exception: some elemental analysis cases)• Instead, MS Interpretation is problem solving, ‘playing chess’, or ‘cracking a code’– Use all available information in a logical and organized manner• Our focus: EI spectra of “small” organic molecules – but interpretation techniques are applicable to spectra obtained with other ionization techniques – Also fragmentation pathways also apply with most ionization techniques (“breakdown curve”)15References on MS Interpretation• McLafferty & Turecek, Interpretation of Mass Spectra, 4th Ed., 1993.– Best book on the subject, must have – We will follow it closely• Smith & Busch,Understanding Mass Spectra, 1999.– Alternative to McLafferty, somewhat easier to read• Lee, A beginner’s Guide to Mass Spectral Interpretation, 1998.– More basic, easy to read. Useful introduction before tackling McLafferty.• Sorrell, Interpreting Spectra of Organic Molecules, 1988.– Older and more general.16MS Interpretation: First Steps• You have a sample to be analyzed. How do you proceed? • Three steps:1. Run the spectra you get against a database (if available)- Don’t stop here!2. Obtain a high resolution spectrum if possible- This will help constrain the elemental compositions3. Follow the standard interpretation procedure (SIP) and make sure that your identification is self-consistent171. Databases• Databases are critical for even the most grizzled MS veterans• EI databases– NIST’s Chemistry WebBook @ http://webbook.nist.gov/chemistry• 6000 molecules, but free and on the web– NIST off-line, Wiley, Palisades• Hundreds of thousands of molecules, $2k-$8k• Protein databases, e.g. MASCOT:– http://www.matrixscience.com/home.html18(mainlib) 1-Propanol10 20 30 40 50 60 70050100152931424559OHReproducibility: 4 Spectra of 1-Propanol in NISTAgain note that it doesn’t correspond to 70 eVsof internal energy(mainlib) 1-Propanol10 20 30 40 50 60 70050100152931424559OH(mainlib) 1-Propanol10 20 30 40 50 60 70050100152931424559OH(replib) 1-Propanol10 20 30 40 50 60 70050100272931334259OH(replib) 1-Propanol10 20 30 40 50 60 70050100273142455759OHContributor: From Japan AIST/NIMC DatabaseContributor: Chemical ConceptsContributor: NIST Mass Spectrometry Data Center, 1990.Contributor: also from NIST19Reproducibility / Signal-to-Noise• Be aware that database (and other) spectra have limited signal-to-noise and reproducibility• McLafferty examples: • +/- for each peak 10% relative to itself• +/- 0.2 absolute (base peak = 100)• Database examples depend on instrument used, how long ago, etc.• Be aware that background / leak / contaminant peaks may be present that are not related to the molecule of interest – Take a “background” spectrum just before your analysis• Make sure m/z values are correct!200.150.100.050.00Signal (Arbitrary units)43.243.143.042.9CHNOCH3N2C2H3OC2H5NC3H7DeCarlo, Kimmel et al., Analytical Chemistry, 2006.2. High Resolution InformationExample High Resolution Aerosol MS212. High Resolution Information• Closest Peaks are 0.0012 amu apart. • What resolution do we need to determine the elemental composition of this peak?From Hoffmann223. Standard Interpretation Procedure• Ask questions in a logical order– Big picture questions first• E.g. which elements are present• Avoid “blind alleys”– More detailed questions later• E.g. molecular subtructures– Put it all together at the end• Postulate a molecule that is consistent with all previous information• Order is useful, we’ll ask you to use it in HWs & exams 233. Standard Interpretation Procedure (SIP) I1. Study all available information (spectroscopic, chemical, sample history). Give explicit directions for obtaining spectrum (better yet, do it yourself).Verify the m/z assignments. Use calibrants if needed (LABS).2. Using isotopic abundances (where possible) deduce the elemental composition of each peak in the spectrum; calculate rings plus double bonds (TODAY).3. Test molecular ion identity; must be the highest mass peak in spectrum, odd-electron ion, and give logical neutral losses. Check with CI or other soft ionization (Lecture I2).24Standard Interpretation Procedure II4. Mark ‘important’ ions: odd-electron and those of highest abundance, highest mass, and/or highest in a group of peaks (Lect. I2).5. Study general appearance of the spectrum; molecular stability, labile bonds (Lect. I3).6. Postulate and rank possible (sub)structuralassignments for (Lect. I3):a) Important low-mass ion seriesb) Important primary neutral fragments from M.+indicated by high-mass ions (loss of largest alkyl favored) plus those secondary fragmentations indicated by MS/MS spectra.c) Important characteristic ions25Standard Interpretation Procedure III7. Postulate molecular structures; test against a reference spectrum, against spectra of similar compounds, or against spectra predicted from mechanisms of ion decompositions (Lect. I4).Remember to followSIP step-by-step in order26TODAY: Elemental Composition• Reminder: use high resolution spectra (if at all possible) and always attempt to identify the peaks of every fragment. • Even with unit resolution, the presence of isotopes of known natural abundances provides a useful & simple method:0.300.250.200.150.100.050.00 Signal (ion current)100806040200 Ion Mass / ChargeC+Mg+Na+NO+Ca+Fe+SA Frag.SA Frag.SA Frag.27Natural Abundances of Stable Isotopes• Note that the isotope of lowest mass is