Organic Mass Spectrometry Interpretation of Mass Spectra

Basic Interpretation Most mass spectra are not trivial to decipher due to: – interferences complexities of fragmentation (exception: some elemental analysis cases) Instead, MS Interpretation is problem solving, ‘playing chess’, or ‘cracking a code’

Basic Interpretation 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 (homework, examples…)

References on MS Interpretation McLafferty & Turecek, Interpretation of Mass Spectra, 4th Ed., 1993. Best book on the subject We will follow it closely Smith & Busch,Understanding Mass Spectra, 1999. Alternative to McLafferty, somewhat easier to read

References on MS Interpretation 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.

MS 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) 2. Obtain a high resolution spectrum if possible This will help constrain the elemental compositions 3. Follow the standard interpretation procedure (SIP) and make sure that your identification is self-consistent

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 substructures Put it all together at the end Postulate a molecule that is consistent with all previous information

Standard Interpretation Procedure (1) 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. (2) Using isotopic abundances (where possible) deduce the elemental composition of each peak in the spectrum; calculate rings plus double bonds.

Standard Interpretation Procedure (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. (4) Mark ‘important’ ions: odd-electron and those of highest abundance, highest mass, and/or highest in a group of peaks.

Standard Interpretation Procedure (5) Study general appearance of the spectrum: molecular stability, labile bonds, etc. (6) Postulate and rank possible sub-structural assignments for: a) Important low-mass ion series b) 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 ions.

Standard Interpretation Procedure (7) Postulate molecular structures; test against a reference spectrum, against spectra of similar compounds, or against spectra predicted from mechanisms of ion decompositions Remember to follow SIP step-by-step in order.

Library 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

Reproducibility of Spectra

Reproducibility of Spectra 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) Library spectra depend on instrument used, how long ago, etc.

Reproducibility of Spectra 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!

High Resolution Information

High Resolution Information

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.

Isotopic Abundances Note that the isotope of lowest mass is the most abundant for all of these elements. – “A”, “A+1”, and “A+2” elements

A + 2 Elements: Cl, Br, O, S, and Si Especially prominent in the spectrum Look for these first!

A + 2 Elements Linear superposition of isotopic patterns If there is more than one atom in the molecule of one of the A+2 elements, the result is even more striking

Structural Isomers vs. MS

A + 2 Elements Oxygen isotopes Absence of A+2 elements The A+2 abundance of O is low (0.2%) Need high abundance accuracy Other isotopic patterns can interfere (C), thus estimate number of oxygen after A+1 and other A+2 elements Absence of A+2 elements Often they are not there. Value of negative info If [(X+2)]/[X] < 3%, the peak X cannot contain the most abundant isotope of Si, S, Cl, or Br

A + 1 Elements 2H/1H is so small that it is considered “A” Increasing number of C atoms linearly increases the probability that one of them is a 13C (see table 2.2) A way to deduce the number of carbon atoms 1.1% of 13C changes ~2% with source • Don’t worry about N for now (next class) “Nitrogen Rule” will come to our rescue

Unknown 2.4 Check for A+2 elements • Isotopic composition of 43 and 58? • Loss between 58 and 43? • Identity of the molecule?

Note about Unknown 2.4 Note the many small peaks below the major ones, due to neutral losses of H and H2 If you don’t see those, the molecule likely has no H Also note that those peaks are less important in determining the structure Start with the higher m/z (overall and in each group of peaks)

Unknown 2.5 Is 0.2% at m/z 80 due to an oxygen atom? Elemental formula of base peak? Identity of the molecule?

Unknown 2.6

A Elements: H, F, P, and I After the number of A+1 and A+2 elements have been assigned, A elements should provide the balance of the mass Use number of atoms consistent with rules of bonding (no CH6 please!) Note that only H can be used until we need to add 19 (F)

Example 1: Example 2:

Unknown 2.7

Unknown 2.8

Rings + Double Bonds Because of the valences, the total number of rings and double bonds in a molecule of the formula CxHyNzOn will be: Calculate: C4H10 , C6H6 , C5H5N , C7H5O For ions, the value may end in 0.5 (‘even electron ion’) More general case AyBnCzDx, where A = H, F, Cl, Br, I; B = O, S; C = N, P; and D = C, Si Does not count double bonds to elements in higher valence states