Analysis of Organic Mass Spectral Data

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Analysis of Organic Mass Spectral Data

• Bioanalytical Chemistry
• Lecture Topic 3

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Mass Spectrum

• X - axis m/z
• mass - based on 12C ? 12.0000
• Z charge on ion
• Y - axis relative abundance
• usually normalized wrt largest line (base peak)
• 0 - 100

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Major Steps in Analysis of Mass Spectral Data

• Identification of molecular ion
• Base peak
• Examination of isotopic distribution pattern
• Negative information
• Determine elemental composition
• Analysis of fragmentation pattern
• Propose possible structures
• Compare postulated species to available reference
  spectra

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Molecular Ion

• Ion whose mass equals that calculated from the
  molecular formula using the masses for each
  element which have the highest natural abundance
  often tallest peak in highest m/z group
• Base peak - most intense peak in spectrum not
  necessarily the molecular ion peak!

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Example Mass Spectrum of Methanol (CH3OH)
CH3OH e- ? CH3OH? 2e-CH3OH ? ? CH2OH
H? CH3OH ? ? CH3 ?OHCH2OH ? H2 CHO
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Example 2 Mass spectra for cyclophosphamide

• Method of sample ionization may change molecular
  ion
• EI M ?
• CI MH

Figure taken from Rubinson, K.A. Chemical
Analysis Boston Little, Brown, 1987.
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Lessons

• Single charge (1) most common so m/z usually
  equates to mass (EI/CI)
• Ions may fragment
• EX CH3OH loses H readily
• Observe CH3O ?

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Example Mass Spectrum of Methanol (CH3OH)
CH3OH e- ? CH3OH? 2e-CH3OH ? ? CH2OH
H? CH3OH ? ? CH3 ?OHCH2OH ? H2 CHO
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Isotopes

• Most abundant isotope of an element is set to
  100
• Abundance of other isotopes are normalized with
  respect to it

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3 Classes of Isotopes

• A - only a single isotope
• EX F, P, I
• A1 - two isotopes with significant relative
  abundance differing by 1 mass unit
• EX H, C, N
• A2 - two isotopes with significant relative
  abundance differing by 2 mass units
• EX Cl, S, O

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Natural Isotopic Abundance of Common Elements in
Organic Compounds
A
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Natural Isotopic Abundance of Common Elements in
Organic Compounds
A1
A2
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Natural Isotopic Abundance of Common Elements in
Organic Compounds
A2
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Isotopes

• Most abundant isotope of an element is set to
  100
• Abundance of other isotopes are normalized with
  respect to it

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Isotopic Distribution Patterns

• If Cl- present then two peaks with ratio 10032.5

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Question

• What Will the Mass Spectrum of Cl2 Look Like?
  (Relative Abundance and m/z for all species)

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Cl2 Revisited

• Two isotopes 35Cl and 37Cl
• Three possible species formed 35Cl35Cl,
  37Cl35Cl, and 37Cl37Cl
• Relative abundance
• 35Cl35Cl 1.0 x 1.0 1.0
• 37Cl35Cl and 35Cl37Cl 1.0 x 0.325 0.325 each
  or 0.66
• 37Cl37Cl 0.325 x 0.325 0.106
• So, answer 3 peaks at 70, 72, and 74 with
  relative intensities of 100, 66, and 10.6

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Problem 1
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Step 1 Identify Molecular Ion
?
?
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Step 2 Normalize Intensity wrt Molecular Ion
A 2
Q Doespattern lookfamiliar?
A 2
A 2
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Step 3 Identify Possible Species

• 84 - 2(35) 14
• Remaining species must be A-type (C, H, etc)
• 14 - 12 2
• Suggests 2H, 1 C, and 2 Cl ?CH2Cl2

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Step 3 Identify Possible Species (contd)
Q Doespattern lookfamiliar?
A2
A2
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Step 3 Identify Possible Species

• 49 - (35) 14
• Remaining species must be A-type (C, H, etc.)
• 14 - 12 2
• Suggests 2H, 1 C, and 1 Cl ?CH2Cl

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Problem 2
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Solution

• Identify M as 62 and (M2) as 64
• 62-35 27
• 27-(212) 3
• so CH2CHCl (vinyl chloride)

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General Characteristics

• Saturated hydrocarbons - regularly spaced
  clusters separated by 14 mass units
• Branching causes breaks in the intensity envelope
• Unsaturated hydrocarbons - fragments offset from
  saturated hydrocarbons by -2
• See mass spec textbooks for pattern info

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Fragmentation Patterns

• Dont forget common sense what moieties can
  your molecular ion lose?
• Usually only one bond broken
• No common losses between 2 and 14

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Fragmentation Patterns

• Cleavage favored at branched C atoms
• Tertiary gt secondary gt primary
• Positive charge on branched C (carbonium ion)
• Ring compounds have strong parent ions
• Intensity related to stability of ring
• Compounds with carbonyl break at this group
• m/z 16 large for primary amines (tables)

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Common Mass Spec Fragments
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Common Mass Spec Fragments
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The Nitrogen Rule

• Observation
• even mass elements have even valence
• odd mass elements have odd valence
• EX Na (11), K (19), Mg2 (12), Ca2 (20), O2-
  (8)
• Consequences
• compound containing C, H, O, and an even number
  of nitrogens (or no nitrogens) will have an even
  molecular weight
• compound containing C, H, O, and an odd number of
  nitrogens will have an odd molecular weight

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Problem

• Which of the following ions will appear at an
  even mass number?
• NH3
• C4H9N
• C2H5NH2
• C5H6N2
• ANS C5H6N2

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Problem 3
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3 Classes of Isotopes

• A - only a single isotope
• EX F, P, I
• A1 - two isotopes with significant relative
  abundance differing by 1 mass unit
• EX H, C, N
• A2 - two isotopes with significant relative
  abundance differing by 2 mass units
• EX Cl, O, S

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Answer

• Compound must contain only C, H, N(A and A1
  isotopes)
• If N present, then Nitrogen rule says odd number
  of nitrogens present
• m/z 15 suggests CH3
• and 73-58 15 so m/z 58 is other piece

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Answer

• m/z 58 - 16 (NH2) 42 (C, H only)
• m/z 42 - (312) 6 consistent with C3H6
• Putting it all together, CH3CH2CH2CH2NH2

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The Numbers Approach

• If compound with formula CwHxNyOz , relative
  intensities of M, M1, and M2 ions will be given
  by

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The Numbers ApproachExact Masses

• Nominal mass vs. exact mass
• non integral
• EX 1H 1.00782522
• High resolution mass spectrometer
• EX ion trap MS
• can differentiate between 12C16O and (14N)2 -
  0.00570 amu

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The Numbers Approach
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Problem

• A molecule containing only C, H, N and/or O, is
  found to have an exact molecular mass of
  203.1522. What is its molecular formula?
• ANS look in table, find C10H21NO3 (n-decyl
  nitrate)

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Computer Identification

• An AID for the analyst
• NOT substitute for training
• 14-36 of possible correct answers recalled with
  database use

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Databases for Reference EI Data

• Wiley Registry of Mass Spectral Data (McLafferty)
• 350,000 EI spectra
• From peer-reviewed literature
• NIST (National Institute of Standards and
  Technology)
• Automated Mass Spectral Deconvolution and
  Identification System (AMDIS) Demo
• 150,000 spectra
• Each spectrum analyzed by human and compute
  experts
• Both contain multiple spectra representing
• Different instruments
• Different experimental conditions

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Probability Based Matching (PBM)

• Data weighting
• Large molecular fragments decompose gt
  probability for higher m/z fragments lower
• Reverse searching
• Are reference peaks in unknown?

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References

• McLafferty, F.W. Turecek, Frantisek
  Interpretation of Mass Spectra (4th ed.)
  University Science Mill Valley, 1993.
• Willard, Dean, Merritt, Settle Instrumental
  Methods of Analysis (6th ed.) Van Nostrand
  New York, Year.

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Web References

• NIST MS Library, avail at URL
  http//www.nist.gov/srd/analy.htm
• NIST Chemistry WebBook, avail at URL
  http//webbook.nist.gov
• Analytical Sciences Digital Library (ASDL), avail
  at URL www.asdlib.org