Why Mass Spectrometry: An Introduction to the IU MSF Jonathan A. Karty, Ph.D. jkarty@indiana.edu

Why Mass Spectrometry: An Introduction to the IU MSF

Jonathan A. Karty, Ph.D.jkarty@indiana.edu

http://msf.chem.indiana.edu

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

Three Questions• Did I make my compound?– 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

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

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 = 1.6605*10-27 kg (1/12 of a 12C atom)– 1 Th = 1 Da/e = 1.0364*10-8 kg/C

Molecular Weight Calculations• The molecular weight is computed by summing the

masses of all atoms in the compound/ion.– Erythromycin (M+H)+: C37H68N1O13

+ = 12.011*37 + 1.008*67 +

14.007 + 15.999*13 = 734.93 Da

• Yet 734.5 is observed by ESI-MS

O

O

O

O

O

OH

O

NH+HOO

OH

OHHO

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 * 12.0000) + (0.011 * 13.0034) = 12.011– For C40: 64.2% 13C0, 28.6% 13C1, 6.2% 13C2

• Spectrum looks like 100% @ 480, 44.5% @ 481, 9.6% @ 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

Unregistered

485484483482481480479

100

90

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10

0

C40 mass spectrum

Monoisotopic Masses• 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 = 734.468– 12C37

1H68 14N1

16O13– 12.000 * 37 + 1.0078 * 68 + 14.0031 + 15.9949 * 13

• Remember to include any ionizing reagent– Electron loss, proton addition, etc.

C37H68NO13+ Mass Spectrum

Average mass = 734.93 u

13C0, 2H0, 18O0

13C1, 2H0, 18O013C2, 2H0, 18O0

13C0, 2H0, 18O1

13C1, 2H1, 18O0

Observed isotope pattern is the convolution of isotope patterns for all atoms

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+] / 0.011 ≈ # carbon atoms– For morphine: (0.1901 / 1) / 0.011 = 17.28 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

Inte

ns

ity

(%

)

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Mass [amu]362 364 366 368 370 372 374 376 378

Inte

ns

ity

(%

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Mass [amu]131 132 133 134 135 136 137 138 139

Two Complex Isotope Patterns

C2H3Cl3

trichloroethane

C12H27SnBrtributyltin bromide

Monoisotopic mass: 131.93Average mass: 133.43

Monoisotopic mass: 370.03Average mass: 369.96

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

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

Agilent 1200/6130 LC-MS

• 1200 HPLC– Binary pump

• 0.05-2 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

Waters CapLC-LCT LC-MS

• CapLC– 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

Bruker Autoflex III

• MALDI-TOF mass spectrometer– 200-150,000 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

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

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 = 0.0001%

• 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) = ±0.0015 Da

Formula Matching Basics• Atomic weights are not integers (except 12C)– 14N = 14.0031 Da; 1H = 1.0078 Da– 16O = 15.9949 Da; 127I = 126.9045 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) = 282.3286

– O, Cl, F, Na decrease it• Morphine (C17H19NO3) = 285.1365

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 285.1365

• 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

Formula Matching Example

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 (0.0061 u) of 306.0820, only 3 have 2 Cl

Elemental Composition ReportTolerance = 20.0 PPM / DBE: min = -1.5, max = 50.0Selected filters: NoneMonoisotopic Mass, Even Electron Ions370 formulas evaluated with 9 results within limitsElements Used:C: 0-40 H: 0-50 N: 0-5 O: 0-5 Cl: 0-2 Error 20 ppm

Mass intensity Calc. Mass mDa PPM i-FIT

306.082 100 306.0816 0.4 1.3 39.7 C17 H18 N Cl2 306.0776 4.4 14.4 376 C12 H18 N3 O2 Cl2 306.0875 -5.5 -18 701.7 C10 H22 N O5 Cl2 306.0798 2.2 7.2 1945.8 C18 H13 N3 Cl 306.0857 -3.7 -12.1 2205.2 C11 H17 N3 O5 Cl 306.0766 5.4 17.6 9102.8 C18 H12 N O4 306.078 4 13.1 9195.6 C19 H8 N5 306.0879 -5.9 -19.3 9289.5 C17 H12 N3 O3 306.0838 -1.8 -5.9 9543.2 C12 H12 N5 O5

error in:Formula

Zoloft C17H18Cl2N

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

Sample LCT Accurate Mass Report

Sample MAT-95 Accurate Mass Report

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!