1. Introduction The pyrolysis products of natural polymers often exhibit very similar neural losses during collision induced dissociation (CID) Some ions exhibit uncommon products such as the neutral gain product of 18 Da Understanding the kinetics of ion-molecule reactions will give more insight into the structure of gas phase ions

2. Background Pyrolysis Extractive Electrospray Ionization (Py-EESI) of cellulose Tandem mass spectrometry (MS/MS) was performed on isolated peaks –Typical losses: -18, -28, -42, -44, -46 –Atypical losses: -15, -10 –Atypical gain: +18 Neutral losses and gains give insight into structural moieties in ions

3. Previous Studies of Cellulose [M+H-H 2 0] + Uninformative neutral loss [M+H-H 2 0] + Uninformative neutral loss [M+H+H 2 0] + Uncommon reaction observed for levoglucosan [M+H+H 2 0] + Uncommon reaction observed for levoglucosan Intensity x 10 4 145 163 181 0 2 4 6 140150160170180m/z + H 2 O - H 2 O MS/MS 163 145 163 185 205 223 265 307 0 2 4 6 100200300400500m/z Intensity x 10 4 Py-EESI of pyrolyzed cellulose

4. Experimental Bruker Esquire 3000  Quadrupole ion trap  Desolvation gas flow rate: 5 L/min CDS Analytical Pyroprobe 5250  20 mg sample volatilized at 400°C  Sample flow rate: 3 L/min Py-EESI  Solvent: 50/49/1 MeOH/H 2 O/Acetic Acid  -4250 V applied to capillary inlet of mass spectrometer  Levoglucosan standard obtained from Sigma-Aldrich

5. Py-EESI Ionization Method i Pyrolysis Gas In (N 2 ) + + + + + + + To mass spectrometer Capillary inlet of mass spectrometer EESI emitter

6. Reactions can occur between trapping and ejection Increase “Reaction Time” to change the amount of time the ions are trapped Evaluating the extent of reaction at different time points gives kinetic information Experimental Design and Scan Function t (ms) Isolation Variable Reaction Time Transfer Ejection Trapping Ionization Pyrolysis V

7. Exponential Growth of m/z 181 Over Time Reaction Time: 0 ms Reaction Time: 300 ms Reaction Time: 600 ms Reaction Time: 900 ms 163 181 0 25 50 75 100 163 181 195 0 25 50 75 100 163 181 0 25 50 75 100 Levoglucosan + methanol - water? Levoglucosan Levoglucosan + water Levoglucosan + methanol?

8. Adduction of Water is Pseudo First-Order The natural log of the concentration vs. time is linear, indicating the reaction is a pseudo-first order reaction The reaction equation depends only on the concentration of one reactant

9. Is the Water Coming from the EESI Solvent? Decreasing the concentration of water at the inlet to the mass spectrometer may decrease the amount water available in the ion trap for adduction Changing the composition of the EESI solvent influences the extent of reaction Use 99/1 Acetonitrile/Acetic Acid as EESI solvent

10. Influence of Changing Solvent Composition Reaction Time: 0 ms Reaction Time: 300 ms Reaction Time: 600 ms Reaction Time: 900 ms 163 0 25 50 75 100 163 0 25 50 75 100 163 0 25 50 75 100 163 0 25 50 75 100 150155160165170175180185190195m/z No increase in relative intensity of m/z 181

11. Water from the EESI Solvent No water adducts are observed when 99/1 Acetonitrile/Acetic Acid is used as an EESI solvent Water in the ion trap may be due to the high concentration of water vapor at the inlet of the mass spectrometer from the EESI solvent Replace water in the EESI solvent with D 2 O to generate D 2 O adduct at m/z 183

12. Replacing H 2 O with D 2 O in EESI Solvent The effect of D 2 O on the appearance of the mass spectrum is not as significant as expected –The relative intensity of [M+D] + (m/z 164) only increases by a small amount –The relative intensity of the D 2 O adduct (m/z 183) does not increase significantly MS Scan of 50/49/1 MeOH/H 2 O/AA 145 155 163 177 181 0 1 2 3 4 Intensity x 10 4 145 149 159 163 177 181 0 2 4 6 140145150155160165170175180 m/z MS Scan of 50/49/1 MeOH/D 2 O/AA

13. 163 181 0 25 50 75 100 163 181 0 25 50 75 100 163 181 0 25 50 75 100 150155160165170175180185190195m/z Reaction Time: 0 ms Reaction Time: 300 ms Reaction Time: 600 ms Reaction Time: 900 ms 163 181 0 25 50 75 100 Does the Ion of m/z 163 Adduct with D 2 O? Levoglucosan + H 2 O No Levoglucosan + D 2 O

14. Influence of D 2 O in EESI Solvent Water in the ion trap is not coming from EESI solvent –No adduction of levoglucosan with D 2 O occurs (m/z 183) –Adduct of levoglucosan with H 2 O is still observed (m/z 181) I m/z163 does not approach zero! Suggests the presence of ions with 2 different structures of m/z 163

15. Two Ion Structures Present? The natural log of the concentration versus time is not linear over the entire reaction time range A linear relationship is observed from 0 to 400 ms After 400 ms, all of structure 1 has reacted; structure 2 is non-reactive with water

16. Discussion of the Reactive Ion Structures EESI of levoglucosan with 50/49/1 Methanol/H 2 O/Acetic Acid generates one ion structure. That structure reacts with water with a rate constant about 50% faster than the reactive structure formed when D 2 O is in the solvent. The difference between the rate constants for the adduction of water with levoglucosan ionized using EESI solvent containing D 2 O vs. H 2 O indicates that the reactive ion structures are not the same

17. Discussion of the Non-Reactive Ion Structures When D 2 O is used in place of H 2 O in the EESI solvent, a second ion structure that does not react with water is generated When EESI is performed using 99/1 Acetonitrile/Acetic Acid the ions generated do not react with water More experiments are required to determine if the ion structures that do not react with water are identical

18. Conclusions Certain structures of protonated levoglucosan undergo pseudo-first order reactions with water Water is present as a background neutral in the vacuum system Changing the composition of EESI solvent changes the structure of the ions generated At least 3 ion structures were observed for protonated levoglucosan, depending on EESI solvent

19. Acknowledgements Thank you to R. J. Reynolds for funding this project Glish Group