Title: Kinetics of Ion-Molecule Reactions of Protonated Levoglucosan and Water to
Differentiate Ion Structure
Chelsea A. Tyler, Sandra E. Spencer, Gary L. Glish
Abstract:
The adduction of water with protonated levoglucosan in a 3D quadrupole ion trap,
an uncommon and thus characteristic reaction, was investigated. This reaction is a
pseudo-first order reaction that only depends on the concentration of levoglucosan; water
is in excess. To determine the source of the water in the ion trap, the composition of the
extractive electrospray ionization (EESI) solvent was manipulated. The extent of the
reaction over time was monitored and the constants of the reaction, the rate constant and
the water concentration, were determined. Depending on the EESI solvent, these
constants differed. This suggests that there are at least 3 ion structures observed for
protonated levoglucosan, depending on the solvent.
Introduction:
Aerosols are defined as solid and liquid particles suspended in a gas1. These
particles can derive from natural or anthropogenic sources. Ambient aerosol analysis is
of interest due to the impact of aerosols on climate change, alternative fuel research,
human health, and forensics.2-7 Mass spectrometry is commonly used for aerosol analysis
because of its speed, specificity and sensitivity. Analysis of compounds by mass
spectrometry requires that the analyte must first be ionized. Electrospray ionization (ESI)
is one of the most commonly used ionization methods because of its ability to ionize a
wide range of compounds.8. A derivative of ESI, extractive electrospray ionization
(EESI), generates ions by directing an electrospray through a nebulized sample.9 In this
method, a high voltage is applied to the electrospray emitter. A solution is pumped
through the emitter, generating charged droplets. Desolvated gaseous ions are generated
by evaporation of solvent from and fission of the charged droplets.
Once ionized, the sample of interest can be analyzed using mass spectrometry.
Although the mass-to-charge ratio of a compound can be determined by mass
spectrometry, little to no structural information can be obtained from a mass spectrum.
Tandem mass spectrometry (MS/MS) is a method used to provide information about the
structure of compounds in the sample. In MS/MS, the compound of interest is isolated
and then fragmented by collisions with neutral molecules. Based on the way the ion
dissociates during collision-induced dissociation (CID), information about its
composition can be inferred.
Pyrolysis in combination with mass spectrometry can be used to generate realtime gaseous molecules of complex materials.10 However, the pyrolysis products of
natural polymers often exhibit very similar and structurally uninformative neutral losses,
such as the neutral loss of a water molecule, during CID. Conversely, some ions undergo
uncommon, and therefore characteristic, reactions. In the experiments detailed in this
paper, the ion-molecule reaction of the adduction of water with levoglucosan is
investigated.
Experimental:
A PyroProbe 5250 (CDS Analytical, Oxford PA) was used to volatilize between
20 and 30 mg of levoglucosan at temperatures ranging from 200 °C to 400 C in nitrogen
pyrolysis gas. Levoglucosan was purchased from Sigma Aldrich (St. Louis, Missouri).
The pyrolysis oven was held at 120 C. The transfer line to the mass spectrometer was
heated to between 70-90 C to prevent condensation of the volatilized sample in the
transfer line.
Extractive electrospray ionization (EESI) was adapted from a design previously
reported.11 An electrospray emitter was used to deliver charged micro droplets of
solvent. Nitrogen gas was used as an electrospray nebulizing gas at a flow rate of
3 L/min. The charged droplets of solvent were directed through the sample produced
from volatilization of levoglucosan into a Bruker Esquire 3000 (Bruker Daltonics,
Billerica MA) quadrupole ion trap mass spectrometer for mass analysis. An extraction
voltage of -4.5 kV, applied to the capillary inlet, and a desolvation gas flow rate of 5
L/min were used for all experiments.
Results and Discussion
The only dissociation product ion observed for levoglucosan is from the neutral
loss of a water molecule. Because this neutral loss pathway is common for organic,
oxygen-containing compounds, very little structural information is gained. However,
levoglucosan also adducts with adventitious water in the ion trap to generate an ion of
m/z 181 [M+H+H2O]+. By trapping the molecular ion at m/z 163 for different times after
isolation, the reaction kinetics for the production of m/z 181 from m/z 163 have been
determined.
The relative intensity of m/z 163 (Im/z163) decreases and the relative intensity of
m/z 181 (Im/z181) increases with increasing trapping time; shown in Figure 1. As shown
in Figure 2A, the ratio of Im/z163/(Im/z163+Im/z181) was plotted over time. The ratio of
Im/z163/(Im/z163+Im/z181) decreases exponentially. The rate law for this reaction can
be linearized by integration. The experimentally determined slope of an integrated rate
law gives the opposite of the rate constant. As shown in Figure 2B, the linear regression
of the natural log of the ratio of Im/z163/(Im/z163+Im/z181) versus reaction time gave the
equation, y = -0.0035x(±0.0001) - 0.1307 with an r2 value of 0.9846. The reaction
appears to be first order based on the integrated rate law although two analytes are
present. This occurs in instances where one of the reactants is in great excess. As a
result, the reaction is pseudo-first order; the reaction equation depends only on the
concentration of one analyte. The adduction of water with levoglucosan is effectively a
first order reaction in terms of levoglucosan; water is in excess. Therefore, the opposite
of the slope of the linear regression line is equal to the rate constant times the
concentration of water (k[H2O]).
Figure 1: Volatilized levoglucosan ionized by EESI using 50/49/1 methanol/water/acetic
acid. A. Reaction time 0 ms. B. Reaction time 300 ms. C. Reaction time 600 ms. D.
Reaction time 900 ms.
A
B
Figure 2: Graphs depicting the reaction over time of volatilized levoglucosan ionized by
EESI with 50/49/1 methanol/H2O/acetic acid. A. The ratio of
Im/z163/(Im/z163+Im/z181) versus reaction time. B. The natural log of the ratio
Im/z163/(Im/z163+Im/z181) versus reaction time.
The water that is available for adduction with protonated water may be coming
from the inlet of the mass spectrometer but also may be present as a background neutral
in the mass spectrometer. If the water that reacts with levoglucosan is from the inlet of
the mass spectrometer, decreasing the concentration of water vapor at the inlet to the
mass spectrometer may decrease the extent of reaction. There are two sources of water
vapor at the inlet to the mass spectrometer, the EESI solvent and humidity from the air.
Because most of the water vapor at the inlet to the mass spectrometer is from the EESI
solvent, removing water from the EESI solvent may decrease the concentration of water
available to react. As seen in Figure 3, the levoglucosan ion generated by an EESI solvent
containing 99/1 acetonitrile/acetic acid does not undergo a ion-molecule reaction with
water; the ratio of Im/z163/(Im/z163+Im/z181) remains constant over time.
Figure 3: Graph depicting the extent of reaction of volatilized levoglucosan ionized by
EESI with 99/1 acetonitrile/acetic acid.
When water was removed from the EESI solvent, protonated levoglucosan did not
react with water, suggesting that the water in the ion trap is coming from the EESI
solvent. However, since changing the solvent prevented the reaction of water with the
levoglucosan ion, it is also likely that the EESI solvent containing 99/1 acetonitrile/acetic
acid ionizes a different structure of levoglucosan. To confirm that the EESI solvent is
providing the water that reacts with protonated levoglucosan, 50/49/1
methanol/D2O/acetic acid was used as an EESI solvent, as shown in Figure 4. If the water
in the ion trap were coming from the EESI solvent, when D2O is substituted for H2O in
the EESI solvent, the levoglucosan molecular ion would ionize to m/z 164 [M+D]+ and
levoglucosan would adduct with D2O, m/z 183 [M+H+D2O]+. However, as shown in
Figure 4, EESI of volatilized levoglucosan using 50/49/1 methanol/D2O/acetic acid does
significantly increase the production of the deuterated analyte, m/z 164 [M+D]+. In
addition, as shown in Figure 5, EESI of volatilized levoglucosan using 50/49/1
methanol/D2O/acetic acid does not result in the adduction of D2O to protonated
levoglucosan, m/z 183, but the adduction of H2O is still observed, m/z 181.
C
A
B
C
Figure 4: Volatilized levoglucosan ionized by EESI using two different solvent systems.
A. Solvent 50/49/1 methanol/H2O/acetic acid B. Solvent 50/49/1 methanol/D2O/acetic
acid
A
B
C
D
Figure 5: Volatilized levoglucosan ionized by EESI using 50/49/1 methanol/D2O/acetic
acid. A. Reaction time 0 ms. B. Reaction time 300 ms. C. Reaction time 600 ms. D.
Reaction time 900 ms.
An exponential decay of the ratio of Im/z163/(Im/z163+Im/z181) versus reaction
time for 50/49/1 methanol/D2O/acetic acid is displayed in Figure 6A. However, unlike
the behavior observed when levoglucosan is ionized by EESI with 50/49/1
methanol/H2O/acetic acid, levoglucosan ionized by EESI with 50/49/1
methanol/D2O/acetic acid reaches a reaction time where it does not react to a further
extent. This suggests the presence of a non-reactive structure that is present after all of
the reactive structure has reacted. As shown in Figure 6B, the natural log of the ratio of
Im/z163/(Im/z163+Im/z181) versus reaction time was plotted. A linear regression curve
was applied from 0 to 400 ms and resulted in the equation,
y = -0.0023x(±0.0001)- 0.1003 with an r2 value of 0.9941. The difference between the
slope of the linear regression for the adduction of water with levoglucosan ionized using
EESI solvent containing D2O vs. H2O indicates that the reactive ion structures are not the
same.
A
B
Figure 6: Graphs depicting the reaction over time of volatilized levoglucosan ionized by
EESI with 50/49/1 methanol/D2O/acetic acid. A. The ratio of
Im/z163/(Im/z163+Im/z181) versus reaction time. B. The natural log of the ratio of
Im/z163/(Im/z163+Im/z181) versus reaction time.
To determine whether the non-reactive ion structures generated using 50/49/1
methanol/D2O/acetic acid and 99/1 acetonitrile/acetic acid as the EESI solvent are the
same, tandem mass spectrometry experiments were conducted to characterize ion
structures. As shown in Figure 7, the compound of interest was isolated using each
solvent system. The dissociation pathways observed are also shown in Figure 7. As
shown in Figure 7A, 7B and 7C, the ion of m/z 163 from 99/1 acetonitrile/acetic acid
exhibits a different CID pattern than the ion from 50/49/1 methanol/H2O/acetic acid and
50/49/1 methanol/D2O/acetic acid. This suggests that the structure of the ion produced
from 99/1 acetonitrile/acetic acid is different form the ion produced from 50/49/1
methanol/H2O/acetic acid and 50/49/1 methanol/D2O/acetic acid. This also suggests that
the structure of 50/49/1 methanol/D2O/acetic acid is primarily the reactive structure and
the unreactive structure is of too low abundance to be seen. In addition, Figure 7A and
7C show similar fragmentation patterns of m/z 163; however, the intensity of the
fragments of m/z 163 in 50/49/1 methanol/D2O/acetic acid is much less and therefore,
multiple rounds of tandem mass spectrometry could not be performed.
A
B
C
Figure 7: Comparison of the collision induced dissociation data of m/z 163 using three
different solvent systems. A. Solvent 50/49/1 methanol/H2O/acetic acid. B. Solvent
50/49/1 methanol/D2O/acetic acid. C. Solvent 99/1 acetonitrile/acetic acid.
Conclusions
The adduction of water with protonated levoglucosan is a pseudo-first order
reaction. Water was removed from the EESI solvent and the ion molecule reaction of
water and protonated levoglucosan no longer occurred, suggesting that the water in the
ion trap may be coming from the EESI solvent. However, replacing H2O with D2O in the
EESI solvent did not lead to the production of a D2O adduct with protonated
levoglucosan, indicating that the source of water is not from the EESI solvent. Thus,
water must be present as a background neutral in the vacuum system. Because only a
fraction of the ions of m/z 163 produced by EESI of volatilized levoglucosan with
50/49/1 methanol/D2O/acetic acid react with water, it may be concluded that two
different ion structures of m/z 163 are present in the ion trap; a non-reactive structure and
a reactive structure. The relative rates between the reactive structure from the EESI
solvent containing D2O and the solvent containing H2O are different, suggesting that the
reactive ion structures are not the same. As shown by the dissociation pathways using
tandem mass spectrometry of the three solvent system, 50/49/1 methanol/D2O/acetic acid
is primarily the reactive structure and the unreactive structure is of too low abundance to
be seen. This suggests that al least 3 ion structures were observed for protonated
levoglucosan, depending on the solvent.
Acknowledgements
The authors acknowledge R. J. Reynolds for funding this research.
Reference:
1. Valsaraj, K. T. and Kommalapati, R. R., Ed. Atmospheric Aerosols and Their
Importance. Atmospheric Aerosols; American Chemical Society: Washington,
DC, 2009.
2. Hauglustaine, D. A. The Importance of Atmospheric Chemistry in the Calculation
of Radiative Forcing on the Climate System. Journal of Geophysical Research
1994, 99, 1173-1186.
3. Zhang, Q.; Jimenez, J. L.; Canagaratna, M. R.; Ulbrich, I. M.; Ng, N. L.;
Worsnop, D. R.; Sun, Y. Understanding Atmospheric Organic Aerosols Via
Factor Analysis of Aerosol Mass Spectrometry: A Review. Analytical and
Bioanalytical Chemistry 2011, 401, 3045-3067.
4. Pandis, S. N. Dynamics of Tropospheric Aerosols. Journal of physical chemistry
(1952) 1995, 99, 9646-9659.
5. Ziemann, Paul J., and Roger Atkinson. Kinetics, Products, and Mechanisms of
Secondary Organic Aerosol Formation. Chemical Society Reviews 2012, 41,
6582-6605.
6. Peters, A.; Wichmann, H.; Tuch, T.; Heinrich, J. Respiratory Effects are
Associated with the Number of Ultrafine Particles. American Journal Respiratory
and Critical Care Medicine 1997, 155, 1376-1383.
7. Arunthari, V. A Prospective, Comparative Trial of Standard and Breath-Actuated
Nebulizer: Efficacy, Safety, and Satisfaction. Respiratory Care 2012, 57, 12421247.
8. Wilm M. Principles of Electrospray Ionization. Molecular & Cellular Proteomics
2011 10: M111.009407
9. Meier, L.; Berchtold, C.; Schmid, S.; Zenobi, R. Extractive Electrospray
Ionization Mass Spectrometry-Enhanced Sensitivity Using an Ion Funnel.
Analytical Chemistry 2012, 84, 2076-2080.
10. Lomax, J. A.; Commandeur, J. M.; Boon, J. J.; Pyrolysis Mass Spectrometry of
Polysaccharides. Biochemical Society Transactions 1991, 19(4), 935-940.
11. Chen, H.; Venter, A.; Cooks, R. G. Extractive Electrospray Ionization for Direct
Analysis of Undiluted Urine, Milk, and Other Complex Mixtures Without Sample
Preparation. Chemical Communications 2006, 2042-2044.