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Minute Papers Due 10/26/2012

Please post this week's minute papers as "comments" to this post. Minute papers should be posted by 5 pm on Friday. Feel free to read your classmate's posts.

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Minute Paper #8 (10/24/2012) – Sarah Anciaux

Title: Tissue Imaging Using Nanospray Desorption Electrospray Ionization Mass Spectrometry

Author: Semmes et al.

Journal: Analytical Chemistry

In this paper the authors propose using a new platform termed nanospray desorption electrospray ionization (nano-DESI) to image a tissue sample. In this platform spatially resolved 2D analysis is achieved under ambient conditions, without sample pretreatment, by addition of a secondary capillary to the DESI platform.

Similar to regular DESI, a charged solvent is directed at a solid sample through a primary capillary. Instead of interacting with the sample, desorbing to the gas state, and going to the MS inlet, as seen in DESI, the sample enters a secondary capillary through a liquid bridge and then self-aspirates as a nano-spray to the inlet. Optimization of this capillary distance and the solvent flow rate allowed for reduction of the sampling region from a previously reported 100 microns down to 8 microns in this study. With this reduced sampling region, the authors were able to take images of rat brain and human kidney samples and found results that were consistent with previously reported data proving the point of concept of the nano-DESI platform.

The authors claim to have successfully proved the point of concept of the nano-DESI by investigating the 2D imaging of biological tissues, but some areas could be addressed further. In the article the authors only investigate one solvent system stated as being optimized for lipids. With liquid extraction the solvent can be very important and have a large effect on what is actually extracted to the liquid phase. It would be interesting to see how important the percent composition of the solvent is on the outcome. For instance, how much can the solvent system be varied without changing the outcome, or relative intensities, of the nano-DESI experiment. The authors also do not address the angles used for the primary and secondary capillaries or the secondary capillaries position relative to the MS inlet. In DESI the angle from the solvent capillary to the stage highly affects the outcome of the desorption, so it would be interesting to see if this parameter is rendered less important in nano-DESI than in DESI. On a similar note I think it would be interesting to vary the angle from the secondary capillary to the stage as well as it appears that geometry is playing a large part in the sampling region for this technique. The final thing I think the authors could address further would be the distance to the MS inlet relative to the secondary capillary. With ESI the angle to the MS inlet from the capillary producing the Taylor cone is very important in producing charged ions that enter the MS inlet, so I would think this placement would also be very important.

Seminar #1 (Minute Paper #6)

Title: Electroanalytical Applications of Scanning Ion Conductance Microscopy

Lecturer: Prof. Lane Baker, Department of Chemistry, Indiana University, Bloomington

It is very important to measure ion currents at the biological interfaces. Baker group has developed an effective tool to carry out electrochemical measurement to characterize the ion transport process through small pores on membrane. They used nano pipette with hole in the center and scanning ion conductance microscopy (SICM) to measure the ion current. The electrode include a pipette electrode (PE), a reference electrode (RE) and a working electrode (WE).

First they used polymer membranes as model platforms. Nanoporous polyimide membranes with cylindrical pores were prepared via the track-etch process. Single nanopores were isolated and optical and electron micrographs of one pore were taken, and current-voltage response at lateral positions was measured with a stationary pipette. When the SICM probe was away from the pore center and over the pore center, different plots of potential applied to working electrode versus the absolute pipette current were obtained. When the SICM probe was positioned over the pore center, the slope of the current -voltage response measured was about 10 times larger than that obtained when they pipette was away from the pore center, indicating some properties directly connected with the position of the pipette. Equivalent circuit model simulating the three-electrode SICM was performed and the result was consistent with the experimental results.

Measurement on the membrane with multiple conical pores of heterogeneous conductance were also performed using a foure-electrode SICM, with a unique conductance observed as the result of its unique pore geometry. Compared to the three-electrode SICM, a counter electrode (CE) was applied to prevent fluctuations in the potential of RE from passage of current across the membrane. The pipet current measured at 0V of the WE was reduced from the pipet current and a correlation between the measured ICM signals and the transport properties of the nanopore was derived. Also they carried out the potential measurement with a theta pipette electrode with an EMF meter. They found that smaller tips has bigger potential response.

Now they are trying to use SICM to study cell interactions and carry out measurement of ion transport in tissues. SICM could be used to image MDCK II cell lines, measure the ion selectivity of Claudins-2 channels between cells and study Claudin-Claudin interactions. Ion replacement experiments were also performed. They are especially interested in the triple points of tight cell junction and the conductance with high spatial resolution of the junction.

I think this technique could be useful in single cell measurements and has potential for real-time measurements, while I'm still wondering if the measurements are reproducible because the fabrication may generate inconsistency among different batches of electrodes and thus affect the electrochemical properties of the electrodes.

Seminar Title: Electroanalytical Applications of Scanning Ion Conductance Microscopy

Speaker: Professor Lane A. Baker, Indiana University
Professor Baker spoke about scanning ion conductance microscopy as an answer to a need for electro chemical measurements at biological interfaces. Cellular ion channels play a critical role in homeostatic processes and disease, so a greater understanding of signaling is important in understanding disease development.

The idea came from the patch clamp method for monitoring ion channels in cells. Here, they place a carbon electrode nanopipette near the cells and are able to do some imaging from the current readings. They have to vibrate the pipette and take the current readings to be able to image the cell. They, however, ran into a limit of resolution by reading current. As they shrunk the electrode down even more, the current signal became smaller as well. So they started measuring potential instead. From this they could then image with very good resolution upwards of 100 nm and better.

This technology is very interesting and raises some questions. I wonder how fast they can scan their electrode. If it is reasonably fast I would like to use it to do some time lapse readings on cells. Maybe stimulate those cells and look at how they respond. It would also be cool to maybe use these to look at GABA response in neurons. I would also like to see this used in some sort of monitoring experiment watching in real time how a cell responds in the human body to some stimulus. Maybe also look at the role of Tau proteins and their interactions in Alzheimer’s disease. Lastly, I want to know more about the double barrel electrodes and how they are used in SICM. It seems as though there are many applications for SICM.

Minute paper # 8, Marzieh Ramezani
Title: Electroanalytical Applications of Scanning Ion Conductance Microscopy
By: Professor Lane Baker, Departmental seminar

Exploring Cell junctions within tissues is important to advance communications between cells that have different functionality. The seminar was started by introducing a new electrochemical tool for measuring the cell junction or any two different biological interface interactions while they are alive and in small scale. Investigation of these stable cell layers seems to be difficult and needs a lot of money for the instrumentation. However, using the new developed technique could probe hundreds of thousands of cell junctions individually all at the same time. For this purpose layers of the interested tissue was grown and nanopipette electrode (PE) was put on the surface of it following by a patch clamp method for monitoring ion channels in cells. Applying a voltage and turning on the circuit enabled them to measure the membrane resistance or the current which is the consequence of the ion transportation.

To control the position of the electrode on the surface, observing how ions flow at the interface, and how the potential varies, the pipette electrode was made smaller and applied in this study. These new nanopipettes routinely achieve 100 nm pore dimension and are easier to make in fabrication process rather than using previous carbon filament electrodes. Then, it was observed that the current was fixed while the electrode was far away from the surface and by becoming closer to the surface, the change in the current was detected which enabled them to track surface species, collect the topographic images and also measure the electrochemical properties of the pipette at the same time. From this, it was found out that measuring conductivity and the resistance is highly dependent on the position of the electrode. In the next step, an extra electrode as the working electrode (WE) was added and by keeping the potential of pipette electrode constant, the potential of the working electrode was measured.

The first sample was a polymembrane insulated single pore in a 100M KCl. By varying the potential of the working electrode from 0V to +3V and -3V different conductive properties of the PE and the WE was detected. It should be noted that observation of conductivity changes would be more difficult when one is working on a biological system with many pores with different geometry. Thus, to investigate this model, a complicated system includes a couple of electrodes as counter electrodes were set up and the conductivity based on the shape of the pores and their distance from each set of electrodes were determined. Also, the potential at epithelial tissues was measured which has advantage to image the tight junctions in small scale with high spatial resolution.

For future studies, they are trying to investigate the ion selectivity of Claudins channels between cells which helps them to estimate the channels density and also which ions are being transported. For this generalization of their work I am wondering how far they can go, since in high voltages like +60 V they could cause problems like polymerization of the cell membrane.


Title: Electroanalytical Applications of Scanning Ion Conductance Microscopy
Author: Lane Baker
From: Seminar

In the seminar, Baker talked about single and multiple nanopores study by utilizing scanning ion conductance microscopy (SICM). SICM makes use of a pipet electrode (PE) containing glass nanopipet to scan the sample surface. The current-voltage properties of the nanopores are detected as signals to determine their position and shape.

Experiments were first done on single nanopore on the polymer membranes, which were made of low-density ion-tracked polyimide, with three electrode SICM system. The target nanopore was then isolated and characterized by optical microscope and scanning electron microscopy (SEM). As to SICM detection, the membrane was placed between two chambers of a perfusion cell, with the pipet electrode and reference electrode (RE) mounting in the upper chamber and the working electrode (WE) in the lower chamber. When applying the nanopipet to the surface of the membrane, pipet ion current changed significantly as the distance between the pipet tip and the surface became smaller. With this distance-modulated signal, a stable distance was chosen to obtain the set current. The nanopipet then scanned through the surface with the fixed distance. Different potential was applied to the WE and the pipet current under each potential was recorded. The result showed that the pipet current decreased greatly with the increase of the potential when the nanopipet was placed over the pore center. However, when the nanoprobe was located over other places on the membrane, the pipet current remained unchanged. Therefore, from the current-voltage response, the position of the nanopore could be identified. Baker further investigated the influence of the shape of the nanopore to the current pipet by comparing the current-voltage properties of cylindrical pore and conical pore. It turned out that cylindrical pore exhibited linear change in the pipet current and the corresponding WE potential while this relationship in conical pore was nonlinear, due to the rectified current flow existing in conical nanopores.

This method was then extended to multiple-pore membranes with four electrode configuration. The extra platinum counter electrode (CE) was set to the upper chamber and acted as a potentiostat. As described above, the position and shape of the nanopores were determined by the current-voltage properties and each pore on the multiple-pore membranes can be studied individually and separately with high resolution.

As mentioned by Baker himself, future work would include applying SICM to biological systems, like a real cell membrane. However, the shape of the pores on the cell membranes or the channel between two cells would not be simply cylindrical or conical. So when testing SICM on polymer membranes, irregular-shaped pores should also be examined. Moreover, as a development, more components such as the CE driver and function generator are added to the system. Although they help to stabilize the potential when performing SICM, they also complicate the system and might limit the actual application of SICM. So as research going on, how to simplify the system should also be considered.

Minute Paper 8
Sarah Gruba
Seminar : Dr.Lane Baker University of Minnesota Chemistry Dept 10/25/12
Electoanalytical applications of scanning ion conductance microscopy

There are two ways in which ions can get across the biological interface. The first is paracellular where the ions go around the cell and the second is transcellular where the ions go through channels. Currently it is believed that these channels are important to look at because of their connection with several diseases. Current methods to look at the ion channel include killing the cell and looking at cross sections or use trans epithelial resistance to measure the ion channels of living cells. The problem with this second technique is that the instrument currently on the market cannot measure individual junctions and requires release from several cells.
Dr. Baker saw this problem and utilized the knowledge behind patch clamp technology to create a two barrel pipette that needed to be fabricated with a laser due to its miniscule size in order to detect one ion channel. A Ag/AgCl electrode was put a little ways up into the channel and when ions were caught in the current between the cathode and anode they would be pulled into the pipette tip and the resistance would go down. They also were able to vibrate the pipette tip in order to image the ion channel and cell at the same time they were measuring the ion release. Having this image and knowing how far away from the ion channel helped them place the electrode a certain distance away from the ion channel during every experiment.
Getting to this point took a lot of work and before they started on real cells with their collaborator Professor Hou who claudin 2 knockout cells, they created a polymer membrane with several ion pores. They did experiments on distance from the ion pore, interference from other ion pores, and affects of the shape of the ion pores and found that their electrode was able to measure the differences caused by all these factors. These results not only showed that their method worked, but that the shape of the pore changed the way the ions released. From there they started work on the knockout cells in which they found differences in release compared to WT cells.
One interesting factor I think that was never mentioned was how long it took for their electrode to actually measure the change. This should have been easy to do in the polymer ion channels because they controlled the potential on the other side so they could have seen how long it took before their electrode sensed it. The reason I think this is applicable is because if the time resolution was small enough you would be able to see the effects of different stimulants on the cells. Also it would have been interesting because you could see if the ions had time to diffuse before getting pulled into the channel.

Electroanalytical Applications of Scanning Ion Conductance Microscopy

Baker, L.
Seminar

The author was motivated by the need for electrochemical measurements at biological interfaces in order to study how ions cross cell membranes and epithelial tissue. To do this, it is necessary to monitor live cells at a small scale. The author developed a new instrument for use on individual cell junctions.

Patch clamp techniques can be used for trancellular ion channels but allow ion leakage when used on tight cell junctions. To address this, the author developed a technique using nanopipettes (20 - 100 nm diameter). The ion flow through the nanopipette is controlled by the distance between the nanopipette tip and the junction surface. This distance was controlled by measuring the modulation of the alternating current generated by vibrating the nanopipette tip. If the tip was far from the surface, no modulation was observed. As the tip approaches the surface, the AC current is modulated and distance between the tip and the surface can be controlled by setting the current to a certain value. This technique was given the name Scanning Ion Conductance Microscopy (SICM).
The current at the surface pore was controlled by three microelectrodes: a pipette electrode inside the nanopipette, a reference electrode on the outside of the membrane and a working electrode on the other side of the membrane.

The SICM technique was tested on pores in polymer membranes. By moving the tip of the nanopipette across the junction surface, the author was able to simultaneously form topographic and ion current images. The author also was able to control the intensity of ion current across the membrane pore by varying the potential on the working electrode.

Upon the addition of a counter electrode outside of the membrane, the author was able to measure multiple pores with heterogeneous conductance, even if the conical shape of the pores gave them a non-linear conductance. The technique proved to have good spatial resolution, able to detect the different ion currents of different pores even when the pores were only separated by a distance roughly equal to the diameters of the pores themselves.

The author discovered that, for even higher resolution current measurements, even smaller pipettes would be required. To avoid this, new two-barreled pipettes (called 'theta' pipettes for the similarity of their cross-sectional shape to the greek letter theta) were developed, with one barrel specialized for maintaining spatial positioning and the other for potential measurements. By measuring potential instead of current, the author was able to have more sensitivity with smaller tip size, instead of the lower sensitivity that would have resulted from smaller tip size in current measurements.

The SICM technique was then applied to biological systems, specifically the claudin-2 protein that control ion conductance in cell junctions. The author was able to measure the ion selectivity of claudin-2 as well as its conductivity. Then, by finding the conductance of an area of membrane containing claudin-2, the author was able to determine the density of claudin-2 on the membrane, which turned out to be roughly ten times less than that which was previously believed.

Minute Paper 8 October 26, 2012
Title: GOFAST: An Integrated Approach for Efficient and Comprehensive Membrane Proteome Analysis
Authors: Yanbao Yu, Ling Xie, Harsha P. Gunawardena, Jainab Khatun, Christopher Maier
Journal: Analytical Chemistry

Membrane proteins are the molecules that surround or attach to the walls of cells and organelles. They play important roles in the function of transport, structure and attachment for the cell in regards to other small molecules. The study of these membrane protein process is therefore of significant biological importance, however techniques in this field are often confronted with a variety of challenges. Due to membrane proteins’ low solubility, the digestion and capture of peptides is severely inefficient, thus decreasing the accuracy and precision with which these proteins can be identified. The authors of this paper introduce a novel technique for identifying membrane proteins.

The technique presented by the authors in this paper is GELFrEE Optimized FASP Technology (GOFAST). This integrated technique combines gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) with filter-aided sample preparation (FASP) and an enzymatic digestion assisted on-filter by microwave. GELFrEE is a sample preparation procedure that has the benefits of being a gel-free separation, accepting of strong detergents and salt interferents, and a high loading capacity. By combining this sample preparation technique with the microwave-assisted on-filter digestion, the peptide sequences available for mass spectrometry detection were increased; therefore the number of identifiable proteins was also raised. Samples were separated after the GOFAST procedure by liquid chromatography-mass spectrometry-mass spectrometry. In a study that included K562 leukemia cell cultures, 2090 proteins were able to be successfully identified.

The authors of this study claim that this sample preparation technique is able to be used on various biological samples and other separations protocols besides two-dimensional separations. Strong anion exchanges, weak cation exchange, and hydrophilic interaction chromatography are said to increase the dynamic range and throughout of GOFAST, however no results were shown to actually solidify this statement. I would attempt to combine these separation techniques into GOFAST to determine whether or not these separation protocols increase the dynamic range of this integrated sample preparation procedure. Another study which they failed to mention would be the selectivity capabilities presented by such a separation technique. If the resolution and separation efficiency are increased for membrane proteins based on this sample preparation technique, does the selectivity capability also increase? I would study whether or not this is the case; as a sample preparation technique to further increase the selective nature of these separations would be highly useful in the genomics field as they probe protein samples for specific sequences.

Title: Dynamic Surface Enhanced Raman Spectroscopy (SERS): Extracting SERS from Normal Raman Scattering
By: K. T. Carron et al.
Journal: Analytical Chemistry

In this paper, the group statistically studied dynamic surface enhanced raman spectroscopy(DSERS) that extracts surface enhanced raman spectroscopy(SERS) from normal raman spectroscopy. Using DSERS, they could remove background signals of solvent and noises, so they could focus on signals of colloids and nanoparticles in solution. For this study, they used scanning electron microscope(SEM) to measure size of colloids, UV-Vis spectroscopy to measure concentration of solution, and, of course, Raman spectroscopy.

They measured Raman spectroscopy of total solution about 1000 times and averaged that. Then, they subtracted the standard deviation from averaged spectroscopy. By doing this, they could remove signals from dominant solvents and variations from laser power fluctuations. Their method canceled the effect of Brownian motion, so they were able to detect spectrum of particles. They could detect 1,2-bis(4-pyridyl)ethylene(BPE) shelled Au nanoparticles in toluene solvent. On the other hand, they also measured Raman spectrum of 4-mercaptopyridine attached Au nanoparticles under various pH condition. Using same statistical technique, they found that there was little change in peaks according to pH. It meant that the peaks from DSERS showed sites that were not affected by pH, like gaps between particle aggregates. Because number of gaps is much smaller than normal surface of particles, they could get Raman spectrum of the very tiny part of materials. They said it as site selection.

Although they said they got Raman spectrum of coated particles, they did not explain the meaning of peaks. They did not explain which peak came from which chemical groups in particles. In figure 2, they said there was an event on the particle, so peak changed. However, they did not say what happened. It would be good to study what chemical changes occurred. They could measure the Raman spectrum of BPE and what caused that change using BPE. For second experiment, site selection, they could study changes in gaps more precisely. They might be able to synthesize connected nanoparticles and compare it to what they got. Thus, they can confirm they were right. Using this technique, they can study lots of fields in colloid chemistry. One thing is that they can study aggregation of nanoparticles and how particles aggregate under various conditions, such as pH, by observing Raman spectrum of aggregated nanoparticles. They could count number of hotspots between particles and figure out differences among nanoparticles under various conditions.

Minute Paper #7 (10/26/12) – Matt Irwin
Presentation: Control and stabilization of morphologies in reactively compatibilized polymer blends: PA6/HDPE as a model system
Presenter: Dr. Ludovic Odoni, Laboratoire Polymères et Matériaux Avancés
Date: 10/24/12

The blending of polymers is of great industrial and academic interest as it provides a method for combining the inherent properties of two or more distinct materials into one “super” polymer system. For example, blends of glassy, brittle polystyrene with a rubbery, ductile poly(lactic acid) could yield a material which is tough and strong. However, achieving blends that are well dispersed at the nanometer length scale is challenging due to the high degree of imcompability between the polymers which thermodynamically drives the system to phase separate. In this presentation, Dr. Odoni presented his group’s recent work to study blends of high density polyethylene (HDPE) with polyamide 6 (PA6) compatibilze via the addition of a high density polyethylene-graft¬¬-amide (HDPE-g-AM) polymer which selectively reacts with PA6 during melt-blending. Techniques used include melt blending, transmission electron microscopy, scanning electron microscopy, and Fourier transform infrared (FTIR) spectroscopy.

In the work presented, the Odoni group performed melt blending at elevated temperatures using different amounts of HDPE, PA6, and HDPE-g-AM to determine the morphologies that can be produced as a function of parameter space. In general, the group found that for all blends with at least ~5 vol% HDPE-g-AM, as the fraction of HDPE was increased, the morphology transitioned from (1) nodular PA6 in an HDPE matrix to (2) stretched PA6 in an HDPE matrix to (3) a cocontniuous blend to (4) stretched HDPE in a PA6 matrix to (5) nodular HDPE in a PA6 matrix. So long as the temperature was above ~200 °C, the group found that the morphology produced was independent of temperature and melt-blending time, suggesting that the reaction of the HDPE-g-AM with the PA6 is kinetically accessible and rapid. In control studies, the group used FTIR to track the concentration of what I believe is the carbonyl group on the amide grafted to the HDPE as the reaction progressed. Based on the results given, it appears that a blend of 60% PA6, 30% HDPE, and 10% HDPE-g-AM produces the most desirable morphology: a cocontniuous blend with roughly 2 – 4 µm. This morphology should be considered the “best” as it results in completely interconnected HDPE and PA6 domains.

While the speaker presented some great techniques on how to use reactive blending to achieve relatively fast production of polymer blends, some questions remain. The presenter noted that his group has not yet determined the minimum concentration of HDPE-g-AM required to compatibilize the blend. As the compatibilizer is frequently the most expensive component of a polymer blend, the minimum concentration needed should be determined before scaling this process up. One potential method for determining this concentration would be to use data from microscopy to develop an empirical model which predicts how the apparent interfacial curvature and domain size of cocontniuous blends scales with the amount of HDPE-g-AM added at fixed ratio of PA6 to HDPE. The model could then be used to predict at what concentration of HDPE-g-AM the domain size diverges to infinity. While not likely to be fully accurate due to its empirical nature, the model would provide a guide for what compositions are worth further investigation experimentally.

Title: Time-Resolved Studies of Ethylene and Propylene Reactions in Zeolite H‑MFI by In-Situ Fast IR Heating and UV Raman Spectroscopy

By: Allotta, P. M. and Stair, P. C.

Journal: ACS Catalysis

The synthesis of gasoline and other valuable hydrocarbon products from methanol is a process of great industrial interest. Methanol is generally thought to undergo this reaction in acidic zeolites via a series of reactions with a collection of evolving intermediate species known as the hydrocarbon pool, in which the reaction of light olefins such as ethylene and propylene plays an important kinetic role1. Much is still unknown, however, about the reaction intermediates formed by light olefins in acidic zeolites and the conditions under which they form. To further elucidate this mechanism, the authors investigated the evolution of species present in ethylene and propylene-exposed zeolites samples heated both conventionally and with a rapid infrared (IR) laser using ultraviolet (UV) Raman spectroscopy.
The authors doused H-MFI catalyst samples with ethylene and propylene prior to heat the samples from room temperature to 300°C using both a conventionally reactor furnace and with an IR laser system and observing the formed products with UV Raman spectroscopy. Due to the rapid and local nature of laser heating, the authors claimed that diffusion of the reaction products to the unheated regions of the catalyst bed was slow relative to the rate of reaction on the laser-heated catalyst, creating a localized reaction environment for the evolution of reaction intermediates. For both the ethylene and propylene doused samples, a large Raman band associated with the presence of polyenes with approximately 6-8 double bonds (1550cm-1) was observed and increased in intensity with temperature for the conventionally heated sample but was only minimally observed for the laser heated sample, causing the authors to conclude the formation of these large polyenes requires the diffusion and reaction of multiple polyethylene and polypropylene dimer and trimer species, which were observed to form at room temperature. Additionally, for both heating methods on both the ethylene and propylene doused samples, the Raman band associated with polyaromatic compounds was observed to experience a downward shift with increasing temperatures from (1622 cm-1 to 1606cm-1). This shift was proposed to be indicative of the growth and the eventual formation of bent polyaromatic compounds as they approached the size restrictions of the zeolite pores. Both of these large species may become trapped in the zeolite pores and play an important role in catalyst deactivation for methanol conversion.
While the authors presented evidence of the formation of large conjugated compounds from light olefins in acidic zeolites, they did not assess the kinetic role these species play in presence of methanol. To further investigate the role of these species, it may be useful to observe the resulting changes in the Raman spectra when douses of methanol are introduced at different points in the heating process. If these species are play an important kinetic role (either as an intermediate or as a poison), then a correlation could be drawn between Raman peak intensity of these species and methanol reactivity. Additionally, it would also be interesting to perform in-situ mass spectroscopy to observe the evolution of gaseous products during this process.

[1] Song, W.; Marcus, D. M.; Fu, H.; Ehresmann, J. O.; Haw, J. F. J. Am. Chem. Soc. 2002, 124, 3844.

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