This research is supported by the National Key Research and Development Program of China (2017YFA0206801, 2018YFA0208600), Natural Science Foundation of Jiangsu Province,&#160;and National Natural Science Foundation of China (91645116). L.X is the Zhong-Wu Specially Appointed Professor of the Jiangsu University of Technology. The authors acknowledge the suggestions from Dr. Hao Hu and Dr. Mingjun Yang.

1. Building model
<ol>
	<li>Build porphycene structure: Open GaussView software by double-click the mouse. Then click Element Fragment button in the menu of the GaussView to choose the needed elements. Construct porphycene. Then click File button to save as the pdb file.</li>
	<li>Solvate the model: Solvate porphycene in a cubic TIP3P21 water box with an edge length of 38 &#197; by issuing the command in the linux operating system: genbox_d -cp prp-vac.pdb -cs spc216.gro -o solv.pdb -maxsol 1484 -box 3.8.</li>
	<li>Build deuterium porphycene: Issue the following command to generate topology file: cns &#60; ppi_solv.inp. Then open prp-wat.psf with vi command and change the mass of H1 from 1.00800 to 2.01600 to replace one intramolecular hydrogen atom of porphycene with deuterium to build single deuterium substituted porphycene.</li>
	<li>Set normal MD simulation parameters: Input method scctb, integral 0.5 fs, and cutoff 12 in the MD input file by opening it with vi command. 
	NOTE: Adopt a cutoff distance of 12 &#197; for calculating both vdW and electrostatic interactions. Simulate the porphycene molecule with DFTB/MIO method22. Set the integration time step as 0.5 fs for MD simulations. Maintain the temperature of the simulation system at 300 K with Langevin thermostat. Then perform the simulations with QM4D software following the steps below.</li>
</ol>
2. Pre-sits
<ol>
	<li>Set up temperature parameters: Input templow 260, temphigh 1100 and ntemp 160 in the input file. 
	NOTE: The temperature range from 260K to 1100K was spread out by QM4D software to 160 temperature points during MD simulations. The template input files are included in Supplementary Files.</li>
	<li>Initiate the pre-sits: Set runtype 100 and step 120,000 in the input file. Then issue the following command: $PATH/qm4d $INPUTFILE &#62; $OUTPUTFILE. 
	NOTE: The total step is 120,000 but can be adjusted depending on the specific need. The suggested parameters of MD simulations are saved in the $INPUTFILE. The same command is also used in the following opt-sits and production simulation steps, with the input file modified accordingly.</li>
	<li>Calculating the decomposed energies
	<ol>
		<li>Extract energy changes: During the pre-sits stage, monitor the energy of each term to calculate the mean values, as shown in Figure 1. Use grep Linux command to extract the energy as follows: 
		grep &#8216;SITS-ener0&#8217; $INPUTFILE | awk &#8216;{a+=$3;b+=$4;c+=$5}END{print a/NR,b/NR,c/NR}.</li>
		<li>Modify average energies in the MD input file: Calculate the average energies based on the output of the command line above, and modify the line vshift0 -30801.95; vshift1 -26.88; vshift2 -13888.28 in the input file with the newly generated averages. 
		NOTE: Numbers -30801.85, -26.88 and -13888.28 are the average energies in the current model system. Please modify the values based on the specific systems.</li>
	</ol>
	</li>
</ol>
3. Opt-sits
<ol>
	<li>Initiate opt-sits: Set runtype 0 in the input file. Then initiate the QM4D program by typing the command as shown in step 2.2 to start the optimization step.</li>
	<li>Monitoring the energy changes and nk values.
	<ol>
		<li>Plot the energy propagation with &#8220;grace&#8221; program and make sure the energy fluctuation can cover the lowest and the highest ends of the temperature range.</li>
		<li>After optimization, save the final nk values of the opt-sits step into a new file, which is named as nk.dat in this protocol.</li>
	</ol>
	</li>
</ol>
4. Running production simulations
<ol>
	<li>Prepare MD input file: Set runtype 1 in the new input file to start the production simulation step. Specify the file name of stored nk file as nkfile nk.dat in the input file. The number of time steps was set as 6,400,000 in the present systems.</li>
	<li>Initiate production MD simulation: Issue the following command to start MD simulations: $PATH/qm4d $INPUTFILE &#62; $OUTPUTFILE. 
	NOTE: Make sure the nk values can be read in by the QM4D software. The simulation time is system dependent so change the simulation step based on specific demands. Select a proper number of time steps to ensure enough simulation time for your own system. This step is likely time consuming, so save restart files to avoid restarting the production from the very beginning once being disrupted.</li>
</ol>
5 Data analysis
<ol>
	<li>Monitoring the distance changes
	<ol>
		<li>Monitor the bond forming and breaking process during the production phase, use the grep command to check the distance changes of H1-N1 and H1-N2 along the simulation time. The same operation can be conducted for H2-N3 and H2-N4. Then plot the distance propagation using the accumulated distance value during the production simulations.</li>
	</ol>
	</li>
	<li>Extracting reaction coordinates
	<ol>
		<li>Extract the reaction coordinates and the energy terms from the production output file generated by QM4D by grep command: 
		grep &#8216;dist 1&#8217; $OUTPUTFILE | awk &#8216;{print $5}&#8217; &#62; distance1; 
		grep &#8216;ener0&#8217; $OUTPUTFILE &#62; ener0.</li>
		<li>Organize data in four columns: q1, q2, U0 and U&#8217; (U0 and U&#8217; are the output normal energy and weighted energy), and write them into the data file at each time frame.</li>
	</ol>
	</li>
	<li>Calculating Free energy
	<ol>
		<li>Calculate the free energy by issuing the following command: 
		sits-pmf 300 $INPUTFILE PMF2 [hist_minx hist_maxx num_binsx] [hist_miny hist_maxy num_binsy] &#62; $OUTPUTFILE. 
		NOTE: sits-pmf is the histogram-based analysis method. [hist_minx hist_maxx num_binsx] defines the range and number of bins for the first reaction coordinate. The second reaction coordinates can be set by [hist_miny hist_maxy num_binsy].</li>
		<li>To project the free energy on the two-dimensional landscape, type the following command: 
		sits-pmf 300 h1-2d.dat PMF2 -0.6 0.6 24 2.45 4.25 36 &#62; sits-pmf.out. 
		NOTE: Use a total of 24 bins and 36 bins to cover the distance changes in two selected reaction coordinates, q1 and q2, respectively. Save the 2D PMF data for each hydrogen/deuterium to the sits-pmf.out file.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The structure of porphycene was shown in Figure 1. The electrostatic embedding QM/MM hybrid potential with SITS method was used to describe the chemical reactions in water23,24. The proton transfer occurs within porphycene3 and thus porphycene is set as QM region and the reminding water is set as MM region. Herein we adopted DFTB/MIO as our QM method to treat the porphycene by balancing the efficiency and accuracy22,25. As a sampling enhancement technique, SITS simulation was shown to broaden the distribution of Us to high-energy regions and meantime preserve sufficient sampling around the energy region at the temperature of interest. For current case, the energy of Us in the &#8220;opt-sits&#8221; step was broadened to wider ranges encompassing the energy of the standard MD simulations in the &#8220;pre-sits&#8221; step as shown in Figure 2. Meantime, the smooth energy changes of Ue indicated that the higher temperature in the QM subsystem would not bring perturbation into the environment. SITS-QM/MM method realized enhanced sampling at the interested QM region without affecting the potential energy of water.
From distance changes in Figure 3, we noticed that H1 transferred from N1 to N2 to form a transit cis state, and then initiated a consecutively rapid H2 transfer to arrive the other trans state again; and vice versa. The dynamics proton transfer process is shown in Supplementary Movie 1. Deuterium D1 transfer between N1 and N2 in the single deuterated porphycene invoked the transfer of H2 between N3 and N4. Asynchronous distance changes indicated the stepwise double proton transfer process for both of porphycene and single deuterium substituted porphycene. The similar distance distributions of D1-N1 and H2-N3 suggested that the cooperative effect on two hydrogen bonds26. Consistent with previously reported primary geometric isotope effect26, the distance of D1-N1 is shorter than the distance of H1-N1 (1.048 &#197; vs. 1.051 &#197;). As shown in Figure 3, we observed around 135, and 65 times of transfer of H or D for porphycene and its isotopomers within 3.2 ns MD simulations, respectively. Deuteriation may exert less effect on the hydrogen bond angles as shown in Figure 4. The sufficient sampling on the two reaction channels enabled us to calculate the free energy changes of each proton transfer. The obvious isotopic effect was observed in the 2D free energy landscape. The transition state has been shifted from (0.01 &#197;, 2.52 &#197;) to (-0.01 &#197;, 2.76 &#197;) as revealed from reaction coordinates (q1, q2) (see Figure 5). The higher q2 value means the nonbonded hydrogen bond were expanded. This may come from the asymmetric scaffold of the deuterated porphycene.
Both proton transfer processes in porphycene and deuterated porphycene can be captured by SITS-QM/MM MD simulations without pre-defining the reaction coordinates. Moreover, SITS-QM/MM MD simulations revealed the structural difference that was introduced by isotopic effect. The hydrogen bond D1-N1 was shortened in comparison with H1-N1. The transition state has been shifted toward higher q2 value because of an asymmetric shape caused by deuteration. Though only subtle difference was detected in covalent hydrogen bond, the distance difference may invoke bigger energy difference around the equilibrium bond distance. We are planning to further validate this observation at higher level QM method in the future studies.
The feasibility of SITS-QM/MM has been well validated in the double channel of reactions without pre-defining reaction coordinates in the present study. This method holds potential of searching reaction products from known reactant states if no prior reaction mechanism is provided. We have adopted DFTB/MIO method in our current implementation of SITS-QM/MM approach, and gained a better understanding of the isotopic effect. It is worth noting that the implemented approach is able to capture the free energy changes, but may not capture dynamic properties without considering the quantum tunneling effect. Still, this protocol acts as a starting point to investigate the chemical reaction mechanisms in the condensed environment. We expect SITS-QM/MM method to be extended to higher level QM methods and thus can exploit more complex systems in the future.

The proton transfer process in porphycenes holds potential applications in developing molecular switches, transistors and information storage devices1,2. In particular, tautomerization in porphycenes through double proton transfer process has attracted wide interest in the fields of spectroscopy and photophysics2. The inner hydrogen atoms of porphycene can migrate from one trans isomer to the other equivalent trans isomer through double proton transfer process as shown in Figure 1. Two mechanisms have been proposed for the double proton transfer process: the concerted and the stepwise mechanism3,4. In the concerted double proton transfer process, both proton atoms move to the transition state synchronously in a symmetric way, whereas one proton completes the transfer before the other proton in a stepwise process. Two hydrogen atoms can transfer simultaneously or stepwise depending on the correlation strength between two hydrogen atoms5.
Isotopic substitution has been used to detect the structural properties of molecules and rate constants of reaction kinetics6. Single deuterium substitution in the inner hydrogen of porphycene leads to an asymmetric shape of the molecule. The hydrogen bond may expand or contract because of mass difference between the hydrogen and deuterium atoms. The isotopic substitution introduces a perturbation in the scaffold of porphycene. The question arises that whether asymmetric structure would affect the proton transfer process. Limbach and coworkers reported that the replacement of hydrogen with deuterium will compress both hydrogen bonds, and the cooperative coupling of two hydrogen bonds in porphycene may favor concerted mechanism7, whereas Yoshikawa stated the deuteration would make the stepwise mechanism contribute more than the concerted mechanism8. Experimental techniques, such as force spectroscopy, have been developed to capture tautomerization details in a single porphycene9. However, it is still challenging to determine the atomic details of proton transfer experimentally because of its transient nature.
Theoretical calculations and simulations can act as complementary tools in elucidating the reaction mechanisms of proton transfer. Among different theoretical methods, molecular dynamics (MD) simulations can monitor dynamic motions of each atom, and has been widely used to reveal complex mechanisms in chemical and enzymatic reactions. However, regular MD simulations tend to suffer from insufficient sampling issue, especially when high energy barrier exists in the process of interest. Therefore, enhanced sampling methods have been developed, which include transition path sampling10,11, umbrella sampling (US)12,13, and integrated tempering sampling (ITS)14,15. Combination of different enhanced sampling methods can further increase sampling efficiency16,17,18. To harness the enhanced sampling algorithms in simulating chemical reactions, we have implemented the selective integrated tempering sampling (SITS) method with quantum mechanical and molecular mechanical (QM/MM) potentials recently19. The proposed SITS-QM/MM method combines the advantages from both methods: the SITS method accelerates the sampling and can explore all possible reaction channels without prior knowledge of the reaction mechanism, and QM/MM provides more accurate description of the bond forming and bond breaking process, which cannot be simulated by MM methods solely. The implemented SITS-QM/MM approach has successfully uncovered concerted double proton transfer, uncorrelated and correlated stepwise double proton transfer mechanism in different systems, without pre-defining reaction coordinates19. For porphycene, the stepwise but correlated proton transfer character has been reported19. The hybrid SITS-QM/MM method was used to investigate the isotopic effect in porphycene in our study, and below are the detailed descriptions of the algorithm and protocol of our method.
We have implemented SITS method with hybrid QM/MM potentials. The effective potential of SITS was defined to include the potential energy at different temperatures with the weighting factors nk to cover wider temperature ranges,
<img alt="Equation 1" src="/files/ftp_upload/60040/60040equ01.jpg" />
where, N is the number of canonical terms, &#946;k is the inverse temperature, and nk is the corresponding weighting factor for each canonical component. UE(R) and&#160;UN(R) represent the enhanced and non-enhanced terms in SITS and are defined as,
<img alt="Equation 2" src="/files/ftp_upload/60040/60040equ02.jpg" />
Us, Use and Ue are the potential energy of sub-system, the interaction between sub-system and the environment, and the potential energy of environment. QM/MM potential is expressed as a hybrid summation of three components,
<img alt="Equation 3" src="/files/ftp_upload/60040/60040equ03.jpg" />
where Uqm, Uqm/mm, and&#160;Umm are the internal energy term of the QM subsystem, the interaction energy between the QM and MM regions, and the interaction energy within the MM subsystem, respectively. The&#160;Uqm/mm term can be further divided into three components, which include the electrostatic, van der Waals, and covalent interaction energy terms between the QM and MM atoms,
<img alt="Equation 4" src="/files/ftp_upload/60040/60040equ04.jpg" />
We assign <img alt="Equation 5" src="/files/ftp_upload/60040/60040equ05.jpg" />, and <img alt="Equation 6" src="/files/ftp_upload/60040/60040equ06.jpg" /> into one&#160;Us term in SITS,
<img alt="Equation 7" src="/files/ftp_upload/60040/60040equ07.jpg" />
The full potential of the system was then decomposed into the energy of subsystem Us, the interaction energy between the subsystem and the environment Use, and the energy of environment Ue. For instance, in the system of the present work, the subsystem is the porphycence, and the environment the water.
The PMF profile along a collective variable&#160;&#964;(R) is derived as,
<img alt="Equation 8" src="/files/ftp_upload/60040/60040equ08.jpg" />
The generally used reaction coordinates for each hydrogen transfer of N1&#8722;H1&#183;&#183;&#183; N2 are q1 = (r1&#8722;r2)/2 and q2 = r1 + r2, where r1 is the distance of N1-H1 and r2 is the distance of H1-N2.
The method has been implemented in the QM/MM MD simulation package QM4D20. The complete source code and documentation can be found here: http://www.qm4d.info/.
Generally, the SITS-QM/MM MD simulations involve four steps: pre-equilibrium (pre-sits); optimization nk (opt-sits); production simulation and data analysis.

<table><tbody><tr><td>operating system</td><td>CentOS Linux release 6.0</td><td></td><td></td></tr><tr><td>QM4D software</td><td>http://www.qm4d.info/</td><td></td><td>in-house program</td></tr><tr><td>Computer desktop</td><td>HP</td><td></td><td></td></tr></tbody></table>

isotopic effect in double proton transfer process of porphycene investigated by enhanced qm/mm method

The single deuterium substitution in porphycene leads to an asymmetric molecular geometry, which may affect the double proton transfer process in the porphycene molecule. In this study, we applied an enhanced QM/MM method called SITS-QM/MM to investigate hydrogen/deuterium (H/D) isotope effects on the double proton transfer in porphycene. Distance changes in SITS-QM/MM molecular dynamics simulations suggested that the deuterium substituted porphycene adopted the stepwise double proton transfer mechanism. The structural analysis and the free energy shifts of double proton transfer process indicated that the asymmetric isotopic substitution subtly compressed the covalent hydrogen bonds and may alter the original transition state location.

The single deuterium substitution effect on double proton transfer process in porphycene was examined in the current protocol (Figure 1). The potential energy of QM sub-system and the water during pre-equilibrium and optimization step were checked to make sure the energy has been broadened to a wider energy range (Figure 2). The representative distance and angle changes (Figure 3 and Figure 4), and the projected free energy changes (Figure 5) were used to characterize the deuterium substitution effect on the geometry and proton transfer process of porphycene.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60040/60040fig01.jpg" /> 
Figure 1. The structures of investigated molecules. 
The structures of porphycene (A) and deuterated porphycene(B). <a href="https://www.jove.com/files/ftp_upload/60040/60040fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60040/60040fig02.jpg" /> 
Figure 2. The potential energy changes during MD simulations. 
The potential energy changes of QM region (A) and environment (B) in pre-sits and opt-sits steps. <a href="https://www.jove.com/files/ftp_upload/60040/60040fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60040/60040fig03.jpg" /> 
Figure 3. The characteristic distance changes. 
(A) The distance changes of H1-N1 and H2-N3 for porphycene, and (B) the distance changes of D1-N1 and H2-N3 for deuterated porphycene during SIRTS-QM/MM simulations; (C) the distribution of distance changes for H1-N1 and H2-N3 for porphycene, and (D) D1-N1 and H2-N3 for deuterated porphycene. <a href="https://www.jove.com/files/ftp_upload/60040/60040fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60040/60040fig04.jpg" /> 
Figure 4. The hydrogen bond angles during the production MD simulations. 
The hydrogen bond angels for (A) prophycene and (B) deuterated porphycene. <a href="https://www.jove.com/files/ftp_upload/60040/60040fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60040/60040fig05.jpg" /> 
Figure 5. The free energy landscape of each hydrogen transfer process that was projected on two reaction coordinates (q1, q2). 
(A) and (B) are 2D free energy landscapes of H1 and H2 transfer in porphycene; (C) and (D) 2D free energy landscape of D1 and H2 transfer in deuterated porphycene. <a href="https://www.jove.com/files/ftp_upload/60040/60040fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary files. Topology file, force field parameter file, coordinates file and input file. <a href="https://www.jove.com/files/ftp_upload/60040/JoVE60040_screen_capture.docx" target="_blank">Please click here to download the file.</a>
Supplementary movie 1. Porphycene. <a href="https://www.jove.com/files/ftp_upload/60040/MovieS1_porphycene.mp4" target="_blank">Please click here to download the video.</a>

Watch this Scientific Journal Video about Isotopic Effect in Double Proton Transfer Process of Porphycene Investigated by Enhanced QM/MM Method at JoVE.com

Isotopic Effect in Double Proton Transfer Process of Porphycene Investigated by Enhanced QM/MM Method

A protocol that uses enhanced QM/MM method to investigate the isotopic effect on the double proton transfer process in porphycene is presented here.

The single deuterium substitution in porphycene leads to an asymmetric molecular geometry, which may affect the double proton transfer process in the ...

isotopic-effect-double-proton-transfer-process-porphycene

Nanyang Technological University

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6.2K Views.  Chinese Academy of Sciences. A protocol that uses enhanced QM/MM method to investigate the isotopic effect on the double proton transfer process in porphycene is presented here.

Video: Isotopic Effect in Double Proton Transfer Process of Porphycene Investigated by Enhanced QM/MM Method

Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy

Monitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards understanding its mechanism. Protonation and conformational changes affect the vibration pattern of amino acid side chains and of the peptide bond, respectively, both of which can be probed by infrared (IR) difference spectroscopy. For proteins whose function can be repetitively and reproducibly triggered by light, it is possible to obtain infrared difference spectra with (sub)microsecond resolution over a broad spectral range using the step-scan Fourier transform infrared technique. With ~102-103 repetitions of the photoreaction, the minimum number to complete a scan at reasonable spectral resolution and bandwidth, the noise level in the absorption difference spectra can be as low as ~10-4, sufficient to follow the kinetics of protonation changes from a single amino acid. Lower noise levels can be accomplished by more data averaging and/or mathematical processing. The amount of protein required for optimal results is between 5-100 &#181;g, depending on the sampling technique used. Regarding additional requirements, the protein needs to be first concentrated in a low ionic strength buffer and then dried to form a film. The protein film is hydrated prior to the experiment, either with little droplets of water or under controlled atmospheric humidity. The attained hydration level (g of water / g of protein) is gauged from an IR absorption spectrum. To showcase the technique, we studied the photocycle of the light-driven proton-pump bacteriorhodopsin in its native purple membrane environment, and of the light-gated ion channel channelrhodopsin-2 solubilized in detergent.

Key steps of protein function, in particular backbone conformational changes and proton transfer reactions, often take place in the microsecond to millisecond time scale. These dynamical processes can be studied by time-resolved step-scan Fourier-transform infrared spectroscopy, in particular for proteins whose function is triggered by light.

Monitoring the dynamics of protonation and protein backbone conformation changes during the function of a protein is an essential step towards ...

Engineering

High Resolution Phonon-assisted Quasi-resonance Fluorescence Spectroscopy

High resolution optical spectroscopy methods are demanding in terms of either technology, equipment, complexity, time or a combination of these. Here we demonstrate an optical spectroscopy method that is capable of resolving spectral features beyond that of the spin fine structure and homogeneous linewidth of single quantum dots (QDs) using a standard, easy-to-use spectrometer setup. This method incorporates both laser and photoluminescence spectroscopy, combining the advantage of laser line-width limited resolution with multi-channel photoluminescence detection. Such a scheme allows for considerable improvement of resolution over that of a common single-stage spectrometer. The method uses phonons to assist in the measurement of the photoluminescence of a single quantum dot after resonant excitation of its ground state transition. The phonon&#39;s energy difference allows one to separate and filter out the laser light exciting the quantum dot. An advantageous feature of this method is its straight forward integration into standard spectroscopy setups, which are accessible to most researchers.

The manuscript describes a method of phonon-assisted quasi-resonant fluorescence spectroscopy that incorporates both laser-limited resolution and photoluminescence (PL) spectroscopy. This method utilizes optical phonons to provide linewidth-limited resolution spectra of atom-like semiconductor structures in the energy domain. The method is also easily realized with a single spectrometer optical spectroscopy setup.

High resolution optical spectroscopy methods are demanding in terms of either technology, equipment, complexity, time or a combination of these. Here we ...

In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging

A method is demonstrated to monitor macroscopic translational diffusion using electron paramagnetic resonance (EPR) imaging. A host-guest system with nitroxide spin probe 3-(2-Iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (IPSL) as a guest inside the periodic mesoporous organosilica (PMO) aerogel UKON1-GEL as a host and ethanol as a solvent is used as an example to describe the protocol. Data is shown from a previous publication, where the protocol has been applied to both IPSL and Tris(8-carboxy-2,2,6,6-perdeutero-tetramethyl-benzo[1,2-d:4,5-d&#8242;]bis(1,3)dithiole) methyl (Trityl) as guest molecules and UKON1-GEL and SILICA-GEL as host systems. 
A method is shown to prepare aerogel samples that cannot be synthesized directly in the sample tube for measurement due to a size change during synthesis. The aerogel is attached to sample tubes using heat shrink tubing and a pressure cooker to reach the necessary temperature without evaporating the solvent in the process. The method does not assume a clearly defined initial distribution of guest molecules at the start of the measurement. Instead, it requires a reservoir on top of the aerogel and experimentally determines the influx rate during data analysis.
The diffusion is monitored continually over a period of 20 hr by recording the 1d spin density profile within the sample. The spectrometer settings for the imaging experiment are described quantitatively. Data analysis software is provided to take the resonator sensitivity profile into account and to numerically solve the diffusion equation. The software determines the macroscopic translational diffusion coefficient by least square minimization of the difference between the experiment and the numerical solution of the diffusion equation.

In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging

A protocol for the in situ monitoring of the diffusion of guest molecules in porous media using electron paramagnetic resonance (EPR) imaging is presented.

A method is demonstrated to monitor macroscopic translational diffusion using electron paramagnetic resonance (EPR) imaging. A host-guest system with ...

Measurement of Ultrafast Vibrational Coherences in Polyatomic Radical Cations with Strong-Field Adiabatic Ionization

We present a pump-probe method for preparing vibrational coherences in polyatomic radical cations and probing their ultrafast dynamics. By shifting the wavelength of the strong-field ionizing pump pulse from the commonly used 800 nm into the near-infrared (1200-1600 nm), the contribution of adiabatic electron tunneling to the ionization process increases relative to multiphoton absorption. Adiabatic ionization results in predominant population of the ground electronic state of the ion upon electron removal, which effectively prepares a coherent vibrational state (&#34;wave packet&#34;) amenable to subsequent excitation. In our experiments, the coherent vibrational dynamics are probed with a weak-field 800 nm pulse and the time-dependent yields of dissociation products measured in a time-of-flight mass spectrometer. We present the measurements on the molecule dimethyl methylphosphonate (DMMP) to illustrate how using 1500 nm pulses for excitation enhances the amplitude of coherent oscillations in ion yields by a factor of 10 as compared to 800 nm pulses. This protocol may be implemented in existing pump-probe setups through the incorporation of an optical parametric amplifier (OPA) for wavelength conversion.

We present a protocol for probing ultrafast vibrational coherences in polyatomic radical cations that result in molecular dissociation.

We present a pump-probe method for preparing vibrational coherences in polyatomic radical cations and probing their ultrafast dynamics. By shifting the ...

Determination of the Photoisomerization Quantum Yield of a Hydrazone Photoswitch

Photoswitching organic molecules that undergo light-driven structural transformations are key components to construct adaptive molecular systems, and they are utilized in a wide variety of applications. In most studies employing photoswitches, several important photophysical properties such as maximum wavelengths of absorption and emission, molar attenuation coefficient, fluorescence lifetime, and photoisomerization quantum yield are carefully determined to investigate their electronic states and transition processes. However, measurement of the photoisomerization quantum yield, the efficiency of photoisomerization with respect to the absorbed photons, in a typical laboratory setting is often complicated and prone to error because it requires the implementation of rigorous spectroscopic measurements and calculations based on an appropriate integration method. This article introduces a set of procedures to measure the photoisomerization quantum yield of a bistable photoswitch using a photochromic hydrazone. We anticipate that this article will be a useful guide for the investigation of bistable photoswitches that are being increasingly developed.

Photoisomerization quantum yield is a fundamental photophysical property that should be accurately determined in the investigation of newly developed photoswitches. Here, we describe a set of procedures to measure the photoisomerization quantum yield of a photochromic hydrazone as a model bistable photoswitch.

Photoswitching organic molecules that undergo light-driven structural transformations are key components to construct adaptive molecular systems, and they ...

A Technical Guide for Performing Spectroscopic Measurements on Metal-Organic Frameworks

Metal-organic frameworks (MOFs) offer a unique platform to understand light-driven processes in solid-state materials, given their high structural tunability. However, the progression of MOF-based photochemistry has been hindered by the difficulty in spectrally characterizing these materials. Given that MOFs are typically larger than 100 nm in size, they are prone to excessive light scatter, thereby rendering data from valuable analytical tools like transient absorption and emission spectroscopy nearly uninterpretable. To gain meaningful insights of MOF-based photo-chemical and physical processes, special consideration must be taken toward properly preparing MOFs for spectroscopic measurements, as well as the experimental setups that garner higher quality data. With these considerations in mind, the present guide provides a general approach and set of guidelines for the spectroscopic investigation of MOFs. The guide addresses the following key topics: (1) sample preparation methods, (2) spectroscopic techniques/measurements with MOFs, (3) experimental setups, (3) control experiments, and (4) post-run stability characterization. With appropriate sample preparation and experimental approaches, pioneering advancements toward the fundamental understanding of light-MOF interactions are significantly more attainable.

Here, we use a polymer stabilizer to prepare metal-organic framework (MOF) suspensions that exhibit markedly decreased scatter in their ground-state and transient absorption spectra. With these MOF suspensions, the protocol provides various guidelines to characterize the MOFs spectroscopically to yield interpretable data.

Metal-organic frameworks (MOFs) offer a unique platform to understand light-driven processes in solid-state materials, given their high structural ...

Molecular Orbital (MO) Theory

Source: Tamara M. Powers, Department of Chemistry, Texas A&#38;M University
This protocol serves as a guide in the synthesis of two metal complexes featuring the ligand 1,1&#39;-bis(diphenylphosphino)ferrocene (dppf): M(dppf)Cl2, where M = Ni or Pd. While both of these transition metal complexes are 4-coordinate, they exhibit different geometries at the metal center. Using molecular orbital (MO) theory in conjunction with 1H NMR and Evans method, we will determine the geometry of these two compounds.

Molecular Orbital MO Theory: Predicting Geometry of Transition Metal Complexes

Source: Tamara M. Powers, Department of Chemistry, Texas A&#38;M University
This protocol serves as a guide in the synthesis of two metal complexes ...

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Nuclear Magnetic Resonance to Study Atomic Level Protein-Protein Interactions

Source: Winkelaar, G., et al. <a href="https://app.jove.com/v/58537">Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST).</a> J. Vis. Exp. (2018).
This video describes the nuclear magnetic resonance spectroscopy technique to study protein-protein interactions between 15N-labeled wild-type and mutant envoplakin proteins and the unlabeled vimentin protein. The successful interaction between wild-type envoplakin and vimentin leads to extensive line broadening and peak disappearance in the NMR spectra, whereas the absence of an interaction between the mutated envoplakin and vimentin results in well-resolved peaks in the NMR spectra.

Source: Winkelaar, G., et al. Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale ...

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Double Resonance Techniques: Overview

Double resonance techniques in Nuclear Magnetic Resonance (NMR) spectroscopy involve the simultaneous application of two different frequencies or radiofrequency pulses to manipulate and observe two distinct nuclear spins. One important application of double resonance is spin decoupling, which selectively suppresses coupling with one type of nucleus while observing the NMR signal from another nucleus, simplifying the spectrum and enhancing resolution.
Spin decoupling is usually achieved by irradiating the sample atoms with a suitable radio frequency (rf) pulse sequence, effectively eliminating all the coupling with one nuclide. This simplifies the observed spectrum, making it easier to analyze and understand the relationships between different nuclei.
Double resonance experiments can be classified as heteronuclear or homonuclear, depending on whether the two sets of nuclei are of different isotopes or the same isotope, respectively. They can also be selective or nonselective, depending on whether the irradiating frequency covers only a portion or all of the resonance frequencies.
In nonselective heteronuclear decoupling, samples are exposed to an appropriate radiofrequency range to remove all coupling with one nuclide. However, increased external field strengths necessitate more powerful irradiation across broader frequency ranges. In certain instruments, continuous irradiation creates enough heat to cause damage to thermally sensitive samples.
To overcome these problems, modern methods using a series of pulses and exact timing delays, like J-modulated spin echo pulse sequences, can be employed to eliminate or adjust coupling effects within the spectrum.
For example, the attached proton transfer (APT) experiment applies a J-modulated spin echo pulse sequence to focus on the phase of detected carbon signals. Carbon atoms attached to an even number of protons exhibit positive signals in the spectrum, while those bonded to an odd number of protons appear as negative signals.
It utilizes a combination of a 180&#176; proton pulse and broadband decoupling to simplify spectral interpretation and assign multiplicities. The 180&#176; pulse is applied to the protons during the APT experiment. Its role is to refocus the spin coupling evolution that naturally occurs due to interactions between protons and carbons.
Specifically, after the 90&#176; pulse creates transverse magnetization, the 180&#176; pulse ensures that the effects of coupling are frozen during the initial period where the decoupler is turned off. This means the chemical shifts evolve only during the time 1/J&#8203; (the coupling constant period) after the first 180&#176; pulse.
By carefully timing this pulse sequence, the experiment isolates carbon multiplicities by utilizing the J-coupling between carbons and their attached protons. After the coupling evolution period (1/J&#8203;), broadband decoupling is turned on. This removes the J-coupling signals, collapsing multiplets into single peaks for each carbon resonance.
The decoupling ensures that carbon signals are free of splitting, allowing for a clear differentiation of signals based on phase (positive or negative) rather than splitting patterns.
The APT technique selectively enhances carbon signals, providing valuable information about carbon-proton connectivity, the number of attached hydrogen atoms, and the overall molecular structure.

Double resonance techniques in Nuclear Magnetic Resonance (NMR) spectroscopy involve the simultaneous application of two different frequencies or ...

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Unit 1

Advanced Nuclear Magnetic Resonance Spectroscopy

KEY TERMS AND CONCEPTS

&sup1;H NMR: Complex Splitting

A proton M that is coupled to a proton X results in doublet signals for M. However, NMR-active nuclei can be simultaneously coupled to more than one nonequivalent nucleus. When M is coupled to a second proton A, such as in styrene oxide, each peak in the doublet is split into another doublet.
Splitting diagrams or splitting tree diagrams are routinely used to depict such complex couplings. While drawing splitting diagrams, the splitting with the larger coupling constant is usually applied first. However, the splittings can be applied in any order, and the same result is obtained.
In propyl bromide, the protons M are coupled to three methyl and two methylene protons, with the coupling constants JAM and JMX. The splitting diagram predicts a quartet of triplets with 12 lines, which would all be visible in the spectrum if JAM &#62;&#62; JMX. However, if JAM &#8773; 2 JMX, some peaks overlap, their intensities are added, and 9 peaks are seen. When JAM &#8773; JMX, as is the case in propyl bromide, 6 peaks are observed. As a result, the relative magnitudes of the J values can cause deviations from the number of peaks and relative intensities predicted by the n+1 rule and Pascal&#39;s triangle.

1H NMR: Complex Splitting

A proton M that is coupled to a proton X results in doublet signals for M. However, NMR-active nuclei can be simultaneously coupled to more than one ...

Isotopic Effect in Double Proton Transfer Process of Porphycene Investigated by Enhanced QM/MM Method

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Chapters in this video

Proton Transfer and Protein Conformation Dynamics in Photosensitive Proteins by Time-resolved Step-scan Fourier-transform Infrared Spectroscopy

High Resolution Phonon-assisted Quasi-resonance Fluorescence Spectroscopy

In Situ Monitoring of Diffusion of Guest Molecules in Porous Media Using Electron Paramagnetic Resonance Imaging

Measurement of Ultrafast Vibrational Coherences in Polyatomic Radical Cations with Strong-Field Adiabatic Ionization

Determination of the Photoisomerization Quantum Yield of a Hydrazone Photoswitch

A Technical Guide for Performing Spectroscopic Measurements on Metal-Organic Frameworks

Molecular Orbital (MO) Theory

Nuclear Magnetic Resonance to Study Atomic Level Protein-Protein Interactions

Double Resonance Techniques: Overview

¹H NMR: Complex Splitting