Name: determination of the photoisomerization quantum yield of a hydrazone photoswitch
Uploaded: 2022-02-07T14:00:00
Description: Watch this Scientific Journal Video about Determination of the Photoisomerization Quantum Yield of a Hydrazone Photoswitch at JoVE.com

This work was supported by the Chung-Ang University Research Grants in 2019 and the National Research Foundation of Korea (NRF-2020R1C1C1011134).

1.&#160;1H NMR spectrum acquisition at photostationary state (PSS)
<ol>
	<li>In a natural quartz NMR tube containing 4.2 mg (0.01 mmol) of hydrazone switch 1, add 1.0 mL of deuterated dimethyl sulfoxide (DMSO-d6). Transfer half of the solution to another NMR tube.</li>
	<li>Place one of the NMR tubes 1 cm in front of a Xenon arc lamp equipped with a 436 nm bandpass filter. Start irradiation to the NMR sample and record a 1H NMR spectrum every d...

<ol>
	<li>Kathan, M., Hecht, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoswitchable+molecules+as+key+ingredients+to+drive+systems+away+from+the+global+thermodynamic+minimum.">Photoswitchable molecules as key ingredients to drive systems away from the global thermodynamic minimum.</a> Chemical Society Reviews. 46, 5536-5550 (2017).</li><li>Feringa, B. L., Browne, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Switches. 2nd ed. , (2011).</li><li>Baroncini, M., Silvi, S., Credi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photo-+and+redox-driven+artificial+molecular+motors.">Photo- and redox-driven artificial molecular motors.</a> Chemical Reviews. 120 (1), 200-268 (2020).</li><li>Goulet-Hanssens, A., Eisenreich, F., Hecht, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enlightening+materials+with+photoswitches.">Enlightening materials with photoswitches.</a> Advanced Materials. 32 (20), 1905966 (2020).</li><li>Bas&#237;lio, N., Pischel, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+delivery+by+controlling+a+supramolecular+host-guest+assembly+with+a+reversible+photoswitch.">Drug delivery by controlling a supramolecular host-guest assembly with a reversible photoswitch.</a> Chemistry-A European Journal. 22 (43), 15208-15211 (2016).</li><li>Wegener, M., Hansen, M. J., Driessen, A. J. M., Szymanski, W., Feringa, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photocontrol+of+antibacterial+activity:+shifting+from+UV+to+red+light+activation.">Photocontrol of antibacterial activity: shifting from UV to red light activation.</a> Journal of the American Chemical Society. 139 (49), 17979-17986 (2017).</li><li>Izquierdo-Serra, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+control+of+endogenous+receptors+and+cellular+excitability+using+targeted+covalent+photoswitches.">Optical control of endogenous receptors and cellular excitability using targeted covalent photoswitches.</a> Nature Communications. 7 (1), 12221 (2016).</li><li>Mourot, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+optical+control+of+nociception+with+an+ion-channel+photoswitch.">Rapid optical control of nociception with an ion-channel photoswitch.</a> Nature Methods. 9 (4), 396-402 (2012).</li><li>Griffiths, K., Halcovitch, N. R., Griffin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+solar+energy+storage+under+ambient+conditions+in+a+MOF-based+solid-solid+phase-change+material.">Long-term solar energy storage under ambient conditions in a MOF-based solid-solid phase-change material.</a> Chemistry of Materials. 32 (23), 9925-9936 (2020).</li><li>Sun, C. -. L., Wang, C., Boulatov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+photoswitches+in+the+storage+of+solar+energy.">Applications of photoswitches in the storage of solar energy.</a> ChemPhotoChem. 3 (6), 268-283 (2019).</li><li>Gu, M., Zhang, Q., Lamon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomaterials+for+optical+data+storage.">Nanomaterials for optical data storage.</a> Nature Reviews Materials. 1 (12), 16070 (2016).</li><li>Roke, D., Wezenberg, S. J., Feringa, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+rotary+motors:+Unidirectional+motion+around+double+bonds.">Molecular rotary motors: Unidirectional motion around double bonds.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (38), 9423-9431 (2018).</li><li>Stranius, K., B&#246;rjesson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+the+photoisomerization+quantum+yield+of+photoswitchable+molecules+in+solution+and+in+the+solid+state.">Determining the photoisomerization quantum yield of photoswitchable molecules in solution and in the solid state.</a> Scientific Reports. 7 (1), 41145 (2017).</li><li>Schneider, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+term+spectral+irradiance+measurements+of+a+1000-watt+xenon+arc+lamp.">Long term spectral irradiance measurements of a 1000-watt xenon arc lamp.</a> NASA-CR. , 132533 (1974).</li><li>Qian, H., Pramanik, S., Aprahamian, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochromic+hydrazone+switches+with+extremely+long+thermal+half-lives.">Photochromic hydrazone switches with extremely long thermal half-lives.</a> Journal of the American Chemical Society. 139 (27), 9140-9143 (2017).</li><li>Shao, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+and+solid-state+emission+toggling+of+a+photochromic+hydrazone.">Solution and solid-state emission toggling of a photochromic hydrazone.</a> Journal of the American Chemical Society. 140 (39), 12323-12327 (2018).</li><li>Shao, B., Qian, H., Li, Q., Aprahamian, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+property+analysis+of+the+solution+and+solid-state+properties+of+bistable+photochromic+hydrazones.">Structure property analysis of the solution and solid-state properties of bistable photochromic hydrazones.</a> Journal of the American Chemical Society. 141 (20), 8364-8371 (2019).</li><li>Moran, M. J., Magrini, M., Walba, D. M., Aprahamian, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Driving+a+liquid+crystal+phase+transition+using+a+photochromic+hydrazone.">Driving a liquid crystal phase transition using a photochromic hydrazone.</a> Journal of the American Chemical Society. 140 (42), 13623-13627 (2018).</li><li>Guo, X., Shao, B., Zhou, S., Aprahamian, I., Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+intracellular+particles+and+precise+control+of+drug+release+using+an+emissive+hydrazone+photochrome.">Visualizing intracellular particles and precise control of drug release using an emissive hydrazone photochrome.</a> Chemical Science. 11 (11), 3016-3021 (2020).</li><li>Yang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+enzymatic+synthesis+of+&#947;-cyclodextrin+using+a+photoremovable+hydrazone+template.">Dynamic enzymatic synthesis of &#947;-cyclodextrin using a photoremovable hydrazone template.</a> Chem. 7 (8), 2190-2200 (2021).</li><li>Yang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multistage+reversible+Tg+photomodulation+and+hardening+of+hydrazone-containing+polymers.">Multistage reversible Tg photomodulation and hardening of hydrazone-containing polymers.</a> Journal of the American Chemical Society. 143 (40), 16348-16353 (2021).</li><li>Connors, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chemical kinetics : the study of reaction rates in solution. , (1990).</li><li>Shao, B., Qian, H., Li, Q., Aprahamian, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+property+analysis+of+the+solution+and+solid-state+properties+of+bistable+photochromic+hydrazones.">Structure property analysis of the solution and solid-state properties of bistable photochromic hydrazones.</a> Journal of the American Chemical Society. 141 (20), 8364-8371 (2019).</li><li>Kuhn, H., Braslavsky, S., Schmidt, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+actinometry+(IUPAC+technical+report).">Chemical actinometry (IUPAC technical report).</a> Pure and Applied Chemistry. 76 (12), 2105-2146 (2004).</li><li>Murov, S. L., Carmichael, I., Hug, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of hotochemistry 2nd ed. Rev. And expanded. , (1993).</li><li>D&#252;rr, H., Bouas-Laurent, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Photochromism: Molecules and Systems. , (2003).</li><li>Kl&#225;n, P., Wirz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Photochemistry of Organic Compounds: From Concepts to Practice. , (2009).</li><li>Harris, J. D., Moran, M. J., Aprahamian, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+molecular+switch+architectures.">New molecular switch architectures.</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (38), 9414-9422 (2018).</li><li>Maafi, M., Brown, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+kinetic+model+for+AB(1&#981;)+systems:+A+closed-form+integration+of+the+differential+equation+with+a+variable+photokinetic+factor.">The kinetic model for AB(1&#981;) systems: A closed-form integration of the differential equation with a variable photokinetic factor.</a> Journal of Photochemistry and Photobiology A: Chemistry. 187, 319-324 (2007).</li><li>Lahikainen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunable+photomechanics+in+diarylethene-driven+liquid+crystal+network+actuators.">Tunable photomechanics in diarylethene-driven liquid crystal network actuators.</a> ACS Applied Materials &amp; Interfaces. 12 (42), 47939-47947 (2020).</li><li>Mallo, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photochromic+switching+behaviour+of+donor-acceptor+Stenhouse+adducts+in+organic+solvents.">Photochromic switching behaviour of donor-acceptor Stenhouse adducts in organic solvents.</a> Chemical Communications. 52, 13576-13579 (2016).</li><li>Feldmeier, C., Bartling, H., Riedle, E., Gschwind, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LED+based+NMR+illumination+device+for+mechanistic+studies+on+photochemical+reactions+-+Versatile+and+simple,+yet+surprisingly+powerful.">LED based NMR illumination device for mechanistic studies on photochemical reactions - Versatile and simple, yet surprisingly powerful.</a> Journal of Magnetic Resonance. 232, 39-44 (2013).</li></ol>

Various strategies to tune the spectral and switching properties of photoswitches have been developed, and the register of photoswitches is rapidly expanding28. It is thus crucial to correctly determine their photophysical properties, and we anticipate the methods summarized in this article will be a helpful guide to experimenters. Provided that the thermal relaxation rate is very slow at room temperature, measurement of PSS compositions at different irradiation wavelengths, molar attenuation coef...

Photochromic organic molecules have attracted considerable attention in a wide range of scientific disciplines as light is a unique stimulus that can drive a system away from its thermodynamic equilibrium non-invasively1. Irradiation of light with appropriate energies allows structural modulation of photoswitches with high spatiotemporal precision2,3,4. Thanks to these advantages, various types of photoswitches based on configurational isomerization of the double bonds (e.g., stilbenes, azobenzenes, imines, fumaramides, thioindigos) and ring opening/closure (e.g., spiropyrans, dithienylethenes, fulgides, donor-acceptor Stenhouse adducts) have been developed and utilized as the core components of adaptive materials at various length scales. Representative applications of photoswitches involve photochromic materials, drug delivery, switchable receptors and channels, information or energy storage, and molecular machines5,6,7,8,9,10,11,12. In most studies presenting newly designed photoswitches, their photophysical properties such as &#955;max of absorption and emission, molar attenuation coefficient (&#949;), fluorescence lifetime, and photoisomerization quantum yield are characterized thoroughly. The investigation of such properties provides key information on the electronic states and transitions that are crucial for understanding the optical properties and isomerization mechanism.
However, accurate measurement of photoisomerization quantum yield-the number of photoisomerization events that occurred divided by the number of photons at the irradiation wavelength absorbed by the reactant-is often complicated in a typical laboratory setting due to several reasons. Determination of the photoisomerization quantum yield is generally achieved by monitoring the advancement of reaction and measuring the number of absorbed photons during irradiation. The primary concern is that the amount of photon absorption per unit time changes progressively because the total absorption by the solution changes over time as the photochemical reaction proceeds. Therefore, the number of consumed reactants per unit time depends on the time section in which it is measured during the irradiation. Thus, one is obliged to estimate the photoisomerization quantum yield that is defined differentially.
A more troublesome problem arises when both the reactant and photoproduct absorb light at the irradiation wavelength. In this case, the photochemical isomerization occurs in both directions (i.e., a photoreversible reaction). The two independent quantum yields for the forward and backward reactions cannot be obtained directly from the observed reaction rate. Inaccurate light intensity is also a common cause of error. For example, the aging of the bulb gradually changes its intensity; irradiance of the Xenon arc lamp at 400 nm decreases by 30% after 1000 h of operation14. The spreading of non-collimated light makes the actual incident irradiance significantly smaller than the nominal power of the source. Thus, it is crucial to accurately quantify the effective photon flux. Of note, thermal relaxation of the metastable form at room temperature should be sufficiently small to be ignored.
This paper introduces a set of procedures to determine the photoisomerization quantum yield of a bistable photoswitch. A number of hydrazone photoswitches developed by the group of Aprahamian, the pioneering research team in the field, have been in the spotlight thanks to their selective photoisomerization and remarkable stability of their metastable isomers15,16,17. Their hydrazone photoswitches comprise two aromatic rings joined by a hydrazone group, and the C=N bond undergoes selective E/Z isomerization upon irradiation at appropriate wavelengths (Figure 1). They have been successfully incorporated as the motile components of dynamic molecular systems18,19,20,21. In this work, we prepared a new hydrazone derivative bearing amide groups and investigated its photoswitching properties for the determination of the photoisomerization quantum yield.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,10-phenanthroline</td>
			<td>Sigma-Aldrich</td>
			<td>131377-2.5G</td>
			<td></td>
		</tr>
		<tr>
			<td>340 nm bandpass filter, 25 mm diameter, 10 nm FWHM</td>
			<td>Edmund Optics</td>
			<td>#65-129</td>
			<td></td>
		</tr>
		<tr>
			<td>436 nm bandpass filter, 25 mm diameter, 10 nm FWHM</td>
			<td>Edmund Optics</td>
			<td>#65-138</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous sodium acetate</td>
			<td>Alfa aesar</td>
			<td>A13184.30</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Samchun</td>
			<td>D1138</td>
			<td>HPLC grade</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide-d6</td>
			<td>Sigma-Aldrich</td>
			<td>151874-25g</td>
			<td></td>
		</tr>
		<tr>
			<td>Gemini 2000; 300 MHz NMR spectrometer</td>
			<td>Varian</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>H2SO4</td>
			<td>Duksan</td>
			<td>235</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating bath</td>
			<td>JeioTech</td>
			<td>CW-05G</td>
			<td></td>
		</tr>
		<tr>
			<td>MestReNova 14.1.1</td>
			<td>Mestrelab Research S.L., https://mestrelab.com/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Natural quartz NMR tube</td>
			<td>Norell</td>
			<td>S-5-200-QTZ-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium ferrioxalate trihydrate</td>
			<td>Alfa aesar</td>
			<td>31124.06</td>
			<td></td>
		</tr>
		<tr>
			<td>Quartz absorption cell</td>
			<td>Hellma</td>
			<td>HE.110.QS10</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-VIS spectrophotometer</td>
			<td>Scinco</td>
			<td>S-3100</td>
			<td></td>
		</tr>
		<tr>
			<td>Xenon arc lamp</td>
			<td>Thorlabs</td>
			<td>SLS205</td>
			<td>Fiber adapter was removed</td>
		</tr>
	</tbody>
</table>

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.

Upon irradiation of 1 in an NMR tube with 436 nm light (Z:E = 54:46 in the initial state), the proportion of 1-E increases due to the dominant Z-to-E isomerization of the hydrazone C=N bond (Figure 1). The isomeric ratio can be readily obtained from the relative signal intensities of distinct isomers in the 1H NMR spectrum (Figure 2). After 5 days of irradiation at 436 nm, the sampl...

Watch this Scientific Journal Video about Determination of the Photoisomerization Quantum Yield of a Hydrazone Photoswitch at JoVE.com

Determination of the Photoisomerization Quantum Yield of a Hydrazone Photoswitch

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 ...

Here we explain a set of protocols for precise measurement of the photoisomerization quantum yield of a photochromic hydrazone as a model photoswitching molecule. The methods introduced here can be applied to other families of bi-stable photoswitches. Protocols for photoswitches with different for photo-physical properties and their selection guidelines are provided in the supplementary information.To begin, place the NMR sample at the distance of one centimeter in front of a xenon arc lamp equipped with a 436-nanometer band pass filter and start irradiation. Record a proton NMR spectrum every day till there is no exchange in the spectra as switch one reaches PSS. For another NMR sample, irradiate the solution using a 340-nanometer band pass filter and record the NMR spectrum as described previously.Open FID files of the NMR spectra at the PSSs with NMR processing software. Integrate a distinctive set of peaks of the distinct isomers and calculate the isomeric ratios. Place the prepared sample at the distance of one centimeter in front of a xenon arc lamp equipped with a 436-nanometer band pass filter and start irradiation.Measure the UV-visible absorption spectrum every two hours until there is no change in the spectra as switch one reaches PSS. For another sample, irradiate the solution using a 340-nanometer band pass filter and measure the UV visible spectrum at PSS in the same way. Deduce the absorbance spectra of the pure 1-Z and 1-E isomers and calculate their molar attenuation coefficients at all wavelengths as described in the text.Heat the silicon oil filled in a heating bath circulator to 131 degrees Celsius and check whether the temperature of the bath is stabilized. Submerge two NMR sample tubes in the heating bath. After one hour of heating, transfer the NMR tubes quickly to a dry ice bath to pause the thermal relaxation caused by latent heat.Thaw the NMR samples at room temperature and ensure dimethyl sulfoxide is defrosted. Then, record the proton NMR spectra of the samples. Again perform the heating and thawing process and record the proton NMR spectra of the samples until there is no change in the proton NMR spectra as switch one reaches thermodynamic equilibrium.Open FID files of the NMR spectra obtained in the course of heating and calculate the concentration of 1-E based on the total sample concentration and the isomeric ratio. Then, plot the averaged concentration of 1-E as a function of the heating time. Perform an exponential fit to the data to obtain the rate constant of thermal relaxation, K, using the equation as described in the text.Plot natural log of K versus reciprocal of T.Perform a linear fit according to the Arrhenius equation as described in the text to extrapolate the rate constant at room temperature and calculate the thermal half-life of 1-E at room temperature using the equation as described in the text. In a 20-milliliter glass vial containing 29.48 milligrams of potassium ferrioxalate trihydrate, add eight milliliters of deionized water. Add one milliliter of 0.5 molar aqueous sulfuric acid to the ferrioxalate solution and dilute to 10 milliliters with deionized water to prepare a 0.006 molar ferrioxalate in 0.05 molar aqueous sulfuric acid solution.In another 20-milliliter glass vial containing 10 milligrams of 1, 10-phenanthroline and 1.356 grams of anhydrous sodium acetate, add 10 milliliters of 0.1 molar aqueous sulfuric acid to make a buffered 0.1%phenanthroline solution. Measure the UV-visible absorption spectrum of the ferrioxalate solution. Determine the fraction of absorbed light at 340 and 436 nanometers using the absorbances of the ferrioxalate solution as described in the text.Place the quartz cuvette containing the ferrioxalate solution one centimeter in front of the xenon arc lamp equipped with a 436-nanometer band pass filter. Start irradiation to the sample for 90 seconds. After irradiation, add 0.35 milliliters of the phenanthroline solution and a magnetic bar into the cuvette, followed by stirring for one hour in the dark to form a ferroin complex.Prepare a quartz cuvette containing two milliliters of non-irradiated ferrioxalate solution and 0.35 milliliters of the phenanthroline solution as a non-irradiated sample. Measure the UV-visible absorption difference between the non-irradiated and irradiated samples. Repeat the procedure for sample preparation and measurement of the UV-visible absorption spectrum described previously with a 340-nanometer band pass filter.Calculate the molar photon flux arriving at the cuvette using this equation. Place the prepared sample one centimeter in front of the xenon arc lamp equipped with a 436-nanometer band pass filter and start irradiation. Measure the UV-visible absorption spectrum with different intervals until there is no change in the spectra as switch one reaches PSS.Once reaching PSS, recover the cuvette from the UV-Vis spectrophotometer and irradiate the solution using a 340-nanometer band pass filter. Measure UV-visible absorption spectrum as described previously. From the obtained UV-Vis absorption spectra, calculate the values of photokinetic factor Ft using the observed absorbances at the irradiation wavelengths.Calculate the unidirectional quantum yields for Z to E and E to Z photoisomerization processes. Upon irradiation at 436 nanometers, the proportion of 1-E increases due to the dominant Z to E isomerization of the hydrazone CN double bond. The isomeric ratio was obtained from the relative signal intensities of distinct isomers in the 1H NMR spectrum.At 436 nanometers, the sample shows 92%of 1-E, while at 340 nanometers, 82%of 1-Z was found. The ismomeric ratios and UV-Vis absorption spectra at PSS are used to deduce the UV-Vis spectra of the pure 1-Z and 1-E isomers. These spectra of the pure isomers suggest that the incomplete photoisomerization is attributed to the reverse photochemical process.For determining the photoisomerization quantum yield, measurement of the E to Z thermal relaxation rate and the effective molar photon flux is required. The rate constant of thermal relaxation extrapolated from the Arrhenius plot was very small at room temperature, and thus, the effect of thermal relaxation in the photoisomerization process could be ignored. The effective molar photon flux arriving at the sample was obtained from ferrioxalate actinometry and the pseudo-quantum yield of photoisomerization at the irradiation wavelength can be calculated.Finally, the unidirectional quantum yields for Z to E and E to Z photoisomerization processes can be calculated from the pseudo-quantum yields. For determination of the photoisomerization quantum yield, it is essential to obtain precise values of the thermal relaxation rate at room temperature and the effective molar photon flux. For those who deal with bi-stable photoswitches other than hydrazones, it is important to use the proper integration method for the photokinetic factor, which is explained in the supplementary information.

determination-photoisomerization-quantum-yield-hydrazone

Research

JoVE Journal

Chemistry

Determination of the Gas-phase Acidities of Oligopeptides

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue located at or near the N-terminus of a helix is often more acidic than that at or near the C-terminus 1-6. Although extensive experimental studies on the acid-base properties of peptides have been carried out in the condensed phase, in particular in aqueous solutions 6-8, the results are often complicated by solvent effects 7. In fact, most of the active sites in proteins are located near the interior region where solvent effects have been minimized 9,10. In order to understand intrinsic acid-base properties of peptides and proteins, it is important to perform the studies in a solvent-free environment.
We present a method to measure the acidities of oligopeptides in the gas-phase. We use a cysteine-containing oligopeptide, Ala3CysNH2 (A3CH), as the model compound. The measurements are based on the well-established extended Cooks kinetic method (Figure 1) 11-16. The experiments are carried out using a triple-quadrupole mass spectrometer interfaced with an electrospray ionization (ESI) ion source (Figure 2). For each peptide sample, several reference acids are selected. The reference acids are structurally similar organic compounds with known gas-phase acidities. A solution of the mixture of the peptide and a reference acid is introduced into the mass spectrometer, and a gas-phase proton-bound anionic cluster of peptide-reference acid is formed. The proton-bound cluster is mass isolated and subsequently fragmented via collision-induced dissociation (CID) experiments. The resulting fragment ion abundances are analyzed using a relationship between the acidities and the cluster ion dissociation kinetics. The gas-phase acidity of the peptide is then obtained by linear regression of the thermo-kinetic plots 17,18.
The method can be applied to a variety of molecular systems, including organic compounds, amino acids and their derivatives, oligonucleotides, and oligopeptides. By comparing the gas-phase acidities measured experimentally with those values calculated for different conformers, conformational effects on the acidities can be evaluated.

The determination of the gas-phase acidities of cysteine-containing oligopeptides is described. The experiments are performed using a triple quadrupole mass spectrometer. The relative acidities of the peptides are measured using collision-induced dissociation experiments, and the quantitative acidities are determined using the extended Cooks kinetic method.

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue ...

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

Synthesis of Cd-free InP/ZnS Quantum Dots Suitable for Biomedical Applications

Fluorescent nanocrystals, specifically quantum dots, have been a useful tool for many biomedical applications. For successful use in biological systems, quantum dots should be highly fluorescent and small/monodisperse in size. While commonly used cadmium-based quantum dots possess these qualities, they are potentially toxic due to the possible release of Cd2+ ions through nanoparticle degradation. Indium-based quantum dots, specifically InP/ZnS, have recently been explored as a viable alternative to cadmium-based quantum dots due to their relatively similar fluorescence characteristics and size. The synthesis presented here uses standard hot-injection techniques for effective nanoparticle growth; however, nanoparticle properties such as size, emission wavelength, and emission intensity can drastically change due to small changes in the reaction conditions. Therefore, reaction conditions such temperature, reaction duration, and precursor concentration should be maintained precisely to yield reproducible products. Because quantum dots are not inherently soluble in aqueous solutions, they must also undergo surface modification to impart solubility in water. In this protocol, an amphiphilic polymer is used to interact with both hydrophobic ligands on the quantum dot surface and bulk solvent water molecules. Here, a detailed protocol is provided for the synthesis of highly fluorescent InP/ZnS quantum dots that are suitable for use in biomedical applications.

In this protocol, the synthesis of Cd-free InP/ZnS quantum dots (QDs) is detailed. InP-based QDs are gaining popularity due to the toxicity of Cd2+ ions that may be released through nanoparticle degradation. After synthesis, QDs are solubilized in water using an amphiphilic polymer for use in biomedical applications.

Fluorescent nanocrystals, specifically quantum dots, have been a useful tool for many biomedical applications. For successful use in biological systems, ...

Determination of Carbonyl Functional Groups in Bio-oils by Potentiometric Titration: The Faix Method

Carbonyl compounds present in bio-oils are known to be responsible for bio-oil property changes upon storage and during upgrading. Specifically, carbonyls cause an increase in viscosity (often referred to as &#39;aging&#39;) during storage of bio-oils. As such, carbonyl content has previously been used as a method of tracking bio-oil aging and condensation reactions with less variability than viscosity measurements. Additionally, carbonyls are also responsible for coke formation in bio-oil upgrading processes. Given the importance of carbonyls in bio-oils, accurate analytical methods for their quantification are very important for the bio-oil community. Potentiometric titration methods based on carbonyl oximation have long been used for the determination of carbonyl content in pyrolysis bio-oils. Here, we present a modification of the traditional carbonyl oximation procedures that results in less reaction time, smaller sample size, higher precision, and more accurate carbonyl determinations. While traditional carbonyl oximation methods occur at room temperature, the Faix method presented here occurs at an elevated temperature of 80 &#176;C.

Here we present a potentiometric titration technique for accurately quantifying carbonyl compounds in pyrolysis bio-oils.

Carbonyl compounds present in bio-oils are known to be responsible for bio-oil property changes upon storage and during upgrading. Specifically, carbonyls ...

Fabrication and Testing of Photonic Thermometers

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being leveraged for developing sophisticated photonic sensors. Silicon photonic sensors seek to exploit the strong confinement of light in nano-waveguides to transduce changes in physical state to changes in resonance frequency. In the case of thermometry, the thermo-optic coefficient, i.e., changes in refractive index due to temperature, causes the resonant frequency of the photonic device such as a Bragg grating to drift with temperature. We are developing a suite of photonic devices that leverage recent advances in telecom compatible light sources to fabricate cost-effective photonic temperature sensors, which can be deployed in a wide variety of settings ranging from controlled laboratory conditions, to the noisy environment of a factory floor or a residence. In this manuscript, we detail our protocol for the fabrication and testing of photonic thermometers.

We describe the process of fabrication and testing of photonic thermometers.

In recent years, a push for developing novel silicon photonic devices for telecommunications has generated a vast knowledge base that is now being ...

Determination of Thermodynamic Properties of Alkaline Earth-liquid Metal Alloys Using the Electromotive Force Technique

A novel electrochemical cell based on a CaF2 solid-state electrolyte has been developed to measure the electromotive force (emf) of binary alkaline earth-liquid metal alloys as functions of both composition and temperature in order to acquire thermodynamic data. The cell consists of a chemically stable solid-state CaF2-AF2 electrolyte (where A is the alkaline-earth element such as Ca, Sr, or Ba), with binary A-B alloy (where B is the liquid metal such as Bi or Sb) working electrodes, and a pure A metal reference electrode. Emf data are collected over a temperature range of 723 K to 1,123 K in 25 K increments for multiple alloy compositions per experiment and the results are analyzed to yield activity values, phase transition temperatures, and partial molar entropies/enthalpies for each composition.

This protocol describes the measurement of the electromotive force of alkaline-earth elements in liquid metal alloys at high temperatures (723-1,123 K) to determine their thermodynamic properties, including activity, partial molar entropy, partial molar enthalpy, and phase transition temperatures, over a wide composition range.

A novel electrochemical cell based on a CaF2 solid-state electrolyte has been developed to measure the electromotive force (emf) of binary alkaline ...

Probing the Structure and Dynamics of Interfacial Water with Scanning Tunneling Microscopy and Spectroscopy

Water/solid interfaces are ubiquitous and play a key role in many environmental, biophysical, and technological processes. Resolving the internal structure and probing the hydrogen-bond (H-bond) dynamics of the water molecules adsorbed on solid surfaces are fundamental issues of water science, which remains a great challenge owing to the light mass and small size of hydrogen. Scanning tunneling microscopy (STM) is a promising tool for attacking these problems, thanks to its capabilities of sub-&#197;ngstr&#246;m spatial resolution, single-bond vibrational sensitivity, and atomic/molecular manipulation. The designed experimental system consists of a Cl-terminated tip and a sample fabricated by dosing water molecules in situ onto the Au(111)-supported NaCl(001) surfaces. The insulating NaCl films electronically decouple the water from the metal substrates, so the intrinsic frontier orbitals of water molecules are preserved. The Cl-tip facilitates the manipulation of the single water molecules, as well as gating the orbitals of water to the proximity of Fermi level (EF) via tip-water coupling. This paper outlines the detailed methods of submolecular resolution imaging, molecular/atomic manipulation, and single-bond vibrational spectroscopy of interfacial water. These studies open up a new route for investigating the H-bonded systems at the atomic scale.

Here, we present a protocol to investigate the structure and dynamics of interfacial water at the atomic scale, in terms of submolecular resolution imaging, molecular manipulation, and single-bond vibrational spectroscopy.

Water/solid interfaces are ubiquitous and play a key role in many environmental, biophysical, and technological processes. Resolving the internal ...

Synthesis and Structure Determination of &#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus highly important to avoid undesired disulfide bond formation during peptide synthesis, because it may result in a completely different peptide structure, and consequently altered bioactivity. However, the correct formation of multiple disulfide bonds in a peptide is difficult to obtain by using standard self-folding methods such as conventional buffer oxidation protocols, because several disulfide connectivities can be formed. This protocol represents an advanced strategy required for the targeted synthesis of multiple disulfide-bridged peptides which cannot be synthesized via buffer oxidation in high quality and quantity. The study demonstrates the application of a distinct protecting group strategy for the synthesis of all possible 3-disulfide-bonded peptide isomers of &#181;-conotoxin PIIIA in a targeted way. The peptides are prepared by Fmoc-based solid phase peptide synthesis using a protecting group strategy for defined disulfide bond formation. The respective pairs of cysteines are protected with trityl (Trt), acetamidomethyl (Acm), and tert-butyl (tBu) protecting groups to make sure that during every oxidation step only the required cysteines are deprotected and linked. In addition to the targeted synthesis, a combination of several analytical methods is used to clarify the correct folding and generation of the desired peptide structures. The comparison of the different 3-disulfide-bonded isomers indicates the importance of accurate determination and knowledge of the disulfide connectivity for the calculation of the three-dimensional structure and for interpretation of the biological activity of the peptide isomers. The analytical characterization includes the exact disulfide bond elucidation via tandem mass spectrometry (MS/MS) analysis which is performed with partially reduced and alkylated derivatives of the intact peptide isomer produced by an adapted protocol. Furthermore, the peptide structures are determined using 2D nuclear magnetic resonance (NMR) experiments and the knowledge obtained from MS/MS analysis.

Synthesis and Structure Determination of &amp;#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Cysteine-rich peptides fold into distinct three-dimensional structures depending on their disulfide connectivity. Targeted synthesis of individual disulfide isomers is required when buffer oxidation does not lead to the desired disulfide connectivity. The protocol deals with the selective synthesis of 3-disulfide-bonded peptides and their structural analysis using NMR and MS/MS studies.

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus ...

Extraction of Lignin with High &#946;-O-4 Content by Mild Ethanol Extraction and Its Effect on the Depolymerization Yield

Lignin valorization strategies are a key factor for achieving more economically competitive biorefineries based on lignocellulosic biomass. Most of the emerging elegant procedures to obtain specific aromatic products rely on the lignin substrate having a high content of the readily cleavable &#946;-O-4 linkage as present in the native lignin structure. This provides a miss-match with typical technical lignins that are highly degraded and therefore are low in &#946;-O-4 linkages. Therefore, the extraction yields, and the quality of the obtained lignin are of utmost importance to access new lignin valorization pathways. In this manuscript, a simple protocol is presented to obtain lignins with high &#946;-O-4 content by relatively mild ethanol extraction that can be applied to different lignocellulose sources. Furthermore, analysis procedures to determine the quality of the lignins are presented together with a depolymerization protocol that yields specific phenolic 2-arylmethyl-1,3-dioxolanes, which can be used to evaluate the obtained lignins. The presented results demonstrate the link between lignin quality and potential for the lignins to be depolymerized into specific monomeric aromatic chemicals. Overall, the extraction and depolymerization demonstrates a trade-off between the lignin extraction yield and the retention of the native aryl-ether structure and thus the potential of the lignin to be used as the substrate for the production of chemicals for higher-value applications.

Extraction of Lignin with High &amp;#946;-O-4 Content by Mild Ethanol Extraction and Its Effect on the Depolymerization Yield

Here, we present a protocol to perform ethanol extraction of lignin from several biomass sources. The effect of the extraction conditions on the lignin yield and &#946;-O-4 content are presented. Selective depolymerization is performed on the obtained lignins to obtain high aromatic monomer products.

Lignin valorization strategies are a key factor for achieving more economically competitive biorefineries based on lignocellulosic biomass. Most of the ...

Synthesis of In37P20(O2CR)51 Clusters and Their Conversion to InP Quantum Dots

This text presents a method for the synthesis of In37P20(O2C14H27)51 clusters and their conversion to indium phosphide quantum dots. The In37P20(O2CR)51 clusters have been observed as intermediates in the synthesis of InP quantum dots from molecular precursors (In(O2CR)3, HO2CR, and P(SiMe3)3) and may be isolated as a pure reagent for subsequent study and use as a single-source precursor. These clusters readily convert to crystalline and relatively monodisperse samples of quasi-spherical InP quantum dots when subjected to thermolysis conditions in the absence of additional precursors above 200 &#176;C. The optical properties, morphology, and structure of both the clusters and quantum dots are confirmed using UV-Vis spectroscopy, photoluminescence spectroscopy, transmission electron microscopy, and powder X-ray diffraction. The molecular symmetry of the clusters is additionally confirmed by solution-phase 31P NMR spectroscopy. This protocol demonstrates the preparation and isolation of atomically-precise InP clusters, and their reliable and scalable conversion to InP QDs.

Synthesis of In37P20O2CR51 Clusters and Their Conversion to InP Quantum Dots

A protocol for the synthesis of In37P20(O2C14H27)51 clusters and their conversion to indium phosphide quantum dots is presented.

This text presents a method for the synthesis of In37P20(O2C14H27)51 clusters and their conversion to indium phosphide quantum dots. The In37P20(O2CR)51 ...