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 day until there is no change in the spectra as switch 1 reaches PSS. After reaching PSS, keep the NMR tube in the dark at room temperature and record the 1H NMR spectrum after 12 h to monitor the progress of thermal relaxation. 
	NOTE: Switch 1 does not show any appreciable change in the 1H NMR spectrum at room temperature due to its bistable nature.</li>
	<li>For the other NMR tube, repeat step 1.2 with a 340 nm bandpass filter to obtain a 1H NMR spectrum at the PSS under 340 nm irradiation.</li>
	<li>Open fid files of the NMR spectra at the PSSs with NMR processing software. Integrate a distinctive set of peaks (H1: C2 proton of quinoline, H2: proton in para-position to the hydrazone group, H3: CH3 of ethyl ester) of the distinct isomers and calculate the isomeric ratio (Figure 2). 
	NOTE: Compositions ([1-Z]:[1-E] ratio) under 436 nm and 340 nm irradiation are 8:92 and 82:18, respectively.</li>
</ol>
2. UV-Vis absorption spectroscopy at PSS
<ol>
	<li>In a glass vial containing 12.6 mg (0.03 mmol) of 1, add 2 mL of spectroscopy grade DMSO. Take 100 &#181;L of the solution and dilute with 1400 &#181;L of DMSO to make 1 mM solution of 1. Transfer 20 &#181;L of 1 mM solution of 1 to a quartz cuvette with 1.0 cm optical path length and dilute with 1980 &#181;L of DMSO to make a 10 &#181;M solution of 1. Seal the cuvette with a PTFE stopper and keep the sample in the dark.</li>
	<li>Prepare another quartz cuvette containing 2 mL of DMSO as a blank sample. Measure the UV-Vis spectrum of the blank sample for baseline correction.</li>
	<li>Place the sample from step 2.1 1 cm in front of a Xenon arc lamp equipped with a 436 nm bandpass filter. Start irradiation to the sample and measure the UV-Vis spectrum every 2 h until there is no change in the spectra as 1 reaches PSS (Figure 3). 
	NOTE: The time taken to reach PSS for the UV-Vis spectroscopy sample is much shorter than for the NMR sample with a higher concentration.</li>
	<li>Repeat step 2.3 with a 340 nm bandpass filter to obtain the UV-Vis spectrum at the PSS under 340 nm irradiation.</li>
	<li>Deduce absorbance spectra of the pure 1-Z and 1-E using Eq (1) and Eq (2) (Figure 4). 
	<img alt="Equation 1" src="/files/ftp_upload/63398/63398eq01.jpg" /> (1) 
	<img alt="Equation 2" src="/files/ftp_upload/63398/63398eq02.jpg" /> (2) 
	Where R436 = the ratio of 1-Z at the PSS under 436 nm irradiation; R340 = the ratio of 1-Z at the PSS under 340 nm irradiation; A436 = the absorbance of 1 in DMSO at the PSS under 436 nm irradiation; A340 = the absorbance of 1 in DMSO at the PSS under 340 nm irradiation.</li>
	<li>Calculate the molar attenuation coefficients of pure 1-Z and 1-E at all wavelengths by dividing the observed absorbance by the sample concentration (10 &#181;M) and the optical path length (1 cm).</li>
</ol>
3. Kinetic studies on thermal relaxation
<ol>
	<li>Heat the silicon oil filled in a heating bath circulator to the desired temperature (131 &#176;C) and check if the temperature of the bath is stabilized. Submerge two NMR samples from step 1.2 in the heating bath. 
	NOTE: The temperature and duration of heating are adjusted depending on the relaxation rate.</li>
	<li>After 1 h of heating, transfer the NMR tubes quickly to a dry ice bath to pause the thermal relaxation caused by latent heat (Figure 5). 
	NOTE: Inaccurate heating temperature or time may lead to serious error in the estimation of the rate constant.</li>
	<li>Thaw the NMR samples obtained from step 3.2 at room temperature and ensure DMSO is defrosted. Record the 1H NMR spectra of the samples.</li>
	<li>Repeat steps 3.1-3.3 until there is no change in the 1H NMR spectra as 1 reaches thermodynamic equilibrium.</li>
	<li>Repeat steps 3.1-3.4 at different temperatures (134, 137, 140, and 143 &#176;C).</li>
	<li>Open fid files of the NMR spectra obtained in the course of heating at 131 &#176;C. Calculate the averaged isomeric ratios as described in step 1.4. Calculate the concentration of 1-E (metastable isomer) based on the total sample concentration (10 mM) and the isomeric ratio.</li>
	<li>Plot the averaged concentration of 1-E&#160;(CE) as a function of the heating time. Perform an exponential fit to the data to obtain the rate constant of thermal relaxation using Eq (3)15,22 (Figure 6). 
	<img alt="Equation 3" src="/files/ftp_upload/63398/63398eq03.jpg" /> (3) 
	Where <img alt="Equation 4" src="/files/ftp_upload/63398/63398eq04.jpg" /> (M) = the concentration of 1-E at the initial state; <img alt="Equation 5" src="/files/ftp_upload/63398/63398eq05.jpg" /> (M) = the concentration of 1-E at the thermodynamic equilibrium at a specific temperature; k&#160;(s-1) = the rate constant of thermal relaxation at a specific temperature; t (s) = the heating time.</li>
	<li>Repeat steps 3.6 to 3.7 using the data obtained at different temperatures.</li>
	<li>Plot ln(k) versus <img alt="Equation 6" src="/files/ftp_upload/63398/63398eq06.jpg" /> and perform a linear fit according to the Arrhenius equation (Eq (4)) to extrapolate the rate constant at room temperature (Figure 7). 
	<img alt="Equation 7" src="/files/ftp_upload/63398/63398eq07.jpg" /> (4) 
	Where A = the pre-exponential factor; Ea (J&#183;mol-1) = the activation energy for thermal relaxation; R = the ideal gas constant (8.3145 J&#183;mol-1K-1); T (K) = the absolute temperature.</li>
	<li>Calculate the thermal half-life of 1-E at room temperature using Eq (5). 
	<img alt="Equation 8" src="/files/ftp_upload/63398/63398eq08.jpg" /> (5) 
	Where &#964;1/2 (s) = the thermal half-life of 1-E at room temperature; k (s-1) = the rate constant of thermal relaxation at room temperature obtained from step 3.9.</li>
	<li>If the rate constant of thermal relaxation is estimated only at a single temperature, calculate the rate constant at room temperature using the following rearranged Eyring equation (Eq (6))18,23. 
	<img alt="Equation 9" src="/files/ftp_upload/63398/63398eq09.jpg" /> (6) 
	<img alt="Equation 10" src="/files/ftp_upload/63398/63398eq10.jpg" /> (7) 
	Where <img alt="Equation 11" src="/files/ftp_upload/63398/63398eq11.jpg" /> (J&#183;mol-1) = the Gibbs energy of activation for thermal relaxation; k1 (s-1) = the rate constant of thermal relaxation estimated at the elevated temperature; k2 (s-1) = the rate constant of thermal relaxation at room temperature (298.15 K); T1 (K) = the absolute temperature at which k1 is obtained; (K) = room temperature (298.15 K).</li>
</ol>
4. Ferrioxalate actinometry
NOTE: All procedures for ferrioxalate actinometry must be performed in the dark or &#62;600 nm light to prevent the influence of ambient light.
<ol>
	<li>In a 20 mL glass vial containing 29.48 mg (0.06 mmol) of potassium ferrioxalate trihydrate, add 8 mL of deionized water. Add 1 mL of 0.5 M aqueous H2SO4 to the ferrioxalate solution and dilute to 10 mL with deionized water to prepare a 0.006 M ferrioxalate in 0.05 M aqueous H2SO4 solution.</li>
	<li>In another 20 mL glass vial containing 10 mg of 1,10-phenanthroline and 1.356 g of anhydrous sodium acetate, add 10 mL of 0.5 M aqueous H2SO4 to make a buffered 0.1% (w/v) phenanthroline solution.</li>
	<li>Transfer 2 mL of the 0.006 M ferrioxalate solution from step 4.1 to a quartz cuvette with 1.0 cm optical path length. Seal the cuvette with a PTFE stopper and keep the sample in the dark.</li>
	<li>Prepare another quartz cuvette containing 2 mL of 0.05 M aqueous H2SO4 as a blank sample. Measure the UV-Vis absorbance of the blank sample for baseline correction.</li>
	<li>Measure the UV-Vis absorbance of the 0.006 M ferrioxalate solution. Determine the fraction of light absorbed using the absorbances of the 0.006 M ferrioxalate solution at 340 and 436 nm and Eq (8) (Figure 8). 
	<img alt="Equation 12" src="/files/ftp_upload/63398/63398eq12.jpg" /> (8) 
	Where f = the fraction of light absorbed by 0.006 M ferrioxalate solution; A&#955; = the absorbance of 0.006 M ferrioxalate solution at wavelength &#955;.</li>
	<li>Prepare two quartz cuvettes with 1.0 cm optical path length and add 2 mL of the 0.006 M ferrioxalate solution.</li>
	<li>Place one of the samples from step 4.6 1 cm in front of the Xenon arc lamp equipped with a 436 nm bandpass filter. Keep the other sample in the dark. Start irradiation to the sample for 90 s. After irradiation, add 0.35 mL of the buffered 0.1% phenanthroline solution and a magnetic bar to both cuvettes followed by stirring for 1 h in the dark to form a [Fe(phen)3]2+ complex. 
	NOTE: Ferrioxalate is photochemically reduced to Fe2+, followed by the nearly quantitative formation of tris-1,10-phenanthroline iron (II) complex.</li>
	<li>Measure the UV-Vis absorption spectrum of the non-irradiated sample from step 4.6 for baseline correction.</li>
	<li>Measure the UV-Vis absorption spectrum of the irradiated sample from step 4.7.</li>
	<li>Repeat steps 4.6-4.9 with a 340 nm bandpass filter (Figure 9). 
	NOTE: Once the ferrioxalate sample is exposed to light, the sample cannot be reused.</li>
	<li>Calculate the molar photon flux arriving at the cuvette using Eq (9). 
	<img alt="Equation 13" src="/files/ftp_upload/63398/63398eq13.jpg" /> (9) 
	Where I (mol&#183;s-1) = the molar photon flux arriving at the cuvette; &#916;A510 = the difference in absorbance at 510 nm between the non-irradiated and irradiated samples; V = the total volume of solution (2.35 mL); &#949;510 = the molar attenuation coefficient of [Fe(phen)3]2+ complex (11100 M-1cm-1)24;&#160;I = the optical path length of quartz cuvette (1.0 cm); t = irradiation time (90 s); f = the absorbed fraction of light obtained from step 4.5; &#934;Fe3+&#160;= the quantum yield of the photoreduction of Fe3+ to Fe2+ (1.22 for 340 nm, 1.11 for 436 nm)25.</li>
</ol>
5. Determination of the photoisomerization quantum yield
<ol>
	<li>Prepare a quartz cuvette with 1.0 cm optical path length containing 2 mL of DMSO as the blank sample. Measure the UV-Vis absorbance of the blank sample for baseline correction.</li>
	<li>Prepare a quartz cuvette with 1.0 cm optical path length containing 2 mL of 10 &#181;M solution of 1 in DMSO obtained from step 2.4 (Z-enriched). Seal the cuvettes with a PTFE stopper.</li>
	<li>Place the sample from step 5.2 1 cm in front of the Xenon arc lamp equipped with a 436 nm bandpass filter. Start irradiation at 436 nm to the sample and measure UV-Vis absorption spectrum with different intervals until there is no change in the spectra as 1 reaches PSS (Figure 10). 
	NOTE: The irradiation setup must be exactly the same as that used for the molar photon flux measurement. The irradiation interval should be adjusted based on the rate of photoisomerization. Generally, 15-20 data points before reaching PSS are suitable.</li>
	<li>Prepare a quartz cuvette with 1.0 cm optical path length containing 2 mL of 10 &#181;M solution of 1 in DMSO obtained from step 2.3 (E-enriched). Seal the cuvettes with a PTFE stopper.</li>
	<li>Replace the 436 nm bandpass filter with the 340 nm bandpass filter and repeat step 5.3 for the sample obtained from step 5.4.</li>
	<li>Calculate the photokinetic factor F(t) using the observed absorbances from step 5.3 and Eq (10)26. 
	<img alt="Equation 14" src="/files/ftp_upload/63398/63398eq14.jpg" /> (10) 
	Where Airr,t = the absorbance at the irradiation wavelength at time t.</li>
	<li>Calculate the pseudo quantum yield Q using the photokinetic factor values obtained from step 5.6 and Eq (11)27. 
	<img alt="Equation 15" src="/files/ftp_upload/63398/63398eq15.jpg" /> (11) 
	Where Q (M-1cm-1) = the pseudo quantum yield defined as ; <img alt="Equation 16" src="/files/ftp_upload/63398/63398eq16.jpg" />; V(L) = the volume of sample; I (mol&#183;s-1) = the molar photon flux arriving at the cuvette; l (cm) = the optical path length; t1, t2 (s) = the two consecutive time points of irradiation; F(t1), F(t2) = the photokinetic factors at time t1 and t2, respectively; Aobs,t1, Aobs,t2, Aobs,&#8734; = the absorbances at specific wavelength at time , t1, and t2 at PSS, respectively. 
	NOTE: Using absorbances at &#955;max of 1-Z is recommended for accuracy.</li>
	<li>Calculate the averaged value of the pseudo quantum yield using the first ten data points.</li>
	<li>Calculate the unidirectional quantum yields for Z-to-E and E-to-Z photoisomerizations using Eq (12) and Eq (13). 
	<img alt="Equation 17" src="/files/ftp_upload/63398/63398eq17.jpg" /> (12) 
	<img alt="Equation 18" src="/files/ftp_upload/63398/63398eq18.jpg" /> (13) 
	Where &#934;Z&#8594;E,&#160;&#934;E&#8594;Z = the unidirectional quantum yields for Z-to-E and E-to-Z photoisomerization processes, respectively; <img alt="Equation 19" src="/files/ftp_upload/63398/63398eq19.jpg" />, <img alt="Equation 20" src="/files/ftp_upload/63398/63398eq20.jpg" /> (M-1cm-1) = the molar attenuation coefficients of 1-Z and 1-E at the irradiation wavelength; <img alt="Equation 21" src="/files/ftp_upload/63398/63398eq21.jpg" />, <img alt="Equation 22" src="/files/ftp_upload/63398/63398eq22.jpg" /> (M) = the concentrations of 1-Z and 1-E at PSS, respectively; Ctot (M) = the total concentration of 1.</li>
	<li>Repeat steps 5.6-5.9 using the data obtained from step 5.5 for calculation of the unidirectional photoisomerization quantum yields under irradiation at 340 nm.</li>
</ol>

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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>

The authors declare no conflicts of interest.

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 ...

determination-photoisomerization-quantum-yield-hydrazone

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Chemistry

3.3K Views.  Chung-Ang University. 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. 

Video: Determination of the Photoisomerization Quantum Yield of a Hydrazone Photoswitch

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 ...

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

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 ...

Disentangling High Strength Copolymer Aramid Fibers to Enable the Determination of Their Mechanical Properties

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to diversify the fiber choices in the United States body armor market, copolymer fibers based on the combination of 5-amino-2-(p-aminophenyl) benzimidazole (PBIA) and the more conventional PPTA were introduced. Little is known regarding the long-term stability of these fibers, but as condensation polymers, they are expected to have potential sensitivity to moisture and humidity. Therefore, characterizing the strength of the materials and understanding their vulnerability to environmental conditions is important for evaluating their use lifetime in safety applications. Ballistic resistance and other critical structural properties of these fibers are predicated on their strength. To accurately determine the strength of the individual fibers, it is necessary to disentangle them from the yarn without introducing any damage. Three aramid-based copolymer fibers were selected for the study. The fibers were washed with acetone followed by methanol to remove an organic coating that held the individual fibers in each yarn bundle together. This coating makes it difficult to separate single fibers from the yarn bundle for mechanical testing without damaging the fibers and affecting their strength. After washing, fourier transform infrared (FTIR) spectroscopy was performed on both washed and unwashed samples and the results were compared. This experiment has shown that there are no significant variations in the spectra of poly(p-phenylene-benzimidazole-terephthalamide-co-p-phenylene terephthalamide) (PBIA-co-PPTA1) and PBIA-co-PPTA3 after washing, and only a small variation in intensity for PBIA. This indicates that the acetone and methanol rinses are not adversely affecting the fibers and causing chemical degradation. Additionally, single fiber tensile testing was performed on the washed fibers to characterize their initial tensile strength and strain to failure, and compare those to other reported values. Iterative procedural development was necessary to find a successful method for performing tensile testing on these fibers.

The primary goal of the study is to develop a protocol to prepare consistent specimens for accurate mechanical testing of high strength copolymer aramid fibers, by removing a coating and disentangling the individual fiber strands without introducing significant chemical or physical degradation.

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to ...

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.

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 ...

Determination of the Photoisomerization Quantum Yield of a Hydrazone Photoswitch

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