This work was supported by the U.S. Department of Energy under Contract No. DE-AC36-08GO28308 with the National Renewable Energy Laboratory. Funding provided by U.S. DOE Office of Energy Efficiency and Renewable Energy Bioenergy Technologies Office. The U.S. Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. Government purposes.

Caution: Please review all relevant material safety data sheets (MSDS) before beginning. Ethanol is flammable. All applicable chemical handling procedures should be followed, as well as all applicable waste disposable and handling procedures.
1. Reagent Solutions
<ol>
	<li>Prepare the hydroxylamine hydrochloride solution (Solution A): Add 7.7 g of hydroxylamine hydrochloride and 50 mL of deionized water to a 250 mL volumetric flask. When all solids have dissolved, dilute to mark with ethanol. This results in a 0.55 M hydroxylamine hydrochloride solution in 80% (v/v) ethanol.</li>
	<li>Prepare the triethanolamine solution (Solution B): Add 17.4 mL of triethanolamine to a 250 mL volumetric flask. Add 10 mL of water, and then dilute up to the mark with ethanol. 95% ethanol can also be used if the addition of water is skipped. This results in a 0.48 M triethanolamine solution in 96 % (v/v) ethanol</li>
	<li>Prepare the hydrochloric acid solution. Either purchase 0.1 N solution or prepare using 10 mL concentrated HCl and 1 L water.</li>
</ol>
2. Bio-oil Sampling and Handling
<ol>
	<li>Make sure the oil sample is at room temperature prior to withdrawing a sample. Thoroughly homogenize (mix by shaking vigorously for at least 1 minute, and visually inspect the sample to ensure it is homogenous. Some bio-oils may require longer shaking times) bio-oil to obtain a representative sample.</li>
	<li>For the bio-oil sample, use 100 to 150 mg of bio-oil.</li>
	<li>Minimize exposure to oxygen and heat to prevent sample degradation prior to analysis.</li>
</ol>
3. Analytical Procedure
<ol>
	<li>Standardization of the Base Solution
	<ol>
		<li>Dry Na2CO3 primary standard in an oven at 105 &#176;C overnight to ensure a dry sample. Allow the Na2CO3 to cool to room temperature before weighing.</li>
		<li>Weigh 100 to 150 mg of sodium carbonate to a titration vessel, record the actual weight, add a stir bar and add enough water to cover the pH electrode bulb and junction.</li>
		<li>Transfer the sample to a titration vessel, washing the reaction vial several times separately with ethanol and water in proportions to make a final 80% solution of ethanol/water. Titrate with the acid solution using an automatic titrator to the endpoint. Record the endpoint. The endpoint is defined as the inflection point on the titration curve.</li>
		<li>Repeat the process twice to obtain three points.</li>
		<li>Use the average value as the normality of the acid solution.</li>
	</ol>
	</li>
	<li>Preparation of Titration Blanks
	<ol>
		<li>For Blank A: add 0.5 mL dimethyl sulfoxide (DMSO) to a 5 mL vial with a spin vane.</li>
		<li>Add 2 mL hydroxylamine hydrochloride solution (solution A).</li>
		<li>Add 2 mL triethanolamine solution (solution B).</li>
		<li>Cap tightly, place in preheated (80 &#176;C) heater block or water bath and stir for 2 hours.</li>
		<li>Titrate with acid solution using an automatic titrator to the endpoint, and record endpoint.</li>
		<li>For Blank B: If mineral acid is suspected to be present in sample, add 0.5 mL DMSO to 5 mL vial with a spin vane.</li>
		<li>Add 2 mL triethanolamine solution (solution B).</li>
		<li>Cap tightly and stir at 80 &#176;C for 2 hours.</li>
		<li>Transfer the sample to a titration vessel, washing the reaction vial several times separately with ethanol and water in proportions to make a final 80% solution of ethanol/water. Titrate with the acid solution using an automatic titrator to the endpoint. Record the endpoint.</li>
		<li>Repeat process three times to obtain three points.</li>
	</ol>
	</li>
	<li>Validation of the Method Using a Known Carbonyl
	<ol>
		<li>Weigh ~ 100 mg of 4-(benzyloxy)benzaldehyde (4-BBA) to a 5 mL vial, record the actual weight, add a spin vane.</li>
		<li>Add 0.5 mL DMSO.</li>
		<li>Dissolve the sample in 2 mL hydroxylamine hydrochloride solution (solution A).</li>
		<li>Add 2 mL triethanolamine solution (solution B).</li>
		<li>Close lid tightly and stir at 80 &#176;C for 2 hours.</li>
		<li>Transfer the sample to a titration vessel, washing the reaction vial several times separately with ethanol and water in proportions to make a final 80% solution of ethanol/water. Titrate with the acid solution using an automatic titrator to the endpoint. Record the endpoint.</li>
		<li>Repeat the process three times, to obtain three points.</li>
	</ol>
	</li>
	<li>Analysis of Bio-oil Using the Method
	<ol>
		<li>Weigh close to 100 mg of the bio-oil to 5 mL vial, record the actual weight and add a spin vane.</li>
		<li>Add 0.5 mL DMSO.</li>
		<li>Dissolve the sample in 2 mL hydroxylamine hydrochloride solution (solution A).</li>
		<li>Add 2 mL triethanolamine solution (solution B).</li>
		<li>Close lid tightly and stir at 80 &#176;C for 2 hours.</li>
		<li>Transfer the sample to a titration vessel, washing the reaction vial several times separately with ethanol and water in proportions to make a final 80% solution of ethanol/water. Titrate with the acid solution using an automatic titrator to the endpoint. Record the endpoint.</li>
		<li>Repeat the process three times, to obtain three points.</li>
		<li>Blank C: If mineral acid is suspected to be present in sample, weigh close to 100 mg of the bio-oil into a 5 mL vial, record the weight and add a spin vane.</li>
		<li>Add 0.5 mL DMSO.</li>
		<li>Add 2 mL triethanolamine solution (solution B).</li>
		<li>Cap tightly and stir at 80 &#176;C for 2 hours.</li>
		<li>Transfer the sample to a titration vessel, washing the reaction vial several times separately with ethanol and water in proportions to make a final 80% solution of ethanol/water. Titrate with the acid solution using an automatic titrator to the endpoint. Record the endpoint.</li>
		<li>Repeat process three times to obtain three points.</li>
	</ol>
	</li>
</ol>
4. Calculations
<ol>
	<li>Standardization of the Base Solution
	<ol>
		<li>Calculate the concentration of the acid solution (mol/L) using the following equation. The weight of the dry sodium carbonate in grams is w1, the purity is written as a fraction (i.e., 99% is 0.99), and the endpoint is in mL. 
		<img alt="Equation 1" src="/files/ftp_upload/55165/55165eq1.jpg" />&#160;&#160;&#160;</li>
	</ol>
	</li>
	<li>Validation of the Method Using a Known Carbonyl
	<ol>
		<li>Calculate the concentration of 4BBA (mol/L) in the sample using the following equation. The weight of the 4BBA in grams is w2, the 4BBA purity is written as a fraction (i.e., 99% is 0.99), the concentration of the acid solution (mol/L) is [acid], the triethanolamine/hydroxylamine&#8226;HCl blank endpoint is EPBA (the average value of three blanks, in mL), and the endpoint is EP (in mL). 
		<img alt="Equation 2" src="/files/ftp_upload/55165/55165eq2.jpg" />&#160;&#160;&#160;</li>
	</ol>
	</li>
	<li>Analysis of Bio-oil
	<ol>
		<li>Calculate the concentration of carbonyls in bio-oils [CO] (mmol/g-bio oil) using the following equation. The weight of the bio-oil in grams is w3, the concentration of the acid solution (mol/L) is [acid], the triethanolamine/hydroxylamine&#8226;HCl blank endpoint is EPBA (the average value of three blanks in mL), and the endpoint is EP (in mL). 
		<img alt="Equation 3" src="/files/ftp_upload/55165/55165eq3.jpg" />&#160;&#160;&#160;</li>
	</ol>
	</li>
	<li>Acid Correction 
	NOTE: The presence of mineral acids or organic acids with pKa &#60; 2 can cause artificially low carbonyl values due to the reaction of the acid with triethanolamine.
	<ol>
		<li>If this is suspected, perform the blanks detailed in sections 3.2.6 and 3.4.8. The weight of bio-oil in the sample in grams is w3, EP is the endpoint of the sample and EPBA is the triethanolamine/hydroxylamine blank. EPBB is the endpoint of Blank B, EPBC is endpoint of Blank C and the weight (g) of oil used in Blank C is wBC: 
		<img alt="Equation 4" src="/files/ftp_upload/55165/55165eq4.jpg" />&#160;&#160;&#160; 
		For bio-oil samples that contain mostly acetic acid, this is an unnecessary step.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Oasmaa, A., Kuoppala, E., Solantausta, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+pyrolysis+of+forestry+residue.+2.+physicochemical+composition+of+product+liquid.">Fast pyrolysis of forestry residue. 2. physicochemical composition of product liquid.</a> Energy Fuels. 17 (2), 433-443 (2003).</li><li>Olarte, 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=Stabilization+of+Softwood-Derived+Pyrolysis+Oils+for+Continuous+Bio-oil+Hydroprocessing.">Stabilization of Softwood-Derived Pyrolysis Oils for Continuous Bio-oil Hydroprocessing.</a> Top. Catal. 59 (1), 55-64 (2016).</li><li>Nicolaides, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The chemical characterization of pyrolytic oils. , (1984).</li><li>Oasmaa, A., Korhonen, J., Kuoppala, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+approach+for+stability+measurement+of+wood-based+fast+pyrolysis+bio-oils.">An approach for stability measurement of wood-based fast pyrolysis bio-oils.</a> Energy Fuels. 25 (7), 3307-3313 (2011).</li><li>Chen, C. L., Lin, S. Y., Dence, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Lignin Chemistry. , 446-457 (1992).</li><li>Scholze, B., Hanser, C., Meier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+water-insoluble+fraction+from+fast+pyrolysis+liquids+(pyrolytic+lignin)+Part+II.+GPC,+carbonyl+groups,+and+13C-NMR.">Characterization of the water-insoluble fraction from fast pyrolysis liquids (pyrolytic lignin) Part II. GPC, carbonyl groups, and 13C-NMR.</a> J. Anal. Appl. Pyrolysis. 58-59, 387-400 (2001).</li><li>Bayerbach, R., Meier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+water-insoluble+fraction+from+fast+pyrolysis+liquids+(pyrolytic+lignin).+Part+IV:+Structure+elucidation+of+oligomeric+molecules.">Characterization of the water-insoluble fraction from fast pyrolysis liquids (pyrolytic lignin). Part IV: Structure elucidation of oligomeric molecules.</a> J. Anal. Appl. Pyrolysis. 85 (1-2), 98-107 (2009).</li><li>Faix, O., Andersons, B., Zakis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Carbonyl+Groups+of+Six+Round+Robin+Lignins.">Determination of Carbonyl Groups of Six Round Robin Lignins.</a> Holzforschung. 52, 268-272 (1998).</li><li>Black, S., Ferrell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Carbonyl+Groups+in+Pyrolysis+Bio-oils+Using+Potentiometric+Titration:+Review+and+Comparison+of+Methods.">Determination of Carbonyl Groups in Pyrolysis Bio-oils Using Potentiometric Titration: Review and Comparison of Methods.</a> Energy Fuels. 30 (2), 1071-1077 (2016).</li><li>Ferrell, J., 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=Standardization+of+Chemical+Analytical+Techniques+for+Pyrolysis+Bio-oil:+History,+Challenges,+and+Current+Status+of+Methods.">Standardization of Chemical Analytical Techniques for Pyrolysis Bio-oil: History, Challenges, and Current Status of Methods.</a> Biofuels, Bioprod. Biorefin. 10, 496-507 (2016).</li></ol>

The authors have nothing to disclose.

Representative titration curves are shown in Figure 2. A blank titration, as well as a titration for a pyrolysis oil sample, are shown. Furthermore, the first derivative of the titration curve (dpH/dV) is shown, which allows for easy recognition of the titration endpoint. The inset table on Figure 2 shows triplicate data for both pyrolysis oil and blank titrations, with average values and standard deviations. The endpoint values shown (in mL) are used in Section 4 to calculate the total ...

While pyrolysis bio-oils are comprised of a large variety of compounds and chemical functional groups, quantification of carbonyl groups is especially important. Carbonyls are known to be responsible for the instability of bio-oil during both storage1 and processing2. The titration method presented here is a simple technique which can reliably quantify the total carbonyl content of bio-oils. Only aldehyde and ketone functional groups are quantified using this method; carboxylic acid and lactone groups are not quantified.
For analysis of bio-oils, quantification of carbonyl groups by titration has traditionally been accomplished using the method of Nicolaides3. This method has been commonly used in the bio-oil literature4,5,6,7. This is a simple procedure where carbonyls are converted to the corresponding oxime (see Figure 1). The liberated HCl reacts with pyridine to force the equilibrium to completion. The conjugate acid of pyridine is titrated with a known amount of NaOH (base titrant). The number of equivalents of NaOH used is stoichiometrically equivalent to the moles of carbonyl present in the bio-oil.
The Nicolaides method, however, has several limitations. It can require reaction times in excess of 48 hours to reach completion. This severely limits sample throughput. It utilizes pyridine, which is toxic. Sample weights of 1 to 2 g are required. Sample weight used is dependent on the amount of hydroxylamine HCl present and the carbonyl content of the sample. If initial estimates of the sample weight used are incorrect, the titration has to be repeated.
Faix et al.8 developed a method that has been modified here to address the issues of the Nicolaides method. The reaction is carried out at 80 &#176;C for 2 hours, thereby increasing sample throughput. Pyridine has been replaced with triethanolamine, which is a less toxic chemical. The sample size can be reduced to 100 to 150 mg. The triethanolamine consumes the liberated HCl, driving the reaction to completion and the unconsumed triethanolamine is titrated directly. A secondary titration of the hydroxylamine is unnecessary. Comparison of these titration methods has shown that the Nicolaides method significantly underestimates carbonyl content of bio-oils9.
The method described here has been modified from the original method8 to be more applicable to the analysis of pyrolysis bio-oils. This method was developed for the analysis of raw pyrolysis bio-oils, but it has been successfully applied to other types of biomass-derived oils, including hydrotreated bio-oils. Additionally, this method has been used to monitor changes in carbonyl content during both aging and upgrading.

<table border="0" cellpadding="0" cellspacing="0" width="818">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:403px;">Analytical balance</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">accurate to 0.1 mg</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:403px;">dry block heater with magnetic stirrer, or hot water bath with magnetic stirrer</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:403px;">Automatic titrator</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td>We used a Metrohm Titrando 809 automatic titrator, though other equivalent systems are acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:403px;">Deionized water</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:403px;">Ethanol (reagent grade)</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td>CAS # 64-17-5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydroxylamine hydrochloride&#160;</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td>CAS # 5470-11-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triethanolamine&#160;</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td>CAS #102-71-6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrochloric acid (37%)&#160;</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td>CAS # 7647-01-0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Carbonate (primary standard)&#160;</td>
			<td style="width:131px;">SigmaAldrich</td>
			<td align="right" style="width:119px;">223484</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-(benzyloxy)benzaldehyde&#160;</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td>CAS # 4397-53-9</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl sulfoxide</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td>CAS # 67-68-5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5 mL glass Reacti-vials with solid lid and teflon spinvane</td>
			<td style="width:131px;">Thermoscientific</td>
			<td style="width:119px;">TS-13223</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200 mL volumetric flask</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Volumetric or mechanical pipettes</td>
			<td style="width:131px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

A typical titration curve consists of a single endpoint, as shown in Figure 2. Typical titrations for both a raw bio-oil sample, and a blank titration, are shown. As the endpoint lies at the inflection point in the titration curve; the endpoint can be easily identified by plotting the first derivative of the titration curve (shown on the right axis, dpH/dV, in Figure 2). Many automatic titration systems have software that calculates the derivative of the ...

Watch this Scientific Journal Video about Determination of Carbonyl Functional Groups in Bio-oils by Potentiometric Titration: The Faix Method at JoVE.com

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

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 ...

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Unit 1

Aldehydes and Ketones

Complexometric Titration, Precipitation Titration, and Gravimetry

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11.0K Views.  National Renewable Energy Laboratory. Here we present a potentiometric titration technique for accurately quantifying carbonyl compounds in pyrolysis bio-oils.

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

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 ...

Preparation and Use of Carbonyl-decorated Carbenes in the Activation of White Phosphorus

Here we present a protocol for the synthesis of two distinct carbonyl-decorated carbenes. Both carbenes can be prepared using nearly identical procedures in multi-gram scale quantities. The goal of this manuscript is to clearly detail how to handle and prepare these unique carbenes such that a synthetic chemist of any skill level can work with them. The two carbenes described are a diamidocarbene (DAC, carbene 1) and a monoamidoaminocarbene (MAAC 2). These carbenes are highly electron-deficient and as such display reactivity profiles that are atypical of more traditional N-heterocyclic carbenes. Additionally, these two carbenes only differ in their electrophilic character and not their steric parameters, making them ideal for studying how carbene electronics influence reactivity. To demonstrate this phenomenon, we are also describing the activation of white phosphorus (P4) using these carbenes. Depending on the carbene used, two very different phosphorus-containing compounds can be isolated. When the DAC 1 is used, a tris(phosphaalkenyl)phosphane can be isolated as the exclusive product. Remarkably however, when MAAC 2 is added to P4 under identical reaction conditions, an unexpected carbene-supported P8&#160;allotrope of phosphorus is isolated exclusively. Mechanistic studies demonstrate that this carbene-supported P8allotrope forms via a [2+2] cycloaddition dimerization of a transient diphosphene which has been trapped by treatment with 2,3-dimethyl-1,3-butadiene.

Here, we present a protocol for the synthesis of two carbonyl-decorated carbenes. The protocol makes these interesting compounds readily available to chemists of all skill levels. In addition to the synthesis of these two carbenes, their use in the activation of white phosphorus is also described.

Here we present a protocol for the synthesis of two distinct carbonyl-decorated carbenes. Both carbenes can be prepared using nearly identical procedures ...

Layer-by-layer Synthesis and Transfer of Freestanding Conjugated Microporous Polymer Nanomembranes

CMP as large surface area materials have attracted growing interest recently, due to their high variability in the incorporation of functional groups in combination with their outstanding thermal and chemical stability, and low densities. However, their insoluble nature causes problems in their processing since usually applied techniques such as spin coating are not available. Especially for membrane applications, where the processing of CMP as thin films is desirable, the processing problems have hindered their commercial application.
Here we describe the interfacial synthesis of CMP thin films on functionalized substrates via molecular layer-by-layer (l-b-l) synthesis. This process allows the preparation of films with desired thickness and composition and even desired composition gradients.
The use of sacrificial supports allows the preparation of freestanding membranes by dissolution of the support after the synthesis. To handle such ultra-thin freestanding membranes the protection with sacrificial coatings showed great promise, to avoid rupture of the nanomembranes. To transfer the nanomembranes to the desired substrate, the coated membranes are upfloated at the air-liquid interface and then transferred via dip coating.

In this paper we describe the interfacial synthesis of conjugated microporous polymers (CMP) on sacrificial substrates, and the dissolution of the substrate for the preparation of freestanding CMP nanomembranes. In addition, we will describe how the fragile nanomembranes can be transferred to other substrates.

CMP as large surface area materials have attracted growing interest recently, due to their high variability in the incorporation of functional groups in ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

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 ...

Preparation of Functional Silica Using a Bioinspired Method

The goal of the protocols described herein is to synthesize bioinspired silica materials, perform enzyme encapsulation therein, and partially or totally purify the same by acid elution. By combining sodium silicate with a polyfunctional bioinspired additive, silica is rapidly formed at ambient conditions upon neutralization. 
The effect of neutralization rate and biomolecule addition point on silica yield are investigated, and biomolecule immobilization efficiency is reported for varying addition point. In contrast to other porous silica synthesis methods, it is shown that the mild conditions required for bioinspired silica synthesis are fully compatible with the encapsulation of delicate biomolecules. Additionally, mild conditions are used across all synthesis and modification steps, making bioinspired silica a promising target for the scale-up and commercialization as both a bare material and active support medium.
The synthesis is shown to be highly sensitive to conditions, i.e., the neutralization rate and final synthesis pH, however tight control over these parameters is demonstrated through the use of auto titration methods, leading to high reproducibility in reaction progression pathway and yield. 
Therefore, bioinspired silica is an excellent active material support choice, showing versatility towards many current applications, not limited to those demonstrated here, and potency in future applications.

Here, we present a protocol to synthesize bioinspired silica materials and immobilize enzymes therein. Silica is synthesized by combining sodium silicate and an amine &#39;additive&#39;, which neutralize at a controlled rate. Material properties and function can be altered either by in situ enzyme immobilization or post-synthetic acid elution of encapsulated additives.

The goal of the protocols described herein is to synthesize bioinspired silica materials, perform enzyme encapsulation therein, and partially or totally ...

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&#8722;

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound electrons with neutral molecules/atoms. State-of-the-art in vacuo anion generation techniques allow application to a broad range of atomic, molecular, and cluster anion systems. These are separated and selected using time-of-flight mass spectrometry. Electrons are removed by linearly polarized photons (photo detachment) using table-top laser sources which provide ready access to excitation energies from the infra-red to the near ultraviolet. Detecting the photoelectrons with a velocity mapped imaging lens and position sensitive detector means that, in principle, every photoelectron reaches the detector and the detection efficiency is uniform for all kinetic energies. Photoelectron spectra extracted from the images via mathematical reconstruction using an inverse Abel transformation reveal details of the anion internal energy state distribution and the resultant neutral energy states. At low electron kinetic energy, typical resolution is sufficient to reveal energy level differences on the order of a few millielectron-volts, i.e., different vibrational levels for molecular species or spin-orbit splitting in atoms. Photoelectron angular distributions extracted from the inverse Abel transformation represent the signatures of the bound electron orbital, allowing more detailed probing of electronic structure. The spectra and angular distributions also encode details of the interactions between the outgoing electron and the residual neutral species subsequent to excitation. The technique is illustrated by the application to an atomic anion (F&#8722;), but it can also be applied to the measurement of molecular anion spectroscopy, the study of low lying anion resonances (as an alternative to scattering experiments) and femtosecond (fs) time resolved studies of the dynamic evolution of anions.

Here, we present a protocol for photoelectron imaging of anionic species. Anions generated in vacuo and separated by mass spectrometry are probed using velocity mapped photoelectron imaging, providing details of anion and neutral energy levels, anion and neutral structure and the nature of the anion electronic state.

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound ...

A Facile Synthetic Method to Obtain Bismuth Oxyiodide Microspheres Highly Functional for the Photocatalytic Processes of Water Depuration

Bismuth oxyhalide (BiOI) is a promising material for sunlight-driven-environmental photocatalysis. Given that the physical structure of this kind of materials is highly related to its photocatalytic performance, it is necessary to standardize the synthetic methods in order to obtain the most functional architectures and, thus, the highest photocatalytic efficiency. Here, we report a reliable route to obtain BiOI microspheres via the solvothermal process, using Bi(NO3)3 and potassium iodide (KI) as precursors, and ethylene glycol as a template. The synthesis is standardized in a 150 mL autoclave, at 126 &#176;C for 18 h. This results in 2-3 &#181;m-sized mesoporous microspheres, with a relevant specific surface area (61.3 m2/g). Shortening the reaction times in the synthesis results in amorphous structures, while higher temperatures lead to a slight increase in the porosity of the microspheres, with no effect in the photocatalytic performance. The materials are photo-active under UV-A/visible light irradiation for the degradation of the antibiotic ciprofloxacin in water. This method has demonstrated to be effective in interlaboratory tests, obtaining similar BiOI microspheres in Mexican and Chilean research groups.

This article describes a synthetic method to obtain bismuth oxyiodide microspheres, which are highly functional to perform the photocatalytic removal of organic pollutants, such as ciprofloxacin, in water under UV-A/visible light irradiation.

Bismuth oxyhalide (BiOI) is a promising material for sunlight-driven-environmental photocatalysis. Given that the physical structure of this kind of ...

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

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

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

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