We thank Loyola University Chicago, Merck, Co., Inc., and the NIH/National Institute of General Medical Sciences (GM128126) for financial support. We thank Mettler-Toledo Autochem Inc. for their support in the production of this article.

1. Open-air reference spectrum
<ol>
	<li>Open the data acquisition software. Click Instrument. Under the Configure tab, click Collect Background. Click Continue. Set scans to 256 and click OK to collect a background. 
	NOTE: Make sure the probe is in the same position in which data collection will take place. Position changes of the probe may impact spectra.</li>
</ol>
2. Solvent reference spectrum
<ol>
	<li>In the data acquisition software, click File. Click New. Click QuickStart.</li>
	<li>Set Duration to 15 min and Sample Interval to 15 s. Click Create to create experiment. 
	NOTE: At this point, the chemical system must be attached to the in situ IR probe to proceed. The following steps are for the preparation of the chemical system to be studied.</li>
	<li>Under inert atmosphere, add Lewis acid to a flame-dried 25 mL 2-neck round bottom flask charged with a stir bar (Figure 1B). Seal the flask with rubber septa and attach an Ar-filled balloon to the flask. Add desired volume of anhydrous solvent via syringe (minimum 3 mL) (Figure 1C). 
	NOTE: FeCl3 is not soluble in dichloroethane (DCE). GaCl3 is soluble in DCE.</li>
	<li>Remove one septum and attach the flask to the in situ IR probe (Figure 1D). Place the flask in a temperature-controlled bath set to desired temperature (Figure 1E).</li>
	<li>Start the experiment in the data acquisition software by clicking the <img alt="Icon" src="/files/ftp_upload/60745/icon.jpg" /> button to begin collecting data, and stop collecting data after 2 min. 
	NOTE: The name of this file is the solvent reference spectrum that you will use in step 3.1.3.</li>
</ol>
3. Titration software setup
<ol>
	<li>Creating new titration experiment
	<ol>
		<li>In the data acquisition software, click File | New | Quick Start. Set Duration to 8 h and Sample Interval to 15 s. 
		NOTE: The data acquisition has the ability to set experiment duration between 15 min and 2 d and sample interval between 15 s and 1 h.</li>
		<li>Click Create to create experiment. In the data acquisition software, go to Spectra tab and click Add Spectra. Click From File and open appropriate solvent reference spectrum obtained in step 2. Check the box with the time signature. Click OK.</li>
		<li>Start experiment in the data acquisition software by clicking the <img alt="Icon" src="/files/ftp_upload/60745/icon.jpg" /> button to begin collecting data.</li>
	</ol>
	</li>
	<li>Click Solvent Subtraction and select appropriate reference spectrum added in step 3.1.3. Stir for 15 min to reach temperature. Use the in situ IR probe to determine temperature.</li>
</ol>
4. Titration procedure
<ol>
	<li>Add 10 &#181;L of carbonyl analyte via syringe (Figure 1F).</li>
	<li>Observe signal response on the data acquisition (Figure 2). System will shift from equilibrium and change with time.</li>
	<li>When the IR signal stabilizes and remains constant, collect IR spectrum. 
	NOTE: The data acquisition collects spectra at a set frequency. Data in our lab are collected every 15 s. We note the time at which the system reaches equilibrium and use the spectrum collected at the time for analysis.</li>
	<li>Repeat steps 4.1-4.3 until desired amount of analyte is added. 
	NOTE: FeCl3 mixture becomes homogeneous once 1 equiv 1 is added and GaCl3 mixture remains homogeneous regardless of amount of 1 added.</li>
</ol>
5. Analysis of IR spectra
<ol>
	<li>Export data for the data acquisition software.
	<ol>
		<li>Click File | Export | Multi-spectrum file.</li>
		<li>Under Format, check CSV and under Data, check Raw. Click Export to export IR data to spreadsheet or mathematical processing software.</li>
	</ol>
	</li>
	<li>Plot desired region of IR spectrum, as shown in Figure 3A,D.</li>
	<li>Examine the spectrum for transitions and/or isosbestic points.</li>
	<li>Separate spectra by progression, as shown in Figure 3B,C for GaCl3 and Figure 3E,F for FeCl3.</li>
</ol>
6. Component analysis
<ol>
	<li>Identify &#955;max of each species of interest, as shown in Figure 4A for GaCl3 and 1 and Figure 4D for FeCl3 and 1, to generate a table of Absorbance vs. equivalent of analyte added, as shown in Figure 4B for GaCl3 and Figure 4E for FeCl3.</li>
	<li>To account for dilution, multiply the absorbance by the total volume of the solution for each spectrum, as shown in Figure 4B for GaCl3 and Figure 4E for FeCl3.</li>
	<li>Plot product of absorbance*volume as a function of equivalents of analyte, as shown in Figure 4C for GaCl3 and Figure 4F for FeCl3.</li>
</ol>
7. Analysis of consumption of species
<ol>
	<li>For in situ-generated species that can be identified, plot a Beer-Lambert relationship, as shown in Figure 5A.</li>
	<li>For known species, measure the impact of concentration on Absorbance at the desired &#955;max and plot a Beer-Lambert relationship.</li>
	<li>Using the two Beer-Lambert relationships, determine the observed in situ amounts of the species of interest, as shown in Figure 5B. 
	NOTE: CMAX = 2 mmol as defined by the amount of FeCl3 present. CADD is the moles of acetone (1) added. CCOORD is the moles of FeCl3-acetone complex (3). COBS is the moles of unbound 1. CND is the moles of 1 not detected. CMAX &#8211; CCOORD is the moles of 3 that have been consumed.</li>
	<li>Plot CND vs. (CMAX &#8211; CCOORD) to determine if there is a correlation, as shown in Figure 5C. 
	NOTE: The slope of this line will be in moles of species 1 per moles of species 3.</li>
</ol>

<ol>
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This article was produced with support by Mettler-Toledo Autochem Inc., which is the producer of the instrument utilized in this article.

Under anhydrous conditions, Lewis acids can have a range of solubilities. The two examples we have presented are GaCl3 and FeCl3 in DCE. GaCl3 is homogeneous at the onset of the titration, while FeCl3 is largely insoluble. Beginning with the homogeneous solution of GaCl3, we completed a titration from 0-4 equiv 1 in 10 &#956;L increments and extracted the IR spectra (Figure 3A). Examination of the transitions that occur over the course of the titrations shows a formation of a single species in the carbonyl region at 1630 cm-1, which grows from 0-1 equiv 1 (Figure 3B) 26,27. When greater than 1 equiv 1 is added to the solution, no change in the peak at 1630 cm-1 occurs and unbound 1 is observed at 1714 cm-1 (Figure 3C). These results are consistent with the formation of 2. When the same titration is performed with FeCl3 (Figure 3D), a peak at 1636 cm-1 forms from 0-1 equiv 1, which is consistent with 3 (Figure 3E). Importantly, the mixture becomes homogenous once 1 equiv 1 is achieved. When the titration proceeds beyond 1 equiv 1, unbound 1 is observed at 1714 cm-1, 3 decreases in intensity, an isosbestic point resolves at 1648 cm-1, and a new peak at 1663 cm-1 forms.
Using the titration IR data, the equivalents of analyte used can be employed to perform Component Analysis of the solution interactions (Figure 4). To account for dilution, we can employ a normalization with respect to volume of the Beer-Lambert equation (eq. 1):
<img alt="Equation 1" src="/files/ftp_upload/60745/60745equ01.jpg" />
where 1) both absorbance (A) and volume (V) are measurable terms; 2) molar absorptivity (&#949;) and pathlength (l) are constant, allowing 3) number of moles (n) to be examined. The normalized absorbance can easily be computed in a spreadsheet (Figure 4B,D), and then this term can be plotted against equivalents of analyte. In Figure 4C, we can see that the signal for 2 increases linearly with respect to 1 until 1 equiv, at which point the signal for 1 increases linearly and 2 is unchanged. In Figure 4F, we see a similar linear increase in the signal of 3 to 1 equiv 1, followed by the presence of 1 beyond 1 equiv added. However, we also observe a linear decrease in the intensity of 3, and we observe less 1 than we should, assuming similar behavior to GaCl3.
Yet more information is available from the IR data for the titration of FeCl3 with 1. The maximum amount of 3 that can form is defined by the amount of FeCl3 added (CMAX = 2 mmol FeCl3 in the example titration). We know the amount of 1 we add to the flask (CADD), and we can measure the amount of unbound 1 we observe at 1714 cm-1 (COBS) and the amount of 3 we observe at 1636 cm-1 (CCOORD) using Beer-Lambert relationships. Lastly, we know we cannot account for all of the 1 added to the flask as free 1 or 3, indicating that some 1 is not detected (CND). We can combine these terms for 1 in the following mass balance (eq. 2):
<img alt="Equation 2" src="/files/ftp_upload/60745/60745equ02.jpg" />
We can use the titration data to calculate the values of these terms in each IR spectrum generated during the titration (Figure 5B). With these values, we can plot the amount of 1 missing (CND) as a function of the amount 3 consumed (CMAX-CCOORD) to determine if there is a correlation (Figure 5C). This correlation is consistent with 3 equiv 1 consuming 1 equiv 3, which may form a complex similar to 4. We have obtained further support for this number of attached ketones via examination of solution conductivity, which is consistent with one or more of the chlorides being displaced to the outer sphere of Fe(III), and X-ray crystallography of an analogous structure with benzaldehyde12. However, it is likely that there is a mixture of different types of highly-ligated structures that are formed in solution, as is indicated by our non-whole number slopes in our consumption analysis in Figure 5, and the crystal structure we observe may simply be the one complex that precipitates.
In addition to the interactions between two species, this method can be used to probe competitive interactions (Figure 7). By establishing the formation and spectral properties of 3 (Figure 7A) and 5 (Figure 7B), the competition of carbonyls for access to the Lewis acid can be observed. By preforming 3 in solution, we can examine how 6 displaces 1 (Figure 7C). When we probe this system, we see that as we add 6 to 3, not all 6 binds to FeCl3. However, we do observe the consumption of 3 with concomitant presence of 1, as well as the formation of 5.
Using this type of competition experiment, we have been able to simulate the state of FeCl3 as a catalyst in carbonyl-olefin metathesis (Figure 8). We previously demonstrated that at low turnovers, carbonyl-olefin metathesis operates via the primary cycle in Figure 8B28. Substrate 7 interacts with FeCl3 to form complex 9 as the resting state of the cycle. Complex 9 then undergoes turnover-limiting [2+2]-cycloaddition to form oxetane complex 10. Retro-[2+2] yields cycloalkene product 8 and 3, which in turn must have the molecule of 1 displaced by a molecule of 7. However, as the [1] increases, 3 is converted to complex 4. Coordinatively saturated 4 then either sequesters FeCl3 or is catalytically competent, resulting in a parallel cycle via ketone complex 11 and oxetane complex 12.
In conclusion, the utilization of in situ IR to monitor the titration of Lewis acids with carbonyl compounds allows chemists to gain insight into Lewis acid/base solution interactions under synthetically relevant conditions. Not only can this technique be employed to identify discrete structures, but it can be employed to observe the transition of one discrete species into another, as well. Findings from this method have been utilized to propose the mechanism of other metathesis reactions29. We are currently using data gathered via this method to facilitate the reactivity of recalcitrant substrates in carbonyl-olefin metathesis, as well as to develop new forms of metathesis reactions. Lastly, the competitive interactions between substrate carbonyls and product carbonyls likely impact other Lewis acid-catalyzed reactions. We are employing this method to examine these other catalytic regimes.

The utilization of Lewis acids to activate substrates containing carbonyls is ubiquitous in organic synthetic methods1,2,3,4. The study of these interactions has relied on solid state X-ray crystallography, as well as in situ NMR spectroscopy2. Limitations of these techniques manifest from artifacts that arise from crystallization, or the inability to probe paramagnetic Lewis acids via NMR analysis. To overcome these issues, chemists have employed infrared (IR) spectroscopy to determine the exact structure of Lewis pairs. Further, IR has been utilized to determine Lewis acidity4,5,6,7,8,9. The Susz lab studied the solid-state interactions of Lewis acids and carbonyls in the stoichiometric regime. Utilizing IR in conjunction with elemental analysis, the Susz group was able to elucidate the structures of neat, 1:1 mixtures of Lewis pairs. This analysis provided a great deal of insight into structural ramifications of the interactions of simple carbonyl compounds with commonly utilized Lewis acids in the solid state, and of particular interest to our lab: FeCl310,11. We posited that we could add to the existing understanding of the interactions of these ou important Lewis pairs via an in situ method that examines synthetically relevant conditions.
In situ IR enables chemists to perform real-time measurements of functional group conversions in situ. These data supply key insights into reaction rates to support hypotheses about the operating mechanisms of a process and to influence of reaction performance. Real-time observations allow chemists to directly track the interconversion of reaction components over the course of the reaction, and the information gleaned can be employed by the synthetic chemist in the development of new compounds and the optimization of synthetic routes and new chemical processes.
Employing in situ IR spectroscopy as a detection method, we probed the substrates and intermediates that participate in the catalytic cycle of metal-mediated carbonyl-olefin metathesis12. The Fe(III)-catalyzed carbonyl-olefin metathesis process, developed by the Schindler lab, exemplifies a powerful method for the production of C=C bonds from functional groups utilized ubiquitously in the construction of complex molecules13,14,15. Since the original report, this process has inspired a plethora of synthetic developments beyond the utilization of Fe(III)16,17,18,19,20,21,22,23,24,25. Importantly, this reaction requires that the Lewis acid catalyst differentiate between a substrate carbonyl and a product carbonyl for successful reactivity. To observe this competitive interaction under synthetically relevant conditions, we combined titration with the continuous observation provided by in situ IR.
We believe this method is of general importance to chemists studying carbonyl-centered reactions catalyzed by Lewis acids. This detailed demonstration aims to help chemists apply this technique to their system of study.

<table>
	<tbody>
		<tr>
			<td>Acetone</td>
			<td>BDH</td>
			<td>BDH1101-19L</td>
			<td>Dried over potassium carbonate</td>
		</tr>
		<tr>
			<td>Balloon</td>
			<td>VWR</td>
			<td>470003-408</td>
			<td>Round Balloons, Assorted Colors, 9&#34; dia.</td>
		</tr>
		<tr>
			<td>Detector LN2 RiR15</td>
			<td>Mettler Toledo</td>
			<td>14474603</td>
			<td></td>
		</tr>
		<tr>
			<td>1,2 Dichloroethane</td>
			<td>Beantown</td>
			<td>223375-2.5L</td>
			<td>Dried over 3&#197; molecular sieves</td>
		</tr>
		<tr>
			<td>Gallium (III) Chloride</td>
			<td>Beantown</td>
			<td>127270-100G</td>
			<td>Anhydrous &#8805;99.999% (trace metals basis)</td>
		</tr>
		<tr>
			<td>25 &#181;L glass syringe</td>
			<td>Hamilton</td>
			<td>80285</td>
			<td></td>
		</tr>
		<tr>
			<td>Inert Argon Gas</td>
			<td>Airgas</td>
			<td></td>
			<td>Ultra High Purity</td>
		</tr>
		<tr>
			<td>Iron (III) Chloride</td>
			<td>Sigma Aldrich</td>
			<td>157740-100G</td>
			<td>Reagent Grade, 97%</td>
		</tr>
		<tr>
			<td>100-mL Jacketed Beaker</td>
			<td>AceGlass</td>
			<td>5340-03</td>
			<td></td>
		</tr>
		<tr>
			<td>3&#197; Molecular Sieves</td>
			<td>Alfa Aesar</td>
			<td>L05335</td>
			<td></td>
		</tr>
		<tr>
			<td>25-mL 2 neck flask</td>
			<td>CTechGlass</td>
			<td>FL-0143-003</td>
			<td></td>
		</tr>
		<tr>
			<td>18G Needle</td>
			<td>BD Biosciences</td>
			<td>305196</td>
			<td>Needles with Regular Bevel, 38.1 mm (11/2&#34;)</td>
		</tr>
		<tr>
			<td>Potassium Carbonate</td>
			<td>Sigma Aldrich</td>
			<td>60109-1KG-F</td>
			<td>Anhydrous</td>
		</tr>
		<tr>
			<td>Prism 8</td>
			<td>GraphPad</td>
			<td></td>
			<td>Mathematical Processing Software</td>
		</tr>
		<tr>
			<td>Probe DST 6.35 x 1.5m X 203 DiComp</td>
			<td>Mettler Toledo</td>
			<td>14474510</td>
			<td>in situ IR probe</td>
		</tr>
		<tr>
			<td>Rice Stir Bar</td>
			<td>Dynalon</td>
			<td>303495</td>
			<td>Diameter: 3 mm (1/8&#34;), Length: 10 mm (3/8&#34;)</td>
		</tr>
		<tr>
			<td>14/20 Rubbber Septa</td>
			<td>VWR</td>
			<td>89097-554</td>
			<td></td>
		</tr>
		<tr>
			<td>5-mL Syringe</td>
			<td>AIR-TITE</td>
			<td>53548-005</td>
			<td>HSW Norm-Ject Sterile Luer-Slip Syringes, Air-Tite</td>
		</tr>
		<tr>
			<td>10-mL Syringe</td>
			<td>AIR-TITE</td>
			<td>53548-006</td>
			<td>HSW Norm-Ject Sterile Luer-Slip Syringes, Air-Tite</td>
		</tr>
		<tr>
			<td>System ReactIR 15</td>
			<td>Mettler Toledo</td>
			<td>1400003</td>
			<td>in situ IR system</td>
		</tr>
		<tr>
			<td>Thermostatic Bath</td>
			<td>Haake</td>
			<td></td>
			<td>Haake A82</td>
		</tr>
	</tbody>
</table>

characterizing lewis pairs using titration coupled with in situ infrared spectroscopy

Lewis acid-activation of carbonyl-containing substrates is a fundamental basis for facilitating transformations in organic chemistry. Historically, characterization of these interactions has been limited to models equivalent to stoichiometric reactions. Here, we report a method utilizing in situ infrared spectroscopy to probe the solution interactions between Lewis acids and carbonyls under synthetically relevant conditions. Using this method, we were able to identify 1:1 complexation between GaCl3 and acetone and a highly ligated complex for FeCl3 and acetone. The impact of this technique on mechanistic understanding is illustrated by application to the mechanism of Lewis acid-mediated carbonyl-olefin metathesis in which we were able to observe competitive binding interactions between substrate carbonyl and product carbonyl with the catalyst.

In this study, in situ IR-monitored titration was used to observe the interactions of 1 and GaCl3 as well as 1 and FeCl3 (Figure 6)12. Using this collection of protocols, we were able to determine that GaCl3 and 1 form 1:1 complex 2 in solution. Alternatively, when FeCl3 and 1 are combined, more complex behavior is observed. Figure 6 displays the equilibria we were examining. Figure 1 displays the physical setup of the titration of FeCl3 with 1. Figure 2 displays the raw feed of data obtained by the in situ IR using the data acquisition software for the titration of FeCl3 with 1. Figure 3 displays the process of extracting the transitions that result from this titration method applied to GaCl3 and FeCl3. Figure 4 displays the extraction of &#955;max data of the titration of GaCl3 with 1 and the titration of FeCl3 with 1. Figure 5 displays the extraction of complex coordination behavior from the titration of FeCl3 with 1. Figure 7 displays an application of these protocols for the examination of competitive access to a Lewis acid. Figure 8 shows the application of these protocols to revising the mechanism of metal catalyzed carbonyl-olefin metathesis.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60745/60745fig01.jpg" /> 
Figure 1: Visual guide to system setup. Necessary components for performing the titration (A). Assembled components prior to attachment to the in situ IR (B). Flask with Ar and ready for solvent addition (C). Flask attached to the in situ IR with solvent (D). Flask under temperature control (E). Ready for addition of analyte (F). <a href="https://www.jove.com/files/ftp_upload/60745/60745fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60745/60745fig02.jpg" /> 
Figure 2: Analyte signal response in the data acquisition interface at 1636 cm-1 for titration of 2 mmol FeCl3 in 12 mL of DCE with 1. The spectrum is collected when the system is at equilibrium, after analyte addition. <a href="https://www.jove.com/files/ftp_upload/60745/60745fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60745/60745fig03.jpg" /> 
Figure 3: Analysis of IR spectra. Spectra collected for the titrations GaCl3 with 0-4 equiv 1 (A) and FeCl3 with 0-4 equiv 1 (D). Breakdown of titration of GaCl3 with 0-1 equiv 1 showing the formation of 2 (B) and with 1-4 equiv 1 showing the presence of 1 (C). Breakdown of titration of FeCl3 with 0-1 equiv 1 showing the formation of 3 (E) and with 1-4 equiv 1 showing the presence of 1, the consumption of 3, and the formation of a new species (F). Reprinted (adapted) with permission from Hanson, C. S., et al12. Copyright 2019 American Chemical Society. <a href="https://www.jove.com/files/ftp_upload/60745/60745fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60745/60745fig04.jpg" /> 
Figure 4: Extraction of &#955;max data from IR for Component Analysis. Spectra collected for the titrations GaCl3 with 0-4 equiv 1 with the &#955;max for 1 and 2 indicated (A) and FeCl3 with 0-4 equiv 1 with the &#955;max for 1 and 3 indicated (D). Table showing representative data normalized to account for dilution for GaCl3 (B) and for FeCl3 (E). Data from (B) plotted for component analysis of titration of GaCl3 with 1 (C) and for component analysis of titration of FeCl3 with 1 (F). Reprinted (adapted) with permission from Hanson, C. S., et al12. Copyright 2019 American Chemical Society. <a href="https://www.jove.com/files/ftp_upload/60745/60745fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60745/60745fig05.jpg" /> 
Figure 5: Consumption analysis of titration of FeCl3 with 1. Segment of IR data used to generate a Beer-Lambert relationship for [3] and the segment of IR data used to determine the consumption of 3 (A). Moles of each 1-containing species measured from IR (B). Plot of moles of 1 not detected vs. moles of 3 consumed (C). Reprinted (adapted) with permission from Hanson, C. S., et al12. Copyright 2019 American Chemical Society. <a href="https://www.jove.com/files/ftp_upload/60745/60745fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60745/60745fig06.jpg" /> 
Figure 6: Lewis acid/base equilibria probed in this study. Titrations of GaCl3 with 1 to form 2 and FeCl3 with 1 to form 3 and 4 are reported. <a href="https://www.jove.com/files/ftp_upload/60745/60745fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/60745/60745fig07.jpg" /> 
Figure 7: Competitive binding experiment. Carbonyl region of IR spectrum of 3 (A) and of IR spectrum of 5 (B). Equilibrium probed in titration of 3 with 6 (C). IR data of titration of 3 with 1 equiv 6 (D). Reprinted (adapted) with permission from Hanson, C. S., et al12. Copyright 2019 American Chemical Society. <a href="https://www.jove.com/files/ftp_upload/60745/60745fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/60745/60745fig08.jpg" /> 
Figure 8: Application of in situIR data in mechanistic proposal. Carbonyl-olefin metathesis reaction of 7 (A). The revised mechanistic proposal of carbonyl-olefin metathesis facilitated by titration coupled with in situ IR spectroscopy (B). Reprinted (adapted) with permission from Hanson, C. S., et al12. Copyright 2019 American Chemical Society. <a href="https://www.jove.com/files/ftp_upload/60745/60745fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Characterizing Lewis Pairs Using Titration Coupled with In Situ Infrared Spectroscopy at JoVE.com

Characterizing Lewis Pairs Using Titration Coupled with In Situ Infrared Spectroscopy

Here, we present a method for the observation of solution interactions between Lewis acids and bases by employing in situ infrared spectroscopy as a detector for titration under synthetically relevant conditions. By examining solution interactions, this method represents a complement to X-ray crystallography, and provides an alternative to NMR spectroscopy.

Lewis acid-activation of carbonyl-containing substrates is a fundamental basis for facilitating transformations in organic chemistry. Historically, ...

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Acid–Base Titration

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9.0K Views.  Loyola University Chicago. Here, we present a method for the observation of solution interactions between Lewis acids and bases by employing in situ infrared spectroscopy as a detector for titration under synthetically relevant conditions. By examining solution interactions, this method represents a complement to X-ray crystallography, and provides an alternative to NMR spectroscopy.

Video: Characterizing Lewis Pairs Using Titration Coupled with In Situ Infrared Spectroscopy

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using conventional synthesis techniques. Coupling soft landing with in situ characterization using secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS) enables analysis of well-defined surfaces under clean vacuum conditions. The capabilities of three soft-landing instruments constructed in our laboratory are illustrated for the representative system of surface-bound organometallics prepared by soft landing of mass-selected ruthenium tris(bipyridine) dications, [Ru(bpy)3]2+ (bpy = bipyridine), onto carboxylic acid terminated self-assembled monolayer surfaces on gold (COOH-SAMs). In situ time-of-flight (TOF)-SIMS provides insight into the reactivity of the soft-landed ions. In addition, the kinetics of charge reduction, neutralization and desorption occurring on the COOH-SAM both during and after ion soft landing are studied using in situ Fourier transform ion cyclotron resonance (FT-ICR)-SIMS measurements. In situ IRRAS experiments provide insight into how the structure of organic ligands surrounding metal centers is perturbed through immobilization of organometallic ions on COOH-SAM surfaces by soft landing. Collectively, the three instruments provide complementary information about the chemical composition, reactivity and structure of well-defined species supported on surfaces.

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of novel materials. Coupled with analysis by in situ secondary ion mass spectrometry (SIMS) and infrared reflection absorption spectroscopy (IRRAS), soft landing provides unprecedented insights into the interactions of well-defined species with surfaces.

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using ...

The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

The exceptional affinity, specificity, and selectivity of antibodies make them extraordinarily attractive vectors for tumor-targeted PET radiopharmaceuticals. Due to their multi-day biological half-life, antibodies must be labeled with positron-emitting radionuclides with relatively long physical decay half-lives. Traditionally, the positron-emitting isotopes 124I (t1/2 = 4.18 d), 86Y (t1/2 = 14.7 hr), and 64Cu (t1/2 = 12.7 hr) have been used to label antibodies for PET imaging. More recently, however, the field has witnessed a dramatic increase in the use of the positron-emitting radiometal 89Zr in antibody-based PET imaging agents. 89Zr is a nearly ideal radioisotope for PET imaging with immunoconjugates, as it possesses a physical half-life (t1/2 = 78.4 hr) that is compatible with the in vivo pharmacokinetics of antibodies and emits a relatively low energy positron that produces high resolution images. Furthermore, antibodies can be straightforwardly labeled with 89Zr using the siderophore-derived chelator desferrioxamine (DFO). In this protocol, the prostate-specific membrane antigen targeting antibody J591 will be used as a model system to illustrate (1) the bioconjugation of the bifunctional chelator DFO-isothiocyanate to an antibody, (2) the radiosynthesis and purification of a 89Zr-DFO-mAb radioimmunoconjugate, and (3) in vivo PET imaging with an 89Zr-DFO-mAb radioimmunoconjugate in a murine model of cancer.

The Bioconjugation and Radiosynthesis of 89Zr-DFO-labeled Antibodies

Due to its multi-day radioactive half-life and favorable decay properties, the positron-emitting radiometal 89Zr is extremely well-suited for use in antibody-based radiopharmaceuticals for PET imaging. In this protocol, the bioconjugation, radiosynthesis, and preclinical application of 89Zr-labeled antibodies will be described.

The exceptional affinity, specificity, and selectivity of antibodies make them extraordinarily attractive vectors for tumor-targeted PET ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Construction of Models for Nondestructive Prediction of Ingredient Contents in Blueberries by Near-infrared Spectroscopy Based on HPLC Measurements

Nondestructive prediction of ingredient contents of farm products is useful to ship and sell the products with guaranteed qualities. Here, near-infrared spectroscopy is used to predict nondestructively total sugar, total organic acid, and total anthocyanin content in each blueberry. The technique is expected to enable the selection of only delicious blueberries from all harvested ones. The near-infrared absorption spectra of blueberries are measured with the diffuse reflectance mode at the positions not on the calyx. The ingredient contents of a blueberry determined by high-performance liquid chromatography are used to construct models to predict the ingredient contents from observed spectra. Partial least squares regression is used for the construction of the models. It is necessary to properly select the pretreatments for the observed spectra and the wavelength regions of the spectra used for analyses. Validations are necessary for the constructed models to confirm that the ingredient contents are predicted with practical accuracies. Here we present a protocol to construct and validate the models for nondestructive prediction of ingredient contents in blueberries by near-infrared spectroscopy.

We present here a protocol to construct and validate models for nondestructive prediction of total sugar, total organic acid, and total anthocyanin content in individual blueberries by near-infrared spectroscopy.

Nondestructive prediction of ingredient contents of farm products is useful to ship and sell the products with guaranteed qualities. Here, near-infrared ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

In Situ Characterization of Boehmite Particles in Water Using Liquid SEM

In situ imaging and elemental analysis of boehmite (AlOOH) particles in water is realized using the System for Analysis at the Liquid Vacuum Interface (SALVI) and Scanning Electron Microscopy (SEM). This paper describes the method and key steps in integrating the vacuum compatible SAVLI to SEM and obtaining secondary electron (SE) images of particles in liquid in high vacuum. Energy dispersive x-ray spectroscopy (EDX) is used to obtain elemental analysis of particles in liquid and control samples including deionized (DI) water only and an empty channel as well. Synthesized boehmite (AlOOH) particles suspended in liquid are used as a model in the liquid SEM illustration. The results demonstrate that the particles can be imaged in the SE mode with good resolution (i.e., 400 nm). The AlOOH EDX spectrum shows significant signal from the aluminum (Al) when compared with the DI water and the empty channel control. In situ liquid SEM is a powerful technique to study particles in liquid with many exciting applications. This procedure aims to provide technical know-how in order to conduct liquid SEM imaging and EDX analysis using SALVI and to reduce potential pitfalls when using this approach.

In Situ Characterization of Boehmite Particles in Water Using Liquid SEM

We present a procedure for real-time imaging and elemental composition analysis of boehmite particles in deionized water by in situ liquid Scanning Electron Microscopy.

In situ imaging and elemental analysis of boehmite (AlOOH) particles in water is realized using the System for Analysis at the Liquid Vacuum Interface ...

Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis

We demonstrate a method of quantification and detection of parasites in aqueous red blood cells (RBCs) by using a simple benchtop Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectrometer in conjunction with Multivariate Data Analysis (MVDA). 3D7 P. falciparum were cultured to 10% parasitemia ring stage parasites and used to spike fresh donor isolated RBCs to create a dilution series between 0&#8211;1%. 10 &#181;L of each sample were placed onto the center of the ATR diamond window to acquire the spectrum. The sample data was treated to improve the signal to noise ratio and to remove the contribution of water, and then the second derivative was applied to resolve spectral features. The data were then analyzed using two types of MVDA: first Principal Component Analysis (PCA) to determine any outliers and then Partial Least Squares Regression (PLS-R) to build the quantification model.

Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis

Here, we present a protocol for the detection and quantification of Plasmodium falciparum in infected aqueous red blood cells using an attenuated total reflection infrared spectrometer and multivariate data analysis.

We demonstrate a method of quantification and detection of parasites in aqueous red blood cells (RBCs) by using a simple benchtop Attenuated Total ...

In Situ Lithiated Reference Electrode: Four Electrode Design for In-operando Impedance Spectroscopy

Extending operating voltage of Li-ion batteries results in higher energy output from these devices. High voltages, however, may trigger or accelerate multiple processes responsible for long-term performance decay. Given the complexity of physical processes occurring inside the cell, it is often challenging to achieve a full understanding of the root causes of this performance degradation. This difficulty arises in part from the fact that any electrochemical measurement of a battery will return the combined contributions of all components in the cell. Incorporation of a reference electrode can solve part of the problem, as it allows the electrochemical reactions of the cathode and the anode to be individually probed. A variation in the voltage range experienced by the cathode, for example, can indicate alterations in the pool of cyclable lithium ions in the full-cell. The structural evolution of the many interphases existing in the battery can also be monitored, by measuring the contributions of each electrode to the overall cell impedance. Such wealth of information amplifies the reach of diagnostic analysis in Li-ion batteries and provides valuable input to the optimization of individual cell components. In this work, we introduce the design of a test cell able to accommodate multiple reference electrodes, and present reference electrodes that are appropriate for each specific type of measurement, detailing the assembly process in order to maximize the accuracy of the experimental results.

The incorporation of reference electrodes in a Li-ion battery provides valuable information to elucidate degradation mechanisms at high voltages. In this article, we present a cell design that accommodates multiple reference electrodes, along with the assembly steps to assure maximum accuracy of the data obtained in electrochemical measurements.

Extending operating voltage of Li-ion batteries results in higher energy output from these devices. High voltages, however, may trigger or accelerate ...

In situ FTIR Spectroscopy as a Tool for Investigation of Gas/Solid Interaction: Water-Enhanced CO2 Adsorption in UiO-66 Metal-Organic Framework

In situ infrared spectroscopy is an inexpensive, highly sensitive, and selective valuable tool to investigate the interaction of polycrystalline solids with adsorbates. Vibrational spectra provide information on the chemical nature of adsorbed species and their structure. Thus, they are very useful for obtaining molecular level understanding of surface species. The IR spectrum of the sample itself gives some direct information about the material. General conclusions can be drawn concerning hydroxyl groups, some stable surface species and impurities. However, the spectrum of the sample is &#34;blind&#34; with respect to the presence of coordinatively unsaturated ions and gives rather poor information about the acidity of surface hydroxyls, species decisive for the adsorption and catalytic properties of the materials. Furthermore, no discrimination between bulk and surface species can be made. These problems are solved by the use of probe molecules, substances that interact specifically with the surface; the alteration of some spectral features of these molecules as a result of adsorption provides valuable information about the nature, properties, location, concentration, etc., of the surface sites.
The experimental protocol for in-situ IR studies of gas/sample interaction includes preparation of a sample pellet, activation of the material, initial spectral characterization through the analysis of the background spectra, characterization by probe molecules, and study of the interaction with a particular set of gas mixtures. In this paper we investigate a zirconium terephthalate metal organic framework, Zr6O4(OH)4(BDC)6 (BDC = benzene-1,4-dicarboxylate), namely UiO-66 (UiO refers to University of Oslo). The acid sites of the UiO-66 sample are determined by using CO and CD3CN as molecular probes. Furthermore, we have demonstrated that CO2 is adsorbed on basic sites exposed on dehydroxylated UiO-66. Introduction of water to the system produces hydroxyl groups acting as additional CO2 adsorption sites. As a result, CO2 adsorption capacity of the sample is strongly enhanced.

In situ FTIR Spectroscopy as a Tool for Investigation of Gas/Solid Interaction: Water-Enhanced CO2 Adsorption in UiO-66 Metal-Organic Framework

The use of FTIR spectroscopy for investigation of the surface properties of polycrystalline solids is described. Preparation of sample pellets, activation procedures, characterization with probe molecules and model studies of CO2 adsorption are discussed.

In situ infrared spectroscopy is an inexpensive, highly sensitive, and selective valuable tool to investigate the interaction of polycrystalline solids ...

Covalent Attachment of Single Molecules for AFM-based Force Spectroscopy

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and surfaces. For a true single molecule experiment, covalent attachment of the probe molecule is essential because only then can hundreds of force-extension traces with one and the same single molecule be obtained. Many traces are in turn necessary to prove that a single molecule alone is probed. Additionally, passivation is crucial for preventing unwanted interactions between the single probe molecule and the AFM cantilever tip as well as between the AFM cantilever tip and the underlying surface. The functionalization protocol presented here is reliable and can easily be applied to a variety of polymers. Characteristic single molecule events (i.e., stretches and plateaus) are detected in the force-extension traces. From these events, physical parameters such as stretching force, desorption force and desorption length can be obtained. This is particularly important for the precise investigation of stimuli-responsive systems at the single molecule level. As exemplary systems poly(ethylene glycol) (PEG), poly(N-isopropylacrylamide) (PNiPAM) and polystyrene (PS) are stretched and desorbed from SiOx (for PEG and PNiPAM) and from hydrophobic self-assembled monolayer surfaces (for PS) in aqueous environment.

Covalent attachment of probe molecules to atomic force microscopy (AFM) cantilever tips is an essential technique for the investigation of their physical properties. This allows us to determine the stretching force, desorption force and length of polymers via AFM-based single molecule force spectroscopy with high reproducibility.

Atomic force microscopy (AFM)-based single molecule force spectroscopy is an ideal tool for investigating the interactions between a single polymer and ...