Name: coulomb explosion imaging as a tool to distinguish between stereoisomers
Uploaded: 2017-08-18T15:00:00
Description: Watch this Scientific Journal Video about Coulomb Explosion Imaging as a Tool to Distinguish Between Stereoisomers at JoVE.com

We thank Robert Berger (Philipps-Universit&#228;t Marburg, Germany) for inspiring discussions about the interpretation of our data and molecular chirality in general. We are grateful to Julia Kiedrowski, Alexander Schie&#223;er and Michael Reggelin from TU Darmstadt (Germany), as well as Benjamin Spenger, Manuel Mazenauer and J&#252;rgen Stohner from ZHAW W&#228;denswil (Switzerland) for providing the sample.
The project was supported by the Hessen State Initiative for Scientific and Economic Excellence under the focus ELCH (Electron dynamics of chiral systems) and the Federal Ministry of Education and Research (BMBF). MS acknowledges financial support by the Adolf Messer foundation.

Caution: Make sure to be familiar with all possible hazards connected with the experiment and in the laboratory. The procedure below includes class-IV lasers, high voltage and vacuum. Consult the material safety data sheet (MSDS) for the species to be investigated.

1. Preparation
<ol>
	<li>Preparatory considerations 
	NOTE: Before the actual experiment starts, two main choices have to be made; the first regarding the possible species under investigations...

<ol>
	<li>G&#252;bitz, G., Schmid, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chiral+Separation+by+Chromatographic+and+Electromigration+Techniques.+A+Review.">Chiral Separation by Chromatographic and Electromigration Techniques. A Review.</a> Biopharm. Drug Disposition. 22, 291-336 (2001).</li><li>Berova, N., Polaravapu, P. L., Nakanishi, K., Woody, R. 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> Comprehensive Chiroptical Spectroscopy. , (2012).</li><li>Bijvoet, J. M., Peerdeman, A. F., van Bommel, A. 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+the+Absolute+Configuration+of+Optically+Active+Compounds+by+means+of+X-rays.">Determination of the Absolute Configuration of Optically Active Compounds by means of X-rays.</a> Nature. 168 (4268), 271-272 (1951).</li><li>Pitzer, 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=Direct+Determination+of+Absolute+Molecular+Stereochemistry+in+Gas+Phase+by+Coulomb+Explosion+Imaging.">Direct Determination of Absolute Molecular Stereochemistry in Gas Phase by Coulomb Explosion Imaging.</a> Science. 341 (6150), 1096-1100 (2013).</li><li>Pitzer, 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=Absolute+Configuration+from+Different+Multifragmentation+Pathways+in+Light-Induced+Coulomb+Explosion+Imaging.">Absolute Configuration from Different Multifragmentation Pathways in Light-Induced Coulomb Explosion Imaging.</a> Chem Phys Chem. 17 (16), 2465-2472 (2016).</li><li>Vager, Z., Naaman, R., Kanter, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coulomb+Explosion+Imaging+of+small+molecules.">Coulomb Explosion Imaging of small molecules.</a> Science. 244 (4903), 426-431 (1989).</li><li>Herwig, P. H., 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=Imaging+the+Absolute+Configuration+of+a+Chiral+Epoxide+in+the+Gas+Phase.">Imaging the Absolute Configuration of a Chiral Epoxide in the Gas Phase.</a> Science. 342 (6162), 1084-1186 (2013).</li><li>D&#246;rner, R., 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=Cold+Target+Recoil+Ion+Momentum+Spectroscopy:+a+'momentum+microscope'+to+view+atomic+collision+dynamics.">Cold Target Recoil Ion Momentum Spectroscopy: a 'momentum microscope' to view atomic collision dynamics.</a> Physics Reports. 330, 95-192 (2000).</li><li>Ullrich, 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=Recoil-ion+and+electron+momentum+spectroscopy:+reaction-microscopes.">Recoil-ion and electron momentum spectroscopy: reaction-microscopes.</a> Rep. Prog. Phys. 66 (9), 1463-1545 (2003).</li><li>Jagutzki, O., 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=Multiple+Hit+Readout+of+a+Microchannel+Plate+Detector+With+a+Three-Layer+Delay-Line+Anode.">Multiple Hit Readout of a Microchannel Plate Detector With a Three-Layer Delay-Line Anode.</a> IEEE Trans Nucl Sci. 49 (5), 2477-2483 (2002).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> RoentDek GmbH MCP Delay Line Detector Manual. , (2017).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> RoentDek GmbH The RoentDek Constant Fraction Discriminators CFD8c, CFD7x, CFD4c, CFD1c and CFD1x. , (2017).</li><li>Zeller, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+He2+quantum+halo+state+using+a+free+electron+laser.">Imaging the He2 quantum halo state using a free electron laser.</a> PNAS. 113 (51), 14651-14655 (2016).</li><li>Pitzer, 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=Stereochemical+configuration+and+selective+excitation+of+the+chiral+molecule+halothane.">Stereochemical configuration and selective excitation of the chiral molecule halothane.</a> J. Phys. B.: At. Mol. Opt. Phys. 49 (23), 234001 (2016).</li><li>Hine, J., Dowell, A. M., Singley, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+Dihalides+as+Intermediates+in+the+Basic+Hydrolysis+of+Haloforms:+IV+Relative+Reactivities+of+Haloforms.">Carbon Dihalides as Intermediates in the Basic Hydrolysis of Haloforms: IV Relative Reactivities of Haloforms.</a> J. Am. Soc. Chem. 78, 479-482 (1956).</li><li>Wales, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+coincidence+detection+algorithm+for+improving+detection+rates+in+coulomb+explosion+imaging.">A coincidence detection algorithm for improving detection rates in coulomb explosion imaging.</a> Nucl. Instr. Meth. in Phys. Res. A. 667, 11-15 (2012).</li><li>Kastirke, 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> Konstruktion und Aufbau einer UHV-tauglichen COLTRIMS-Kammer. , (2014).</li><li>Calegari, F., 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=Charge+migration+induced+by+attosecond+pulses+in+bio-relevant+molecules.">Charge migration induced by attosecond pulses in bio-relevant molecules.</a> J. Phys. B.: At. Mol. Opt. Phys. 49 (14), 142001 (2016).</li><li>Gaie-Levrel, F., Garcia, G. A., Schwell, M., Nahon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VUV+state-selected+photoionization+of+thermally-desorbed+biomolecules+by+coupling+an+aerosol+source+to+an+imaging+photoelectron/photoion+coincidence+spectrometer:+case+of+the+amino+acids+tryptophan+and+phenylalanine.">VUV state-selected photoionization of thermally-desorbed biomolecules by coupling an aerosol source to an imaging photoelectron/photoion coincidence spectrometer: case of the amino acids tryptophan and phenylalanine.</a> Phys. Chem. Chem. Phys. 13, 7024-7036 (2010).</li><li>Ablikim, U., 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=Identification+of+absolute+geometries+of+cis+and+trans+molecular+isomers+by+Coulomb+Explosion+Imaging.">Identification of absolute geometries of cis and trans molecular isomers by Coulomb Explosion Imaging.</a> Sci. Rep. 6, 38202 (2016).</li></ol>

Due to the variety of components, a COLTRIMS setup requires a rather high level of technical expertise, especially in the areas of vacuum technique, particle detection, fast electronics and data analysis. Before turning to the investigation of complex species, it should thus be thoroughly checked if the setup is running properly, e.g. by performing and analyzing a measurement on a diatomic or triatomic species.
Optimizing the intensity and duration of the laser pulses and the overlap ...

Chirality is a feature of our nature that has been fascinating researchers for more than 150 years. In the 19th century, Pasteur, van&#39;t Hoff and others discovered that molecules can occur in two mirror image structures that are not super-imposable - like our left and right hands. This property was termed &#39;chiral&#39;, from the Greek word for &#39;hand&#39;.
So far, no difference in thermodynamic properties or in energy levels of left- and right-handed forms (the two &#39;enantiomers&#39;) has been found. In order to analyze the handedness of a given sample and to separate the enantiomers, interaction with other chiral molecules can be used, as is for example done in various chromatographical approaches.1 Chiroptical methods such as (vibrational) circular dichroism, (V)CD, and optical rotatory dispersion, ORD, are routinely employed to distinguish between enantiomers.2
When it comes to the determination of the microscopic structure, these techniques require additional information, e.g. from quantum-chemical calculations. The only technique that is widely accepted to directly determine the absolute configuration is anomalous X-ray diffraction.3
It has recently been shown that the absolute configuration of simple chiral species can be determined by Coulomb Explosion Imaging.4,5 In this approach, molecules in the gas phase are multiply ionized so that the remaining cores strongly repel each other. This repulsion leads to fast fragmentation (&#39;explosion&#39;) of the molecules. The direction and the magnitude of the fragment momenta correlate to the structure of the molecule &#8211; for small molecules, the momentum directions correspond surprisingly well to the bond axes. Coulomb Explosion for molecular structure determination has been pioneered using molecular ion beams from an accelerator.6 This beam foil technique has recently also been applied for chiral recognition.7
Contrary to anomalous X-ray diffraction, the sample must not be crystalline but provided in the gas phase. This makes the Coulomb Explosion approach ideal for volatile species and thus complementary to X-ray diffraction. In certain cases, the handedness can even be determined for individual molecules.
In practice, the exact reconstruction of the molecular structure has proven difficult even for methane derivatives, e.g. molecules with a central carbon and different substituents. This is attributed to the fact that the interaction between the fragments is not exactly Coulombic and that not all bonds break simultaneously. In order to obtain stereochemical information, especially to distinguish between enantiomers, this reconstruction is fortunately not necessary. Instead, the momentum vectors of different fragments can be correlated to yield a quantity that is distinct for the left- and right-handed molecules. To get reliable results, at least four fragment momenta have to be recorded.
In order to measure this momentum information, the fragments from one &#8211; and only one &#8211; molecular break-up have to be detected in a single measurement step. This condition is usually referred to as &#39;coincident detection&#39;. In addition, the emission directions have to be analyzed, which amounts in practice to record the time and the position of the fragment impact in a list-mode data format.
In atomic and molecular physics, techniques have been developed that implement this approach of measurement by employing electrostatic spectrometers for mass separation and time- and position-sensitive multi-hit detectors. The most prominent example is the COLTRIMS (Cold Target Recoil Ion Momentum Spectroscopy) setup &#8211; also known as Reaction Microscope.8,9 A sketch for this kind of experiment is given in Figure 1. Contrary to a standard COLTRIMS that can record electrons as well, Coulomb Explosion Imaging requires only the ion detector.
Spectrometer and detector are mounted under ultra-high vacuum (&#60;1 x 10-9 hPa) to avoid creation of ions from residual gas. Single molecules of the sample are provided via a gaseous free molecular jet created by supersonic expansion: By virtue of the vapor pressure, the molecules expand through a small nozzle (around 50 &#181;m diameter) into the vacuum. This part of the experiment, the source chamber, is separated from the interaction region by usually two skimmers and differentially pumped stages. An additional differentially pumped section is located behind the interaction region to dump the gas jet and thus avoid background gas in the interaction region.
The ionizing radiation intersects with the molecular jet under 90&#176;. Most laboratories nowadays use femtosecond laser pulses, although synchrotron radiation, fast ions or electron impact are possible &#39;projectiles&#39; to induce Coulomb Explosion.
The following protocol makes the assumption that a running setup for coincident imaging of ions and a femtosecond laser are available in the lab. The peak intensity needed to induce Coulomb Explosion into four or even five fragments must be on the order of 6 x 1014 W/cm2. To avoid exceedingly long measurements, the repetition rate of the laser should be 10 kHz or more. This is crucial because, on the one hand, coincident detection can only be ascertained if the probability for fragmentation in the laser focus is significantly below 1 per laser pulse (ideally not more than 10%). The total fragmentation rate, on the other hand, should not be lower than a few kHz because the share of relevant multifragmentation pathways is usually less than 10-4. As encouraging fact, it should be mentioned that in principle already a single fragmentation event is sufficient to identify the configuration of an enantiopure sample, and that detection of a few hundred allows to determine the abundance of the enantiomers in a sample of unknown enantiomeric composition.

<table border="0" cellpadding="0" cellspacing="0" width="599">
	<tbody>
		<tr height="31">
			<td height="31" style="height:31px;width:179px;">CHBrCl2</td>
			<td style="width:93px;">SigmaAldrich</td>
			<td style="width:147px;">139181-10G</td>
			<td style="width:180px;">or other suitable sample</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:179px;">femtosecond laser system</td>
			<td>KMLabs</td>
			<td style="width:147px;">Wyvern500</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:179px;">High-reflective mirrors</td>
			<td>EKSMA</td>
			<td style="width:147px;">042-0800</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:179px;">mirror mounts</td>
			<td>Newport</td>
			<td style="width:147px;">U100-A-LH-2K&#160;&#160;</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:179px;">focusing mirror (protected silver, f = 75 mm)</td>
			<td>Thorlabs&#160;</td>
			<td style="width:147px;">CM254-075-P01</td>
			<td style="width:180px;">(if available: f = 60 mm)</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:179px;">COLTRIMS spectrometer, including electronics and data acquisition system</td>
			<td>RoentDek</td>
			<td style="width:147px;">custom</td>
			<td style="width:180px;">contrary to the standard COLTRIMS, only one detector is needed</td>
		</tr>
	</tbody>
</table>

coulomb explosion imaging as a tool to distinguish between stereoisomers

This article shows how the COLTRIMS (Cold Target Recoil Ion Momentum Spectroscopy) or the "reaction microscope" technique can be used to distinguish between enantiomers (stereoisomers) of simple chiral species on the level of individual molecules. In this approach, a gaseous molecular jet of the sample expands into a vacuum chamber and intersects with femtosecond (fs) laser pulses. The high intensity of the pulses leads to fast multiple ionization, igniting a so-called Coulomb Explosion that produces several cationic (positively charged) fragments. An electrostatic field guides these cations onto time- and position-sensitive detectors. Similar to a time-of-flight mass spectrometer, the arrival time of each ion yields information on its mass. As a surplus, the electrostatic field is adjusted in a way that the emission direction and the kinetic energy after fragmentation lead to variations in the time-of-flight and in the impact position on the detector.
Each ion impact creates an electronic signal in the detector; this signal is treated by high-frequency electronics and recorded event by event by a computer. The registered data correspond to the impact times and positions. With these data, the energy and the emission direction of each fragment can be calculated. These values are related to structural properties of the molecule under investigation, i.e. to the bond lengths and relative positions of the atoms, allowing to determine molecule by molecule the handedness of simple chiral species and other isomeric features.

In this part, we show results obtained for halomethanes. These species are ideal for proof-of-principle experiments due to their simplicity and high vapor pressure. In the meantime, the more complex species halothane has been investigated using single soft-x-ray photons from a synchrotron source to induce multiple ionization.14
CHBrClF
Bromochlo...

Watch this Scientific Journal Video about Coulomb Explosion Imaging as a Tool to Distinguish Between Stereoisomers at JoVE.com

Coulomb Explosion Imaging as a Tool to Distinguish Between Stereoisomers

For small chiral species, Coulomb Explosion Imaging provides a new approach to determine the handedness of individual molecules.

This article shows how the COLTRIMS (Cold Target Recoil Ion Momentum Spectroscopy) or the "reaction microscope" technique can be used to distinguish ...

The overall goal of this experiment is to use Coulomb explosion to image the handedness of individual molecules in the gas phase. This method is a new approach to address important questions in stereochemistry. In particular, it allows to determine the absolute configuration of small chiral species and their photoinduced dynamics.The main advantage of this technique is that energy and emission direction of all charge fragments from a molecular breakup can be recorded in coincidence. We have developed this multicoincidence imaging technique over a period for more than 25 years to learn more about and to measure the secrets of stereochemistry on the basis of single molecules. The heart of the experiment is a setup for coincident imaging of ions and a femtosecond laser such as this COLTRIMS apparatus.The femtosecond laser beam enters from an adjacent room. The basics of the COLTRIMS technique are depicted in this animation. A molecular beam enters the vacuum chamber through a nozzle and a skimmer.A laser beam crosses the molecular beam at right angles and leads to ionization and subsequent fragmentation. The electric field in the spectrometer guides the ions to the detector where the times and the positions of their impacts are recorded. Prepare the setup for operation and check the vacuum in the interaction chamber.The pressure should be less than 10 to the negative nine hectopascal. With the laser on and at low intensity perform the beam alignment. Be sure the beam enters the experiment chamber and is reflected from the chamber's focusing mirror.Use a beam card to check that the incoming and outgoing beams coincide spatially. Block the laser beam before continuing. Prepare to turn on the spectrometer and detector power supplies.First, turn off the vacuum gauges in the interaction chamber. Next, connect the amplified signal output of the microchannel plates to a fast oscilloscope. Now turn on the high voltage power supplies for the detector and the electric field in the spectrometer.Return to work with the laser beam by first unblocking it. Have a rotatable polarization filter ready to adjust the beam intensity. Place a power meter in the beam just before the chamber and use the filter to adjust the beam intensity to below 10 to the 14th watts per square centimeter.For this setup, this corresponds to 100 milliwatts on the power meter. Remove the power meter and observe the oscilloscope trace. It is crucial to check the signal quality and the detector settings.If too much noise is recorded the true coincidences are very difficult to find. The signal frequency should be about 5%of the laser repetition rate. The signal should have one peak of several hundred millivolts without any ringing.The width of the signals from the microchannel plate should not exceed 10 nanoseconds. Now turn to the the data acquisition software. There, display an image of the detector, which should be a circle with a diffused spot at its center due to the laser.At this point, provide one bar of argon from a gas cylinder as the source for the gas jet. Observe the detector image live in the data acquisition software while adjusting the focusing mirror. If a narrow spot appears overlap between the molecular jet and the laser beam has been located.The goal is to maximize the jet beam overlap and increase the counts in the jet spot. After optimizing the jet beam overlap, prepare the sample for use in the experiment. This sample is already in a cylinder that is compatible with the setup.First, cool the cylinder in liquid nitrogen for one to two minutes to avoid sample losses. Connect the cooled cylinder to the jet system and tighten the connection to make it vacuum-proof. Next, open the valve to pump the system for a few seconds to remove air.Wait for the sample to return to room temperature before opening the valve to the jet nozzle. The pressure in the source chamber should increase. Return to the data acquisition software.At the computer, verify that a jet spot is still visible, and identify the most prominent peaks in the time-of-flight spectrum. Continue to work in the data acquisition software. For analysis, make a plot using the time-of-flight of the first detected ion along the x-axis and the time-of-flight of the second detected ion along the y-axis.When two fragments have been detected in coincidence, they appear as regions with many counts. If two fragments add up to the parent mass, they fulfill momentum conservation and show up as sharp diagonal lines. Perform a time-of-flight analysis of four particles with a plot created by summing the times-of-flight for the first and second hits to use as the x-axis value and summing the times-of-flight for the third and fourth hits to use for the y-axis value.The positions and shapes of the resulting structures reveal information of the masses and momenta. Adjust the laser power so that the number of counts in the spectrum is optimized. When optimizing the rate for the relevant breakup we have to kep in mind that the overall rate should not exceed 10%of the laser repetition rate to avoid false coincidences.These data are from a synthetic racemic mixture of bromochlorofluoromethane. Only events where a fragmentation into five singly charged ions were recorded are shown. The horizontal axis is calculated using a triple product of the momentum vectors of the halogen ions.The S enantiomer is at the left of the histogram. The diagram illustrates its associated angle data. The R enantiomer is at the right of the histogram along with a diagram to illustrate its angle.The laser repetition rate was 100 kilohertz and the measurement took about 11 hours. This animation demonstrates how it is possible to use the recorded momentum vector data of the fragments to transform their random orientation in the jets into a molecular coordinate system. After watching this video, you should have an idea how Coulomb explosion imaging is performed using the COLTRIMS technique.Recording the momentum vectors of four or five fragments in coincidence allows a detailed view into the molecule. Extracting this information from the raw data however, requires a lot of effort in the analysis step. The secrets of life science in the world of molecules occurs on a time scale of a billionth of a billionth of a second and our method can reveal and visualize these fast dynamics.

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Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in potentially heterogeneous specimens. Nucleic acids can be detected via several established approaches, including analytical methods that are spectrophotometric (e.g., A260), fluorometric (e.g., binding of fluorescent dyes), or colorimetric (nucleoside-specific chromogenic chemical reactions).1 Though it cannot readily distinguish RNA from DNA, the A260/A280 ratio is commonly employed, as it offers a simple and rapid2 assessment of the relative content of nucleic acid, which absorbs predominantly near 260 nm and protein, which absorbs primarily near 280 nm. Ratios &lt; 0.8 are taken as indicative of 'pure' protein specimens, while pure nucleic acid (NA) is characterized by ratios &gt; 1.53.
However, there are scenarios in which the protein/NA content cannot be as clearly or reliably inferred from simple uv-vis spectrophotometric measurements. For instance, (i) samples may contain one or more proteins which are relatively devoid of the aromatic amino acids responsible for absorption at &asymp;280 nm (Trp, Tyr, Phe), as is the case with some small RNA-binding proteins, and (ii) samples can exhibit intermediate A260/A280 ratios (~0.8 &lt; ~1.5), where the protein/NA content is far less clear and may even reflect some high-affinity association between the protein and NA components. For such scenarios, we describe herein a suite of colorimetric assays to rapidly distinguish RNA, DNA, and reducing sugars in a potentially mixed sample of biomolecules. The methods rely on the differential sensitivity of pentoses and other carbohydrates to Benedict's, Bial's (orcinol), and Dische's (diphenylamine) reagents; the streamlined protocols can be completed in a matter of minutes, without any additional steps of having to isolate the components. The assays can be performed in parallel to differentiate between RNA and DNA, as well as indicate the presence of free reducing sugars such as glucose, fructose, and ribose (Figure 1).

A suite of colorimetric assays is described for rapidly distinguishing protein, RNA, DNA, and reducing sugars in potentially heterogeneous biomolecular samples.

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in ...

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Real-time monitoring allows for fast optimization of reactions performed using continuous-flow processing. Here the preparation of 3-acetylcoumarin is used as an example. The apparatus for performing in-situ Raman monitoring is described, as are the steps required to optimize the reaction.

By using inline monitoring, it is possible to optimize reactions performed using continuous-flow processing in a simple and rapid way. It is also possible ...

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Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further ...

Spin Saturation Transfer Difference NMR (SSTD NMR): A New Tool to Obtain Kinetic Parameters of Chemical Exchange Processes

This detailed protocol describes the new Spin Saturation Transfer Difference Nuclear Magnetic Resonance protocol (SSTD NMR), recently developed in our group to study processes of mutual-site chemical exchange that are difficult to analyze by traditional methods. As the name suggests, this method combines the Spin Saturation Transfer method used for small molecules, with the Saturation Transfer Difference (STD) NMR method employed for the study of protein-ligand interactions, by measuring transient spin saturation transfer along increasing saturation times (build-up curves) in small organic and organometallic molecules undergoing chemical exchange.
Advantages of this method over existing ones are: there is no need to reach coalescence of the exchanging signals; the method can be applied as long as one signal of the exchanging sites is isolated; there is no need to measure T1 or reach steady state saturation; rate constant values are measured directly, and T1 values are obtained in the same experiment, using only one set of experiments.
To test the method, we have studied the dynamics of the hindered rotation of N,N-dimethylamides, for which much data is available for comparison. The thermodynamic parameters obtained using SSTD are very similar to the reported ones (spin-saturation transfer techniques and line-shape analysis). The method can be applied to more challenging substrates that cannot be studied by previous methods.
We envisage that the simple experimental set up and the wide applicability of the method to a great variety of substrates will make this a common technique amongst organic and organometallic chemists without extensive expertise in NMR.

Spin Saturation Transfer Difference NMR SSTD NMR: A New Tool to Obtain Kinetic Parameters of Chemical Exchange Processes

A detailed protocol describing the SSTD NMR method is presented here to help new users apply this new method to obtain the kinetic parameters of their own systems undergoing chemical exchange.

This detailed protocol describes the new Spin Saturation Transfer Difference Nuclear Magnetic Resonance protocol (SSTD NMR), recently developed in our ...

NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential nutrients. The objective of this work is to demonstrate the qualitative and quantitative analysis of encapsulated fish oil supplements using high-resolution 1H and 13C NMR spectroscopy utilizing two different NMR instruments; a 500 MHz and an 850 MHz instrument. Both proton (1H) and carbon (13C) NMR spectra can be used for the quantitative determination of the major constituents of fish oil supplements. Quantification of the lipids in fish oil supplements is achieved through integration of the appropriate NMR signals in the relevant 1D spectra. Results obtained by 1H and 13C NMR are in good agreement with each other, despite the difference in resolution and sensitivity between the two nuclei and the two instruments. 1H NMR offers a more rapid analysis compared to 13C NMR, as the spectrum can be recorded in less than 1 min, in contrast to 13C NMR analysis, which lasts from 10 min to one hour. The 13C NMR spectrum, however, is much more informative. It can provide quantitative data for a greater number of individual fatty acids and can be used for determining the positional distribution of fatty acids on the glycerol backbone. Both nuclei can provide quantitative information in just one experiment without the need of purification or separation steps. The strength of the magnetic field mostly affects the 1H NMR spectra due to its lower resolution with respect to 13C NMR, however, even lower cost NMR instruments can be efficiently applied as a standard method by the food industry and quality control laboratories.

Here, high-resolution 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy was used as a rapid and reliable tool for quantitative and qualitative analysis of encapsulated fish oil supplements.

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential ...

Electrochemical Impedance Spectroscopy as a Tool for Electrochemical Rate Constant Estimation

Electrochemical impedance spectroscopy (EIS) was used for advanced characterization of organic electroactive compounds along with cyclic voltammetry (CV). In the case of fast reversible electrochemical processes, current is predominantly affected by the rate of diffusion, which is the slowest and limiting stage. EIS is a powerful technique that allows separate analysis of stages of charge transfer that have different AC frequency response. The capability of the method was used to extract the value of charge transfer resistance, which characterizes the rate of charge exchange on the electrode-solution interface. The application of this technique is broad, from biochemistry up to organic electronics. In this work, we are presenting the method of analysis of organic compounds for optoelectronic applications.

Electrochemical impedance spectroscopy (EIS) of species that undergo reversible oxidation or reduction in solution was used for determination of rate constants of oxidation or reduction.

Electrochemical impedance spectroscopy (EIS) was used for advanced characterization of organic electroactive compounds along with cyclic voltammetry (CV). ...

Color Spot Test As a Presumptive Tool for the Rapid Detection of Synthetic Cathinones

Synthetic cathinones are a large class of new psychoactive substances (NPS) that are increasingly prevalent in drug seizures made by law enforcement and other border protection agencies globally. Color testing is a presumptive identification technique indicating the presence or absence of a particular drug class using rapid and uncomplicated chemical methods. Owing to their relatively recent emergence, a color test for the specific identification of synthetic cathinones is not currently available. In this study, we introduce a protocol for the presumptive identification of synthetic cathinones, employing three aqueous reagent solutions: copper(II) nitrate, 2,9-dimethyl-1,10-phenanthroline (neocuproine) and sodium acetate. Small pin-head sized amounts (approximately 0.1-0.2 mg) of the suspected drugs are added to the wells of a porcelain spot plate, and each reagent is then added dropwise sequentially before heating on a hotplate. A color change from very light blue to yellow-orange after 10 min indicates the likely presence of synthetic cathinones. The highly stable and specific test reagent has the potential for use in the presumptive screening of unknown samples for synthetic cathinones in a forensic laboratory. However, the nuisance of an added heating step for the color change result limits the test to laboratory application and decreases the likelihood of an easy translation to field testing.

Here we present a simple, inexpensive, and selective chemical spot test protocol for the detection of synthetic cathinones, a class of new psychoactive substances. The protocol is suitable for use in various areas of law enforcement that encounter illicit material.

Synthetic cathinones are a large class of new psychoactive substances (NPS) that are increasingly prevalent in drug seizures made by law enforcement and ...

Automation of a Positron-emission Tomography (PET) Radiotracer Synthesis Protocol for Clinical Production

The development of new positron-emission tomography (PET) tracers is enabling researchers and clinicians to image an increasingly wide array of biological targets and processes. However, the increasing number of different tracers creates challenges for their production at radiopharmacies. While historically it has been practical to dedicate a custom-configured radiosynthesizer and hot cell for the repeated production of each individual tracer, it is becoming necessary to change this workflow. Recent commercial radiosynthesizers based on disposable cassettes/kits for each tracer simplify the production of multiple tracers with one set of equipment by eliminating the need for custom tracer-specific modifications. Furthermore, some of these radiosynthesizers enable the operator to develop and optimize their own synthesis protocols in addition to purchasing commercially-available kits. In this protocol, we describe the general procedure for how the manual synthesis of a new PET tracer can be automated on one of these radiosynthesizers and validated for the production of clinical-grade tracers. As an example, we use the ELIXYS radiosynthesizer, a flexible cassette-based radiochemistry tool that can support both PET tracer development efforts, as well as routine clinical probe manufacturing on the same system, to produce [18F]Clofarabine ([18F]CFA), a PET tracer to measure in vivo deoxycytidine kinase (dCK) enzyme activity. Translating a manual synthesis involves breaking down the synthetic protocol into basic radiochemistry processes that are then translated into intuitive chemistry &#34;unit operations&#34; supported by the synthesizer software. These operations can then rapidly be converted into an automated synthesis program by assembling them using the drag-and-drop interface. After basic testing, the synthesis and purification procedure may require optimization to achieve the desired yield and purity. Once the desired performance is achieved, a validation of the synthesis is carried out to determine its suitability for the production of the radiotracer for clinical use.

Automation of a Positron-emission Tomography PET Radiotracer Synthesis Protocol for Clinical Production

Positron-emission tomography (PET) imaging sites that are involved in multiple early clinical research trials need robust and versatile radiotracer manufacturing capabilities. Using the radiotracer [18F]Clofarabine as an example, we illustrate how to automate the synthesis of a radiotracer using a flexible, cassette-based radiosynthesizer and validate the synthesis for clinical use.

The development of new positron-emission tomography (PET) tracers is enabling researchers and clinicians to image an increasingly wide array of biological ...

Solid Phase 11C-Methylation, Purification and Formulation for the Production of PET Tracers

Routine production of radiotracers used in positron emission tomography (PET) mostly relies on wet chemistry where the radioactive synthon reacts with a non-radioactive precursor in solution. This approach necessitates purification of the tracer by high performance liquid chromatography (HPLC) followed by reformulation in a biocompatible solvent for human administration. We recently developed a novel 11C-methylation approach for the highly efficient synthesis of carbon-11 labeled PET radiopharmaceuticals, taking advantage of solid phase cartridges as disposable &#34;3-in-1&#34; units for the synthesis, purification and reformulation of the tracers. This approach obviates the use of preparative HPLC and reduces the losses of the tracer in transfer lines and due to radioactive decay. Furthermore, the cartridge-based technique improves synthesis reliability, simplifies the automation process and facilitates compliance with the Good Manufacturing Practice (GMP) requirements. Here, we demonstrate this technique on the example of production of a PET tracer Pittsburgh compound B ([11C]PiB), a gold standard in vivo imaging agent for amyloid plaques in the human brains.

Solid Phase 11C-Methylation, Purification and Formulation for the Production of PET Tracers

We report an efficient carbon-11 radiolabeling technique to produce clinically relevant tracers for Positron Emission Tomography (PET) using solid phase extraction cartridges. 11C-methylating agent is passed through a cartridge preloaded with precursor and successive elution with aqueous ethanol provides chemically and radiochemically pure PET tracers in high radiochemical yields.

Routine production of radiotracers used in positron emission tomography (PET) mostly relies on wet chemistry where the radioactive synthon reacts with a ...

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 ...