Name: analysis of volatile and oxidation sensitive compounds using a cold inlet system and electron impact mass spectrometry
Uploaded: 2014-09-05T15:00:00
Description: Watch this Scientific Journal Video about Analysis of Volatile and Oxidation Sensitive Compounds Using a Cold Inlet System and Electron Impact Mass Spectrometry at JoVE.com

JS is indebted to Prof. B. Hoge of the Inorganic Chemistry department, Bielefeld University, for the idea of establishing the presented inlet system. The analyzed phosphane was a generous gift from Prof. B. Hoge. Sample preparation of the analyzed compound was performed by M. Wiesemann. Photographs of the phospane were taken by Dr. J. Bader. The mechanical workshop of the faculty of chemistry is acknowledged for the manufacturing of the interface and the glass workshop of the faculty of chemistry for the manufacturing of the lockable test tubes with flanges. Prof. B. Hoge and Prof. H. Gr&#246;ger are acknowledged for funding of this publication.

1. Sample Preparation
<ol>
	<li>Use custom-made lockable test tubes with a flange (Figure 1) for transportation and transfer of the samples into the mass spectrometer. Prior to filling with sample, evacuate the lockable test tubes attached to a multiple manifold Schlenk line and remove residual water by heating with a heat gun. Vent the test tube with dry Argon and evacuate again, while heating.</li>
	<li>Immerse the lockable test tube into a cold trap filled with liquid nitrogen (CAUTION: Be careful w...

<ol>
	<li>Field, F. H., Franklin, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Electron Impact phenomena and the Properties of Gaseous Ions Revised Edition. , (1957).</li><li>Schaeffer, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Improved+Mass+Spectrometer+Ion+Source.">An Improved Mass Spectrometer Ion Source.</a> Rev. Sci. Instrum. 25, 660-662 (1954).</li><li>Yamashita, M., Fenn, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospray+Ion+Source+-+Another+Variation+of+the+Free-Jet+Theme.">Electrospray Ion Source - Another Variation of the Free-Jet Theme.</a> J. Phys. Chem. 88, 4451-4459 (1984).</li><li>Lubben, A. T., McIndoe, J. S., Weller, A. 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H., Shiea, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+reactive+1,2,3-hexatriene-5-one+monomer+by+low-temperature+atmospheric+pressure+ionization+mass+spectrometry.">Detection of reactive 1,2,3-hexatriene-5-one monomer by low-temperature atmospheric pressure ionization mass spectrometry.</a> Rapid Communications in Mass Spectrometry. 12, 931-934 (1998).</li><li>Wang, C. 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The acquisition of mass spectra from compounds which decompose under standard sample preparation procedures is presented in this protocol. The presented technique is designed for the analysis of metal organyls, silanes and phosphane, which are highly susceptible to oxidation and/or hydrolysis, making it interesting especially for inorganic chemists. In order to achieve optimal results, vacuum or air-free conditions have to be preserved throughout the analysis. Therefore the protocol should be followed meticulously. In ca...

The analysis of compounds, such as metal organyls, silanes, or phosphanes by mass spectrometry is not always feasible. Several of these compounds are known to decompose rapidly when in contact with air. Therefore the most crucial steps when measuring mass spectra are sample preparation, the transfer of the analyte into the mass spectrometer and ion generation in the absence of air. In this protocol, we describe a strategy to meet these requirements and present an inlet system, which makes it possible to obtain mass spectra of volatile compounds previously not to be analyzed by mass spectrometry due to their difficult handling and rapid decomposition under ambient conditions. Thereby, unambiguous identification of novel or existing volatile metal organyls, silanes and phosphanes, susceptible to oxidation or hydrolysis, can now be performed with the assistance of mass spectrometry. There are two requirements which have to be met in order to analyze compounds which are susceptible to oxidation or hydrolysis: sample preparation and ion generation under inert conditions. The last premise can be easily met using a mass spectrometer with an ion source operating under vacuum. This is the case with most matrix-assisted-laser-desorption/ionization (MALDI) mass spectrometers and with all electron impact ionization (EI) mass spectrometers1,2. Electrospray ionization (ESI) is not readily compatible for the analysis of compounds susceptible to oxidation or hydrolysis, as the ionization process takes place under ambient conditions3. However, for some compounds which react not vigorously with oxygen or water, the drying and nebulizing gas with which most ESI sources are operated is sufficient for analysis by mass spectrometry4. This is also the case for Ionization strategies similar to ESI, e.g., low-temperature ESI, low-temperature atmospheric pressure ionization, and low-temperature liquid secondary ion mass spectrometry5-7. In contrast, sample preparation and transfer into the ion source under inert conditions is much more challenging. Both MALDI and ESI instruments have been coupled with glove boxes in order to enable sample preparation of compounds susceptible to oxidation and/or hydrolysis in an inert atmosphere4,8. The mass spectrometer is interfaced to the glove box either with a transfer capillary (ESI) or directly attached to the glove box (MALDI). The coupling of a glove box to a mass spectrometer via a transfer capillary would also be possible using another ionization strategy &#8211; liquid injection field desorption/ionization (LIFDI) &#8211; with which the analysis of sensitive compounds was reported9,10.
Additionally, MALDI and LIFDI are not suitable for the analysis of highly volatile compounds. MALDI requires the co-crystallization of the analyte with a matrix and LIFDI requires the deposition of the analyte onto an emitter from a solution. With both ionization strategies it is very likely that the analyte will evaporate along with the solvent. In contrast to MALDI instruments, EI mass spectrometers usually offer several methods for introducing the sample into the ion source: the direct inlet probe (small amounts of solids, oils, or waxes are deposited into an aluminum crucible which is introduced using a push rod), a septum inlet (for liquids), or coupling with a gas chromatograph. Again, at least part of the sample transfer takes place under ambient conditions and is difficult to perform under an inert atmosphere.
In the 1960&#8217;s, a sample inlet system was presented which enables the introduction of samples under vacuum into the ion source of an EI instrument &#8211; the all-glass heated inlet system (AGHIS)11,12. Here, the sample was located inside a sealed piece of glass capillary, which was inserted into the AGHIS. Subsequently, the AGHIS was evacuated and the glass container with the sample was broken. The AGHIS was then heated to evaporate the sample which reached the ion source of an EI mass spectrometer by means of a leak. When the glass capillary with the sample was prepared inside a glove box, the sample could be introduced into the mass spectrometer without any contact to air. However, the AGHIS is an apparatus which is not commercially available and difficult to assemble even for a skilled glassblower workshop. Due to the large dimensions switching between direct inlet using a push rod and AGHIS is not straight forward.
In our mass spectrometry lab, we developed a similar inlet system in the style of the AGHIS. However, as it is not possible to heat the inlet system, the analyte has to exhibit a certain volatility in order to enter the ion source of the mass spectrometer. The volatility of the analyte has to be sufficient, to allow for the transfer of the compound under vacuum at liquid nitrogen temperature &#8211; either by boiling or sublimation. The custom-made inlet system consists of a stainless steel plate, which is positioned at the direct inlet system, a stainless steel tube with a needle valve, and a flange, to which a lockable test tube containing the sample can be attached. The installation of the cold inlet system requires no modifications to the mass spectrometer (Autospec X, Vacuum Generators, now Waters Corp., Manchester, UK) &#8211; switching between cold inlet system and direct inlet using a push rod can be performed easily within seconds.
The presented inlet system is of particular use when metal organyls, silanes, or phosphanes, susceptible to oxidation or hydrolysis, have to be analyzed. These compounds are commonly analyzed using nuclear magnetic resonance (NMR) spectroscopy or&#160;infrared (IR) spectroscopy. Unfortunately, these methods allow not always for an unambiguous identification of a compound because they yield incomplete information, e.g., when elements such as chlorine or bromine are part of the molecule. Gas electron diffraction on the other hand is able to provide detailed information about the analyte, however, the method is very time consuming, sample preparation is difficult, and only few groups are able to conduct these analysis13,14. Here, the cold inlet system for the analysis of metal organyls, silanes, or phosphanes, susceptible to oxidation or hydrolysis by EI mass spectrometry is of great use for (in)organic chemists enabling the unambiguous identification of novel compounds by supplying them with information regarding the mass of a molecule and of characteristic fragment ions. The only prerequisite for the measurement of mass spectra for a substance is a certain volatility at reduced pressure.

<table border="0" cellpadding="0" cellspacing="0" width="971">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Name of Reagent/Material</td>
			<td style="width:71px;">Company</td>
			<td style="width:155px;">Catalog Number</td>
			<td style="width:435px;">Comments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:311px;">VG Autospec X</td>
			<td colspan="2">Micromass Co. UK Ltd (now Waters)</td>
			<td style="width:435px;">other EI mass spectrometers with direct inlet using a push rod should also be compatible with this technique</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Lockable test tubes with flange</td>
			<td style="width:71px;">-</td>
			<td style="width:155px;">-</td>
			<td style="width:435px;">costum made, teflon tap should be used for locking the test tube</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:311px;">Interface for lockable test tubes</td>
			<td style="width:71px;">-</td>
			<td style="width:155px;">-</td>
			<td style="width:435px;">costum made , interface is prepared from stainless steel. Needle valve has to be included into the interface-design!</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:311px;">Schlenk line</td>
			<td style="width:71px;">-</td>
			<td style="width:155px;">-</td>
			<td style="width:435px;">costum made, has to include vacuum pump for evacuation of thest tubes and cold trap with liquid nitrogen for trapping of the sample</td>
		</tr>
	</tbody>
</table>

analysis of volatile and oxidation sensitive compounds using a cold inlet system and electron impact mass spectrometry

This video presents a protocol for the mass spectrometrical analysis of volatile and oxidation sensitive compounds using electron impact ionization. The analysis of volatile and oxidation sensitive compounds by mass spectrometry is not easily achieved, as all state-of-the-art mass spectrometric methods require at least one sample preparation step, e.g., dissolution and dilution of the analyte (electrospray ionization), co-crystallization of the analyte with a matrix compound (matrix-assisted laser desorption/ionization), or transfer of the prepared samples into the ionization source of the mass spectrometer, to be conducted under atmospheric conditions. Here, the use of a sample inlet system is described which enables the analysis of volatile metal organyls, silanes, and phosphanes using a sector field mass spectrometer equipped with an electron impact ionization source. All sample preparation steps and the sample introduction into the ion source of the mass spectrometer take place either under air-free conditions or under vacuum, enabling the analysis of compounds highly susceptible to oxidation. The presented technique is especially of interest for inorganic chemists, working with metal organyls, silanes, or phosphanes, which have to be handled using inert conditions, such as the Schlenk technique. The principle of operation is presented in this video.

An EI mass spectrum of tris(trifluoromethyl)phosphane is presented in Figure 3, a compound, which decomposes rapidly when in contact with air (Figure 4). The presented interface allows for the straight forward measurement of mass spectra for these compounds. The operation of the novel interface is easy and fast and presents no obstacle when operating the mass spectrometer with the routinely applied direct inlet using the push rod.
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Watch this Scientific Journal Video about Analysis of Volatile and Oxidation Sensitive Compounds Using a Cold Inlet System and Electron Impact Mass Spectrometry at JoVE.com

Analysis of Volatile and Oxidation Sensitive Compounds Using a Cold Inlet System and Electron Impact Mass Spectrometry

This video presents a protocol for the mass spectrometrical analysis of volatile and oxidation sensitive compounds using electron impact ionization. The presented technique is especially of interest for inorganic chemists, working with metal organyls, silanes, or phosphanes which have to be handled using inert conditions, such as the Schlenk technique.

This video presents a protocol for the mass spectrometrical analysis of volatile and oxidation sensitive compounds using electron impact ionization. The ...

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Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

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This paper presents a protocol for the visualization of gaseous streams of an ambient ionization source using schlieren photography and mass spectrometry.

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A method for species identification of botanical material by direct analysis in real time-high resolution mass spectrometry and multivariate statistical analysis is presented.

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A protocol for determining the effectiveness of photocatalysts in degrading indoor air concentration (ppb) model volatile organic carbons such as 2-propanol is described.

We demonstrate a versatile protocol to be used for determining the effectiveness of photocatalysts in degrading indoor air concentration (ppb) volatile ...

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Chemical analysis of volatile and semivolatile compounds dissolved in liquid samples can be challenging. The dissolved components need to be brought to the gas phase, and efficiently transferred to a detection system. Fizzy extraction takes advantage of the effervescence phenomenon. First, a carrier gas (here, carbon dioxide) is dissolved in the sample by applying overpressure and stirring the sample. Second, the sample chamber is decompressed abruptly. Decompression leads to the formation of numerous carrier gas bubbles in the sample liquid. These bubbles assist the release of the dissolved analyte species from the liquid to the gas phase. The released analytes are immediately transferred to the atmospheric pressure chemical ionization interface of a triple quadrupole mass spectrometer. The ionizable analyte species give rise to mass spectrometric signals in the time domain. Because the release of the analyte species occurs over short periods of time (a few seconds), the temporal signals have high amplitudes and high signal-to-noise ratios. The amplitudes and areas of the temporal peaks can then be correlated with concentrations of the analytes in the liquid samples subjected to fizzy extraction, which enables quantitative analysis. The advantages of fizzy extraction include: simplicity, speed, and limited use of chemicals (solvents).

Fizzy extraction is a new laboratory technique for analysis of volatile and semivolatile compounds. A carrier gas is dissolved in the liquid sample by applying overpressure and stirring the sample. The sample chamber is then decompressed. The analyte species are liberated to the gas phase due to effervescence.

Chemical analysis of volatile and semivolatile compounds dissolved in liquid samples can be challenging. The dissolved components need to be brought to ...

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A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

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A protocol is described for the manual synthesis of oligo-peptoids followed by sequence analysis by mass spectrometry.

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...

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Here we describe a high-throughput method for comprehensive drug surveillance that allows for the improved resolution and detection of large panels of drugs of abuse and their metabolites, with quality control based on multisegment injection-capillary electrophoresis-mass spectrometry.

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug ...

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, metal-binding interactions, and conformational shape. Coupling ESI with ion mobility-mass spectrometry (IM-MS) provides an instrumental technique that allows for simultaneous measurement of a peptide&#8217;s mass-to-charge (m/z) and collision cross section (CCS) that relate to its stoichiometry, protonation state, and conformational shape. The overall charge of a peptide complex is controlled by the protonation of 1) the peptide&#8217;s acidic and basic sites and 2) the oxidation state of the metal ion(s). Therefore, the overall charge state of a complex is a function of the pH of the solution that affects the peptides metal ion binding affinity. For ESI-IM-MS analyses, peptide and metal ions solutions are prepared from aqueous-only solutions, with the pH adjusted with dilute aqueous acetic acid or ammonium hydroxide. This allows for pH dependence and metal ion selectivity to be determined for a specific peptide. Furthermore, the m/z and CCS of a peptide complex can be used with B3LYP/LanL2DZ molecular modeling to discern binding sites of the metal ion coordination and tertiary structure of the complex. The results show how ESI-IM-MS can characterize the selective chelating performance of a set of alternative methanobactin peptides and compare them to the copper-binding peptide methanobactin.

Ion mobility-mass spectrometry and molecular modeling techniques can characterize the selective metal chelating performance of designed metal-binding peptides and the copper-binding peptide methanobactin. Developing new classes of metal chelating peptides will help lead to therapeutics for diseases associated with metal ion misbalance.

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, ...

Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of protein therapeutics and the determination of the conformational transition pathways critical for biological functions, ranging from molecular recognition to enzymatic catalysis. Hydrogen/deuterium exchange (HDX) reaction coupled with top-down mass spectrometric (MS) analysis provides a means to characterize protein higher-order structures and dynamics in a conformer-specific manner. The conformational resolving power of this technique is highly dependent on the efficiencies of separating protein states at the intact protein level and minimizing the residual non-deuterated protic content during the HDX reactions. 
Here we describe a capillary electrophoresis (CE)-based variant of the HDX MS approach that aims to improve the conformational resolution. In this approach, proteins undergo HDX reactions while migrating through a deuterated background electrolyte solution (BGE) during the capillary electrophoretic separation. Different protein states or proteoforms that coexist in solution can be efficiently separated based on their differing charge-to-size ratios. The difference in electrophoretic mobility between proteins and protic solvent molecules minimizes the residual non-deuterated solvent, resulting in a nearly complete deuterating environment during the HDX process. The flow-through microvial CE-MS interface allows efficient electrospray ionization of the eluted protein species following a rapid mixing with the quenching and denaturing modifier solution at the outlet of the sprayer. The online top-down MS analysis measures the global deuteration level of the eluted intact protein species, and subsequently, the deuteration of their gas-phase fragments. This paper demonstrates this approach in differential HDX for systems, including the natural protein variants coexisting in milk.

Presented here is a protocol for a capillary electrophoresis-based hydrogen/deuterium exchange (HDX) approach coupled with top-down mass spectrometry. This approach characterizes the difference in higher-order structures between different protein species, including proteins in different states and different proteoforms, by conducting concurrent differential HDX and electrophoretic separation.

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of ...