The authors wish to acknowledge start-up funding from The University of Toledo, as well as funds from the American Chemical Society&#39;s Herman Frasch Foundation in partial support of this work. Mr. Thomas Kina is acknowledged for his assistance with developing a suitable pressure gauge for measuring the reaction pressures. Mr. Steve Modar is thanked for useful discussions.

CAUTION: 1) The following protocols have been deemed safe through repeated trials. However, caution should be exercised when sealing vials, throughout the reaction, and especially when opening the reactions, as inhomogeneity in the reaction vials may lead to equipment failure. Vials should be inspected for physical defects prior to use. Vials should be placed behind some form of blast shield or hood sash immediately after sealing to prevent incidents should the vials fail. 2) Although there is little chance for asphyxiation due to the small quantities of CO2 used, reactions should be set-up as well as opened in a well-ventilated area or in a fume hood. 3) Dry ice is a cryogen and can cause serious tissue damage. Care should therefore be exercised while manipulating it to avoid frostbite, such as limiting direct contact or using cryogenic gloves. 4) Dry ice will condense water vapor, meaning that prior to use, the dry ice should be mechanically exfoliated to ensure the mass is of CO2(s) only. This can be achieved by simply rubbing the dry ice between one&#8217;s fingers, or more safely, rubbing it between one&#8217;s fingers with a protective layer such as a glove or towel.
1. Reaction in a 7.5 mL Vial (Air Not Excluded)
<ol>
	<li>Add a stir bar to a dry 7.5 mL vial.</li>
	<li>Add palladium acetate (6.7 mg, 0.03 mmol) to the vial.</li>
	<li>Add silver trifluoroacetate (99.9 mg, 0.45 mmol) to the vial.</li>
	<li>Add phenyl iodide (92.3 mg, 0.45 mmol) to the vial.</li>
	<li>Add tert-amyl amine (26.3 mg, 0.30 mmol) to the vial.</li>
	<li>Add acetic acid (1.0 mL) to the vial. 
	Note: The ratio of solution volume to vial size is important, as the immediate sublimation of CO2 upon addition of dry ice can mechanically displace solvent if too much is used relative to the size of the reaction vessel.</li>
	<li>Add deionized water (21.7 &#956;L, 12.1 mmol) to the vial.</li>
	<li>Weigh dry ice (26.3 mg, 0.60 mmol), and immediately add the dry ice to the vial, while ensuring to also immediately seal the vial with a PTFE-lined cap. 
	Note: The whole operation should be performed within approximately 5 seconds to prevent sublimation and escape of the small amount of CO2 added (this is slowed by the formation of frozen acetic acid around the dry ice). The amount of CO2 added will be an approximate value, and in our hands a deviation of a few mg is permissible.</li>
	<li>Stir the sealed reaction vial for 15 minutes at room temperature.</li>
	<li>Transfer the reaction vessel to a pre-heated plate at 110 &#176;C and stir for 14 hours before allowing to cool.</li>
	<li>Upon cooling, carefully open the vial to vent CO2.</li>
	<li>Remove all of the volatiles in vacuo. 
	Note: This operation can be performed in the vial, or the solution can be transferred to a larger round bottom flask.</li>
	<li>Add 1.2 M HCl(aq) (6 mL) to the reaction mixture and stir open to air for 15 minutes.</li>
	<li>Transfer the aqueous fraction to a separatory funnel, washing with additional 1.2 M HCl (4 mL), and extract with a 1:1 diethyl ether/hexanes mixture (3 x 8 mL). 
	Note: This organic wash contains excess phenyl iodide and other neutral by-products, and can be disposed of.</li>
	<li>Neutralize and make basic the aqueous solution by addition of saturated NH4OH(aq) (10 mL is a good starting point).</li>
	<li>Extract the aqueous layer with dichloromethane (2 x 10 mL).</li>
	<li>Dry the combined organic fractions over Na2SO4, then filter into a tared sample vial.</li>
	<li>Evaporate the solvent in vacuo, giving the product (2-Methyl-4-phenyl-butanamine) as a yellow oil.</li>
</ol>
2. Reaction in a 7.5 mL Vial (Purging Conditions &#8211; Air Excluded)
<ol>
	<li>Add a stir bar to a dry 7.5 mL vial.</li>
	<li>Add palladium acetate (6.7 mg, 0.03 mmol) to the vial.</li>
	<li>Add silver trifluoroacetate (99.9 mg, 0.45 mmol) to the vial.</li>
	<li>Add phenyl iodide (92.3 mg, 0.45 mmol) to the vial.</li>
	<li>Add tert-amyl amine (26.3 mg, 0.30 mmol) to the vial.</li>
	<li>Add acetic acid (1.0 mL) to the vial. 
	Note: The ratio of solution volume to vial size is important, as the immediate sublimation of CO2 upon addition of dry ice can mechanically displace solvent if too much is used relative to the size of the reaction vessel.</li>
	<li>Add deionized water (21.7 &#956;L, 12.1 mmol) to the vial.</li>
	<li>Tare the vial on a balance, add approximately 98 mg of dry ice, and then allow the CO2 to sublimate off until a final mass of approximately 26 mg is achieved, followed by immediately sealing the vial with a PTFE-lined cap. 
	Note: If desirable, this step can be performed with a greater mass of dry ice to further exclude air from the vial. It is noteworthy that this may introduce water, and thus may not be the most effective strategy for water sensitive reactions.</li>
	<li>Stir the sealed reaction vial for 15 minutes at room temperature.</li>
	<li>Transfer the reaction vessel to a pre-heated plate at 110 &#176;C and stir for 14 hours before allowing to cool.</li>
	<li>Upon cooling, carefully open the vial to vent CO2.</li>
	<li>Remove all of the volatiles in vacuo. 
	Note: This operation can be performed in the vial, or the solution can be transferred to a larger round bottom flask.</li>
	<li>Add 1.2 M HCl(aq) (6 mL) to the reaction mixture, and stir open to air for 15 minutes.</li>
	<li>Transfer the aqueous fraction to a separatory funnel, washing with additional 1.2 M HCl (4 mL), and extract with a 1:1 diethyl ether/hexanes mixture (3 x 8 mL). 
	Note: This organic wash contains excess phenyl iodide and other neutral by-products, and can be disposed of.</li>
	<li>Neutralize and make basic the aqueous solution by addition of saturated NH4OH(aq) (10 mL is a good starting point).</li>
	<li>Extract the aqueous layer with dichloromethane (2 x 10 mL).</li>
	<li>Dry the combined organic fractions over Na2SO4, then filter into a tared sample vial.</li>
	<li>Evaporate the solvent in vacuo, giving the product (2-Methyl-4-phenyl-butanamine) as a yellow oil.</li>
</ol>
3. Reaction in a 40 mL Vial (Air Not Excluded)
<ol>
	<li>Add a stir bar to a dry 40 mL vial.</li>
	<li>Add palladium acetate (33.5 mg, 0.15 mmol) to the vial.</li>
	<li>Add silver trifluoroacetate (499.5 mg, 2.25 mmol) to the vial.</li>
	<li>Add phenyl iodide (461.5 mg, 2.25 mmol) to the vial.</li>
	<li>Add tert-amyl amine (131.5 mg, 1.5 mmol) to the vial.</li>
	<li>Add acetic acid (5.0 mL) to the vial. 
	Note: The ratio of solution volume to vial size is important, as the immediate sublimation of CO2 upon addition of dry ice can mechanically displace solvent if too much is used relative to the size of the reaction vessel.</li>
	<li>Add deionized water (108.5 &#956;L, 6.02 mmol) to the vial.</li>
	<li>Weigh dry ice (131.5 mg, 3.0 mmol), and immediately add the dry ice to the vial, while ensuring to also immediately seal the vial with a PTFE-lined cap. 
	Note: The whole operation should be performed within approximately 5 seconds to prevent sublimation and escape of the small amount of CO2 added (this is slowed by the formation of frozen acetic acid around the dry ice). The amount of CO2 added will be an approximate value, and in our hands a deviation of a few mg is permissible.</li>
	<li>Stir the sealed reaction vial for 15 minutes at room temperature.</li>
	<li>Transfer the reaction vessel to a pre-heated plate at 110 &#176;C and stir for 14 hours before allowing to cool.</li>
	<li>Upon cooling, carefully open the vial to vent CO2.</li>
	<li>Remove all of the volatiles in vacuo. 
	Note: This operation can be performed in the vial, or the solution can be transferred to a larger round bottom flask.</li>
	<li>Add 1.2 M HCl(aq) (30 mL) to the reaction mixture and stir open to air for 15 minutes.</li>
	<li>Transfer the aqueous fraction to a separatory funnel, washing with additional 1.2 M HCl (20 mL), and extract with a 1:1 diethyl ether/hexanes mixture (3 x 8 mL). 
	Note: This organic wash contains excess phenyl iodide and other neutral by-products, and can be disposed of.</li>
	<li>Neutralize and make basic the aqueous solution by addition of saturated NH4OH(aq) (10 mL is a good starting point).</li>
	<li>Extract the aqueous layer with dichloromethane (2 x 20 mL).</li>
	<li>Dry the combined organic fractions over Na2SO4, then filter into a tared sample vial.</li>
	<li>Evaporate the solvent in vacuo, giving the product (2-Methyl-4-phenyl-butanamine) as a yellow oil.</li>
</ol>
4. Reaction in a 35 mL Pressure Tube (Air not excluded)
<ol>
	<li>Add a stir bar to a dry 35 mL pressure tube.</li>
	<li>Add palladium acetate (6.7 mg, 0.03 mmol) to the pressure tube.</li>
	<li>Add silver trifluoroacetate (132.5 mg, 0.6 mmol) to the pressure tube.</li>
	<li>Add phenyl iodide (183.6 mg, 0.9 mmol) to the pressure tube.</li>
	<li>Add 2-methyl-N-(3-methylbenzyl)butan-2-amine (57.4 mg, 0.3 mmol) to the pressure tube.</li>
	<li>Add acetic acid (1.0 mL) to the vial, followed by 1,1,1,3,3,3,-hexafluoroisopropanol (1.0 mL). 
	Note: The ratio of solution volume to vial size is important, as the immediate sublimation of CO2 upon addition of dry ice can mechanically displace solvent if too much is used relative to the size of the reaction vessel.</li>
	<li>Add deionized water (21.7 &#956;L, 1.2 mmol) to the pressure tube.</li>
	<li>Weigh dry ice (1.32 g, 30 mmol), and immediately add the dry ice to the pressure tube, while ensuring to also immediately seal the pressure tube with the appropriate Teflon screw cap. 
	Note: The whole operation should be performed within approximately 5 seconds to prevent sublimation and escape of the small amount of CO2 added (this is slowed by the formation of frozen acetic acid around the dry ice). The amount of CO2 added will be an approximate value, and in our hands a deviation of a few mg is permissible.</li>
	<li>Stir the sealed reaction vessel for 15 minutes at room temperature.</li>
	<li>Transfer the reaction vessel to a pre-heated plate at 90 &#176;C and stir for 24 hours before allowing to cool.</li>
	<li>Upon cooling, put a towel or padded glove over the cap, and carefully open the pressure tube to vent CO2.</li>
	<li>Remove all of the volatiles in vacuo. 
	Note: This operation can be performed in the pressure tube with an appropriate adaptor, or the solution can be transferred to a larger round bottom flask.</li>
	<li>Add 1.2 M HCl(aq) (12 mL) to the reaction mixture and stir open to air for 15 minutes.</li>
	<li>Transfer the aqueous fraction to a separatory funnel, washing with additional 1.2 M HCl (8 mL), and extract with a 1:1 diethyl ether/hexanes mixture (3 x 8 mL). 
	Note: This organic wash contains excess phenyl iodide and other neutral by-products and can be disposed of.</li>
	<li>Neutralize and make basic the aqueous solution by addition of saturated NH4OH(aq) (10 mL is a good starting point).</li>
	<li>Extract the aqueous layer with dichloromethane (2 x 10 mL).</li>
	<li>Dry the combined organic fractions over Na2SO4, then filter into a tared sample vial.</li>
	<li>Evaporate the solvent in vacuo, giving the product (2-Methyl-N-(3-methylbenzyl)-4-phenylbutan-2-amine) as a yellow oil.</li>
</ol>

The use of CO2 as a directing group for C-H activation of Lewis basic substrates is currently the focus of United States Provisional Patent #62/608,074.

Using the van der Waals Equation of State, the approximate pressure of these systems can be calculated45
Eq. 1:&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;<img alt="Equation" src="/files/ftp_upload/58281/58281eq1.jpg" />
Under the conditions in Protocol 1, we can assume 26.3 mg of CO2 gives n =5.98 x 10-4 mols
<img alt="Equation 1b" src="/files/ftp_upload/5828...

The use of gaseous compounds in chemical reactions typically requires specialized equipment and procedures1,2. At bench scale, some gases can be added directly from a tank using a high pressure regulator3. An alternative method is to condense the gas under cryogenic conditions4,5. Although useful, these strategies require the use of specialized pressure reactors with valves, which can be cost prohibitive for running numerous reactions in parallel. This can therefore greatly slow the rate at which reaction screening can proceed. As a result, chemists have found it desirable to introduce these compounds using alternative methods. Ammonia can be added to reactions using different ammonium carboxylate salts, taking advantage of the weak equilibrium between these salts and free ammonia6. Transfer hydrogenation is an important strategy for reduction reactions of olefins, carbonyl, and nitro groups that circumvents the use of flammable hydrogen gas with compounds such as ammonium formate or hydrazine as carriers of H27. Another gas of interest in this area is carbon monoxide8 &#8211; CO can be generated in situ by liberation from metal carbonyl complexes9,10, or alternatively it can be generated by decarbonylation from sources such as formates and formamides11,12,13 or chloroform14,15.
One gas which has not enjoyed significant development in this respect is carbon dioxide16. One reason for this is that many transformations that involve CO2 also require high temperatures and pressures, and thus are automatically relegated to specialized reactors17,18. Recent efforts to develop more reactive catalysts, however, have facilitated running many of these reactions under atmospheric pressures of CO219,20,21,22. We recently discovered a reaction in which carbon dioxide could be used to mediate the &#947;-C(sp3)&#8211;H arylation of aliphatic amines23. This strategy was expected to combine the benefits of a static directing group approach including amide24,25,26,27,28, sulfonamide29,30,31,32, thiocarbonyl33,34, or hydrazone35-based directing groups (chemical robusticity), with the ease of a transient directing group (decreased step economy)36,37,38,39.
Although the reaction could occur under atmospheric pressure of CO2, the need for a Schlenk set-up to screen reactions proved prohibitively slow. Furthermore, increasing the pressure slightly led to improved reaction yields, but could not be easily achieved using a Schlenk line. We therefore sought an alternative strategy, and subsequently identified that dry ice could be easily used as a solid source of CO2 that could be added to a variety of reaction vessels to introduce the necessary amount of carbon dioxide to achieve moderate pressures (Figure 1). Though underutilized in synthesis, a similar strategy is fairly common as a method to generate liquid CO2 for chromatography and extraction applications40,41,42,43,44. Utilizing this strategy allowed our group to rapidly screen large numbers of reactions in parallel, while the ability to access moderate CO2 pressures of between 2-20 atmospheres were critical to improving the yields of the reactions. Under these conditions, both primary (1&#176;) and secondary (2&#176;) amines can be arylated with electron rich and electron poor aryl halides.

<table>
	<tbody>
		<tr>
			<td>7.5 mL Sample Vial with Screw Cap (Thermoset)</td>
			<td>Qorpak</td>
			<td>GLC-00984</td>
			<td>Can be reused.</td>
		</tr>
		<tr>
			<td>40 mL Sample Vial with Screw Cap (Thermoset)</td>
			<td>Qorpak</td>
			<td>GLC-01039</td>
			<td>Can be reused.</td>
		</tr>
		<tr>
			<td>Pressure Tube, #15 Thread, 7&#34; Long, 25.4 mm O.D.</td>
			<td>Ace Glass</td>
			<td>8648-06</td>
			<td>Can be reused.</td>
		</tr>
		<tr>
			<td>Pie-Block for 2 Dram Vials</td>
			<td>ChemGlass</td>
			<td>CG-1991-P14</td>
			<td>Can be reused.</td>
		</tr>
		<tr>
			<td>Pie-Block for 10 Dram Vials</td>
			<td>ChemGlass</td>
			<td>CG-1991-P12</td>
			<td>Can be reused.</td>
		</tr>
		<tr>
			<td>3.2 mm PTFE Disposable Stir Bars</td>
			<td>Fisher</td>
			<td>14-513-93</td>
			<td>Can be reused.</td>
		</tr>
		<tr>
			<td>C-MAG HS 7 Control Hotplate</td>
			<td>IKA</td>
			<td>20002695</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical Weighing Balance</td>
			<td>Sartorius</td>
			<td>QUINTIX2241S</td>
			<td></td>
		</tr>
		<tr>
			<td>Double-Ended Micro-Tapered Spatula</td>
			<td>Fisher Scientific</td>
			<td>21-401-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Hei-VAP Advantage - Hand Lift Model with G5 Dry Ice Condenser Rotary Evaporator</td>
			<td>Heidolph</td>
			<td>561-01500-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Bump Trap 14/20 Joint</td>
			<td>ChemGlass</td>
			<td>CG-1322-01</td>
			<td></td>
		</tr>
		<tr>
			<td>tert-Amyl amine</td>
			<td>Alfa Aesar</td>
			<td>B24639-14</td>
			<td>Used as received.</td>
		</tr>
		<tr>
			<td>2-Methyl-N-(3-methylbenzyl)butan-2-amine</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Prepared from reductive amination of tert-amyl amine and 3-tolualdehyde in the presence of sodium borohydride in methanol.</td>
		</tr>
		<tr>
			<td>Palladium Acetate</td>
			<td>Chem-Impex International, Inc.</td>
			<td>4898</td>
			<td>Used as received.</td>
		</tr>
		<tr>
			<td>Silver Trifluoroacetate</td>
			<td>Oakwood Chemicals</td>
			<td>007271</td>
			<td>Used as received.</td>
		</tr>
		<tr>
			<td>Phenyl Iodide</td>
			<td>Oakwood Chemicals</td>
			<td>003461</td>
			<td>Used as received.</td>
		</tr>
		<tr>
			<td>Acetic Acid</td>
			<td>Fisher Chemical</td>
			<td>A38</td>
			<td>Used as received.</td>
		</tr>
		<tr>
			<td>1,1,1,3,3,3-Hexafluoroisopropanol</td>
			<td>Oakwood Chemicals</td>
			<td>003409</td>
			<td>Used as received.</td>
		</tr>
		<tr>
			<td>Deionized Water</td>
			<td></td>
			<td></td>
			<td>Obtained from in-house deionized water system.</td>
		</tr>
		<tr>
			<td>Dry Ice</td>
			<td>Carbonic Enterprises Dry Ice Inc.</td>
			<td></td>
			<td>Non-food grade dry ice.</td>
		</tr>
		<tr>
			<td>Concentrated Hydrochloric Acid</td>
			<td>Fisher Chemical</td>
			<td>A144SI</td>
			<td>Diluted to a 1.2 M solution prior to use.</td>
		</tr>
		<tr>
			<td>Diethyl Ether, Certified</td>
			<td>Fisher Chemical</td>
			<td>E138</td>
			<td>Used as received.</td>
		</tr>
		<tr>
			<td>Hexanes, Certified ACS</td>
			<td>Fisher Chemical</td>
			<td>H292</td>
			<td>Used as received.</td>
		</tr>
		<tr>
			<td>Saturated Ammonium Hydroxide</td>
			<td>Fisher Chemical</td>
			<td>A669</td>
			<td>Used as received.</td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Fisher Chemical</td>
			<td>D37</td>
			<td>Used as received.</td>
		</tr>
		<tr>
			<td>Sodium Sulfate, Anhydrous</td>
			<td>Oakwood Chemicals</td>
			<td>044702</td>
			<td>Used as received.</td>
		</tr>
		<tr>
			<td>250 mL Separatory Funnel</td>
			<td></td>
			<td></td>
			<td>Prepared in-house by staff glassblower.</td>
		</tr>
		<tr>
			<td>100 mL Round Bottom Flask</td>
			<td></td>
			<td></td>
			<td>Prepared in-house by staff glassblower.</td>
		</tr>
		<tr>
			<td>Scientific Disposable Funnel</td>
			<td>Caplugs</td>
			<td>2085136030</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate Glass Scintillation Vials, 20 mL</td>
			<td>Fisher Scientific</td>
			<td>03-337-15</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mm O.D. Thin Walled Precision NMR Tubes</td>
			<td>Wilmad</td>
			<td>666000575</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform-d</td>
			<td>Cambridge Isotope Laboratories, Inc.</td>
			<td>DLM-7</td>
			<td>Used as received.</td>
		</tr>
	</tbody>
</table>

achieving moderate pressures in sealed vessels using dry ice as a solid co2 source

Herein is presented a general strategy to perform reactions under mild to moderate CO2 pressures with dry ice. This technique obviates the need for specialized equipment to achieve modest pressures, and can even be used to achieve higher pressures in more specialized equipment and sturdier reaction vessels. At the end of the reaction, the vials can be easily depressurized by opening at room temperature. In the present example CO2 serves as both a putative directing group as well as a way to passivate amine substrates, thereby preventing oxidation during the organometallic reaction. In addition to being easily added, the directing group is also removed under vacuum, obviating the need for extensive purification to remove the directing group. This strategy allows the facile &#947;-C(sp3)-H arylation of aliphatic amines and has the potential to be applied to a variety of other amine-based reactions.

Following these protocols, it is possible to charge a reaction vial with an appropriate amount of carbon dioxide to achieve chemical reactions that require CO2 atmospheres. The pressure achieved in Step 1 is calculated to be approximately 3 atmospheres (see discussion for determination of this value), although due to partial solvation, the observed pressure is in the vicinity of 2 atmospheres at room temperature, and should be approximately 2.6 atmospheres under the reaction co...

Watch this Scientific Journal Video about Achieving Moderate Pressures in Sealed Vessels Using Dry Ice As a Solid CO2 Source at JoVE.com

Achieving Moderate Pressures in Sealed Vessels Using Dry Ice As a Solid CO2 Source

Here we present a protocol for performing reactions in simple reaction vessels under low-to-moderate pressures of CO2. The reactions can be performed in a variety of vessels simply by administering the carbon dioxide in the form of dry ice, without the need for costly or elaborate equipment or set-ups.

Achieving Moderate Pressures in Sealed Vessels Using Dry Ice As a Solid CO2 Source

Herein is presented a general strategy to perform reactions under mild to moderate CO2 pressures with dry ice. This technique obviates the need for ...

achieving-moderate-pressures-sealed-vessels-using-dry-ice-as-solid

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Chemistry

9.9K Views.  University of Toledo. Here we present a protocol for performing reactions in simple reaction vessels under low-to-moderate pressures of CO2. The reactions can be performed in a variety of vessels simply by administering the carbon dioxide in the form of dry ice, without the need for costly or elaborate equipment or set-ups.

Video: Achieving Moderate Pressures in Sealed Vessels Using Dry Ice As a Solid CO2 Source

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

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 ...

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

High-pressure Sapphire Cell for Phase Equilibria Measurements of CO2/Organic/Water Systems

The high pressure sapphire cell apparatus was constructed to visually determine the composition of multiphase systems without physical sampling. Specifically, the sapphire cell enables visual data collection from multiple loadings to solve a set of material balances to precisely determine phase composition. Ternary phase diagrams can then be established to determine the proportion of each component in each phase at a given condition. In principle, any ternary system can be studied although ternary systems (gas-liquid-liquid) are the specific examples discussed herein. For instance, the ternary THF-Water-CO2 system was studied at 25 and 40&#160;&#176;C and is described herein. Of key importance, this technique does not require sampling. Circumventing the possible disturbance of the system equilibrium upon sampling, inherent measurement errors, and technical difficulties of physically sampling under pressure is a significant benefit of this technique. Perhaps as important, the sapphire cell also enables the direct visual observation of the phase behavior. In fact, as the CO2 pressure is increased, the homogeneous THF-Water solution phase splits at about 2 MPa. With this technique, it was possible to easily and clearly observe the cloud point and determine the composition of the newly formed phases as a function of pressure.
The data acquired with the sapphire cell technique can be used for many applications. In our case, we measured swelling and composition for tunable solvents, like gas-expanded liquids, gas-expanded ionic liquids and Organic Aqueous Tunable Systems (OATS)1-4. For the latest system, OATS, the high-pressure sapphire cell enabled the study of (1) phase behavior as a function of pressure and temperature, (2) composition of each phase (gas-liquid-liquid) as a function of pressure and temperature and (3) catalyst partitioning in the two liquid phases as a function of pressure and composition. Finally, the sapphire cell is an especially effective tool to gather accurate and reproducible measurements in a timely fashion.

High-pressure Sapphire Cell for Phase Equilibria Measurements of CO2/Organic/Water Systems

The high pressure sapphire cell apparatus is a unique tool to study, without sampling, phase behavior under a wide range of pressures. Using a cathetometer, very precise volume measurements can be recorded to measure liquid expansion and phase composition. Thus, this synthetic method enables the study of (1) phase equilibria of multi-component mixtures and (2) the partition behavior of catalyst or model compounds as a function of pressure.

The high pressure sapphire cell apparatus was constructed to visually determine the composition of multiphase systems without physical sampling. ...

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over large or small sample areas. It has been applied in many areas of science, including materials science, geoscience, studying works of cultural heritage, and in chemical biology. In the case of chemical biology, for example, visualizing the metal distributions within cells allows us to study both naturally-occurring metal ions in the cells, as well as exogenously-introduced metals such as drugs and nanoparticles. Due to the fully hydrated nature of nearly all biological samples, cryo-fixation followed by imaging under cryogenic temperature represents the ideal imaging modality currently available. However, under the circumstances that such a combination is not easily accessible or practical, aldehyde based chemical fixation remains useful and sometimes inevitable. This article describes in as much detail as possible in the preparation of adherent mammalian cells by chemical fixation for X-ray fluorescent imaging.

Here, we present a protocol on how to determine the quantity and distribution of metals in a sample using synchrotron X-ray fluorescence. We focus on adherent cells, and describe the chemical fixation method to prepare this sample. We then describe how to mount and image the sample using synchrotron X-rays.

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over ...

Real-time Monitoring of Reactions Performed Using Continuous-flow Processing: The Preparation of 3-Acetylcoumarin as an Example

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 to ensure consistent product quality over time using this technique. We here show how to interface a commercially available flow unit with a Raman spectrometer. The Raman flow cell is placed after the back-pressure regulator, meaning that it can be operated at atmospheric pressure. In addition, the fact that the product stream passes through a length of tubing before entering the flow cell means that the material is at RT. It is important that the spectra are acquired under isothermal conditions since Raman signal intensity is temperature dependent. Having assembled the apparatus, we then show how to monitor a chemical reaction, the piperidine-catalyzed synthesis of 3-acetylcoumarin from salicylaldehyde and ethyl acetoacetate being used as an example. The reaction can be performed over a range of flow rates and temperatures, the in-situ monitoring tool being used to optimize conditions simply and easily.

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 ...

Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a technique used to measure the size distribution of polymer solutions or colloidal particles. Although this technique is widely used for the assessment of polymer solutions, it is difficult to measure the particle size in concentrated solutions due to the multiple scattering effect or strong light absorption. Therefore, the concentrated solutions should be diluted before measurement. Implementation of the confocal optical component in a dynamic light scattering microscope1 helps to overcome this barrier. Using such a microscopic system, both transparent and turbid systems can be analyzed under the same experimental setup without a dilution. As a representative example, a size distribution measurement of a temperature-responsive polymer solution was performed. The sizes of the polymer chains in an aqueous solution were several tens of nanometers at a temperature below the lower critical solution temperature (LCST). In contrast, the sizes increased to more than 1.0 &#181;m when above the LCST. This result is consistent with the observation that the solution turned turbid above the LCST.

A protocol for the direct measurement of particle size distribution in concentrated solutions using dynamic light scattering microscopy is presented.

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a ...

Quantifying X-Ray Fluorescence Data Using MAPS

The quantification of X-ray fluorescence (XRF) microscopy maps by fitting the raw spectra to a known standard is crucial for evaluating chemical composition and elemental distribution within a material. Synchrotron-based XRF has become an integral characterization technique for a variety of research topics, particularly due to its non-destructive nature and its high sensitivity. Today, synchrotrons can acquire fluorescence data at spatial resolutions well below a micron, allowing for the evaluation of compositional variations at the nanoscale. Through proper quantification, it is then possible to obtain an in-depth, high-resolution understanding of elemental segregation, stoichiometric relationships, and clustering behavior.
This article explains how to use the MAPS fitting software developed by Argonne National Laboratory for the quantification of full 2-D XRF maps. We use as an example results from a Cu(In,Ga)Se2 solar cell, taken at the Advanced Photon Source beamline 2-ID-D at Argonne National Laboratory. We show the standard procedure for fitting raw data, demonstrate how to evaluate the quality of a fit and present the typical outputs generated by the program. In addition, we discuss in this manuscript certain software limitations and offer suggestions for how to further correct the data to be numerically accurate and representative of spatially resolved, elemental concentrations.

Here, we demonstrate the use of the X-ray fluorescence fitting software, MAPS, created by Argonne National Laboratory for the quantification of fluorescence microscopy data. The quantified data that results is useful for understanding the elemental distribution and stoichiometric ratios within a sample of interest.

The quantification of X-ray fluorescence (XRF) microscopy maps by fitting the raw spectra to a known standard is crucial for evaluating chemical ...

Minimum Burning Pressures of Water-based Emulsion Explosives

This manuscript describes a protocol to measure the minimum pressure required for sustained burning of water-based emulsion explosives. Pumping water-based emulsion explosives for blasting applications can be very hazardous, as demonstrated by a number of pump accidents around the globe in the last decades, including some that resulted in fatalities. In Canada, the recognition of this hazard has led to the development of pumping guidelines that were endorsed by both the explosives industry and the Explosive Regulatory Division of the Canadian government. In these guidelines, it was noted that the minimum burning pressures (MBP) measured in a laboratory would provide a good guide to characterize the behaviour of these products in pumping systems. The same guidelines also call for the design of pump systems that prevent, whenever possible, pressures from exceeding the MBP of the product being pumped. At the time of publication of these guidelines, a methodology existed for measuring such MBP values but it had never been validated to measure the MBP of ammonium nitrate water-based emulsions (AWEs). AWEs are now used much more widely than any other water-based explosives and precursors in on-site bulk loading operations.
The Canadian Explosives Research Laboratory (CanmetCERL) has been conducting research over the last ten years to develop a validated testing protocol to measure and interpret representative MBP values for AWEs. The test, as it is performed today, will be described and the critical components will be justified by reference to recent published data. Results of MBP measurements, for a range of AWE products, will be presented. Inclusion of the MBP test in the test standards for the authorization of high explosives in Canada will also be discussed.

We present an apparatus based on hot-wire ignition in a pressurized enclosure and an associated methodology to measure the minimum pressure required to induce sustained combustion in water-based emulsion explosives. This method improves product characterization to allow one to use them more safely during pumping and mixing operations.

This manuscript describes a protocol to measure the minimum pressure required for sustained burning of water-based emulsion explosives. Pumping ...

Solid-phase Synthesis of [4.4] Spirocyclic Oximes

A convenient synthetic route for spirocyclic heterocycles is well sought after due to the molecule&#39;s potential use in biological systems. By means of solid-phase synthesis, regenerating Michael (REM) linker strategies, and 1,3-dipolar cycloaddition, a library of structurally similar heterocycles, both with and without a spirocyclic center, can be constructed. The main advantages of the solid-support synthesis are as follows: first, each reaction step can be driven to completion using a large excess of reagents resulting in high yields; next, the use of commercially available starting materials and reagents keep the costs low; finally, the reaction steps are easy to purify via simple filtration. The REM linker strategy is attractive because of its recyclability and traceless nature. Once a reaction scheme is completed, the linker can be reused multiple times. In a typical solid-phase synthesis, the product contains either a part of or the whole linker, which can prove undesirable. The REM linker is &#34;traceless&#34; and the point of attachment between the product and the polymer is indistinguishable. The high diastereoselectivity of the intramolecular 1,3-dipolar cycloaddition is well documented. Limited by the insolubility of the solid support, the reaction progression can only be monitored by a change in the functional groups (if any) via infrared (IR) spectroscopy. Thus, the structural identification of intermediates cannot be characterized by conventional nuclear magnetic resonance (NMR) spectroscopy. Other limitations to this method stem from the compatibilities of the polymer/linker to the desired chemical reaction scheme. Herein we report a protocol that allows for the convenient production of spirocyclic heterocycles that, with simple modifications, can be automated with high-throughput techniques.

Here we present a protocol to demonstrate an efficient method for the synthesis of spirocyclic heterocycles. The five-step process utilizes solid-phase synthesis and regenerating Michael linker strategies. Generally difficult to synthesize, we present a customizable method for the synthesis of spirocyclic molecules otherwise inaccessible to other modern approaches.

A convenient synthetic route for spirocyclic heterocycles is well sought after due to the molecule&#39;s potential use in biological systems. By means of ...

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 ...