Name: tuning a parallel segmented flow column and enabling multiplexed detection
Uploaded: 2015-12-15T15:00:00
Description: Watch this Scientific Journal Video about Tuning a Parallel Segmented Flow Column and Enabling Multiplexed Detection at JoVE.com

This work was supported by UWS and ThermoFisher Scientific. One of the authors (DK) acknowledges the receipt of an Australian Postgraduate Award.

Note: This protocol contains instructions on how to use a PSF column on a HPLC system coupled with multiple detectors for multiplexed detection. The protocol has been written assuming the reader has basic knowledge and experience in chromatography and various HPLC detection methods.
Caution: Please refer to material safety data sheets (MSDS) for all materials and reagents before use (i.e., MSDS for methanol). Ensure the use of all appropriate safety practices when handling solvents an...

<ol>
	<li>Camenzuli, M., Ritchie, H. J., Ladine, J. R., Shalliker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+design+of+a+new+concept+chromatography+column.">The design of a new concept chromatography column.</a> Analyst. 136 (24), 5127-5130 (2011).</li><li>Camenzuli, M., Ritchie, H. J., Ladine, J. R., Shalliker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+separation+performance+using+a+new+column+technology:+Parallel+segmented+outlet+flow.">Enhanced separation performance using a new column technology: Parallel segmented outlet flow.</a> J. Chromatogr, A. 1232, 47-51 (2012).</li><li>Camenzuli, M., Ritchie, H. J., Ladine, J. R., Shalliker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+flow+management+in+preparative+chromatographic+separations:+A+preliminary+investigation+into+enhanced+separation+using+a+curtain+flow+inlet+fitting+and+segmented+flow+outlet.">Active flow management in preparative chromatographic separations: A preliminary investigation into enhanced separation using a curtain flow inlet fitting and segmented flow outlet.</a> 35 (3), 410-415 (2012).</li><li>Camenzuli, M., Ritchie, H. J., Shalliker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradient+elution+chromatography+with+segmented+parallel+flow+column+technology:+A+study+on+4.6mm+analytical+scale+columns.">Gradient elution chromatography with segmented parallel flow column technology: A study on 4.6mm analytical scale columns.</a> J. Chromatogr., A. 1270, 204-211 (2012).</li><li>Camenzuli, M., Ritchie, H. J., Shalliker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+HPLC+separation+performance+using+parallel+segmented+flow+chromatography.">Improving HPLC separation performance using parallel segmented flow chromatography.</a> Microchem. J. 111, 3-7 (2013).</li><li>Shalliker, R. A., Ritchie, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segmented+flow+and+curtain+flow+chromatography:+Overcoming+the+wall+effect+and+heterogeneous+bed+structures.">Segmented flow and curtain flow chromatography: Overcoming the wall effect and heterogeneous bed structures.</a> J. Chromatogr, A. 1335, 122-135 (2014).</li><li>Camenzuli, M., Ritchie, H. J., Shalliker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+active+flow+technology+HPLC+columns+as+a+platform+for+multiplexed+detection.">Evaluating active flow technology HPLC columns as a platform for multiplexed detection.</a> Microchem. J. 110, 473-479 (2013).</li><li>Camenzuli, 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=Parallel+segmented+outlet+flow+high+performance+liquid+chromatography+with+multiplexed+detection.">Parallel segmented outlet flow high performance liquid chromatography with multiplexed detection.</a> Anal. Chim. Acta. 803, 154-159 (2013).</li><li>Shalliker, R. A., Camenzuli, M., Pereira, L., Ritchie, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+segmented+flow+chromatography+columns:+Conventional+analytical+scale+column+formats+presenting+as+a+'virtual'+narrow+bore+column.">Parallel segmented flow chromatography columns: Conventional analytical scale column formats presenting as a 'virtual' narrow bore column.</a> J. Chromatogr., A. 1262, 64-69 (2012).</li><li>Soliven, A., 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=Improving+the+performance+of+narrow-bore+HPLC+columns+using+active+flow+technology.">Improving the performance of narrow-bore HPLC columns using active flow technology.</a> Microchem. J. 116, 230-234 (2014).</li><li>Camenzuli, M., Ritchie, H. J., Dennis, G. R., Shalliker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+segmented+flow+chromatography+columns+with+multiplexed+detection:+An+illustration+using+antioxidant+screening+of+natural+products.">Parallel segmented flow chromatography columns with multiplexed detection: An illustration using antioxidant screening of natural products.</a> Microchem. J. 110, 726-730 (2013).</li></ol>

This study involves the characterization and profiling of coffee using HPLC with multiplexed detection employing a parallel segmented flow (PSF) column. Multiplexed HPLC using PSF columns enables the characterization and identification of key chemical entities by reducing the data complexity of the sample whilst obtaining a greater degree of molecule-specific information within a fraction of the time it takes using conventional multi-detection processes. The PSF column not only allows a platform for multiplexed detection...

Active Flow Technology Columns
Active flow technology (AFT) chromatography columns were recently developed to overcome inefficiency in separations associated with flow heterogeneity1-6 and also to enable multiplexed detection. In this particular communication we detail the operational process of the parallel segmented flow (PSF) column with multiplexed detection. The key functional advantages of the PSF column are: (1) the flow from the radial central region of the column bed is isolated from the peripheral or wall region of flow, (2) the volume of mobile phase that must be processed by a detection source is reduced, and (3) detection sources can be multiplexed to expand sample information without instilling detection delays across each detection process, or subsequently necessitating the splitting of a post column flow stream7,8. The key feature in the design of the PSF column that enables the advantage of multiplexed detection is the novel outlet fitting and frit assembly. Figure 1 is a photograph of the AFT column compared to a conventional column. It is important to understand that the splitting process obtained using parallel segmented flow columns is not the same as post column flow stream splitting. In a post column flow stream split the entire sample band from the leading edge to the extremities of the tail is sampled equally (i.e., axially), thereby, each flow stream is equal with respect to efficiency and sensitivity; the magnitude of the sensitivity thereby being divided by the number splits. In PSF, however, the splitting process samples the band radially, not axially. As such, the central port samples the peak apex &#8212; the most concentrated region of the peak. Thus, the sensitivity here is highest as the peak is not diluted by the diffuse tailing region. The sample eluting from the peripheral ports is not as efficient as in the central zone, but, since the band is sampled radially, rather than axially, the width of the peak is narrower than would be the case for a sampling process that divides the peak in the axial direction, i.e., a post column split. Hence the sensitivity with a concentration dependent detector is not reduced.
In the PSF column, the outlet fitting comprises multiple exit ports and on the inside of this end fitting there is housed an annular frit. The inner portion of this annular frit channels flow out of the column via the radial central outlet port, while the outer radial portion of the outlet frit channels flow out of the column via the peripheral or wall region outlet flow ports. The inner and outer portions of the outlet frit are separated by an impermeable barrier that prevents cross flow between these flow regions2. As a consequence of this design the central radial flow stream through the column bed is separated from the wall region flow inside the column. The relative portion of flow from these two regions can be varied to almost any desired ratio through pressure management in order to optimize various functional aspects of the column technology, such as separation efficiency or detection sensitivity. In essence, this design effectively establishes within the larger format column a &#39;virtual&#39; column, having a narrower internal diameter, and hence the column functions as a true wall-less column, overcoming column bed heterogeneity and the wall effects9,10.
The major benefits of PSF columns are improvements in column efficiency, minimization of the solvent processing for the detection source(s) and enabling multiplexed detection. However, an added advantage is that since the tailing and fronting regions of any band are removed from the overall elution profile the solute at elution or detection is present in a higher concentration than would otherwise be observed for the same solute injection and concentration load on a conventional column, depending on the segmentation ratio employed. As a consequence there is often observed a gain in signal intensity for separations conducted on PSF columns2. In fact, if the segmentation ratio is adjusted such that 25% of the flow exits from each of the four exit ports, the signal intensity that is observed using Ultra Violet (UV) detection shows practically the exact same signal intensity as apparent using a conventional column where the entire (100%) of the mobile phase is analyzed7. Furthermore, fine tuning of the outlet ratio between the central and wall flow regions allows the column efficiency to be optimized. The gains in column efficiency observed using AFT columns cannot be stated to a single value, since these efficiency gains are a function of three factors: (1) the flow rate, (2) the segmentation ratio, and (3) the solute retention factor. Nevertheless, gains in efficiency compared to conventional columns are almost always observed, and sometimes these gains are more than 100% in the number of theoretical plates1,2. The ability to tune the segmentation ratio allows the analyst to effectively tailor the diameter of the &#39;virtual&#39; column, and this is an important factor in relation to the detection process. For example, a virtual 2.1 mm internal diameter (i.d.) column is established from a physical 4.6 mm i.d. column when the segmentation ratio is 21% of mobile phase eluting from the radial central exit port. Under these conditions, the virtual 2.1 mm i.d. column performs with an efficiency that may be more than 70% greater than the conventional 2.1 mm i.d. column, depending on flow rate, and solute retention factor10.
The current PSF column design that is used for multiplexed detection incorporates a 4-port outlet fitting, but the column can be fitted with a 2-port end-fitting also, however, this limits detection to only two detectors. The basic operation of these columns is, however, the same, except that four detectors can be coupled simultaneously to the 4-port outlet PSF column broadening the scope for multiplexed detection. Aside from pre- and post- column connective tubing, the only additional requirements to operate a PSF column is tubing that can be connected to the peripheral outlet ports, and a means by which the amount of mobile phase passing through each tube can be measured, typically either a mass measurement or a volumetric measurement. For ease of tuning, the internal diameter of all outlet flow tubing should be the same. The flow ratio between the peripheral and radial central exit ports is then varied through the use of pressure management, simply by changing the length of the tubing located on the peripheral outlet fitting, or the length of tubing post detector on the radial central outlet port.
Multiplexed Detection Using PSF Columns 
An important advantage of PSF columns is that each of the outlet exit ports can be connected directly to a detection source, thus enabling multiplexed detection. In a well-designed detection system a single analysis with multiplexed detection can provide substantial information in regards to the nature of the components within the sample. Importantly, destructive and non-destructive tests can be conducted at exactly the same time, without detection delay. This allows the absolute assignment of, for example, anti-oxidants using DPPH&#8226; reagent, with components observed to elute with UV and/or mass spectrometry (MS) detection responses7,11. Therefore, four independent detectors can be operated simultaneously with appropriate portions of flow directed to each detector via any of the four outlet ports. Since the flow through these ports can be easily adjusted the amount of solute reaching any of the detectors can be adjusted to suit the sensitivity of the given detector source. It should be noted, however, that the most efficient solute migration is observed through the radial central outlet port. Each of the peripheral ports offer equivalent separation efficiency, which when set to 25% through each port, is only slightly less efficient than a conventional column. As such, it is important that the quantitative detector be set to analyze sample from the radial central exit port.
When setting up a PSF column for the purpose of multiplexing detection there are a number of considerations that need to be made to achieve efficient and high quality results; that is tube dimensions for each port, choice of which port for a type of detector and flow adjustment.
Tube Dimensions for Each Port
In chromatography the length of post column tubing plays a crucial role in the efficiency and performance of the separation. Large dead-volume as a result of long or wide i.d. tubing from column outlet to detector will result in a loss of efficiency, resolution and sensitivity. Thus, appropriate tubing dimensions must be utilized when setting up the PSF column to attain the maximum potential in providing efficient separation whilst providing the benefits of multiplexing.
Port to Detector 
Figure 2 is an illustration of an example setup of multiplexed detection (Ultra Violet-Visible (U-Vis), mass spectrometer (MS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH&#8226;) detection). The illustration shows the central port is attached to the MS detector, whereas the DPPH&#8226; and UV-Vis detectors are attached to the peripheral ports. Since the MS is the most sensitive detector of the three, flow to this detector was directed from the central outlet port. As DPPH&#8226; detection is selective to the presence of antioxidants, and the least sensitive and most tolerant to band broadening, flow to this detector was directed from a peripheral port. The UV-Vis was a secondary &#39;generic&#39; detector, so flow to this detector was directed from a second peripheral port.
Flow Adjustment
Once the appropriate tubing has been attached from a port to detector, the exiting flow from each of the detectors can be adjusted to the required amount. A simple way to measure the amount of flow exiting from each detector is to weigh the amount of mobile phase that elutes through each port over a given period of time. The percentage flow can thus be determined, and the flow ratios can be adjusted by either shortening or lengthening the tubing attached to the outlet line on the detectors accordingly to suit the requirements of the detectors of choice. Different detectors have different requirements of flow, for example, the flow cell of a fluorescence detector (FLD) is not flow rate limited, but care must be taken to avoid over-pressurization of the flow cell. Hence the control of flow through the FLD is usually attained by adjusting the pressure drop across the other detectors and the remainder of the flow then passes through the FLD. A detector that is sensitive to the amount of flow that is delivered is the MS. Generally, current high end mass spectrometers can readily process around 1 to 1.5 ml/min of moderately aqueous mobile phase. Above this flow rate, flooding of the source may make the MS inoperable. However, detection sensitivity in most mass spectrometers is benefited by using lower flow rates; hence the PSF flow splitting capabilities are extremely useful for applications involving MS detection. High column volumetric flow rates can be utilized, but with low volume loads transported to the MS detector. Tuning of the flow to the MS detector, however, must be made by adjusting the pressure drop prior to the MS detector, rather than post MS. Here, the use of narrow bore tubing (0.1 mm i.d.) is very useful, as the pressure can be readily adjusted without adding inappropriate dead volume.
Depending on the type of detector the adjustment of the segmentation ratio can be done either pre or post detector. If a non-destructive detector, such as UV-Vis is used, the flow percentage would be measured and tuned post detector. If a single destructive detector is used in the multiplex set up the flow percentage is determined by back calculating with respect to other port flow percentages. If a reagent based detector is used such as DPPH&#8226;, the flow percentage is measured post detector without the addition of reagent; and if two or more destructive detectors are used, then the flow ratio is measured pre-detector. Detection systems that may require additional instrumentation, such DPPH&#8226;, will have extra system pressure that may alter the flow percentage once attached to the detection system. Therefore, careful consideration should be paid to system pressure of a destructive detector, when adjusting flow percentage pre-detector. Irrespective of the flow ratio that is set through any of the ports, quantitative information should be obtained through appropriate standardization. Once the flow ratios are set, however, they are robust, and they do not change even under gradient elution conditions7,
The detailed video protocol accompanying this manuscript is intended to show how to use and tune a PSF column functioning in a multiplexed mode of detection.

<table border="0" cellpadding="0" cellspacing="0" width="686">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HPLC instrument</td>
			<td style="width:167px;"></td>
			<td style="width:119px;"></td>
			<td style="width:185px;">Multiple detectors of choice for multiplexed detection. Detectors of choice may require additional instrumentation i.e. pump.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Parallel Segmented Flow HPLC column</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">Not Defined</td>
			<td>Soon to be commercialised</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methanol</td>
			<td style="width:167px;">Any brand</td>
			<td style="width:119px;"></td>
			<td>HPLC Grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PEEK tubing</td>
			<td style="width:167px;">Any brand</td>
			<td style="width:119px;"></td>
			<td>Various lengths and i.d.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Column stoppers</td>
			<td style="width:167px;">Any brand</td>
			<td style="width:119px;"></td>
			<td>For blocking unused peripheral ports.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PEEK tube cutter</td>
			<td style="width:167px;">Any brand</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Analytical Scale Balance</td>
			<td style="width:167px;">Any brand</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stop watch</td>
			<td style="width:167px;">Any brand</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Eluent collection vessels</td>
			<td style="width:167px;">Any brand</td>
			<td style="width:119px;"></td>
			<td>1-2 mL Sample vials can be used as eluent collection vessels</td>
		</tr>
	</tbody>
</table>

tuning a parallel segmented flow column and enabling multiplexed detection

Active flow technology (AFT) is new form of column technology that was designed to overcome flow heterogeneity to increase separation performance in terms of efficiency and sensitivity and to enable multiplexed detection. This form of AFT uses a parallel segmented flow (PSF) column. A PSF column outlet end-fitting consists of 2 or 4 ports, which can be multiplexed to connect up to 4 detectors. The PSF column not only allows a platform for multiplexed detection but also the combination of both destructive and non-destructive detectors, without additional dead volume tubing, simultaneously. The amount of flow through each port can also be adjusted through pressure management to suit the requirements of a specific detector(s). To achieve multiplexed detection using a PSF column there are a number of parameters which can be controlled to ensure optimal separation performance and quality of results; that is tube dimensions for each port, choice of port for each type of detector and flow adjustment. This protocol is intended to show how to use and tune a PSF column functioning in a multiplexed mode of detection.

A multiplexed HPLC analysis was carried out using an AFT column in PSF mode (Figure 1) and set up as illustrated in Figure 2. This type of setup allowed a coffee sample to be analyzed simultaneously using UV-Vis, DPPH&#8226; and MS in Total Ion Count (TIC) mode. The compounds of the coffee sample that responded to DPPH&#8226; could then be easily matched up to the UV-Vis and MS - TIC responses based on the alignment of retention time as illustrated in Figure...

Watch this Scientific Journal Video about Tuning a Parallel Segmented Flow Column and Enabling Multiplexed Detection at JoVE.com

Tuning a Parallel Segmented Flow Column and Enabling Multiplexed Detection

Here, we present a protocol for the operation and tuning of parallel segmented flow chromatography columns to enable multiplexed detection.

Active flow technology (AFT) is new form of column technology that was designed to overcome flow heterogeneity to increase separation performance in terms ...

tuning-parallel-segmented-flow-column-enabling-multiplexed

Research

JoVE Journal

Chemistry

Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 &#956;m polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 &#956;m microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.

We describe a procedure for profiling salivary proteins using multiplexed microsphere-based antibody arrays. Monoclonal antibodies were covalently linked to fluorescent dye-encoded 4.5 &#x03BC;m polymer microspheres using carbodiimide chemistry. The modified microspheres were deposited in fiber-optic microwells to measure protein levels in saliva using fluorescence sandwich immunoassays.

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array ...

Exploring the Radical Nature of a Carbon Surface by Electron Paramagnetic Resonance and a Calibrated Gas Flow

While the first Electron Paramagnetic Resonance (EPR) studies regarding the effects of oxidation on the structure and stability of carbon radicals date back to the early 1980s the focus of these early papers primarily characterized the changes to the structures under extremely harsh conditions (pH or temperature)1-3. It is also known that paramagnetic molecular oxygen undergoes a Heisenberg spin exchange interaction with stable radicals that extremely broadens the EPR signal4-6. Recently, we reported interesting results where this interaction of molecular oxygen with a certain part of the existing stable radical structure can be reversibly affected simply by flowing a diamagnetic gas through the carbon samples at STP7. As flows of He, CO2, and N2 had a similar effect these interactions occur at the surface area of the macropore system.
This manuscript highlights the experimental techniques, work-up, and analysis towards affecting the existing stable radical nature in the carbon structures. It is hoped that it will help towards further development and understanding of these interactions in the community at large.

Stable radicals that are present in carbon substrates interact with paramagnetic oxygen through a Heisenberg spin exchange. This interaction can be significantly reduced under STP conditions by flowing a diamagnetic gas over the carbon system. This manuscript describes a simple method to characterize the nature of those radicals.

While the first Electron Paramagnetic Resonance (EPR) studies regarding the effects of oxidation on the structure and stability of carbon radicals date ...

Transport of Surface-modified Carbon Nanotubes through a Soil Column

Carbon nanotubes (CNTs) are widely manufactured nanoparticles, which are being utilized in a number of consumer products, such as sporting goods, electronics and biomedical applications. Due to their accelerating production and use, CNTs constitute a potential environmental risk if they are released to soil and groundwater systems. It is therefore essential to improve the current understanding of environmental fate and transport of CNTs. The transport and retention of CNTs in both natural and artificial media have been reported in literature, but the findings widely vary and are thus not conclusive. There are a number of physical and chemical parameters responsible for variation in retention and transport. In this study, a complete procedure of selected multiwalled carbon nanotubes (MWCNTs) is presented starting from their surface modification to a complete set of laboratory column experiments at critical physical and chemical scenarios. Results indicate that the stability of the commercially available MWCNTs are critical with their attached surface functional group which can also influence the transport and retention of MWCNT through the surrounding medium.

Surface properties of a nanoparticle are important for their interaction with the surrounding medium. Therefore the surface modification of carbon nanotubes can be critical for their transport and retention through porous media. Here, lab scale column experiments are used to understand the possible transport and retention of these nanoparticles.

Carbon nanotubes (CNTs) are widely manufactured nanoparticles, which are being utilized in a number of consumer products, such as sporting goods, ...

Ethylene Polymerizations Using Parallel Pressure Reactors and a Kinetic Analysis of Chain Transfer Polymerization

We demonstrate a method for high-throughput catalyst screening using a parallel pressure reactor starting from the initial synthesis of a nickel &#945;-diimine ethylene polymerization catalyst. Initial polymerizations with the catalyst lead to optimized reaction conditions, including catalyst concentration, ethylene pressure and reaction time. Using gas-uptake data for these reactions, a procedure to calculate the initial rate of propagation (kp) is presented. Using the optimized conditions, the ability of the nickel &#945;-diimine polymerization catalyst to undergo chain transfer with diethylzinc (ZnEt2) during ethylene polymerization was investigated. A procedure to assess the ability of the catalyst to undergo chain transfer (from molecular weight and 13C NMR data), calculate the degree of chain transfer, and calculate chain transfer rates (ke) is presented.

A protocol for the high-throughput analysis of polymerization catalyst, chain transfer polymerizations, polyethylene characterization, and reaction kinetic analysis is presented.

We demonstrate a method for high-throughput catalyst screening using a parallel pressure reactor starting from the initial synthesis of a nickel ...

Highly Stereoselective Synthesis of 1,6-Ketoesters Mediated by Ionic Liquids: A Three-component Reaction Enabling Rapid Access to a New Class of Low Molecular Weight Gelators

In organic chemistry ionic liquids (ILs) have emerged as safe and recyclable reaction solvents. In the presence of a base ILs can be deprotonated to form catalytically active N-Heterocyclic Carbenes (NHCs). Here we have used ILs as precatalysts in the addition of &#945;,&#946;-unsaturated aldehydes to chalcones to form 1,6-ketoesters, incorporating an anti-diphenyl moiety in a highly stereoselective fashion. The reaction has a broad substrate scope and several functional groups and heteroaromatics can be integrated into the ketoester backbone in generally good yields with maintained stereoselectivity. The reaction protocol is robust and scalable. The starting materials are inexpensive and the products can be obtained after simple filtration, avoiding solvent-demanding chromatography. Furthermore, the IL can be recycled up to 5 times without any loss of reactivity. Moreover, the 1,6-ketoester end product is a potent gelator in several hydrocarbon based solvents. The method enables rapid access to and evaluation of a new class of low molecular weight gelators (LMWGs) from recyclable and inexpensive starting materials.

Ionic liquids (ILs) mediate fast, simple and cheap access to 1,6-ketoesters in high diastereoselectivities and good yields. The reaction protocol is robust and the 1,6-ketoesters can be obtained in gram scale after a simple filtration protocol. Moreover, the 1,6-ketoesters are potent gelators in hydrocarbon solvents.

In organic chemistry ionic liquids (ILs) have emerged as safe and recyclable reaction solvents. In the presence of a base ILs can be deprotonated to form ...

Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented. A major difficulty in adapting PCD to modern HPLC systems and columns is the need for large volume reaction coils that enable reagent mixing and then the derivatization reaction to take place. This large post column dead volume leads to band broadening, which results in a loss of observed separation efficiency and indeed detection in sensitivity. In reaction flow post column derivatization (RF-PCD) the derivatization reagent(s) are pumped against the flow of mobile phase into either one or two of the outer ports of the reaction flow column where it is mixed with column effluent inside a frit housed within the column end fitting. This technique allows for more efficient mixing of the column effluent and derivatization reagent(s) meaning that the volume of the reaction loops can be minimized or even eliminated altogether. It has been found that RF-PCD methods perform better than conventional PCD methods in terms of observed separation efficiency and signal to noise ratio. A further advantage of RF-PCD techniques is the ability to monitor effluent coming from the central port in its underivatized state. RF-PCD has currently been trialed on a relatively small range of post column reactions, however, there is currently no reason to suggest that RF-PCD could not be adapted to any existing one or two component (as long as both reagents are added at the same time) post column derivatization reaction.

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented.

A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is ...

Curtain Flow Column: Optimization of Efficiency and Sensitivity

Active Flow Technology (AFT) is a form of column technology that increases the separation performance of a HPLC column through the use of a specially purpose built multiport end-fitting(s). Curtain Flow (CF) columns belong to the AFT suite of columns, specifically the CF column is designed so that the sample is injected into the radial central region of the bed and a curtain flow of mobile phase surrounding the injection of solute prevents the radial dispersion of the sample to the wall. The column functions as an 'infinite diameter' column. The purpose of the design is to overcome the radial heterogeneity of the column bed, and at the same time maximize the sample load into the radial central region of the column bed, which serves to increase detection sensitivity. The protocol described herein outlines the system and CF column set up and the tuning process for an optimized infinite diameter 'virtual' column.

Here, we present a protocol for the operation and optimization of Active Flow Technology (AFT) column in Curtain flow (CF) mode for enhanced separation performance.

Active Flow Technology (AFT) is a form of column technology that increases the separation performance of a HPLC column through the use of a specially ...

A Protocol for Electrochemical Evaluations and State of Charge Diagnostics of a Symmetric Organic Redox Flow Battery

Redox flow batteries have been considered as one of the most promising stationary energy storage solutions for improving the reliability of the power grid and deployment of renewable energy technologies. Among the many flow battery chemistries, non-aqueous flow batteries have the potential to achieve high energy density because of the broad voltage windows of non-aqueous electrolytes. However, significant technical hurdles exist currently limiting non-aqueous flow batteries to demonstrate their full potential, such as low redox concentrations, low operating currents, under-explored battery status monitoring, etc. In an attempt to address these limitations, we recently reported a non-aqueous flow battery based on a highly soluble, redox-active organic nitronyl nitroxide radical compound, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO). This redox material exhibits an ambipolar electrochemical property, and therefore can serve as both anolyte and catholyte redox materials to form a symmetric flow battery chemistry. Moreover, we demonstrated that Fourier transform infrared (FTIR) spectroscopy could measure the PTIO concentrations during the PTIO flow battery cycling and offer reasonably accurate detection of the battery state of charge (SOC), as cross-validated by electron spin resonance (ESR) measurements. Herein we present a video protocol for the electrochemical evaluation and SOC diagnosis of the PTIO symmetric flow battery. With a detailed description, we experimentally demonstrated the route to achieve such purposes. This protocol aims to spark more interests and insights on the safety and reliability in the field of non-aqueous redox flow batteries.

We present the protocols for electrochemically evaluating a symmetric non-aqueous organic redox flow battery and for diagnosing its state of charge using FTIR.

Redox flow batteries have been considered as one of the most promising stationary energy storage solutions for improving the reliability of the power grid ...

A Continuous-flow Photocatalytic Reactor for the Precisely Controlled Deposition of Metallic Nanoparticles

In this work, a novel photocatalytic reactor for the pulsed and controlled excitation of the photocatalyst and the precise deposition of metallic nanoparticles is developed. Guidelines for the replication of the reactor and its operation are provided in detail. Three different composite systems (Pt/graphene, Pt/TiO2, and Au/TiO2) with monodisperse and uniformly distributed particles are produced by this reactor, and the photodeposition mechanism, as well as the synthesis optimization strategy, are discussed. The synthesis methods and their technical aspects are described comprehensively. The role of the ultraviolet (UV) dose (in each excitation pulse) on the photodeposition process is investigated and the optimum values for each composite system are provided.

For a continuous and scalable synthesis of noble-metal-based nanocomposites, a novel photocatalytic reactor is developed and its structure, operation principles, and product quality optimization strategies are described.

In this work, a novel photocatalytic reactor for the pulsed and controlled excitation of the photocatalyst and the precise deposition of metallic ...

Preparation and Application of a New Bacterial Biosensor for the Presumptive Detection of Gunshot Residue

MicRoboCop is a biosensor that has been designed for a unique application in forensic chemistry. MicRoboCop is a system made up of three devices that, when used together, can indicate the presence of gunshot residue (GSR) by producing a fluorescence signal in the presence of three key analytes (antimony, lead, and organic components of GSR). The protocol describes the synthesis of the biosensors using Escherichia coli (E. coli), and the analytical chemistry methods used to evaluate the selectivity and sensitivity of the sensors. The functioning of the system is demonstrated by using GSR collected from the inside of a spent cartridge casing. Once prepared, the biosensors can be stored until needed and can be used as a test for these key analytes. A positive response from all three analytes provides a presumptive positive test for GSR, while each individual device has applications for detecting the analytes in other samples (e.g., a detector for lead contamination in drinking water). The main limitation of the system is the time required for a positive signal; future work may involve studying different organisms to optimize the response time.

A protocol is presented using synthetic biology techniques to synthesize a set of bacterial biosensors for the analysis of gunshot residue, and to test the functioning of the devices for their intended use using fluorescence spectroscopy.

MicRoboCop is a biosensor that has been designed for a unique application in forensic chemistry. MicRoboCop is a system made up of three devices that, ...