One of the authors (DK) acknowledges the receipt of an Australian Postgraduate Award.

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 and High Performance Liquid Chromatography (HPLC) eluent. Ensure appropriate use of engineering controls of HPLC, analytical balance and detector instrumentation, and ensure the use of personal protective equipment (safety glasses, gloves, lab coat, full length pants, and closed-toe shoes).
Note: This protocol contains instructions on how to use a CF column on a HPLC system coupled with a UV-Vis detector. The protocol has been written assuming the reader has basic knowledge and experience in chromatography.
1. Setup of HPLC Instrument
Note: This section can be altered to suit the analysts&#39; needs, i.e., choice of solvents, detector wavelength and flow rate that are appropriate to the sample of interest.
<ol>
	<li>Prepare the HPLC instrument with 100% ultrapure water (e.g., Milli-Q water) for line A and 100% methanol for line B as the mobile phase and purge the pumps as per manufacturer requirement.</li>
	<li>Set the UV-Vis detector to 254 nm.</li>
	<li>Choose either a pre-injection flow split mode of set-up, or a dual pump flow set-up. For the split-flow mode proceed to Step 2, for the dual pump mode proceed to Step 3.</li>
</ol>
2. Split-flow System Setup
<ol>
	<li>Disconnect the pump line from the injector valve of the auto-sampler.</li>
	<li>Attach a T-piece to the pump line.</li>
	<li>Attach a 15 cm piece of 0.13 mm i.d. tubing to each port of the T-piece.</li>
	<li>Connect one tube from the T-piece to injector valve of auto-sampler.</li>
	<li>Set the pump to 1.0 ml min-1.</li>
	<li>Before connecting pump lines to the inlet of the CF column, tune the segmentation ratio of the flow to 40%:60% (center line:peripheral line) as follows in step 2.7.</li>
	<li>Tuning of CF inlet ratio on split-flow system
	<ol>
		<li>Measure the mass of two empty collection vessels using an analytical balance and label one collection vessel central and the other one peripheral (one for the line from the auto-sampler to center port and one for the line from the T-piece to peripheral port).</li>
		<li>For 1.0 min, collect the exiting mobile phase from the line coming from the injector (at the point that will be connected to the column) into the collection vessel, whose mass was measured in 2.7.1.</li>
		<li>Re-weigh the collection vessel on the analytical scale and determine the mass of mobile phase collected.</li>
		<li>Repeat steps 2.7.2 to 2.7.3 for the eluent exiting the line from the T-piece that is to be connected to the peripheral port.</li>
		<li>Determine the percentage of flow (ml min-1) from each line of flow according to the following equations: 
		<img alt="Equation 1" src="/files/ftp_upload/53471/53471eq1.jpg" /></li>
		<li>Adjust the flow ratio to 40%:60% (&#177; 2%) (line from injector to central port:line from T-piece to peripheral port). If the line from the injector to the central port flow percentage is above 40%, increase the pressure drop by decreasing the internal diameter of the tubing, or increasing its length. If the line from injector to central port flow percentage is below 40%, increase internal diameter of the tubing or decrease the length of the tube.</li>
		<li>Once the flow ratios are tuned turn the pump flow off.</li>
		<li>Connect the line from the injector to the central port of the column inlet and the line from the T-piece to the peripheral port of the column inlet.</li>
		<li>Slowly ramp the flow rate to 1.0 ml min-1 at 100% line B.</li>
		<li>Equilibrate the column (4.6 mm i.d. x 100 mm length) by allowing 100% Methanol (line B) mobile phase to flow through the column at 1.0 ml min-1 for 10 min. This time is scaled according to the dimensions of other columns the user may employ.</li>
		<li>For the tuning of the CF outlet go to Step 4. &#39;Tuning of CF outlet flow&#39;.</li>
	</ol>
	</li>
</ol>
3. Dual Pump System Setup
<ol>
	<li>Connect the HPLC system pump to the injector and then connect the line from the injector to the central inlet port of the column.</li>
	<li>Connect the additional pump directly to the inlet peripheral port of the column. Note that this second pump by-passes the injector.</li>
	<li>Ramp the flow rate of the system pump attached to the central port to 0.4 ml min-1 (representative of 40% of the total flowrate of 1.0 ml min-1) at 100% Methanol (line B).</li>
	<li>At the same time as Step 3.3, ramp the flow rate of the peripheral pump to 0.6 ml min-1 (representative of 60% of the total flowrate of 1.0 ml min-1) at 100% Methanol (line B).</li>
	<li>Equilibrate the column (4.6 mm i.d. x 100 mm length) by allowing 100 % Methanol (line B) mobile phase to flow through the column at 1.0 ml min-1 for 10 min. This time is scaled according to the dimensions of other columns the user may employ.</li>
	<li>For the tuning of the CF outlet go to Step 4. &#39;Tuning of CF outlet flow&#39;.</li>
</ol>
4. Tuning of CF Outlet Flow
<ol>
	<li>Connect the central outlet port to the UV-Vis detector using a 15 cm piece of 0.13 mm i.d. tubing.</li>
	<li>Connect a 15 cm piece of 0.13 mm i.d. tubing to the peripheral outlet port of the CF column.</li>
	<li>Weigh the mass of two empty collection vessels on the analytical balance and label one vessel central and the other peripheral.</li>
	<li>For 1.0 min, collect the exiting mobile phase from the UV-Vis detector (central flow) into the collection vessel label central, whose mass was measured in 4.2.</li>
	<li>Re-weigh the collection vessel containing the collected eluent on the analytical scale and determine the mass of mobile phase collected.</li>
	<li>Repeat steps 4.4 to 4.5 for the eluent exiting the line from the peripheral outlet port.</li>
	<li>Determine the percentage of flow from each line of flow according to the following equations: 
	<img alt="Equation 2" src="/files/ftp_upload/53471/53471eq2.jpg" /></li>
	<li>Adjust the flow ratio to 21%:79% (&#177; 2%) (central outlet flow from UV-Vis:peripheral outlet flow from line ). If the central flow percentage from the UV-Vis is above 21%, increase the pressure drop by decreasing the internal diameter of the tubing attached to the exit of the UV-Vis detector, or increasing its length. If the central flow percentage from the UV-Vis is below 21%, increase internal diameter of the tubing attached to the exit of the UV-Vis detector, or decrease the length of the tube. Each time the length of tubing has been changed, repeat steps 4.3 to 4.7. 
	Note: The CF column in &#39;virtual&#39; 2.1 mm i.d. mode is ready for analysis.</li>
</ol>

<ol>
	<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=The+use+of+parallel+segmented+outlet+flow+columns+for+enhanced+mass+spectral+sensitivity+at+high+chromatographic+flow+rates.">The use of parallel segmented outlet flow columns for enhanced mass spectral sensitivity at high chromatographic flow rates.</a> Rapid Commun. Mass Sp. 26 (8), 943-949 (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=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., 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>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>Kocic, D., 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=High+through-put+and+highly+sensitive+liquid+chromatography-tandem+mass+spectrometry+separations+of+essential+amino+acids+using+active+flow+technology+chromatography+columns.">High through-put and highly sensitive liquid chromatography-tandem mass spectrometry separations of essential amino acids using active flow technology chromatography columns.</a> J. Chromatogr. A. 1305, 102-108 (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>Shellie, R., Haddad, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+two-dimensional+liquid+chromatography.">Comprehensive two-dimensional liquid chromatography.</a> Anal. Bioanal. Chem. 386 (3), 405-415 (2006).</li><li>Abia, J. A., Mriziq, K. S., Guiochon, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radial+heterogeneity+of+some+analytical+columns+used+in+high-performance+liquid+chromatography.">Radial heterogeneity of some analytical columns used in high-performance liquid chromatography.</a> J. Chromatogr. A. 1216 (15), 3185-3191 (2009).</li><li>Knox, J. H., Laird, G. R., Raven, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+radial+and+axial+dispersion+in+liquid+chromatography+in+relation+to+the+&amp;#34;infinite+diameter+effect&amp;#34.">Interaction of radial and axial dispersion in liquid chromatography in relation to the &amp;#34;infinite diameter effect&amp;#34.</a> J. Chromatogr. A. 122, 129-145 (1976).</li><li>Miyabe, K., Guiochon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+the+column+radial+heterogeneity+from+an+analysis+of+the+characteristics+of+tailing+peaks+in+linear+chromatography.">Estimation of the column radial heterogeneity from an analysis of the characteristics of tailing peaks in linear chromatography.</a> J. Chromatogr. A. 830 (1), 29-39 (1999).</li><li>Shalliker, R. A., Scott Broyles, B., Guiochon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axial+and+radial+diffusion+coefficients+in+a+liquid+chromatography+column+and+bed+heterogeneity.">Axial and radial diffusion coefficients in a liquid chromatography column and bed heterogeneity.</a> J. Chromatogr. A. 994 (1-2), 1-12 (2003).</li><li>Gritti, F., Guiochon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+thermal+heterogeneity+of+the+column+on+chromatographic+results.">Effects of the thermal heterogeneity of the column on chromatographic results.</a> J. Chromatogr. A. 1131 (1-2), 151-165 (2006).</li><li>Shalliker, R. A., Wong, V., Broyles, B. S., Guiochon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+bed+compression+in+an+axial+compression+liquid+chromatography+column.">Visualization of bed compression in an axial compression liquid chromatography column.</a> J. Chromatogr. A. 977 (2), 213-223 (2002).</li><li>Tallarek, U., Albert, K., Bayer, E., Guiochon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+transverse+and+axial+apparent+dispersion+coefficients+in+packed+beds.">Measurement of transverse and axial apparent dispersion coefficients in packed beds.</a> AICHE J. 42 (11), 3041-3054 (1996).</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> J. Sep. Sci. 35 (3), 410-415 (2012).</li><li>Shalliker, R. A., Broyles, B. S., Guiochon, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+evidence+of+two+wall+effects+in+liquid+chromatography.">Physical evidence of two wall effects in liquid chromatography.</a> J. Chromatogr. A. 888 (1-2), 1-12 (2000).</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>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>Foley, D., 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=Precision+and+Reliability:+an+Intercontinental+Study+of+Curtain+Flow+Chromatography.">Precision and Reliability: an Intercontinental Study of Curtain Flow Chromatography.</a> Thermo Scientific. , (2013).</li><li>Pravadali-Cekic, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidimensional+Approaches+for+the+Analysis+of+Complex+Samples+using+HPLC.">Multidimensional Approaches for the Analysis of Complex Samples using HPLC.</a> University of Western Sydney. , (2014).</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>Foley, D., 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=Curtain+flow+chromatography+('the+infinite+diameter+column')+with+automated+injection+and+high+sample+through-put:+The+results+of+an+inter-laboratory+study.">Curtain flow chromatography ('the infinite diameter column') with automated injection and high sample through-put: The results of an inter-laboratory study.</a> Microchem. J. 110, 127-132 (2013).</li></ol>

This work was supported by UWS and ThermoFisher Scientific.

This study involved the inter-laboratory analysis of CF chromatography columns to test the analytical performance in terms of efficiency and sensitivity. The CF column was set up with a dual pumping system as described in section &#39;3. Dual pump system set up&#39; to achieve a flow ratio of 40:60 (center:peripheral) on the inlet of the CF column. The 40:60 (center:peripheral) flow ratio was achieved by setting the flow rate of each pump to the value that represents 40% and 60% of the total flow rate, respectively. The ...

In recent years column technology for High Performance Liquid Chromatography (HPLC) has advanced greatly; peak capacities have increased considerably thanks largely to the use of smaller particle sizes and the more efficient core shell particles. Since separations are generally more efficient, a flow-on effect has been an increase in sensitivity since peaks are now sharper and hence taller1-8.
Nevertheless, radial bed heterogeneity is still a limiting factor in the performance of all columns, but this is not a new story since chromatographers have known this for many years. Column beds are heterogeneous in both the radial direction9-12, and along the column axis10,12-15. The wall-effect especially is an important contributor to the loss of separation performance7,16-18. Shalliker and Ritchie7 recently reviewed aspects of column bed heterogeneity and hence this need not be discussed here further. Although suffice to say, that the variation in column bed packing density and the wall effects lead to a distortion of the solute plug, such that bands elute through the column in plugs that resemble partially filled soup bowls rather than thin flat solid discs7 that are usually depicted in basic teaching texts. When experiments were undertaken such that the solute migration through the bed could be visualized the plug profiles inside the column were partly hollow and the tailing section of the band is largely the wall component of the sample plug. The end result is that it takes many more plates to separate these &#39;partially hollow&#39; plugs than would be required if the discs were solid and flat12,14,17. To overcome the band broadening issues associated with wall effects and the variation in radial packing density, a new form of column technology known as Active Flow Technology (AFT) was designed7,19. The purpose of this design was to remove wall effects through the physical separation of solvent eluting along the wall region, from that of mobile phase eluting in the radial central region of the column19. There are two main types of AFT columns; Parallel Segmented Flow (PSF) columns and Curtain Flow (CF) columns 7. Since this protocol is aimed at the use and optimization of CF columns, PSF columns will not be further discussed.
Curtain Flow (CF)
Curtain Flow (CF) column formats utilize AFT end-fittings at both the inlet and the outlet of the column. AFT end-fittings consist of an annular frit located inside a multiport fitting. The frit is made up of three parts: a porous radial central portion that is aligned with the central port of the end fitting, a porous outer portion that is aligned with the peripheral port(s) of the end fitting, and an impermeable ring that separates the two porous portions preventing any cross-flow between the radial central and outer regions of the frit19. Figure 1 illustrates the design of the AFT frit and Figure 2 illustrates the CF column format. In this mode of operation (CF) the sample is injected into the radial central port of the inlet fitting, whilst additional mobile phase is introduced through the peripheral port of the inlet to &#39;curtain&#39; the migration of solutes through the radial central region of the column. Hence the sample enters the bed in the radial central region of the column with the outer region of the column having mobile phase only passed through it. Studies have shown that a volumetric flow rate ratio of around 40:60 (central:peripheral port) for the inlet end-fitting of a 4.6 mm internal diameter (i.d.) column is optimal6,7,16. The AFT outlet of the CF column allows the adjustment of the central and peripheral flow to their relative portion and can be varied to almost any desired ratio through pressure management. The optimization of a CF column can significantly improve various functional aspects of the column technology, such as separation efficiency or detection sensitivity. In this manner a &#39;wall-less&#39;, &#39;infinite-diameter&#39; or &#39;virtual&#39; column is established6,10,18,20. The purpose of CF columns is to actively manage the migration of sample through the column to prevent the sample from reaching the wall region. Thus, the solute concentration upon exit to the detector is maximized, increasing sensitivity of around 2.5 times greater than the conventional column format when using Ultraviolet (UV) detection16, and even greater when using mass spectral detection6.
CF columns are ideally suited for low concentration samples, since detection sensitivity is increased. Further, they are ideal when coupled to flow rate limited detectors, such as the mass spectrometer (MS)6. An AFT column in a 4.6 mm i.d. format, for example can be tuned to deliver the same volume of solvent to a detector as a standard 2.1 mm i.d. format column when operated at the same linear velocities, by adjusting exiting central flow to 21%. Likewise the AFT column could also be tuned to deliver the same volume load to a detector as a 3.0 mm i.d. column, by adjusting exiting central flow to 43%. In fact any &#39;virtual&#39; column format could be produced to suit the analytical requirement6,18,22. Using these specially designed end-fittings at the inlet and the outlet ensures that a true wall-less column is established.
There are two ways to set up the solvent delivery system to the central and peripheral ports of the inlet: split-flow system6 and two pump system6,7. Figure 3 illustrates each of these CF system set ups.
Split-flow system
In a split-flow system (Figure 3A) the pump flow leading to the injector is split pre-injector using a zero-dead volume T-piece, where one flow stream of mobile phase is connected to the injector, which is then connected to the central port of the inlet end-fitting of the column. The second flow stream of mobile phase by-passes the injector and is connected to the peripheral port on the inlet of the column. During the splitting of flow, the flow stream percentage is adjusted to 40:60 (center:peripheral) before the lines are connected to the column, i.e., from injector to center and pump to peripheral.
Two pump system
The CF column requires two flow streams at the inlet end-fitting of the column. Depending on the type of autosampler/injector of the HPLC instrument, split-flow set up may not be possible, and so CF can then be achieved through 2 pumps (Figure 3B21). Each pump is allocated and connected to either the central or peripheral port and the flow rate is set to represent 40% of flow for central port and 60% for peripheral port. For example, if the total flow rate is 1.0 ml min-1, the central pump flow rate is set to 0.4 ml min-1 and the peripheral pump is set to 0.6 ml min-1.
The choice of which mode of operation is largely dependent on the HPLC instrumentation and chromatographic mode of operation. For example in some autosamplers a change in pressure between sample load position and sample inject position may occur disrupting the split-flow ratio and thus in this case a dual pump set up would be recommended for optimal CF performance. Regardless of the solvent delivery system set up chosen for the inlet of the CF column, the CF outlet optimization remains the same. The outlet central port of the CF column is attached to the Ultraviolet-Visible (UV-Vis) detector with the smallest volume possible of tubing to minimize the effects of post-column dead volume. Since, CF columns emulate narrow-bore columns, dead volume between the column outlet and the detector is detrimental to the separation performance of the CF column. It is critical to ensure the smallest amount of volume of tubing between the central port and the UV-Vis detector to minimize the effects of dead volume such as band broadening, loss in efficiency and sensitivity. Hence, the use of narrow bore tubing (0.1 mm i.d.) is advised to readily allow pressure adjustments without adding inappropriate dead volume. Tubing is also attached to the peripheral port and directed to waste. Upon the outlet of the CF column, the segmentation ratio can be adjusted to any ratio that fits the purpose of the analyst. When a 4.6 mm i.d. CF is used, for example, it is often convenient to set the ratio as either 43:57 or 21:79 (center:peripheral) to emulate a &#39;virtual&#39; 3.0 mm i.d. column or 2.1 mm i.d. column, respectfully. That way the separation performance is readily bench-marked. The segmentation ratio is measured by weighing the amount of flow exiting from the detector that is connected to the central port and flow exiting the peripheral port over a period time. The percentage flow through each port can then be determined and the ratios can be adjusted by altering the length of tubing attached or using tubing that has a different internal diameter (i.d.).
This video protocol details the operation and optimization procedures of a CF column for enhanced chromatographic performance.

<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;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Additional Pump</td>
			<td style="width:167px;"></td>
			<td style="width:119px;"></td>
			<td>Required if 2 pump CF system set up is to be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Curtain 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.&#160;</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>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">T-piece</td>
			<td>Any brand</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

AFT columns were developed using a specialized frit design (Figure 1) in the multiport column end-fittings to overcome the column bed heterogeneity and improve separation performance. An inter-laboratory study on the separation performance of CF chromatography columns (Figure 2) was carried out with a dual pump system set up (Figure 3B) as described in section 3 of this protocol23. A three component test mixture was analyzed un...

Watch this Scientific Journal Video about Curtain Flow Column: Optimization of Efficiency and Sensitivity at JoVE.com

Curtain Flow Column: Optimization of Efficiency and Sensitivity

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 ...

curtain-flow-column-optimization-of-efficiency-and-sensitivity

Research

JoVE Journal

Chemistry

6.5K Views.  University of Western Sydney. 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. 

Video: Curtain Flow Column: Optimization of Efficiency and Sensitivity

Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins

The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. Kinesins display differences across the superfamily both in ATP turnover and in microtubule interaction. These differences tailor specific kinesins to various functions such as cargo transport, microtubule sliding, microtubule depolymerization and microtubule stabilization. To understand the mechanism of action of a kinesin it is important to understand how the chemical cycle of ATP turnover is coupled to the mechanical cycle of microtubule interaction. To dissect the ATP turnover cycle, one approach is to utilize fluorescently labeled nucleotides to visualize individual steps in the cycle. Determining the kinetics of each nucleotide transition in the ATP turnover cycle allows the rate-limiting step or steps for the complete cycle to be identified. For a kinesin, it is important to know the rate-limiting step, in the absence of microtubules, as this step is generally accelerated several thousand fold when the kinesin interacts with microtubules. The cycle in the absence of microtubules is then compared to that in the presence of microtubules to fully understand a kinesin&#8217;s ATP turnover cycle. The kinetics of individual nucleotide transitions are generally too fast to observe by manually mixing reactants, particularly in the presence of microtubules. A rapid mixing device, such as a stopped-flow fluorimeter, which allows kinetics to be observed on timescales of as little as a few milliseconds, can be used to monitor such transitions. Here, we describe protocols in which rapid mixing of reagents by stopped-flow is used in conjunction with fluorescently labeled nucleotides to dissect the ATP turnover cycle of a kinesin.

Kinesins are characterized by nucleotide-dependent interaction with microtubules: a cycle of ATP turnover coupled to a cycle of microtubule interaction. Here, we describe protocols to analyze the kinetics of individual nucleotide transitions in the ATP turnover cycle of a kinesin using fluorescently labeled nucleotides and stopped-flow fluorescence.

The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. ...

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, ...

Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional units (e.g., domains). Traditional consensus-building models fail to account for interpositional dependencies &#8211; functionally required covariation of residues that tend to appear simultaneously throughout evolution and across the phylogentic tree. These relationships can reveal important clues about the processes of protein folding, thermostability, and the formation of functional sites, which in turn can be used to inform the engineering of synthetic proteins. Unfortunately, these relationships essentially form sub-motifs which cannot be predicted by simple &#8220;majority rule&#8221; or even HMM-based consensus models, and the result can be a biologically invalid &#8220;consensus&#8221; which is not only never seen in nature but is less viable than any extant protein. We have developed a visual analytics tool, StickWRLD, which creates an interactive 3D representation of a protein alignment and clearly displays covarying residues. The user has the ability to pan and zoom, as well as dynamically change the statistical threshold underlying the identification of covariants. StickWRLD has previously been successfully used to identify functionally-required covarying residues in proteins such as Adenylate Kinase and in DNA sequences such as endonuclease target sites.

Synthetic protein sequences based on consensus motifs typically ignore co-evolving residues, that imply interpositional dependencies (IPDs). IPDs can be essential to activity, and designs that disregard them may result in suboptimal results. This protocol uses StickWRLD to identify IPDs and help inform rational protein design, resulting in more efficient results.

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional ...

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.

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 ...

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 ...

Improved Heterojunction Quality in Cu2O-based Solar Cells Through the Optimization of Atmospheric Pressure Spatial Atomic Layer Deposited Zn1-xMgxO

Atmospheric pressure spatial atomic layer deposition (AP-SALD) was used to deposit n-type ZnO and Zn1-xMgxO thin films onto p-type thermally oxidized Cu2O substrates outside vacuum at low temperature. The performance of photovoltaic devices featuring atmospherically fabricated ZnO/Cu2O heterojunction was dependent on the conditions of AP-SALD film deposition, namely, the substrate temperature and deposition time, as well as on the Cu2O substrate exposure to oxidizing agents prior to and during the ZnO deposition. Superficial Cu2O to CuO oxidation was identified as a limiting factor to heterojunction quality due to recombination at the ZnO/Cu2O interface. Optimization of AP-SALD conditions as well as keeping Cu2O away from air and moisture in order to minimize Cu2O surface oxidation led to improved device performance. A three-fold increase in the open-circuit voltage (up to 0.65 V) and a two-fold increase in the short-circuit current density produced solar cells with a record 2.2% power conversion efficiency (PCE). This PCE is the highest reported for a Zn1-xMgxO/Cu2O heterojunction formed outside vacuum, which highlights atmospheric pressure spatial ALD as a promising technique for inexpensive and scalable fabrication of Cu2O-based photovoltaics.

Improved Heterojunction Quality in Cu2O-based Solar Cells Through the Optimization of Atmospheric Pressure Spatial Atomic Layer Deposited Zn1-xMgxO

Here we present a protocol for synthesizing Zn1-xMgxO/Cu2O heterojunctions in open-air at low temperature via atmospheric pressure spatial atomic layer deposition (AP-SALD) of Zn1-xMgxO on cuprous oxide. Such high quality conformal metal oxides can be grown on a variety of substrates including plastics by this cheap and scalable method.

Atmospheric pressure spatial atomic layer deposition (AP-SALD) was used to deposit n-type ZnO and Zn1-xMgxO thin films onto p-type thermally oxidized Cu2O ...

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

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

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

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

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 ...

Continuous Flow Chemistry: Reaction of Diphenyldiazomethane with p-Nitrobenzoic Acid

Continuous flow technology has been identified as instrumental for its environmental and economic advantages leveraging superior mixing, heat transfer and cost savings through the &#34;scaling out&#34; strategy as opposed to the traditional &#34;scaling up&#34;. Herein, we report the reaction of diphenyldiazomethane with p-nitrobenzoic acid in both batch and flow modes. To effectively transfer the reaction from batch to flow mode, it is essential to first conduct the reaction in batch. As a consequence, the reaction of diphenyldiazomethane was first studied in batch as a function of temperature, reaction time, and concentration to obtain kinetic information and process parameters. The glass flow reactor set-up is described and combines two types of reaction modules with &#34;mixing&#34; and &#34;linear&#34; microstructures. Finally, the reaction of diphenyldiazomethane with p-nitrobenzoic acid was successfully conducted in the flow reactor, with up to 95% conversion of the diphenyldiazomethane in 11 min. This proof of concept reaction aims to provide insight for scientists to consider flow technology&#39;s competitiveness, sustainability, and versatility in their research.

Continuous Flow Chemistry: Reaction of Diphenyldiazomethane with p-Nitrobenzoic Acid

Flow chemistry carries environmental and economic advantages by leveraging superior mixing, heat transfer and cost benefits. Herein, we provide a blueprint to transfer chemical processes from batch to flow mode. The reaction of diphenyldiazomethane (DDM) with p-nitrobenzoic acid, conducted in batch and flow, was chosen for proof of concept.

Continuous flow technology has been identified as instrumental for its environmental and economic advantages leveraging superior mixing, heat transfer and ...

Synthesis of Platinum-nickel Nanowires and Optimization for Oxygen Reduction Performance

Platinum-nickel (Pt-Ni) nanowires were developed as fuel cell electrocatalysts, and were optimized for the performance and durability in the oxygen reduction reaction. Spontaneous galvanic displacement was used to deposit Pt layers onto Ni nanowire substrates. The synthesis approach produced catalysts with high specific activities and high Pt surface areas. Hydrogen annealing improved Pt and Ni mixing and specific activity. Acid leaching was used to preferentially remove Ni near the nanowire surface, and oxygen annealing was used to stabilize near-surface Ni, improving durability and minimizing Ni dissolution. These protocols detail the optimization of each post-synthesis processing step, including hydrogen annealing to 250 &#176;C, exposure to 0.1 M nitric acid, and oxygen annealing to 175 &#176;C. Through these steps, Pt-Ni nanowires produced increased activities more than an order of magnitude than Pt nanoparticles, while offering significant durability improvements. The presented protocols are based on Pt-Ni systems in the development of fuel cell catalysts. These techniques have also been used for a variety of metal combinations, and can be applied to develop catalysts for a number of electrochemical processes.

&#160; The protocol describes the synthesis and electrochemical testing of platinum-nickel nanowires. Nanowires were synthesized by the galvanic displacement of a nickel nanowire template. Post-synthesis processing, including hydrogen annealing, acid leaching, and oxygen annealing were used to optimize nanowire performance and durability in the oxygen reduction reaction.

Platinum-nickel (Pt-Ni) nanowires were developed as fuel cell electrocatalysts, and were optimized for the performance and durability in the oxygen ...