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. 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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><tbody><tr><td>HPLC instrument</td><td></td><td></td><td></td></tr><tr><td>Additional Pump</td><td></td><td></td><td>Required if 2 pump CF system set up is to be used.</td></tr><tr><td>Curtain Flow HPLC column</td><td>Thermo Fisher Scientific</td><td>Not Defined</td><td>Soon to be commercialized</td></tr><tr><td>Methanol</td><td>Any brand</td><td></td><td>HPLC Grade</td></tr><tr><td>PEEK tubing</td><td>Any brand</td><td></td><td>Various lengths and i.d.&amp;nbsp;</td></tr><tr><td>PEEK tube cutter</td><td>Any brand</td><td></td><td></td></tr><tr><td>Analytical Scale Balance</td><td>Any brand</td><td></td><td></td></tr><tr><td>Stop watch</td><td>Any brand</td><td></td><td></td></tr><tr><td>Eluent collection vessels</td><td>Any brand</td><td></td><td>1-2 ml sample vials can be used as eluent collection vessels</td></tr><tr><td>T-piece</td><td>Any brand</td><td></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 ...

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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

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

Bioengineering

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 ...

A Modular Microfluidic Technology for Systematic Studies of Colloidal Semiconductor Nanocrystals

Colloidal semiconductor nanocrystals, known as quantum dots (QDs), are a rapidly growing class of materials in commercial electronics, such as light emitting diodes (LEDs) and photovoltaics (PVs). Among this material group, inorganic/organic perovskites have demonstrated significant improvement and potential towards high-efficiency, low-cost PV fabrication due to their high charge carrier mobilities and lifetimes. Despite the opportunities for perovskite QDs in large-scale PV and LED applications, the lack of fundamental and comprehensive understanding of their growth pathways has inhibited their adaptation within continuous nanomanufacturing strategies. Traditional flask-based screening approaches are generally expensive, labor-intensive, and imprecise for effectively characterizing the broad parameter space and synthesis variety relevant to colloidal QD reactions. In this work, a fully autonomous microfluidic platform is developed to systematically study the large parameter space associated with the colloidal synthesis of nanocrystals in a continuous flow format. Through the application of a novel translating three-port flow cell and modular reactor extension units, the system may rapidly collect fluorescence and absorption spectra across reactor lengths ranging 3 - 196 cm. The adjustable reactor length not only decouples the residence time from the velocity-dependent mass transfer, it also substantially improves the sampling rates and chemical consumption due to the characterization of 40 unique spectra within a single equilibrated system. Sample rates may reach up to 30,000 unique spectra per day, and the conditions cover 4 orders of magnitude in residence times ranging 100 ms - 17 min. Further applications of this system would substantially improve the rate and precision of the material discovery and screening in future studies. Detailed within this report are the system materials and assembly protocols with a general description of the automated sampling software and offline data processing.

Detailed herein are the operation and assembly protocols of a modular microfluidic screening platform for the systematic characterization of colloidal semiconductor nanocrystal syntheses. Through fully adjustable system arrangements, highly efficient spectra collection may be carried out across 4 orders of magnitude reaction time scales within a mass transfer-controlled sampling space.

Colloidal semiconductor nanocrystals, known as quantum dots (QDs), are a rapidly growing class of materials in commercial electronics, such as light ...

Engineering

Simple In-House Ultra-High Performance Capillary Column Manufacturing with the FlashPack Approach

Capillary ultra-high performance liquid chromatography (UHPLC) is currently a method of choice for the sample separation step in LC-MS-based proteomics. However, capillary columns are much less robust in comparison to their higher flow countertypes. Because of easy contamination and blocking, they often need replacement. That makes them a markedly expensive part of the total LC-MS analysis cost. In-house packing of UHPLC capillary columns saves a lot of money and allows customization. However, the standard packing procedure in the 100-bar pressure bomb works well only for HPLC columns but is too slow for UHPLC sorbents. Here we provide a description of an optimized FlashPack protocol applied to the same 100-bar pressure bomb setup. The method is based on packing from ultra-high sorbent concentration slurry and is developed for in-house manufacturing of UHPLC capillary columns of unlimited length in reasonable time.

Here we present a protocol for the optimized FlashPack capillary column packing procedure. Application of an optimized protocol to a common 100-bar pressure bomb setup allows 10-times faster packing and manufacturing of long ultra-high performance capillary columns.

Capillary ultra-high performance liquid chromatography (UHPLC) is currently a method of choice for the sample separation step in LC-MS-based proteomics. ...

Biochemistry

Design and Optimization Strategies of a High-Performance Vented Box

This study aims to solve the problems of air flow chaos and poor performance in a vented box caused by the heterogeneous distribution of airflow through the design of the internal structure of the vented box with constant energy consumption. The final goal is to evenly distribute the airflow inside the vented box. Sensitivity analysis was performed for three structural parameters: the number of pipes, the number of holes in the middle pipe, and the number of each increment from the inside to the outside pipe. A total of 16 random array sets of three structural parameters with four levels were determined using the orthogonal experimental design. Commercial software was used for the construction of a 3D model for the selected experimental points, and this data was used to obtain the airflow velocities, which were then used to obtain the standard deviation of each experimental point. According to the range analysis, the combination of the three structural parameters was optimized. In other words, an efficient and economical optimization method considering the performance of the vented box was established, and it could be widely used to extend the storage time of fresh food.

Here, we present the range analysis method to optimize the sample points generated by an orthogonal experimental design to ensure that fresh food can be stored in a vented box for a long time by regulating the airflow pattern.

This study aims to solve the problems of air flow chaos and poor performance in a vented box caused by the heterogeneous distribution of airflow through ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

Biology

High-Throughput Assays of Critical Thermal Limits in Insects

Upper and lower thermal limits of plants and animals are important predictors of their performance, survival, and geographic distributions, and are essential for predicting responses to climate change. This work describes two high-throughput protocols for measuring insect thermal limits: one for assessing critical thermal minima (CTmin), and the other for assessing heat knock down time (KDT) in response to a static heat stressor. In the CTmin assay, individuals are placed in an acrylic-jacketed column, subjected to a decreasing temperature ramp, and counted as they fall from their perches using an infrared sensor. In the heat KDT assay, individuals are contained in a 96 well plate, placed in an incubator set to a stressful, hot temperature, and video recorded to determine the time at which they can no longer remain upright and move. These protocols offer advantages over commonly used techniques. Both assays are low cost and can be completed relatively quickly (~2 h). The CTmin assay reduces experimenter error and can measure a large number of individuals at once. The heat KDT protocol generates a video record of each assay and thus removes experimenter bias and the need to continuously monitor individuals in real time.

Thermal limits can predict the environments organisms tolerate, which is valuable information in the face of rapid climate change. Described here are high-throughput protocols to assess critical thermal minima and heat knockdown time in insects. Both protocols maximize the throughput and minimize the cost of the assays.

Upper and lower thermal limits of plants and animals are important predictors of their performance, survival, and geographic distributions, and are ...

Cross Cylindrical Flow: Measuring Pressure Distribution and Estimating Drag Coefficients

Source: David Guo, College of Engineering, Technology, and Aeronautics (CETA), Southern New Hampshire University (SNHU), Manchester, New Hampshire
The pressure distributions and drag estimations for cross cylindrical flow have been investigated for centuries. By ideal inviscid potential flow theory, the pressure distribution around a cylinder is vertically symmetric. The pressure distribution upstream and downstream of the cylinder is also symmetric, which results in a zero-net drag force. However, experimental results yield very different flow patterns, pressure distributions and drag coefficients. This is because the ideal inviscid potential theory assumes irrotational flow, meaning viscosity is not considered or taken into account when determining the flow pattern. This differs significantly from reality.
In this demonstration, a wind tunnel is utilized to generate a specified airspeed, and a cylinder with 24 ports of pressure is used to collect pressure distribution data. This demonstration illustrates how the pressure of a real fluid flowing around a circular cylinder differs from predicted results based on the potential flow of an idealized fluid. The drag coefficient will also be estimated and compared to the predicted value.

Cross Cylindrical Flow: Pressure Distribution &amp; Drag Coefficients

Source: David Guo, College of Engineering, Technology, and Aeronautics (CETA), Southern New Hampshire University (SNHU), Manchester, New Hampshire
The ...

Education

JoVE Science Education

Aeronautical Engineering

Efficiency of Liquid-liquid Extraction

Source: <a href="https://www.jove.com/author/Kerry+M._Dooley">Kerry M. Dooley</a> and Michael G. Benton, Department of Chemical Engineering, Louisiana State University, Baton Rouge, LA
Liquid-liquid extraction (LLE) is a separation technique used instead of distillation when either: (a) the relative volatilities of the compounds to be separated are very similar; (b) one or more of the mixture components are temperature sensitive even near ambient conditions; (c) the distillation would require a very low pressure or a very high distillate/feed ratio.1The driving force for mass transfer is the difference in solubility of one material (the solute) in two other immiscible or partially miscible streams (the feed and the solvent). The feed and solvent streams are mixed and then separated, allowing the solute to transfer from the feed to the solvent. Normally, this process is repeated in successive stages using counter-current flow. The solute-rich solvent is called the extract as it leaves, and the solute-depleted feed is the raffinate. When there is a reasonable density difference between the feed and solvent streams, extraction can be accomplished using a vertical column, although in other cases a series of mixing and settling tanks may be used.
In this experiment, the operational goal is to extract isopropanol (IPA, ~10 - 15 wt. %, the solute) from a mixture of C8-to-C10 hydrocarbons using pure water as solvent. A York-Scheibel type (vertical mixers and coalescers, one each per physical stage) extraction column is available. Like most extractors, the overall efficiency (number theoretical stages/physical stages) of this column is quite low, especially in comparison to many distillation columns. The low efficiencies arise from both slow mass transfer (two liquid resistances instead of one as in distillation) and often also from maldistribution of the phases. The effect of agitator speed on both the solute recovery in the extract and the overall column efficiency will be evaluated.

Liquid-liquid Extraction and York-Scheibel Column Operation

Source: Kerry M. Dooley and Michael G. Benton, Department of Chemical Engineering, Louisiana State University, Baton Rouge, LA
Liquid-liquid extraction ...