This work was supported by UWS and ThermoFisher Scientific. 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 describes 3 methods of reaction flow post-column derivatization (RF-PCD) techniques, each with a different reagent specific to the nature of a chemical compound of interest. For the analysis of ROS go to section &#34;1. Detection of ROS using DPPH&#8226;&#34;, for the analysis of primary amines see section &#34;2. Detection of primary amines using fluorescamine&#34;, and for the analysis of phenolic compounds go to section &#34;3. Detection of phenols using 4-aminoantipyrene and potassium ferricyanide&#34;. Use ultra-pure water (e.g., Milli-Q water) throughout.
Note: Connection of the RF column is achieved in almost the same way as a conventional HPLC column with the major difference being the number of end fittings on a RF column. Fittings used to connect a standard HPLC column to the HPLC system are able to be used to connect a RF column to the HPLC system.
1. Detection of ROS Using DPPH&#8226;
<ol>
	<li>Set up of HPLC instrument
	<ol>
		<li>Prepare the HPLC instrument with 100% water on line A and 100% methanol on line B as the mobile phase. Purge the pumps as per manufacturer&#39;s requirements.</li>
		<li>Set up the HPLC instrumental components and the additional derivatization pump as illustrated in Figure 4A.</li>
		<li>Set the UV-Vis detector to analyze at a wavelength of 520 nm.</li>
	</ol>
	</li>
	<li>Set up of RF column
	<ol>
		<li>Connect the inlet of the RF column to the HPLC instrument.</li>
		<li>Connect a 15 cm length of 0.13 mm i.d. tubing to outlet central port of the RF column.</li>
		<li>Connect an outlet peripheral port to the UV-Vis detector using a 15 cm length of 0.13 mm i.d. tubing.</li>
		<li>Connect the DPPH&#8226; pump line to a peripheral port on the outlet of the RF column.</li>
		<li>Block the unused peripheral port on the outlet of the RF column using a column stopper.</li>
		<li>Bring the flow rate of the HPLC pump to 1 ml min-1 at 100% line B - 100% Methanol.</li>
		<li>Equilibrate the column with the 100 % methanol mobile phase for 10 min for a 4.6 mm i.d. x 100 mm length column. This time should be scaled according to the dimensions of other columns the user may employ.</li>
	</ol>
	</li>
	<li>Preparation of DPPH&#8226; reagent
	<ol>
		<li>Prepare a 0.1 mg/ml solution of DPPH&#8226; in methanol.</li>
		<li>Sonicate the flask containing the DPPH&#8226; reagent for 10 min.</li>
		<li>Cover the flask in foil to prevent exposure to light.</li>
		<li>Purge the DPPH&#8226; pump with the prepared DPPH&#8226; reagent as per the manufacturer&#39;s requirements.</li>
	</ol>
	</li>
	<li>Tuning the RF column outlet
	<ol>
		<li>Accurately weigh two clean and dry vessels. Label one vessel as central and the other as peripheral.</li>
		<li>Collect the effluent exiting the central port into the vessel labeled central for 1.0 min.</li>
		<li>Re-weigh the central port vessel and calculate the weight of the flow from the central port as follows: 
		Weight of Central Port (g) = Final Weight of Central Port Vessel (g) - Initial Weight of Central Port Vessel (g)</li>
		<li>Repeat steps 1.4.2 and 1.4.3 for the effluent exiting the UV-Vis that is attached to the peripheral port of the RF column and calculate weight for the peripheral port vessel as follows: 
		Weight of Peripheral Port (g) = Final Weight of Peripheral Port Vessel (g) - Initial Weight of Peripheral Port Vessel (g)</li>
		<li>Calculate the percentage of the flow coming from the central and peripheral ports as follows: 
		% Central Port = Weight of Central Port (g) / (Weight of Central Port (g) + Weight of Peripheral Port (g)) x 100 
		% Peripheral Port = Weight of Peripheral Port (g) / (Weight of Central Port (g) + Weight of Peripheral Port (g)) x 100</li>
		<li>Ensure the segmentation ratio between the central flow and the peripheral flow is 30:70 (central:peripheral). If the central flow is above 30%, decrease the amount of flow exiting by adding a length of 0.13 mm i.d. tubing to the outlet of the central port. If the central flow is below 30% decrease the length of 0.13 mm tubing from the central port.</li>
		<li>Repeat step 1.4.1 to 1.4.6 until a segmentation ratio of 30:70 (central:peripheral) is achieved.</li>
		<li>Set the flow rate of the DPPH&#8226; reagent pump to 0.5 ml min-1. 
		Note: The RF column set up with DPPH&#8226; reagent is now ready for analysis. Samples may now be injected.</li>
	</ol>
	</li>
	<li>Post run shutdown conditions
	<ol>
		<li>Once all of the samples have been injected, indicating that the run has finished, stop the derivatization reagent pump flow.</li>
		<li>Remove the DPPH&#8226; reagent pump line from the peripheral port and stopper the port.</li>
		<li>Equilibrate the column with the mobile phase in which it is to be stored by allowing the mobile phase to pass through the column at 1 ml min-1 for 10 min.</li>
		<li>Stop the flow of the mobile phase pump on the HPLC instrument.</li>
		<li>Replace the DPPH&#8226; reagent with methanol and purge the additional pump. 
		Note: The HPLC system can now be shutdown.</li>
	</ol>
	</li>
</ol>
2. Detection of Primary Amines Using Fluorescamine
<ol>
	<li>Preparation of mobile phase
	<ol>
		<li>Prepare 1 L of a of 10 mM ammonium acetate solution, adjusting the pH of the solution to 9.0 with 5 M ammonium hydroxide prior to dilution to volume.</li>
		<li>Add 52.6 ml of acetonitrile (ACN) to the ammonium acetate buffer to attain a premixed mobile phase of 95:05 (buffer:ACN).</li>
	</ol>
	</li>
	<li>Set up of HPLC instrument
	<ol>
		<li>Prepare the HPLC instrument with the pre-mixed prepared buffer on line A as the mobile phase. Purge the pump as per manufacturer&#39;s requirements.</li>
		<li>Set up the HPLC instrumental components and the additional derivatization pump as illustrated in Figure 4A.</li>
		<li>Attach a pulse dampener coil to the derivatization pump.</li>
		<li>Set-up the fluorescence detector (FLD) with an excitation wavelength of 390 nm and emission wavelength of 475 nm.</li>
	</ol>
	</li>
	<li>Set up of RF column
	<ol>
		<li>Connect the inlet of the RF column to the HPLC instrument.</li>
		<li>Connect a 15 cm length of 0.13 mm i.d. tubing to outlet central port of the RF column.</li>
		<li>Connect an outlet peripheral port of the RF column to the FLD detector using a 15 cm length of 0.13 mm i.d. tubing.</li>
		<li>Connect the derivatization pump line to a peripheral port on the outlet of the RF column.</li>
		<li>Block the unused peripheral port on the outlet of the RF column using a column stopper.</li>
		<li>Bring the flow rate of the HPLC pump to 1 ml min-1 at 100% line A - 10 mM Ammonium Acetate buffer pH 9, premixed with 5% ACN.</li>
		<li>Equilibrate the column with the 100 % line A mobile phase for 10 min&#160;for a 4.6 mm i.d. x 100 mm length column. This time may be scaled according to the dimensions of other columns the user may employ.</li>
	</ol>
	</li>
	<li>Preparation of fluorescamine reagent
	<ol>
		<li>Make 100 ml of a 0.1 mg ml-1 fluorescamine reagent.</li>
		<li>Sonicate for 1 min.</li>
		<li>Cover with foil to prevent exposure to light.</li>
		<li>Purge the reagent pump with the prepared fluorescamine reagent as per the manufacturer&#39;s requirements.</li>
	</ol>
	</li>
	<li>Tuning the RF column outlet
	<ol>
		<li>Accurately weigh two clean and dry vessels. Label one vessel as central and the other as peripheral.</li>
		<li>Collect the effluent exiting the central port into the vessel labeled central for 1.0 min.</li>
		<li>Re-weigh the central vessel and calculate the weight of the flow from the central port as follows in step 1.4.3.</li>
		<li>Repeat steps 2.5.2 to 2.5.3 for the effluent exiting the FLD that is attached to the peripheral port of the RF column and calculate weight for peripheral as follows in step 1.4.4.</li>
		<li>Calculate the percentage of the flow coming from the central and peripheral ports as follows in step 1.4.5.</li>
		<li>Ensure the segmentation ratio between the central flow and the peripheral flow is 43:57 (central:peripheral). If the central flow is above 43%, decrease the amount of flow exiting by adding a length of 0.13 mm i.d. tubing to the outlet of the central port. If the central flow is below 43%, decrease the length of 0.13 mm tubing from the central port.</li>
		<li>Repeat step 2.5.1 to 2.5.6 until a segmentation ratio of 43:57 (central:peripheral) is achieved.</li>
		<li>Set the flow rate of the mobile phase pump to 0.7 ml min-1.</li>
		<li>Set the derivatization pump to flow at 0.1 ml min-1. 
		Note: The RF column set up with fluorescamine reagent is now ready for analysis. Samples may now be injected.</li>
	</ol>
	</li>
	<li>Post run shutdown conditions
	<ol>
		<li>Once all of the samples have been injected, indicating that the run has finished, stop the derivatization pump.</li>
		<li>Remove the derivatization pump line from the peripheral port and stopper.</li>
		<li>Equilibrate the column with the mobile phase in which it is to be stored by allowing the mobile phase to pass through the column at 1 ml min-1 for 10 min.</li>
		<li>Stop the flow of the mobile phase pump.</li>
		<li>Replace the fluorescamine reagent with acetonitrile and purge the derivatization pump. 
		Note: The HPLC system can now be shutdown.</li>
	</ol>
	</li>
</ol>
3. Detection of Phenols Using 4-Aminoantipyrene and Potassium Ferricyanide
<ol>
	<li>Preparation of mobile phase
	<ol>
		<li>Prepare 1 L of a of 100 mM ammonium acetate solution, adjusting the pH of the solution to 9.0 with 5 M ammonium hydroxide prior to dilution to volume.</li>
		<li>Add 52.6 ml of methanol to the ammonium acetate buffer to attain a premixed mobile phase of 95:5 (buffer:methanol).</li>
	</ol>
	</li>
	<li>Set up of HPLC instrument
	<ol>
		<li>Prepare the HPLC instrument with the pre-mixed prepared buffer on line A as the mobile phase. Purge the pump as per manufacturer&#39;s requirements.</li>
		<li>Set up the HPLC instrumental components and the two additional reagent pumps as illustrated in Figure 4B.</li>
		<li>Attach a pulse dampener coil to each of the reagent pumps.</li>
		<li>Set the UV-Vis detector to analyze at a wavelength of 500 nm.</li>
	</ol>
	</li>
	<li>Set up of RF column
	<ol>
		<li>Connect the inlet of the RF column to the HPLC instrument.</li>
		<li>Connect a 15 cm length of 0.13 mm i.d. tubing to outlet central port of the RF column.</li>
		<li>Connect an outlet peripheral port of the RF column to the UV-Vis detector using a 15 cm length of 0.13 mm i.d. tubing.</li>
		<li>Connect each reagent (i.e., 4-aminoantipyrene and potassium ferricyanide) pump line to a peripheral port on the outlet of the RF column.</li>
		<li>Bring the flow rate of the HPLC pump to 1 ml min-1 at 100% line A - 100 mM Ammonium Acetate buffer pH 9, premixed with 5% methanol.</li>
		<li>Equilibrate the column with the 100 % line A mobile phase for 10 min&#160;for a 4.6 mm i.d. x 100 mm length column. This time may be scaled according to the dimensions of other columns the user may employ.</li>
	</ol>
	</li>
	<li>Preparation of 4-aminoantipyrene reagent
	<ol>
		<li>Prepare an ammonium acetate buffer with a pH of 9 by following step 3.1.1.</li>
		<li>Weigh 150 mg 4-aminoantipyrene and dissolve in 100 ml of prepared ammonium acetate buffer (pH 9).</li>
		<li>Sonicate for 1 min.</li>
		<li>Cover with foil to prevent exposure to light.</li>
		<li>Purge the first reagent pump with the prepared 4-aminoantipyrene reagent as per the manufacturer&#39;s requirements.</li>
	</ol>
	</li>
	<li>Preparation of potassium ferricyanide reagent
	<ol>
		<li>Weigh 150 mg of potassium ferricyanide and dissolve in 100 ml of ammonium acetate buffer (pH 9) prepared as per step 3.1.1.</li>
		<li>Sonicate for 1 min.</li>
		<li>Cover in foil to prevent exposure to light.</li>
		<li>Purge the second reagent pump with the prepared potassium ferricyanide reagent as per the manufacturer&#39;s requirements.</li>
	</ol>
	</li>
	<li>Tuning of RF column
	<ol>
		<li>Accurately weigh two clean and dry vessels. Label one vessel as central and the other as peripheral.</li>
		<li>Collect the effluent exiting the central port into the vessel labeled central for 1.0 min.</li>
		<li>Re-weigh the central vessel and calculate the weight of the flow from the central port as follows in step 1.4.3.</li>
		<li>Repeat steps 3.6.2 to 3.6.3 for the effluent exiting the UV-Vis that is attached to the peripheral port of the RF column and calculate weight for peripheral as follows in step 1.4.4.</li>
		<li>Calculate the percentage of the flow coming from the central and peripheral ports as follows in step 1.4.5.</li>
		<li>Ensure the segmentation ratio between the central flow and the peripheral flow is 50:50 (central:peripheral). If the central flow is above 50%, decrease the amount of flow exiting by adding a length of 0.13 mm i.d. tubing to the outlet central port. If the central flow is below 50%, decrease the length of 0.13 mm tubing from the central port.</li>
		<li>Repeat step 3.6.1 to 3.6.6 until a segmentation ratio of 50:50 (central:peripheral) is achieved.</li>
		<li>Set the flow rate of the 4-aminoantipyrene pump to 0.5 ml min-1.</li>
		<li>Set the flow rate of the potassium ferricyanide pump to 0.25 ml min-1. 
		Note: The RF column set up with the two-component reagents is now ready for analysis. Samples may now be injected.</li>
	</ol>
	</li>
	<li>Post run shutdown conditions
	<ol>
		<li>Once all of the samples have been injected the run has finished, indicating that the run has finished, stop both of the reagent pumps.</li>
		<li>Remove the reagent pump lines from the peripheral ports and replace them with a 15 cm piece of 0.13 mm tubing.</li>
		<li>Equilibrate the column with the mobile phase in which it is to be stored by allowing the mobile phase to pass through the column at 1 ml min-1 for 10 min.</li>
		<li>Stop the flow of the mobile phase pump on the HPLC instrument.</li>
		<li>Replace both the reagents on the reagent pumps with methanol and purge the additional pumps as per manufacturer requirement. 
		Note: The HPLC system can now be shutdown.</li>
	</ol>
	</li>
</ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reaction+Detectors+in+Modern+Liquid+Chromatography.">Reaction Detectors in Modern Liquid Chromatography.</a> Chromatographia. 15, 161-166 (1982).</li><li>Pravadil-Cekic, S., 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=Using+Reaction+Flow+Chromatography+for+the+Analysis+of+Amino+Acid:+Derivatisation+With+Fluorescamine+Reagent.">Using Reaction Flow Chromatography for the Analysis of Amino Acid: Derivatisation With Fluorescamine Reagent.</a> Microchem. J. , (2015).</li><li>Camenzuli, M., Ritchie, H. J., Dennis, G. R., Shalliker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+segmented+flow+chromatography+columns+with+multiplexed+detection:+An+illustration+using+antioxidant+screening+of+natural+products.">Parallel segmented flow chromatography columns with multiplexed detection: An illustration using antioxidant screening of natural products.</a> Microchem. J. 110, 726-730 (2013).</li><li>Koleva, I. I., Niederlander, H. A. G., van Beek, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+On-Line+HPLC+Method+for+Detection+of+Radical+Scavenging+Compounds+in+Complex+Mixtures.">An On-Line HPLC Method for Detection of Radical Scavenging Compounds in Complex Mixtures.</a> Anal Chem. 72, 2323-2328 (2000).</li><li>Selim, 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=A+Two-component+Post-column+Derivatisation+Method+Utilsing+Reaction+Flow+Chromatography.">A Two-component Post-column Derivatisation Method Utilsing Reaction Flow Chromatography.</a> Microchem. J. 116, 87-91 (2014).</li><li>Bigley, F. P., Grob, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Phenols+in+Water+and+Wastewater+by+Post-column+Reaction+Detection+High-performance+Liquid+Chromatography.">Determination of Phenols in Water and Wastewater by Post-column Reaction Detection High-performance Liquid Chromatography.</a> J. Chromatogr. A. 350, 407-416 (1985).</li><li>Camenzuli, M., Ritchie, H. J., Dennis, G. R., Shalliker, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reaction+flow+chromatography+for+rapid+post+column+derivatisations:+The+analysis+of+antioxidants+in+natural+products.">Reaction flow chromatography for rapid post column derivatisations: The analysis of antioxidants in natural products.</a> J. Chromatogr. A. 1303, 62-65 (2013).</li></ol>

The authors have nothing to disclose.

RF-PCD allows for the efficient mixing of the derivatization reagent with the HPLC effluent post-column without the use of reaction coils, minimizing the effects of band broadening and improving separation performance. RF-PCD methods have also shown improvements in signal response with respect to detection method. Camenzuli et al.28 was the first to report the use of reaction flow columns with DPPH&#8226; for the detection of ROS in an espresso coffee sample. Their study involved the analys...

High performance liquid chromatography (HPLC) coupled with post column derivatization (PCD) is a powerful tool that is useful in solving a number of issues in the analytical laboratory. It can be used to detect compounds that are otherwise undetectable with the suite of detectors available1,2, increase the signal of the target analyte, which allows lower limits of detection and quantitation3-5 or selectively derivatize a target analyte in order to avoid matrix effects6. Commonly used PCD reactions include the reaction of amines, such as amino acids, with ortho-phthaladehyde7-9, ninhydrin9,10 or fluorescamine11,12, the derivatization of reactive oxygen species (ROS) with the 2,2-diphenyl-1-picrylhydrazil radical (DPPH&#8226;)13,14 or 2,2&#39;-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS)15,16, and the use of the iodide-azide reagent to derivatize sulfur containing compounds17,18.
There are, however, numerous drawbacks to the use of PCD reactions with HPLC systems6. Principally among these is the use of reaction coils between the point of addition of the derivatization reagent(s) and the detector, which allow time for mixing and the reaction to occur8. These reaction loops often have volumes of 500 &#181;l or more, which is significant compared to the volume of the rest of the HPLC system19. The use of these high volume reaction loops results in increased peak broadening compared to what would be observed without the presence of the reaction loop. This results in shorter, wider peaks that have higher limits of quantitation and detection and negatively effects chromatographic resolution. Figures 1 and 2 highlight the deterioration of peak shape that result from the addition of various post column reaction loop volumes. This analysis was performed with a mobile phase composition of 94% methanol and 6% Milli-Q water. The flow rate of the mobile phase was 1 ml/min, the injection volume was 20 &#181;l and the analysis wavelength was 265 nm. Coils of varying dead volumes from 20 &#181;l to 1,000 &#181;l were inserted between the column and the detector to simulate the effects of reaction loop dead volume in PCD methods. These loops were prepared from stainless steel tubing of 0.5 mm internal diameter. The experiment was performed on a HPLC system consisting of a controller (SCL-10AVP), a Low Pressure Gradient Valve (FCL-10ALVP), a Pump (LC-20AD), an Injector (SIL-10ADVP), and a pda detector (SPD-M10ADVP). The mobile phase was pumped through a degasser prior to introduction into the HPLC system. The separation was performed using a 250 mm x 4.6 mm i.d. 5 &#181;m column. Experimental conditions were chosen to be typical of PCD reactions that have recently been published in the literature.
The simplest, most common post column reactor setup is termed a non-segmented tubular reactor which is effectively a long, thin tube through which the liquid can flow and the reaction can take place. In this system peak broadening is dependent on not just the dead volume added to the system, but also the internal diameter of the tube as highlighted by Iijima et al.8. Furthermore, coil geometry plays a part in the observed brand broadening. Stewart 20 stated that coiling of the reactor changes the secondary flow profiles, resulting in better mixing, meaning that the dead volume may be minimized. It has been stated that peak broadening is not significant when using an open tubular knitted coil 21 . When the peak broadening is excessively large, other types of reactors may also be considered 20,22. These may include bed reactors or segmented flow reactors. These reactors are particularly useful for slow reactions that would otherwise require large reaction loops. As non-segmented tubular reactors are the most common types of reactors used in PCD applications, the rest of this article deals with this type of reactor setup.
The design of the reaction flow (RF) column incorporates a multi-port end fitting that allows mobile phase to exit (or enter) the column through either a single port located at the radial central region of the column or three ports located at the outer wall region of the column (see Figure 3). These two streams are separated using an end fitting containing a central porous frit that is surrounded by an impermeable ring that is in turn surrounded by an outer porous frit that extends out to the column wall. Due to the central impermeable ring cross flow is not possible between the two porous regions.
During reaction flow chromatography, the derivatization reagent(s) are pumped against the direction of mobile phase flow into one or two of the outer ports of the reaction flow column. The column eluent is mixed with the derivatization reagent(s) in the outer frit and passed to the detector through a free outer port. Reaction flow can be used for either a single reagent derivatization (1 port for the derivatization reagent, 1 port to pass the column eluent to the detector and 1 port blocked) or a dual reagent system (2 ports for the derivatization reagents and 1 port to pass the column eluent to the detector). The flow from the central stream can either be used to detect the underivatized column eluent, effectively multiplexing detection23, or passed to waste.
One major tuning technique that is available when running RF-PCD chromatography is the ratio of the central and peripheral flows. The optimum ratio for each derivatization depends on a number of factors such as whether the central flow will be detected or passed to waste. Therefore once the optimum ratio has been determined, it should be ensured that the correct flow ratio is achieved prior to each run being performed.
It has been found that the use of a frit to mix the column eluent stream and the derivatization reagent in RF-PCD results in more efficient mixing compared with traditional mixing techniques that typically employ a zero dead volume T-piece or low dead volume W-piece to mix the two streams. This has allowed for the use of relatively small reaction loops, or even the elimination of the reaction loop altogether. The reduction of the reaction loop size results in sharper peaks compared to traditional post column derivatization methods. This means that, despite the fact that not all of the column eluent is derivatized, greater signal to noise ratios are observed and therefore lower limits of detection and quantitation can be achieved.
Reaction flow chromatography has been developed to overcome difficulties with the adaption of PCD reactions to modern HPLC columns and systems, particularly the loss in efficiency caused by band broadening due to large post column dead volumes caused by the need to employ large volume reaction loops. The more efficient mixing processes in RF-PCD compared to conventional PCD mean that smaller reaction loop volumes may be employed leading to an increase in observed separation efficiency. Furthermore RF-PCD chromatography shows both increased signal and decreased noise compared to conventional PCD techniques resulting in lower limits of detection and quantitation compared to conventional PCD methods. An additional advantage of RF-PCD compared to conventional PCD methods is the ability to monitor the underivatized stream that elutes from the central port of the RF column as well as the derivatized stream that elutes from the peripheral region of the column. RF-PCD is a relatively new but promising technique that displays many advantages over traditional PCD methods.
Connection of the RF column is achieved in almost the same way as a conventional HPLC column with the major difference being the number of end fittings on a RF column. Fittings used to connect a standard HPLC column to the HPLC system are able to be used to connect a RF column to the HPLC system.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>HPLC instrument</td>
			<td>Agilent</td>
			<td>1290 Series HPLC</td>
			<td></td>
		</tr>
		<tr>
			<td>Additional Pump(s) for derivatisation system</td>
			<td>Shimadzu</td>
			<td>LC-20A</td>
			<td></td>
		</tr>
		<tr>
			<td>RF colum</td>
			<td></td>
			<td></td>
			<td>Non-commercial</td>
		</tr>
		<tr>
			<td>PEEK tubing</td>
			<td>Sigma Aldrich</td>
			<td>Z227307</td>
			<td></td>
		</tr>
		<tr>
			<td>Column stoppers</td>
			<td></td>
			<td></td>
			<td>Provided with column</td>
		</tr>
		<tr>
			<td>PEEK tube cutter</td>
			<td>Sigma Aldrich</td>
			<td>Z290882</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical Scale Balance</td>
			<td></td>
			<td></td>
			<td>4-point analytical balance</td>
		</tr>
		<tr>
			<td>Stop watch</td>
			<td></td>
			<td></td>
			<td>Non-Scientific equiptment</td>
		</tr>
		<tr>
			<td>Eluent collection vials</td>
			<td></td>
			<td></td>
			<td>Any Small vial with a flat bottom will do e.g. HPLC vials</td>
		</tr>
		<tr>
			<td>HPLC Vials</td>
			<td></td>
			<td></td>
			<td>Will depend on instrument used</td>
		</tr>
		<tr>
			<td>Vessels for mobile phase and derivatisation solution(s)</td>
			<td>Sigma Aldrich</td>
			<td>Z232211</td>
			<td></td>
		</tr>
		<tr>
			<td>General Laboratory glassware</td>
			<td></td>
			<td></td>
			<td>Volumetric Flasks, pippettes, etc. Quantity and volumes will depend on sample preparation method</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>DPPH</td>
			<td>Sigma Aldrich</td>
			<td>D9132</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Acetate</td>
			<td>Sigma Aldrich</td>
			<td>17836</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonia</td>
			<td>Sigma Aldrich</td>
			<td>320145</td>
			<td>Corrosive</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma Aldrich</td>
			<td>34998</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescamine</td>
			<td>Sigma Aldrich</td>
			<td>F9015</td>
			<td></td>
		</tr>
		<tr>
			<td>4-aminoantipyrene&#160;</td>
			<td>Acros Organics BVBA</td>
			<td>AC103151000</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium ferricyanide&#160;</td>
			<td>AnalaR</td>
			<td>B10204-30</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The first PCD method that was adapted for use by RF-PCD was the derivatization of antioxidants using the 2,2-diphenyl-1-picrylhydrazil radical (DPPH&#8226;)24. This reaction was introduced by Koleva et al.25 and has been widely used since. The detection relies on the decolorization of the DPPH&#8226; radical in the presence of reactive oxygen species, hence the presence of antioxidants results in a drop in the observed absorbance. The DPPH&#8226; reaction of...

Watch this Scientific Journal Video about Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns at JoVE.com

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

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14.9K Views.  University of Western Sydney. A protocol for the use of reaction flow high performance liquid chromatography columns for methods employing post column derivatization (PCD) is presented.

Video: Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry UPLC-MS

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to ...

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

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

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

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

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

Isolation and Preparation of Bacterial Cell Walls for Compositional Analysis by Ultra Performance Liquid Chromatography

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan&#8217;s central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

The bacterial cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by peptides. Ultra Performance Liquid Chromatography provides high resolution and throughput for novel discoveries of peptidoglycan composition. We present a procedure for the isolation of cell walls (sacculi) and their subsequent preparation for analysis via UPLC.

The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the ...

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 ...

Curtain Flow Column: Optimization of Efficiency and Sensitivity

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

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

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

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 ...

Synthesis of Programmable Main-chain Liquid-crystalline Elastomers Using a Two-stage Thiol-acrylate Reaction

This study presents a novel two-stage thiol-acrylate Michael addition-photopolymerization (TAMAP) reaction to prepare main-chain liquid-crystalline elastomers (LCEs) with facile control over network structure and programming of an aligned monodomain. Tailored LCE networks were synthesized using routine mixing of commercially available starting materials and pouring monomer solutions into molds to cure. An initial polydomain LCE network is formed via a self-limiting thiol-acrylate Michael-addition reaction. Strain-to-failure and glass transition behavior were investigated as a function of crosslinking monomer, pentaerythritol tetrakis(3-mercaptopropionate) (PETMP). An example non-stoichiometric system of 15 mol% PETMP thiol groups and an excess of 15 mol% acrylate groups was used to demonstrate the robust nature of the material. The LCE formed an aligned and transparent monodomain when stretched, with a maximum failure strain over 600%. Stretched LCE samples were able to demonstrate both stress-driven thermal actuation when held under a constant bias stress or the shape-memory effect when stretched and unloaded. A permanently programmed monodomain was achieved via a second-stage photopolymerization reaction of the excess acrylate groups when the sample was in the stretched state. LCE samples were photo-cured and programmed at 100%, 200%, 300%, and 400% strain, with all samples demonstrating over 90% shape fixity when unloaded. The magnitude of total stress-free actuation increased from 35% to 115% with increased programming strain. Overall, the two-stage TAMAP methodology is presented as a powerful tool to prepare main-chain LCE systems and explore structure-property-performance relationships in these fascinating stimuli-sensitive materials.

A novel methodology is presented to synthesize and program main-chain liquid-crystalline elastomers using commercially available starting monomers. A wide range of thermomechanical properties was tailored by adjusting the amount of crosslinker, while the actuation performance was dependent on the amount of applied strain during programming.

This study presents a novel two-stage thiol-acrylate Michael addition-photopolymerization (TAMAP) reaction to prepare main-chain liquid-crystalline ...

A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography (HPLC)

Glucosinolates are a well-studied and highly diverse class of natural plant compounds. They play important roles in plant resistance, rapeseed oil quality, food flavoring, and human health. The biological activity of glucosinolates is released upon tissue damage, when they are mixed with the enzyme myrosinase. This results in the formation of pungent and toxic breakdown products, such as isothiocyanates and nitriles. Currently, more than 130 structurally different glucosinolates have been identified. The chemical structure of the glucosinolate is an important determinant of the product that is formed, which in turn determines its biological activity. The latter may range from detrimental (e.g., progoitrin) to beneficial (e.g., glucoraphanin). Each glucosinolate-containing plant species has its own specific glucosinolate profile. For this reason, it is important to correctly identify and reliably quantify the different glucosinolates present in brassicaceous leaf, seed, and root crops or, for ecological studies, in their wild relatives. Here, we present a well-validated, targeted, and robust method to analyze glucosinolate profiles in a wide range of plant species and plant organs. Intact glucosinolates are extracted from ground plant materials with a methanol-water mixture at high temperatures to disable myrosinase activity. Thereafter, the resulting extract is brought onto an ion-exchange column for purification. After sulfatase treatment, the desulfoglucosinolates are eluted with water and the eluate is freeze-dried. The residue is taken up in an exact volume of water, which is analyzed by high-pressure liquid chromatography (HPLC) with a photodiode array (PDA) or ultraviolet (UV) detector. Detection and quantification are achieved by conducting comparisons of the retention times and UV spectra of commercial reference standards. The concentrations are calculated based on a sinigrin reference curve and well-established response factors. The advantages and disadvantages of this straightforward method, when compared to faster and more technologically advanced methods, are discussed here.

A Straightforward Method for Glucosinolate Extraction and Analysis with High-pressure Liquid Chromatography HPLC

Here, we describe in great detail an established and robust protocol for the extraction of glucosinolates from ground plant materials. After an on-column sulfatase treatment of the extracts, the desulfoglucosinolates are eluted and analyzed by high-pressure liquid chromatography.

Glucosinolates are a well-studied and highly diverse class of natural plant compounds. They play important roles in plant resistance, rapeseed oil ...

A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and related diseases, but their precise measurement is still a challenge. Many protocols have been described for neurotransmitter measurement by a variety of instruments, including high-pressure liquid chromatography (HPLC). However, there are shortcomings, such as complicated operation or hard-to-detect multiple targets, which cannot be avoided, and presently, the dominant analysis technique is still HPLC coupled with sensitive electrochemical or fluorimetric detection, due to its high sensitivity and good selectivity. Here, a detailed protocol is described for the pretreatment and detection of catecholamines with high pressure liquid chromatography with electrochemical detection (HPLC-ECD) in real urine samples of infants, using electrospun composite nanofibers composed of polymeric crown ether with polystyrene as adsorbent, also known as the packed-fiber solid phase extraction (PFSPE) method. We show how urine samples can be easily precleaned by a nanofiber-packed solid phase column, and how the analytes in the sample can be rapidly enriched, desorbed, and detected on an ECD system. PFSPE greatly simplifies the pretreatment procedures for biological samples, allowing for decreased time, expense, and reduction of the loss of targets. 
Overall, this work illustrates a simple and convenient protocol for solid-phase extraction coupled to an HPLC-ECD system for simultaneous determination of three monoamine neurotransmitters (norepinephrine (NE), epinephrine (E), dopamine (DA)) and two of their metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC)) in infants' urine. The established protocol was applied to assess the differences of urinary catecholamines and their metabolites between high-risk infants with perinatal brain damage and healthy controls. Comparative analysis revealed a significant difference in urinary MHPG between the two groups, indicating that the catecholamine metabolites may be an important candidate marker for early diagnosis of cases at risk for brain damage in infants.

We present a convenient solid-phase extraction coupled to high-pressure liquid chromatography (HPLC) with electrochemical detection (ECD) for simultaneous determination of three monoamine neurotransmitters and two of their metabolites in infants' urine. We also identify the metabolite MHPG as a potential biomarker for the early diagnosis of brain damage for infants.

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and ...

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 ...