Name: post column derivatization using reaction flow high performance liquid chromatography columns
Uploaded: 2016-04-26T13:00:00
Description: Watch this Scientific Journal Video about Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns at JoVE.com

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

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 meth...

<ol>
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Chromatogr. A. 1217, 7690-7698 (2010).</li><li>Raudonis, R., Raudone, L., Jakstas, V., Janulis, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+post-column+free+radical+scavenging+and+ferric+reducing+antioxidant+power+assays+for+screening+of+antioxidants+in+strawberries.">Comparative evaluation of post-column free radical scavenging and ferric reducing antioxidant power assays for screening of antioxidants in strawberries.</a> J. Chromatogr. A. 1233, 8-15 (2012).</li><li>Zakrzewski, R. <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+Methimazole+in+Pharmaceutical+Preparations+using+an+HPLC+Method+Coupled+with+an+Iodine-Azide+Post-Column+Reaction.">Determination of Methimazole in Pharmaceutical Preparations using an HPLC Method Coupled with an Iodine-Azide Post-Column Reaction.</a> J. Liq. Chrom. Rel. Technol. 32, 383-398 (2008).</li><li>Zakrzewski, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+a+reversed-phase+HPLC+method+with+post-column+iodine-azide+reaction+for+the+determination+of+thioguanine.">Development and validation of a reversed-phase HPLC method with post-column iodine-azide reaction for the determination of thioguanine.</a> J. Anal. 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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. 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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>
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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...

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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 ...

The overall goal of this method is to improve the efficiency and sensitivity of high performance liquid chromatography post column derivatization, or PCD method, through the use of a reaction flow column. This method can help answer key questions in fields such as pharmaceutical, biomedical, and environmental sciences, where compounds that have low response to HPLC detectors are analyzed. The main advantage of this technique is that no reaction coils are needed.As mixing of the column effluent and derivatizing reagent occurs more efficiently than conventional methods. This method has been used to provide insight into antioxidants, amino acids, and phenols. It can also be applied to other class compounds, such as thiols, metals, antibiotics, and toxins.Although the entire sample is not derivatized, due to lower band broadening, the concentration of analyte within the effluent flow stream is higher than in conventional post column derivatization analysis. To begin this procedure, prepare the HPLC instrument with 100%water on Line A and 100%methanol on Line B as the mobile phase, purging the pumps as per the manufacturer's requirements. Set up the HPLC instrumental components and the additional derivatization pump.Following this, set the UV/VIS detector to analyze at a wavelength of 520 nanometers. Connect the inlet of the reaction flow, or RF, column to the HPLC instrument. Connect an outlet peripheral port to the UV/VIS detector using a 15 centimeter length of 0.13 millimeter inner diameter tubing.Next, connect the DPPH pump line to a peripheral port on the outlet of the RF column. Block the unused peripheral port on the outlet of the RF column using a column stopper. Connect a 15 centimeter length of 0.13 millimeter inner diameter tubing to the outlet central port of the RF column.Bring the flow rate of the HPLC pump to one millimeter per minute at 100%methanol. Then equilibrate the column with 100%methanol for 10 minutes. At this point, prepare a 0.1 milligram per milliliter solution of DPPH and methanol.Sonicate the flask containing the DPPH reagent for 10 minutes. Purge the DPPH pump with the prepared DPPH reagent as per the manufacturer's requirements. Following this, take two dry and clean vessels and label one as central"and the other as peripheral"Accurately weigh the two vessels.Collect the effluent exiting the central port into the vessel labeled central"for one minute. After reweighing the central port vessel, calculate the weight of the flow from the central port. Repeat the previous steps for the effluent exiting the UV/VIS that is attached to the peripheral port of the RF column.Calculate the weight for the peripheral port vessel. Next, calculate the percentage of the flow coming from the central and peripheral ports. Repeat the previous steps until the flow ratio is correct, then set the flow rate of the DPPH pump to 0.5 milliliters per minute.Once the run has finished, stop the derivatization reagent pump flow. Remove the DPPH reagent pump line from the peripheral port and stopper the port. Equilibrate the column with the mobile phase in which it is to be stored, allowing the mobile phase to pass through the column at one millimeter per minute for 10 minutes.Then, stop the flow of the bubble phase pump on the HPLC instrument. Finally, replace the DPPH reagent with methanol and purge the additional pump. Two chromatograms of a ristretto coffee sample, derivatized with a DPPH radical using both conventional PCD and RF-PCD instrumentation, are shown here.The calculated limits of quantitation and detection for each of the amino acids analyzed in both RF-PCD and conventional PCD modes are listed here. A chromatogram of the four amino acids analyzed using the conventional PCD method, the RF-PCD method, and the un-derivatized stream from the RF-PCD method is shown here. A comparison of the signals obtained for the peaks due to glycine and leucine using both the conventional PCD and RF-PCD methods is displayed here.A comparison of the peak width of the tryptophan peak when analyzing using the conventional PCD method, the RF-PCD method, and the un-derivatized stream from the RF-PCD method is shown here. A 21-component test sample that contained some components that show a response to the derivatization scheme and some that do not were separated, derivatized, and detected. The same mixture was also separated and detected un-derivatized for comparison.A comparison of the peak shape of para-Cresol, both derivatized using the RF-PCD column and un-derivatized, is shown here. Once mastered, this technique can be set up in the same time as conventional post column derivatization analysis. When performing this procedure, it is important to remember to be as close to the prescribed flow ratios as possible.Following this procedure, other post column derivatization reagents, such as OPA, ninhydrin, or halogens can be used in order to analyze other compounds. After watching this video, you should have a good idea of how to set up and tune a reaction flow column.

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