Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns

Summary

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

Abstract

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.

Introduction

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 available^1,2, increase the signal of the target analyte, which allows lower limits of detection and quantitation^3-5 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-phthaladehyde^7-9, ninhydrin^9,10 or fluorescamine^1....

Protocol

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 in....

Results

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^•)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^• radical in the presence of reactive oxygen species, hence the presence of antioxidants results in a drop in the observed absorbance. The DPPH^• reaction often employs large .......

Discussion

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^• for the detection of ROS in an espresso coffee sample. Their study involved the analys.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HPLC instrument	Agilent	1290 Series HPLC
Additional Pump(s) for derivatization system	Shimadzu	LC-20A
RF colum			Non-commercial
PEEK tubing	Sigma Aldrich	Z227307
Column stoppers			Provided with column
PEEK tube cutter	Sigma Aldrich	Z290882
Analytical Scale Balance			4-point analytical balance
Stop watch			Non-Scientific equiptment
Eluent collection vials			Any Small vial with a flat bottom will do, e.g., HPLC vials
HPLC Vials			Will depend on instrument used
Vessels for mobile phase and derivatization solution(s)	Sigma Aldrich	Z232211
General Laboratory glassware			Volumetric Flasks, pippettes, etc. Quantity and volumes will depend on sample preparation method.
Methanol	Sigma Aldrich	34860
DPPH	Sigma Aldrich	D9132
Ammonium Acetate	Sigma Aldrich	17836
Ammonia	Sigma Aldrich	320145	Corrosive
Acetonitrile	Sigma Aldrich	34998
Fluorescamine	Sigma Aldrich	F9015
4-aminoantipyrene	Acros Organics BVBA	AC103151000
Potassium ferricyanide	AnalaR	B10204-30