Chemiluminescent Reactions and Liquid Chromatography

The applicability of chemiluminescence reactions as a means of detecting compounds in liquid chromatography (LC) is based to a large degree on post column reactions. A primer on liquid chromatography (and high performance LC) can be found here; however, a brief description follows. This describes, in the main, HPLC chromatographic systems.

Components of High Performance Liquid Chromatography

Liquid phase samples (mixtures) are injected onto an LC column usually using a syringe and specially devised injection valve. The sample is swept onto the chromatographic column by the flowing mobile phase and chromatographic separation occurs as the mixture travels down the column. Normal HPLC detectors detect the elution of a compound from the end of the column based on some physical characteristic such as ultraviolet light absorption, ability to fluoresce, or the difference in index of refraction between the analyte and the mobile phase itself. The majority of HPLC systems work this way.

An example schematic of an HPLC system is shown below:

Schematic of an HPLC instrument

Need for HPLC Chemiluminescence Detection

The use of chemiluminescence detection for HPLC comes from the need to detect compounds either very sensitively (at very low concentrations) or very selectively, that is, a target compound that must be determined in the presence of co-eluting compounds that just can not be successfully separated from the analyte. Since chemiluminescence derives from the generation of light cause by a chemical reaction, there is no source lamp light that must be filtered out (as in the case of fluorescence detection) in order to detect the analyte emission. This means that the photons coming from the de-exciting analyte molecule are detected against a black background, and this detection can be accomplished by a photomultiplier which can detect a large percentage of the emitted photons.

Methods of HPLC Post Column Chemiluminescence Detection

IF a target analyte can be determined via HPLC chemiluminescence then it probably has one of three characteristics: 1) it either chemiluminesces when mixed with a specific reagent; 2) it catalyzes chemiluminescence between other reagents; or 3) is suppresses chemiluminescence between other reagents. Examples of all three will be given below using the well explored luminol reaction. Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) reacts with oxidants like hydrogen peroxide (H2O2) in the presence of a base and a metal catalyst to produce an excited state product (3-aminophthalate, 3-APA) which gives off light at approximately 425 nm. If luminol is the target analyte (seldom) then a schematic of a post column detector based on its solution phase reaction would look like this:
Luminol Based HPLC Schematic
In this case one reagent pump would send a solution containing a dissolved metal ion like copper(II) or iron(III) to the mixer at the end of the LC column, while the other reagent pump would send a solution containing the oxidant such as H2O2 or hypochlorite (another oxidant) and a base. Depending on the catalyst used (which basically controls the time necessary for maximum light emission to develop AND the decay profile of that emission) the distance from the mixer to the detection cell is carefully determined to allow for the most sensitive detection-in this case the detection of luminol arriving from the LC column where it could have been separated from interfering compounds. More realistically, some important chemical species can be derivatized using luminol itself or luminol like reagents that can be detected in the same or similar ways. What follows is a method of chemiluminescence detection in which the suppression of a background chemiluminescence signal could be used to determined a compound that elutes from the LC column. For instance, many organic molecules will complex metal cations and thereby make them less available as catalysts in the luminol reaction. This is a nifty way to determine the concentration of the organic molecule: Mix a constant concentrations of a metal cation, luminol, base, and an oxidant. This will create a baseline light signal that is relatively constant. With the LC column output fed into the mixer, the amount of light detected will DECREASE when an organic analyte (which can complex with the metal ion) elutes from the column. The amount of light decrease depends directly on the amount of the analyte. This is true as long as the amount of metal cation is not completely complexed. At this point the light decrease will no longer be linearly related to the amount of organic analyte.

Basically the same schematic seen above is seen here with the metal catalyst coming from the first reagent pump and feeding into a second mixer placed upstream of the first mixer. This is to allow the eluting organic molecules (e.g., analytes like amino acids) to have time to tie up the metal catalyst before they are mixed with the other reagents. The second reagent pump adds luminol, base and oxidant. When that metal/organic complex gets to the second mixer and ultimately to the detection cell, the baseline light intensity will drop off. Voila! An "antisignal"-proportional to the amount of the (analyte) organic molecules eluting from the column!

HPLC Luminol Supression Schematic

These notes were written by Dr. Thomas G. Chasteen at Sam Houston State University, Huntsville, Texas.

Chemical Reaction Properties of different chemiluminescence solution phase reactions
Gas Phase Chemiluminescence

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