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What Is Chemiluminescence ?


Chemiluminescence (CL) refers to the production of light during a chemical reaction. If this takes place inside a living organism it is called bioluminescence. The most familiar terrestrial example of this "cold light" is in the common firefly. An enzyme called luciferase (a name meaning "light-bearing") triggers a reaction that produces energy emitted as light - the flashing beacon from the insect's lower abdomen. Bioluminescence is also found in some fungi and earthworms. It is most common in the oceans, where many organisms, from fish to worms living at great depths, have glowing organs.

Chemists have exploited these light-emitting reactions as markers in a large number of laboratory and clinical tests. The same types of reaction produce the light from emergency "light sticks" sold to campers and in the glowing necklaces seen at concerts and sporting events.

Chemiluminescence takes a special place among other spectroscopic techniques because of its inherent sensitivity, although it can suffer from lack of selectivity.

It requires:

• no excitation source (as do fluorescence and phosphorescence)
• only a single light detector such as a photomultiplier tube
• no monochromator and often not even a filter

Although not as widely applicable as fluorescence spectroscopy, the detection limits for chemiluminescent methods can be 10 to 100 times better (i.e. lower) than using other luminescence techniques.

Luminol Chemiluminescence

In a typical CL system, a "fuel" is chemically oxidized to produce an excited state species. In the extensively-studied luminol system, oxidation takes place in basic aqueous solution, using an oxidizer such as peroxide, perborate, permanganate, or hypochlorite. The blue emitting species here is 3-aminophthalate, whose emission peak lies in the blue region of the visible spectrum near 440 nm.

Forensic scientists use chemiluminescence to identify blood spatter at a suspected crime scene. A mixture of luminol and hydrogen peroxide is sprayed over the surface and any iron(II) present (a constituent of red blood cells, which are very hard to remove from any surface) catalyses the oxidation of luminol by hydrogen peroxide under alkaline conditions. The excited species produced relaxes back to its ground state and a tell-tale blue chemiluminescent glow is produced.

The presence of a catalyst is critical to the deployment of CL as an analytical technique. Many metal cations catalyze the reaction to increase light emission or increase the speed of the oxidation producing the emitter and therefore the onset/intensity of light production. Some metals, however, suppress chemiluminescence at different concentrations. This can be used as the basis for a variety of different analytical determinations.

For example, luminol CL can be useful to (i) determine hydrogen peroxide or the progress of reactions that produce H2O2, (ii) to determine the concentrations of metal cations and (iii) to determine analytes that effect the concentration of metal catalysts.

The Chemistry of Luminol Emission

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In the presence of a base, hydrogen peroxide, and a metal catalyst, the luminol molecule undergoes oxidation and liberates nitrogen gas. The 3-aminopthalate anion is formed in an electronically excited state that relaxes back to the ground state with the release of pale blue light, with the emission peaking near 440 nm.

“Firefly”-A Propeller-Based Chemiluminescence Analyzer

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The Chemiluminescence PCB shown opposite is housed in a small enclosure that connects to the user's PC via a USB cable. External connections to a PMT from the CL PCB are via the mini-DIN 6 connector, whose pinout is given below. Valve control signals are also available on this connector to control load/inject cycling of a switching valve in a CL FIA experiment. A LabVIEWTM vi controls instrument parameters and handles data acquisition during a chemiluminescence experiment.
Pin
Colour
Function
1
Brown
GND
2
Red
Current in (PMT)
3
Orange
PMT -HV bias DAC
4
Yellow
Valve A
5
Green
Valve B
6
Blue
+5V

Photomultiplier Tubes (PMT's)


A PMT is an extremely sensitive photon detector. It consists of a photocathode, a string of electrodes called "dynodes", and a final collection electrode called the anode.

The first step in the detection process is conversion of a photon into a photoelectron - this takes place at the photocathode. The dynodes are connected to a resistor chain that is biased to maintain an ~80-100 volts potential difference between each dynode pair. Electrons undergo acceleration in the inter-dynode space and when they collide with the next dynode, a shower of secondary electrons is produced. These in turn travel to the next dynode and so on.

Each dynode collision by one electron results in (roughly) 3 secondary electrons, so in a device with n dynodes, there is an amplification by a factor 3n. The anode electrode collects the electron current; thereafter a current amplifier is used to convert the current into a voltage.

To operate a PMT, a source of HV of up to -1000 volts relative to ground is required. An integral -HV base for a PMT that incorporates the power supply as well as the resistive divider chain is available from companies such as Hamamatsu. Shown at left is the Hamamatsu C8991 base, whose external connections are indicated in the diagram below.


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A 1P28 photomultiplier tube

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PMT base, incorporating a high voltage bias supply and a resistive divider chain

-HV bias to the PMT is provided by a C8991 base via its integral high voltage power supply. The C8991 is powered by a 12-15V DC plugpack. A 0-1V control voltage from the CL PCB (miniDIN, pin #3) sets the high voltage bias (and hence the gain) of the PMT. The gain factor is -1000:1, ie a 600 mV control voltage results in a -600V bias.

The Chemiluminescence LabVIEWTM vi


The LabVIEWTM front panel for the chemiluminescence analyzer is shown below. This vi and its associated Propeller code has three important functions - (i) it controls a solenoid valve to fill a sample injection loop with analyte and injects it (load/inject cycling) according to user timing requirements, (ii) it sets the photomultiplier -HV bias and (iii) it converts the PMT current to a voltage and digitizes the readings. The light blue trace shown here is not real CL data; the cover over the PMT was opened and closed repeatedly to admit low-level light to check for a PMT response.
Operation of the instrument is straightforward. The load and inject times (in seconds) are set via front panel controls at top left of screen. Interval/msec sets the sampling interval - this can be set fairly short (25 msec) - or longer, if desired. The ADC is a Linear Technology LTC2440 and this can be configured to sample at various acquisition rates (higher rates give slightly higher noise) in the ADC options box. The corresponding ADC period should be shorter than the user's chosen sampling interval.

The I-V gain parameter controls the current-to-voltage conversion gain and is a number from 0-7. A higher number corresponds to a lower current gain and a lower number to a higher gain. Each successively lower I-V gain setting results in an ~ 3.3x increase in the gain, giving this control an effective range of 3000:1. PMT -HV controls the PMT voltage and Limit -HV sets the upper limit of this voltage - this value will not be exceeded.

Some experimentation with the PMT -HV and I-V gain settings is necessary depending on the amount of chemiluminescence produced at different analyte concentrations. To protect the PMT its output voltage (as read by the output box and displayed in the graph) should be restricted to less than 1000 mV at all times.

A separate 12-15V plug pack is required to run the PMT. This should initially be turned off and a check made to ensure that room light is excluded before switching on.

To run the vi the forward arrow button is pressed. Once underway a trace appears on screen and the control voltage for the PMT bias can be turned up as needed. Care should be taken as the PMT is extremely sensitive to light and any stray light will produce a large background. During a run the elapsed time and the PMT output (now a voltage, after current-to-voltage conversion) is displayed on screen. If needed the vertical and horizontal scales on the waveform chart can be edited according to the user's requirements. File saving to an ascii file can be activated by setting File? to ON.

A run can be stopped at any time by pressing the large stop button. At the conclusion of an experiment the PMT plug-pack must be powered down to protect the PMT from inadvertent damage due to exposure to room light.

Detection of Ultra-Trace Levels of Cobalt (Co) by Chemiluminescence

The data opposite gives an excellent indication of the high sensitivity of chemiluminescence detection. Here, Co(II) is detected by its chemiluminescent reaction in the luminol/hydrogen peroxide system. The vertical axis is the CL signal and the horizontal axis is elapsed time.

In the experimental setup, a stream of luminol merges with a peroxide stream into which manual Co(II) sample injections can been made. In the bottom (red) trace the PMT bias voltage is set to -790V and a series of 7 blanks are injected.

The green trace shows the result for a series of 6 injections, now each containing just 1 ppb of Co(II). In the top (blue) trace the concentration of the injected samples (total 5) has been increased to 10 ppb; in addition the PMT voltage was reduced slightly to -740V.

Note that the traces shown here have been offset relative to each other for the sake of readability. Even at just 1 ppb of Co, the CL signal is clearly enhanced relative to the blank, indicating that the detection limit attainable by the technique is a few hundred parts-per-trillion (ppt).

The technique is so sensitive that even the blank produces a CL response. Co is well known to be one of the easiest elements to detect with this chemistry, but many different metal ions would also produce a CL response. One downside to the use of chemiluminescent reactions can thus be their lack of species selectivity.