By: Katelyn Hansen A2

Table of Contents
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . .3-5
History. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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. . . . . . . . . . .. .6
What is Chemiluminescence?. . . . . . . . . . . . . . . . . . . . . . . . .

7-16
Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . .. . . . .17-19
Bibliography. . . . . . . . . . . . . . . . . .. . . . . . . . .. . . . . .

. . . . . . .. . .20

Introduction
Chemiluminescence, like atomic emission spectroscopy (AES), uses
quantitative measurements of the optical emission from excited chemical
species to determine analyte concentration; however, unlike AES,
chemiluminescence is usually emission from energized molecules instead of
simply excited atoms. The bands of light determined by this technique
emanate from molecular emissions and are therefore broader and more complex
then bands originating from atomic spectra. Furthermore, chemiluminescence
can take place in either the solution or gas phase, whereas AES is almost
strictly as gas phase phenomenon.

Though liquid phase chemiluminescence plays a significant role in
laboratories using this analytical technique (often in combination with
liquid chromatography), we will concentrate on gas phase chemiluminescence
reactions since the instrumental components are somewhat simpler. These
detectors are also often used as detectors for gas chromatography.

Like fluorescence spectroscopy, chemiluminescence’s strength lies in
the detection of electromagnetic radiation produced in a system with very
low background. And on top of this, because the energy necessary to excite
the analytes to higher electronic, vibrational, and rotational states (from
which they can decay be emission) does not come from an external light
source like a laser or lamp, the problem of excitation source scattering is
completely avoided. The major limitation to the detection limits achievable
by chemiluminescence involves the dark current of the photomultiplier (PMT)
necessary to detect the analyte light emissions.If the excitation energy
for analytes in chemiluminescence doesn’t come from a source lamp or laser,
where does it come from? The energy is produced by a chemical reaction of
the analyte and a reagent. An example of a reaction of this sort is shown
below:
A chemiluminescence reaction
pic
In gas phase chemiluminescence, the light emission (represented as
Planck’s constant times nu-the light’s frequency) is produced by the
reaction of an analyte (dimethyl sulfide in the above example) and a
strongly oxidizing reagent gas such as fluorine (in the example above) or
ozone, for instance. The reaction occurs on a time scale such that the
production of light is essentially instantaneous; therefore, most
analytical systems simply mix analytes and the reagent in a small volume
chamber directly in front of a PMT. If the analytes are eluting from a gas
chromatographic column then the end of the column is often fed directly
into the reaction chamber itself. Since as much of the energy released by
the reaction should (in the analyst’s eye) be used to excite as many of the
analyte molecules as possible, loss of energy via gas phase collisions is
undesirable, and therefore a final consideration is that the gas pressure
in the reaction chamber be maintained at a small pressure (~ 1 torr) by a
vacuum pump in order to minimize the effects of little deactivation. It
must be stated that the ambiguous specification of “products” in the above
reaction is often necessary because of the nature and complexity of the
reaction. In some reactions, the chemiluminescent emitters are relatively
well known. In the above reaction the major emitter is electronically and
vibrationally excited HF; however, in the same reaction, other emitters
have been determined whose identities are not known and these also
contribute to the total light detected by the PMT. To the analytical
chemist the ambiguity about the actual products in the reaction is, in most
case, not important. All the analyst cares about is the sensitivity of the
instrument (read detection limits for target analytes), its selectivity-
that is, response for an analyte as compared to an interfering compound,
and the linear range of response.

Here is a schematic of the components necessary for a gas phase
chemiluminescence detector interfaced to a capillary gas chromatograph.

Schematic of a GC chemiluminescence detector
pic

HISTORY
The term “chemiluminescence” was first thought up by Eilhardt Weidemann in
1888, and refers to the emission of light from a chemical reaction. In its
simplest form it can be represented by;
pic
Where I* is an excited state intermediate. This is termed “direct
chemiluminescence”. In certain cases where the excited state is an
inefficient emitter, its energy may be passed on to another species (a
sensitiser, F) for light emission to be observed. This called “indirect
chemiluminescence”;
pic
These reactions can occur in the gas, liquid and solid phases, the
reactions studied at Deakin University for Chemical Analysis are all in the
liquid phase. The light emitted from chemiluminescent reactions has
differing degrees of intensity, lifetime and wavelength. The wavelength can
extend across the spectrum from near ultraviolet, through the visible and
into the near infrared. Solution phase chemiluminescent reactions which
have found analytical application often produce light in the visible
region.


WHAT IS IT REALLY?
Chemiluminescence is the generation
electromagnetic radiation as light by the release of energy from a
chemical reaction. While e lig
the light, in principle, be emitted in the ultraviolet, visible
or in to those emitting visible light are
the most common.They are also most interesting and useful.

Chemiluminescent reactions can be grouped into three types:
1. Chemical reactions using synthetic compounds and usually involving a
highly oxidized species such as peroxide are commonly termed
chemiluminescent reactions.


2. Light-emitting reactions arising from a living organism, such as the
firefly or jellyfish, are commonly termed bioluminescent reactions.


3. Light-emitting reactions which take place by the use of electrical
current are designated electrochemiluminescent reactions.


Chemiluminescent reaction usually involve the cleavage or fragmentation of
the O-O bond an organic peroxide compound. Peroxides, especially cyclic
peroxides, are prevalent in light emitting reactions because the relatively
weak peroxide bond is easily cleaved and the resulting molecular
reorganization liberates a large amount of energy. In order to achieve the
highest levels of sensitivity, a chemiluminescent reaction must be as
efficient as possible in generating photons of light. Each chemiluminescent
compound or group can produce no more than one photon of light. A perfectly
efficient reaction would have a chemiluminescence quantum yield (pic ) of
one, i.e. one photon/molecule reacted according to the equation:
pic
The chemiexcitation quantum yield (pic) is the probability of generating
an electronic excited state in a reaction and has a value between 0 and 1,
with 0 being a completely dark reaction and, when 1, all product molecules
are generated in the excited state. The most useful chemiluminescent
reactions will have apicof about 10-3 or greater. The fluorescence
quantum yield (pic) is the probability of the excited state emitting a
photon by fluorescence rather than decaying by other processes. This
property, which can have values between 0 and 1 is frequently at least 0.1.

The reaction quantum yield (pic) is the fraction of starting molecules
which undergo the luminescent reaction rather than a side reaction. This
value is usually about 1.

It is possible to increase the yield of chemiluminescence when the emitter
is poorly fluorescent (lowpic). A highly fluorescent acceptor is used in
these cases in order to transfer the excitation energy from the primary
excited state compound to the fluorescent acceptor/emitter. The
chemiluminescence quantum yield is then determined by the equation:
pic
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 analyst and the mobile phase itself. The majority of HPLC
systems work this way.


An example diagram of an HPLC system is shown below:
pic
Need for HPLC Chemiluminescence Detection
The use of chemiluminescence detection for HPLC comes from the need to
perceive compounds either very sensitively (at very low absorptions) 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 based chemiluminescence detection
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:

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.


Detection based on luminol suppression
What follows is a method of chemiluminescence detection in which the
suppression of a background chemiluminescence signal could be used to
determine a compound that elutes from the LC column. For instance, many
organic molecules will complex metal cat-ions 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
concentration of a metal cat-ion, 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 cat-
ion 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. An “anti-signal”-proportional to the amount of the
(analyte) organic molecules eluting from the column.

pic
The following liquid phase chemiluminescence reactions are currently being
studied at Deakin University for their application to chemical analysis;
| |Ttion of tris(2,2′-bipyridyl)ruthenium(III) by suitable|
| |substrates such as amines and the oxalate anion.|
| |The reduction of acidic potassium permanganate in the presence of|
| |”polyphosphates” by certain tertiary amines.|
| |The peroxyoxalate reaction; the reaction of certain oxalate|
| |esters and oxamides with hydrogen peroxide in the presence of a |
| |suitable fluorescent species (sensitiser).|
Schematic of a simple liquid chromatographic separation
Liquid phase chemiluminescence reactions have found applicability to the
determination of a wide range of analytics from trace metals to
pharmaceuticals. Analytically these reactions are attractive due to;
| |The potential for excellent limits of detection because of the|
| |absence of source noise and scatter.|
| |High selectivity due to the limited number of available reactions.|
| |Simple, robust and inexpensive instrumentation suitable to both|
| |batch and flow analytical techniques.|
To date the most successful analytical application area for solution phase
chemiluminescence has been in the biomedical and clinical fields. And at
Deakin, liquid phase chemiluminescence reactions have been used as the
basis for sensitive and selective discovery for a range of analytical
practices including flow injection analysis (FIA), sequential injection
analysis (SIA), high performance liquid chromat- ography (HPLC) and
capillary electro- phoresis. Flow injection analysis (FIA) is a well
established, powerful, sample handling technique for laboratory analysis
and process analytical chemistry. It is an unsegmented flow technique where
samples (10-200 micro L) are injected into a moving liquid carrier stream
and are transported to a flow through detector via conduits constructed of
teflon tubing (0.3-0.8 mm internal diameter). The sample is modified by
reaction with reagents merging with the main carrier stream. The response
at the detector is in the form of a peak, the dimensions of which are
directly related to analyte concentration. Automated flow injection systems
have been applied to on-line process analysis in industrial and
environmental situations with a great deal of success. Flow injection
analysis is also ideally suited to monitoring solution phase
chemiluminescence’s reactions due to the capability to mix sample and
reagent in close proximity to a detector.

The animation below shows simple two-line FIA manifold utilizing
chemiluminescence’s detection. The reagent merges with the carrier stream
at a T-piece (marked T on the diagram) just prior to a flow cell, typically
a glass or Teflon coil.

Sequential injection analysis (SIA) employs a multi-position valve operated
in synchronization with a pump, typically either a peristaltic or syringe
type. The ports of the valve are connected to sample and reagent
reservoirs, aliquots of sample and reagent are sequentially aspirated into
a holding coil connected to the common port of the valve. The resulting
stack of sample and reagent zones is then propelled towards the detector by
the pump operating in the forward mode. The flow reversal leads to a mixing
of the sample and reagent zones to create a zone of product whose
properties are measured at the detector. The order in which the sample and
reagent zones are stacked in the holding coil depends upon the type of
chemistry being utilized.

Here are some reactions and their color of chemiluminescence:
|Reaction |Color (?max)|Quantum|
|||yield*|
|Oxidation of luminol in aqueous alkali |blue|0.01 |
||(425 nm) ||
|Oxidation of luminol in dimethyl |green-yellow|0.05 |
|sulphoxide|(500 nm) ||
|Oxidation of lucigenin in alkaline|blue-green|0.016 |
|hydrogen peroxide|(440 nm) ||
|Oxidation of lophine in alcoholic|yellow|-|
|sodium hydroxide|(525 nm) ||
|Peroxyoxalate reaction|sensitiser|0.05 – 0.5 |
||dependant||
|Reduction of|orange|-|
|tris(2,2′-bipyridyl)ruthenium (III) by |(610 nm) ||
|certain amines, alkaloids and oxalate |||
|Oxidation of some alkaloids by acidic |red|-|
|potassium permaganate in the presence |(680 nm) ||
|of polyphosphates|||
|ATP-dependant oxidation of D-luciferin |||
|with firefly luciferase|||
|pH 8.6|green-yellow|0.88 |
||(560 nm) ||
|pH 7.0|red (615 nm)||
1The intensity of emission of a reaction is dependant on the quantum
yield. The quantum yield is a measure of the efficiency of the
chemiluminescence reaction. Quantum yields vary from 10-15 (ultra-weak
chemiluminescence) to nearly 1 (bioluminescent processes).

Examples of Chemiluminescence
A buffered luminol solution with a copper(II) catalyst is added to the
funnel on the left. A hydrogen peroxide solution is added to the funnel on
the right. On releasing the rubber tubing the solutions mix. The chemical
reaction generates energy which is seen as blue light. This process is
chemiluminescence. When the reacting solution mixes with a dye,
fluorescein, the light energy from the reaction causes the fluorescein to
give off a greenish light in a process called fluorescence.

Commercially available light sticks contain a solution in a glass vial.

When the vial is broken, a second solution mixes with the first and light
is generated. Different dyes give off different colors when they are caused
to fluoresce by the light of the chemiluminescent reaction. Light sticks
give off light from 3 to 8 hours, depending on the concentrations of
reactants.

Chemiluminescence, or chemical light, is the production of light from
a non-heat generating chemical reaction. In nature, our model for
chemiluminescence has been the firefly, which uses a biochemical reaction
to produce light in an extremely efficient manner. Now with Inefficient
chemical reactions, coupled with short lifetimes of fluorescent molecules
(some can be as short as one billionth of a second), originally made the
commercial production of chemical light products highly impractical. In the
last two decades, chemical reactions using a fluorescent molecule, a key
intermediate and a catalyst have enabled OMNIGLOW to produce sustainable,
instantaneous, highly visible light in many colors and much intensity.

Summary of the report and how it is used
Analytical methods based on chemiluminescence have taken their strong
position among the more mundane analytical techniques because of a
triumvirate of strengths: sensitivity, selectivity, and in many cases, a
wide linear detection range. This is true even though chemiluminescence is
not as widely applicable as absorption, emission, or even fluorescence
methods of detection since so few molecules undergo chemiluminescent
reactions. Because chemiluminescence, light emission generated from a
chemical reaction, requires no light source for excitation, the analytical
signal appears out of an essentially black background, and the only
background signal is that of the photomultiplier tube’s (PMT) dark current.

Therefore light source warm-up and drift and interference from light
scattering are absent. In the case of systems where red and near infrared
light are observed in analytical detection, red sensitive PMT’s dark
current can be minimized by cooling; with blue light emission detection,
cooling is not required. Detection limits routinely orders of magnitude
lower than fluorescence methods are achievable. In addition, interfering
molecules are often less of a problem too since chemiluminescence reactions
can be so selective.

The initial reports in the scientific literature of lophine and then
lucigenin chemiluminescence in the last quarter of the nineteenth century
blossomed in this century into reports involving many different
chemiluminescent reagents. The most common or well known solution phase
systems involve luminol (or its derivatives), oxalate esters, lucigenin (or
its derivatives), ruthenium tris-bipyridine, and luciferin. Gas phase
examples include the ozone- and fluorine-induced, sodium vapor, and
chlorine dioxide chemiluminescence detectors for gas chromatography.

Because many of the solution phase systems use hydrogen peroxide or organic
peroxides as oxidant and these can be generated many ways in liquid systems
and because many of the solution phase reagents mentioned above can be
tagged onto a large variety of analytes, high performance liquid
chromatographic (HPLC) solution phase chemiluminescence is more common and
variously applied than gas phase chemiluminescence reactions.

In a very general way, the requirements for the analytically useful
production of light from a chemical reaction are:
1) Excess chemical energy produced by the reaction must be relatively
efficiently used to populate the excited state of the emitter
2) The excited species must have few mechanism of deactivation except light
emission. In many systems, the initially excited state molecule is used as
a conduit of energy to excite a second or third molecule which, instead, is
the actually emitting species. In solution-phase systems, pH and catalysts
must also be considered; however, in gas phase systems reaction cell
pressure and temperature can be important factors. A more detailed
description of these phenomenon and their applications can be found in the
literature in a number of places.

The use of chemiluminescence as a detection method following
analytical separation makes up a significant share of its application. For
example, liquid phase chemiluminescence has been applied to high
performance liquid chromatography and very recently to capillary
electrophoresis.

Gas phase analytical chemiluminescence reactions have in the main
been employed with gas chromatography to detect trace chemical species or
target analytes in complex matrices . Other workers have recently employed
“separationless” chemiluminescence methods to determine total sulfur
content in gasoline and coal and nitrate and nitrite in flow injection
analysis . A very recent supercritical fluid chromatographic interface to a
chemiluminescent nitrogen detector has also been reported for the
examination of polymers and pharmaceuticals although SFC/chemiluminescence
techniques have appeared before.

This glancing survey of liquid and gas phase chemiluminescence is not
meant to imply that these are the only roles for this method. Researchers
have, for instance, recently used in vivochemiluminescence initiated by UV-
A irradiation of mouse skin as a means of determining skin oxidative stress
processes. Others have used chemiluminescence as a means of following
antioxidant evaluation in mouse kidney and brain and plasma, as a means of
DNA detection and sequencing, detection of polymerase chain reaction-
derived nucleic acids, and alkaline phosphatase determination.

Finally, chemiluminescence has long been used as a means of measuring
concentrations of short lived species in gas mixtures and in the atmosphere
and to that end chemiluminescent techniques have been used to determine
ozone and hydrogen peroxide in the atmosphere, to detect the possible
emitter in the reaction of tetrakis ethylene with oxygen, and to map out
the “hot bands” of HNO produced in the reaction of NO with HCO among many
other gas phase applications. The Journal of Bioluminescence and
Chemiluminescence is obviously an excellent source in this field and
periodically publishes literature searches sorted by year and author.


Bibliography
Campbell, A.K. “Chemiluminescence: Principles and Applications in
Biology and Medicine”; VCH, Ellis Horwood Ltd.: New York, 1988.

DeLuca, M.A. “Bioluminescence and Chemiluminescence”; Academic
Press:Orlando, FL, 1978.

Fontijn, A. Ed. “Gas-phase Chemiluminescence and Chemi-ionization”;
Elsevier; New York, 1985.

Nieman, T. “Chemiluminescence: Theory and Instrumentation, Overview”,
inEncyclopedia of Analytical Science, pp 608-613; Academic
Press:Orlando, FL, 1995.

Nieman, T. “Chemiluminescence: Techniques, Liquid-Phase
Chemiluminescence”, in Encyclopedia of Analytical Science, pp
613-621;Academic Press: Orlando, FL, 1995.

Pringle, M.J. “Analytical Applications of Chemiluminescence”; in
RecentAdvances in Clinical Chemistry, Vol 30; pp. 89-
183, 1993; AcademicPress: New York, 1993.

———————–
1 Deakin University. (Available online)
http://www.deakin.org.au/~swlewis/prop.htm, January 22, 2004.