Chemiluminescence-based detection: principles and analytical applications in flowing streams and in immunoassays

Chemiluminescence-based detection: principles and analytical
applications in flowing streams and in immunoassays
W.R.G. Baeyens a,*, S.G. Schulman b, A.C. Calokerinos c, Y. Zhao a,
A. Ma Garcı´a Campan˜a d, K. Nakashima e, D. De Keukeleire f
a Uni6ersity of Ghent, Faculty of Pharmaceutical Sciences, Department of Pharmaceutical Analysis,
Laboratory of Drug Quality Control, Harelbekestraat 72 B-9000, Ghent, Belgium
b Uni6ersity of Florida, College of Pharmacy, Medicinal Chemistry, J. Hillis Miller Health Center, Gaines6ille 32610, FL, USA
c Uni6ersity of Athens, Chemistry Department, Laboratory of Analytical Chemistry, Panepistimiopolis 15771, Athens, Greece
d Uni6ersity of Granada, Faculty of Sciences, Department of Analytical Chemistry, Fuentenue6a s:n, E-18071 Granada, Spain
e Nagasaki Uni6ersity, School of Pharmaceutical Sciences, 1 -14 Bunkyo-machi, 852 Nagasaki, Japan
f Uni6ersity of Ghent, Faculty of Drug Sciences, Department of Pharmaceutics, Laboratory of Pharmacognosy and Phytochemistry,
Harelbekestraat 72 B-9000, Ghent, Belgium
Received 25 July 1997; accepted 17 November 1997
Abstract
The present paper provides the principles of chemiluminescence (CL) and its powerful applications in analytical
chemistry, mainly in the area of flow injection analysis, column liquid chromatographic and capillary electrophoretic
separating systems, and its potential in immunoassays. CL is light produced by a chemical reaction. The most
common advantages of chemiluminescent reactions are the relatively simple instrumentation required, the very low
detection limits and wide dynamic ranges, which have contributed to the interest of CL detection in flow injection
analysis, high performance liquid chromatography, including miniaturized systems, and, most recently, the exploding
area of capillary electrophoresis. The latter powerful microanalytical separation technique offers high numbers of
theoretical plates and relatively short analysis times requiring only small sample volumes, the migrating system
comprising aqueous buffer solutions. In non-isotopic immunoassays, covering a great variety of applications in
human and veterinary medicine, forensic medicine, agriculture and food industry, the radioisotope is replaced by a
fluorescence or chemiluminescent label. The use of CL as a detection principle permits quantitative determination of
various compounds at low concentrations. Disadvantages of the CL-based technique may include lack of sufficient
selectivity and sensitivity to various physicochemical factors. © 1998 Elsevier Science B.V. All rights reserved.
Keywords: Chemiluminescence; Liquid chromatography; Flow injection analysis; Capillary electrophoresis
* Corresponding author. Tel.: 32 2648097; fax: 32 92648196; e-mail: willy.baeyens@rug.ac.be
0731-7085:98:$19.00 © 1998 Elsevier Science B.V. All rights reserved.
PII S0731-7085(98)00062-4
W.R.942 G. Baeyens et al. : J. Pharm. Biomed. Anal. 17 (1998) 941–953
1. Introduction
In slightly over three decades, luminescence spectrometry
has transcended its origins as a curiosity
in the physical laboratory to become a firmly
established and widely employed branch of analytical
chemistry. Owing to elegant new instrumentation
and especially to new techniques, some of
which entirely new and some borrowed from other
disciplines, fluorescence, phosphorescence, CL and
bioluminescence spectrometries can be routinely
applied to qualitative and quantitative analytical
problems.
Molecular luminescence techniques have several
characteristics that make them useful for many
kinds of analyses. Their advantages include low
limits of detection (for highly luminescent
molecules), amenability to remote detection (by
means of a laser or fiber optic probes), applicability
to complex samples, and the generation of several
types of information—excitation and emission
spectra, decay times, polarization data—useful for
molecular identification. In liquid chromatography
(LC) luminescence detection represents an interface
between the selectivity of an elegant separation
method and an ultrasensitive detection method.
Due to the general trend in analytical sciences to
study smaller samples at increasingly lower concentrations,
the need for improved detection technology
increases. Moreover, the problem of waste
disposal is gradually forcing analytical separating
systems into miniaturization, e.g. narrow-bore and
capillary liquid chromatography versus conventional
HPLC, miniaturized high performance thin
layer chromatography (HPTLC), and, during recent
years, capillary electrophoresis (CE). As CL
(and fluorescence) may help solve the problem of
detection limits in column liquid and in capillary
electrophoretic miniaturized set-ups, not using hazardous
labels, the technique is bound to be thoroughly
explored for the coming decade.
The principles of CL and some of its powerful
applications in the wide area of analytical chemistry,
often biomedical and environmental analysis,
including direct methods and detection of CL
emission in flow injection, chromatographic and
CE separating systems as well as in immunoassays
will be treated in the present paper.
2. Chemiluminescene: Principles and
considerations in separation systems
[9,31,36,37,47]
CL is light produced by a chemical reaction; the
energy levels are identical with those involved in
fluorescence phenomena, the only difference being
the mode of excitation. The distinction between CL
and bioluminescence lies in the origin of the reactions.
Reactions that are biological in origin (e.g.
the firefly luciferin–luciferase–ATP reaction) are
called bioluminescence, the remainder, CL.
CL reactions generally yield a product in an
electronically excited state producing visible light.
As a general rule, only quite exothermic reactions
can generate the required energies. Therefore, most
CL reactions use oxygen, hydrogen peroxide, or
similar potential oxidants. As a principle in bioluminescence
and CL reactions, at least two reagents,
A and B react to form a product C, some fraction
of which is present in an electronically excited state,
C*, which may subsequently relax to the ground
state emitting a photon:
A B“C*“C hn
Hence, the luminescence process takes place at
the rate of a chemical reaction and the factors that
affect emission intensity are in fact a combination
of chemical reaction rate and luminescence considerations.
Most common cited advantages of CL
reactions are the relatively simple instrumentation
required, the low detection limits and wide dynamic
ranges, having contributed to the interest in CL
detection in HPLC and in flow injection analysis
(FIA).
CL is often described as a dark-field technique:
the absence of a strong background light level, as
in absorption spectroscopy, reduces the background
signal, improving detection limits. Small
changes in a small signal are more easily observed
than small changes in the presence of a large signal.
As a principle, it is possible to measure CL in a
fluorometer by turning off the excitation source.
What is actually needed, is a photomultiplier tube,
sufficiently sensitive in the spectral region of interest.
However, some disadvantages are to be considered
as well. A CL reagent may yield significant
W.R.G. Baeyens et al. : J. Pharm. Biomed. Anal. 17 (1998) 941–953 943
emission not just for one unique analyte, i.e. a
lack of selectivity may occur. Moreover, CL emission
intensities are sensitive to a variety of environmental
factors such as temperature, solvent,
ionic strength, pH, and other species present in
the system. As a result, separation conditions
(HPLC, CE,…) may not always match the optimum
CL emission conditions. Third, as the emission
intensity from a CL reaction varies with time
(light flash composed of a signal increase after
reagent mixing, passing through a maximum, then
decreasing back to the base-line), the CL emission
versus time profile differs widely from one compound
to another and care has to be taken to
detect the signal in the flowing stream during
strictly defined periods. For many CL systems,
there is a low background level of emission in the
absence of analyte. Hence, CL signals in flow
systems, increasing proportionally to the analyte
concentration, appear as sharp peaks superimposed
on a low constant blank signal, measured
as viewed by the time window when the mixture
of analyte and reagent(s) pass through the detector
cell. Due to the small portion of CL emission
that is only measured from this time profile, reactions
with complex kinetics can give nonlinear
plots of response versus analyte concentration [3].
There exists a wide variety of typical CL labels
and reactions that have been used for analytical
purposes, amongst which chemical, biochemical,
biomedical, pharmaceutical, food, environmental
and toxicological applications, for which the
reader is referred to the specific literature
[3,6,15,26,37,38,45,46,48–52]. In HPLC systems,
several chemiluminescent reactions have been utilized,
including peroxyoxalate (oxalate ester hydrogen
peroxide), firefly luciferase, lucigenin and
luminol (5-amino-2,3-dihydro-1,4-phthalazinedione)
reactions. The classical peroxyoxalate reaction
(Fig. 1), most commonly used for
post-column detection in conventional and microcolumn
HPLC, however, requires the use of organic
solvents, due to the limited aqueous
solubility of most oxalate esters, which may cause
precipitation problems when dealing with hydrophilic
reversed-phase eluents. On the other
hand, in the presence of a suitable fluorophore,
high quantum efficiencies may be reached, and,
importantly, a wide range of ‘excitable’
fluorophores can be analyzed when compared to
other CL systems. TDPO (bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)
phenyl] oxalate), for example,
has been used to sensitively determine
fluorescent compounds separated with an acidic
mobile phase [39]; femtomole concentrations of
catecholamines were determined with this reagent
after on-column fluorogenic derivatization with
ethylene diamine and subsequent post-column CL
reaction detection [22]. In CE separations applying
peroxyoxalate CL detection, extra problems
occur due to, on one hand, the influence of organic
solvents on the migration behaviour of the
analytes in the electrophoretic buffer, and, on the
other hand, to the possible effects of high electric
field strengths on the stability of the oxalate
reagent mixture. Luminol, in the presence of a
catalyst, reacts with hydrogen peroxide in alkaline
solution to emit light of about 425–435 nm; when
adding the catalyst and hydrogen peroxide in a
post-column mode, CL can be measured in an
HPLC system. Luminol derivatives such as isoluminol
and N-(4-aminobutyl)-N-ethylisoluminol
(ABEI), may also be applied to label carboxylic
acids and amines (e.g. amino acids); after separation
and post-column reagent addition, the labeled
analytes may be detected via their CL
emission. Continuous flow CL-based detection of
various analytes, including many drugs, has been
extensively studied for the determination of tetra-
Fig. 1. Principle of aryl oxalate–peroxide reaction generating
CL emission from fluorophores.
W.R.944 G. Baeyens et al. : J. Pharm. Biomed. Anal. 17 (1998) 941–953
Fig. 2. FIA manifold for the determination of glucose in plasma.
cyclines [19,40], cyclamate [34], and catecholamines
[15].
In principle, it should be feasible to chemically
excite various fluorescent species, native fluorescers
as such, or fluorescent derivatives generated
after suitable labeling reactions, as described
for various liquid chromatographic
[4,5,7,8,16,18,23,24,53,54] and CE [12,55]
separations.
3. Flow injection analysis and liquid
chromatography
As mentioned above, the major attractions of
CL reactions for analytical applications are the
excellent detection limits and the wide dynamic
ranges that can be achieved with relatively simple
instrumentation. CL reactions are being more
widely exploited in analytical chemistry as better
sample handling and on-line separation techniques
become available, in the first place FIA
and HPLC. Both are unsegmented, liquid phase,
continuous flow techniques that are used in conjunction
with flow-though detectors, compatible
with CL reactions [3,25,26].
The use of CL detection in a flowing stream
requires a method to deliver and mix the CL
reagents with the analyte stream or column
effluent and a suitable flow-cell that allows detection
of the CL emission at an appropriate time
period after the initiation of the reaction.
FIA is a simple and elegant analytical technique
in which a discrete liquid sample is injected into a
liquid carrier stream which transports it to a
flow-through detector. During transport, the sample
can undergo on-line physical or chemical
treatment. In its simplest form, FIA is used to
solely transport a sample to a detector. The first
reaction that was adapted to a FIA procedure was
the copper(II)-catalyzed oxidation of luminol for
the determination of hydrogen peroxide; later on,
other metal ions and catalysts, for example cobalt,
were determined. Signal-to-noise ratios need to be
maximized by simplex optimization of some key
variables, such as reagent concentrations, flow
rates, pH, sample volume and length of mixing
coils. Fig. 2 shows a more complex chemical
reaction and sample matrix via a four-line manifold
incorporating an on-line dialyser and immobilized
enzyme reactor for the determination of
glucose in blood plasma [42].
As a given CL reagent may yield significant
emission not for just one unique analyte, but for a
variety of compounds, lack of selectivity may
occur, an obvious disadvantage for FIA applications.
Selective reactors, such as enzyme reactors,
positioned before the CL reaction may overcome
W.R.G. Baeyens et al. : J. Pharm. Biomed. Anal. 17 (1998) 941–953 945
this problem. For HPLC applications, on the
other hand, lack of detector selectivity is not a
real issue: detector response to one single substance
might possibly be considered as a restriction
as well. In a flow system, the entire emission
versus time profile is not constant but varies with
time. The CL intensity detected in a flowing
stream application would be the integrated portion
of the emission profile that occurs during the
time interval intersected by the observation cell.
To maximize detector sensitivity, adjustment of
solution flow rate (and:or detector cell volume) is
required so that the observation time window is
near the peak in the CL emission versus time
profile [42].
FIA is most useful for optimizing the reaction
conditions for CL emission, and, as such, is an
important tool for the quantitative analysis of real
samples. Some compounds may be directly determined
by simple oxidation reactions. Metal ions,
catalysts, ligands, oxidants and reductants, hydrogen
peroxide, inorganic chlorine compounds can
be determined by this approach. For complex
samples, however, it is obvious that selectivity
may remain a problem and, therefore, optimized
FIA procedures should be applied in post-column
derivatization reactions established for HPLC
procedures.
In the HPLC area, probably the most widely
used analytical technique nowadays for the separation
and analysis of mixtures, the need for a
highly sensitive and simple separation technique
has been particularly obvious in biochemical and
biomedical research, where progress has often
been hampered by time-consuming, tedious or
inadequately sensitive or specific methodology.
The explosive growth and great popularity of
HPLC was catalysed by advances in column (silica)
technology and instrumentation. The reversed-
phase mode of HPLC (RPLC), using a
non-polar stationary phase and a polar mobile
phase, has emerged as the most widely applied
HPLC technique. It is estimated that \80% of
the current HPLC separations are performed using
this technique. Operational simplicity, high
efficiency, column stability, and the ability to
simultaneously analyse a broad spectrum of both
closely related and widely different compounds
have made this technique the most universal mode
of HPLC. Progress is still continuing, particularly
in the area of micro-HPLC.
An ideal detector should have good sensitivity
to all eluting components. It should be reasonably
linear so that it can be used in quantitative analysis.
It should not significantly degrade the separation
obtained in the column and should be
reliable and easy to operate. As unfortunately, a
practical universal detector for HPLC has not yet
been developed, a device must be chosen providing
adequate sensitivity for each particular problem.
For the liquid chromatographer interested in
trace analysis, there are few detectors capable of
measuring picogram or femtogram quantities of
material in the column effluent. Conventional absorption
detectors can measure chromophores in
the nanogram range, but these measurements are
frequently confounded by matrix interferences.
Among the optical methods of detection, fluorescence
represents a means of increasing both the
selectivity and the sensitivity of analysis. Selectivity
is enhanced, since not all compounds that
absorb radiation will emit.
When eliminating the light source from the
fluorimeter and employing chemical excitation,
the factors that limit the sensitivity of classical
fluorescence measurements, including straylight,
background emission, light source instability, are
reduced, resulting in extraordinary limits of detection,
at least for several fluorophores. The beneficial
effects of substituting chemical excitation for
photoexcitation, thus achieving CL, can be used
for the excitation, thus, the determination of
many classes of fluorophores, and also for the
detection of small amounts of hydrogen peroxide
produced by photochemical or enzymatic
reactions.
Commonly used reagents for post-column CL
reaction in HPLC include luminol, whose reaction
has been used in conjunction with low capacity
cation-exchange resins for the determination of
transition metal ions catalyzing the CL reaction
[6,42]. The peroxyoxalate reaction, using bis-
(2,4,6-trichlorophenyl)oxalate (TCPO) and, to a
lesser extent, bis-(2,4-dinitrophenyl)oxalate
(DNPO), still comprises a useful, versatile and
efficient CL system for the detection of LC eluW.
R.946 G. Baeyens et al. : J. Pharm. Biomed. Anal. 17 (1998) 941–953
ates. In general, fluorophores having low oxidation
potentials are most efficiently excited in the
peroxyoxalate reaction. The amino-substituted
polycyclic aromatic hydrocarbons, for example,
apparently as a result of their low oxidation potentials
and ability to form charge transfer complexes,
are among the most efficient fluorophores
to be chemically excited.
Under optimal conditions and for efficient
fluorophores, limits of detection can be improved
by more than two orders of magnitude by substituting
chemical excitation for light excitation. The
major disadvantage for post-column CL detection
in column effluents is the requirement to add the
oxalate ester and the hydrogen peroxide reagents
separately in order to excite the eluting
fluorophores. Apart from mixing problems, the
fact that the additional pumps must be as pulsefree
as possible and that the mixing tees should be
designed so as to contribute as little as possible to
band broadening, it should be kept in mind that
CL reactions display a luminescence growth curve
followed by a decay of the signal intensity that is
caused by the exhaustion of the light-generating
agent(s), as described earlier. In a flow system, the
half-life of the CL signal is a very important
parameter. For given values of the various flow
rates (that obviously should be extremely constant),
the dead volume between the mixing tee
and the flow-cell, and the volume of the flow-cell
itself, the CL half-life determines the percentage
of the emitted light that will be measured. The
relative short CL half-life of the DNPO as compared
with the TCPO system should be taken into
account when constructing a detection set-up.
With respect to the proper solvent selection in
peroxyoxalate CL systems, TCPO being the most
commonly used aryl oxalate, it should be mentioned
that esters and ethers are the best solvents
for this reagent. However, because ethers react
with oxygen to form peroxides, TCPO decomposes
rather rapidly if dissolved in an ether. Most
commonly used esters are not readily miscible
with water. In other words, CL monitoring of
partly aqueous solutions—most HPLC eluents—
requires the presence of a third solvent to create a
homogeneous system. In practice, ethyl acetate is
often selected as solvent for TCPO, and hydrogen
peroxide is dissolved in tetrahydrofuran or acetone.
Conventionally, the two reagent solutions
are mixed on-line, and the mixture is then added
to the HPLC effluent [27] (Fig. 3). Occasionally,
TCPO and hydrogen peroxide are premixed; however,
the sensitivity of such a system does not
remain constant for over 1 day.
Application of microcolumn LC in conjunction
with peroxyoxalate CL detection of fluorophores
in effluents was suggested, Fig. 4 showing a schematic
representation of a measuring device [2].
In principle, the conversion of a FIA:CL set-up
to an HPLC:CL set-up only requires the inclusion
of an HPLC column between the injector and the
CL reactor, taking into account the practical necessities
of switching tubing, connectors, pumps
and the higher pressures applied in the latter
systems.
4. Capillary electrophoresis
CE is a most powerful separation tool in analytical
sciences because of the high number of
theoretical plates and the relative short analysis
times offered. It is a fundamental microanalytical
technique (extremely small sample requirement)
that is more than complementary to conventional
chromatographic techniques, and well suited for
biomedical, biochemical, pharmaceutical, food,
environmental, toxicological research, protein
analysis, DNA sequencing, chiral analysis, purity
testing, etc.
Fig. 3. Schematic diagram for a post-column CL-based
(TCPO) detection system in HPLC (D, damper; F, flow-cell; I,
injector; MC, mixing coil; P, pump; PM, photomultiplier; R,
recorder).
W.R.G. Baeyens et al. : J. Pharm. Biomed. Anal. 17 (1998) 941–953 947
Fig. 4. Scheme for the mixing of column eluate with reagents
required for CL generation. A capillary of about 100 mm i.d.
is inserted against the end-frit of the column and brought
through a Valco mixing cross by which the TCPO and hydrogen
peroxide solutions are added. The glass 74-ml flow cell is
also fixed to the mixing cross, the column end tubing ends into
the flow-cell and the reagents and the LC eluate are premixed
in the first part of the flow-cell.
the capillary by differential electroosmotic flow
has been mentioned in literature, as well as a
system applying sample injection by a rotary injector,
three syringe pumps and two mixing parts
where the fluorescent reagent is mixed with the
buffer in a three-way connector [28].
Thanks to the many modes of CE, the most
diverse classes of molecules—not restricted to
charged species—can nowadays be investigated,
the lack of sensitive detection originally having
been a serious limitation. As a result of the small
capillary and cell dimensions, and due to the
nanoliter sample volumes, relatively concentrated
analytical solutions (\mg ml 1) or pre-concentration
methods are to be used, absorptiometric
detection (UV) being most common. The potentials
of CL detection are currently being considered
for CE purposes, apart from other (often
home-made) detecting principles [56]. In principle,
any fluorescing substance or appropriately labeled
molecule can be measured in CE experiments
after suitable chemical excitation, implying the
detecting device to add the chosen ‘chemical system’,
solubility problems and decay curves of the
light flash to be taken into account, but removing,
on the other hand, the light source for excitation
which influences detection limits and often causes
base-line problems. As discussed earlier, it is not
possible for any given fluorescing species to be
chemically excited by any of the existing chemiluminescent
systems. As in biomedical liquid chromatography
and flow injection analysis, highly
sensitive detection (B10 7 M injected solutions)
is envisaged considering the use of peroxyoxalate,
firefly luciferase, luminol, acridinium ester or various
oxidative reactions. Reactor design for postcolumn
derivatization in CE is clearly under
development which will be of value for the development
of CL detectors. Many CL-based reactors
are still in the optimization phase due to reproducibility
problems when measuring the lightflash,
not to speak of the band-broadening
problem. Moreover, the importance of micromachining
in CE instrumentation—the technology
used to produce integrated circuits—is being cited
in recent literature.
As in HPLC work, the needs and priorities of
analysis will determine the derivatization mode:
Validated CE methods are applied routinely in
many pharmaceutical analytical laboratories,
where applications include purity testing, quantitative
determinations of formulation content, and
chiral analysis [1]. As in liquid chromatography,
pre-electrophoretic labeling may be performed so
as to have access to a different detection technique,
e.g. fluorescence [28–30]. Post-separation
treatments or labeling procedures are possible as
well [28], though instrumentally complicated due
to the high voltage bridge installed over the separating
capillary. Post-capillary fluorescence detection
devices have been described for capillary
zone electrophoresis systems, comprising a capillary
reactor to mix the tagging reagent with the
migrating zones, avoiding zone broadening. A
reactor in which the reagent buffer is pumped into
W.R.948 G. Baeyens et al. : J. Pharm. Biomed. Anal. 17 (1998) 941–953
application of existing separating systems for the
intact species, stability of the analyte before or
after labeling, availability of reacting devices, sensitivity
and so on. As detectors for CE should be
selective and sensitive, the response signals reproducibly
related with the solute concentration over
a wide linear range, independent of the buffer, the
‘cell’ not contributing to extra-column broadening,
the system being reliable and easy in use, it is
clear that some of these performance criteria are
to be considered when choosing a detector for a
particular application. Selectivity will be most important
(unless a ‘universal’ detecting device is
envisaged), followed by sensitivity, linearity range
and noise.
As described above, in CL detection the analytes
(native fluorescers, labeled compounds, key
species in a more or less complex CL reaction) are
introduced into a system that chemically brings
along the required excitation energy so as to emit
light, without the need for any excitation source
(lamp, laser) as in (photo) fluorescence-based setups.
Although CL reactions are, in general, rather
inefficient in the production of light, the lumigenic
reaction can be monitored over its entire course,
and the resulting light output can be integrated.
Consequently, chemiluminescent determinations
can be very sensitive.
Hara et al. [20,21], applied high performance
CE of proteins in a pH 3.5 phosphate buffer using
50 m i.d. fused silica capillary tubes, and found
Eosine Y to migrate together with protein as a
supramolecular complex in the presence or absence
of molybdate, silver(I) and mercury(II).
This finding provided the authors not only the
possibility to overcome the problems encountered
in protein estimation, such as the appearance of
multiple peaks in the fluorescence detection mode
after protein labeling with a fluorophore, low
sensitive detection and adsorption of the protein
onto the inner wall of the capillary tube, but also
to measure Eosine Y instead of protein much
more sensitively. Their experiments were carried
out starting from a standard HPCE set-up with
on-column detection (by burning off the polymer
coating) via UV-visible or fluorescence emission
measurements, the latter by using a specially designed
detection holder, and by peroxyoxalate-induced
CL detection (TCPO–H2O2). Although the
system showed some promise, the sensitivity obtained
was not sufficient, prompting the authors
to further improve sensitivity, which allowed them
to reach, for example, a bovine serum albumin
detection limit of 4 fmol (applied) using Rose
Bengal as a dye.
Dadoo et al. [13] described highly sensitive CL
detection in a CE set-up based on the luminol
reaction. The authors were stimulated by CL detection
results obtained in HPLC work and applied
the luminol CL reaction in a CE set-up, so
as to provide to CE analysis the essential sensitivity,
several orders of magnitude greater than that
available from UV absorption measurements. A
photon counting system was used for detection in
order to measure the low light levels generated in
the capillary column. Detection limits of 400 amol
and lower were obtained for some compounds.
Ruberto and Grayeski [35] presented a detection
interface designed for the addition of postcolumn
reagents to evaluate CL as a detection
method for CE. Their interface utilizes a reactor
that introduces the reagents into the migrating
system in a sheathing flow profile. They studied
reaction conditions including pH, concentration
and flow rates of the reagents for acridinium CL
to evaluate the effect on the detector response.
They estimated detection limits for the interface
to be in the low fmole to upper amole range for
acridiniums. The interface was evaluated as a
potential source of zone dispersion by investigating
its effects on bandwidth. ‘Chemical band narrowing’
due to the fast kinetics of the applied CL
reaction was observed. The CL-producing oxidation
reaction of the acridinium ester by hydrogen
peroxide in the presence of a base was considered
suitable as a derivatizing tag for amino acids,
peptides and proteins in CE analysis. It is clear
that the positive charge will provide greater mobility
in the applied electric fields. It is worth
mentioning that the acridinium reaction has a
high CL efficiency yielding improved detectability;
its rate can be adjusted for measurements in flowing
systems, which require reaction completion in
a few seconds to minimize overlapping bands.
Moreover, acridiniums have been modified to inW.
R.G. Baeyens et al. : J. Pharm. Biomed. Anal. 17 (1998) 941–953 949
clude functional groups suitable for the derivatization
of biomolecules. The authors conclude that
increased sensitivity can be achieved due to the
larger detection volumes provided by the interface
without the band broadening expected from these
volumes. If the kinetics of the CL reaction are fast,
light production ceases before any significant diffusion
occurs; low detection limits can be obtained
with acridinium CL due to the reduced background
present from chemical excitation.
Wu and Huie [43] employed peroxyoxalate CL
detection in a home-made CE apparatus using a
two-step approach for the CE separation and
dynamic elution (elution under pressure) of the
analytes. In this way, the authors avoided the
problems associated with incompatibilities between
mixed aqueous-organic solvents and electrically-
driven separations by switching off the CE
power supply at an appropriate time and connecting
the CE capillary to a syringe pump to effect
dynamic elution. They separated three dansylated
amino acids and examined the effects of dynamic
flow-rate and reagent concentration on the CL
signal intensity employing a post-column CL detection
reactor which consisted of various fusedsilica
capillaries held within a stainless-steel tee
and a detection cell. Dynamic elution of the electrophoretic
buffer and transport of the CLreagents
under pressure were achieved using two
syringe pumps. The CL emission generated within
the post-column mixing region was detected in a
window by burning off 2 mm length of the reaction
capillary polyimide coating, the light being
collected via one end of an optical fiber bundle,
and detected using a photomultiplier tube. Average
detection limits for dansylated amino acids of
about 1.2 fmol were reported.
Zhao et al. [44] designed a post-column reactor
for CL detection in the CE separation of isoluminol
thiocarbamyl derivatives of amino acids. A
detection limit of 500 attomoles for derivatized
valine was reported.
It is worth mentioning that most CL detectors
only offer poor separation efficiencies which actually
limit full exploitation of this detection technique
in CE. Increased band broadening caused by
turbulences at the column end often are at the
basis of these drawbacks. Zhao et al. [44] were
capable of producing a high separation efficiency;
therefore it needs to be mentioned that—at least
taking into account the few relevant CE-CL publications
available so far—only carefully designed
and optimized CL detectors may contribute to
acceptable plate numbers making the technique
more attractive to the analytical scientist.
Dadoo et al. [14] designed a CL detector for CE
in which the signal is generated at the column
outlet. The analytes emerging from the column
react with the reagents to produce visible light that
is carried by a fiber optic to a photomultiplier
tube. They adapted the luminol and firefly luciferase
reactions for use in their detection scheme,
yielding detection limits of 2 10 8 M for luminol
and 5 10 9 M for ATP, approximately 3
orders of magnitude lower (better) than those
obtained with absorbance. The authors state that
concentration detection limits in the nanomolar
range should be routinely possible using a proposed
end-column detector.
5. Immunoassays [11,26,37,46]
Immunoassay methods cover quite an important
field of analytical chemistry. Their power to
quantitate rather specifically an almost limitless
number of analytes has proved their value in the
area of biomedical analysis. Nanomolar and picomolar
amounts of large biopolymers present in
biological matrices, unassayable by other techniques,
have provided biochemists with much essential
information. Diagnostic methodologies
have evolved, with immunoassays becoming centrally
important in the analysis of drugs, pesticides,
hormones and proteins.
Immunoanalytical methods are based upon the
competitive binding hat occurs between a labeled
and an unlabeled ligand for highly specific receptor
sites on antibodies. Analysis is effected by
measuring some physical or chemical property
associated with the label, thereby allowing the
construction of a standard curve that represents a
measured physical signal as a function of the
concentration of the unlabelled ligand. Unknown
ligand (analyte) concentrations are extracted from
this calibration curve.
W.R.950 G. Baeyens et al. : J. Pharm. Biomed. Anal. 17 (1998) 941–953
Distinction of the signal corresponding to either
the bound or free labeled analyte from that of the
total labeled analyte population can be accomplished
in two ways. The first involves physical
separation of the bound fraction from the free
fraction of labeled analyte. This can be accomplished
by chemical precipitation, using a salt
such as ammonium sulfate or a polymer such as
poly(ethylene glycol), followed by centrifugation.
Alternatively, one might employ ‘solid-phase’
chemisorption, where the analyte or antibody is
attached to a solid surface (beads, tube wall, dip
sticks, etc.) and distribution of reactants between
the liquid phase and the solid phase is followed by
their physical separation. These techniques,
known as heterogeneous immunoassays, are required
in radioimmunoassay, where the radiactivities
of bound and free labeled analytes cannot be
distinguished from one another. Also, the majority
of immunoassay techniques in which the label
is an enzyme, such as ELISA (enzyme-linked immunosorbent
assay), are heterogeneous methods.
Homogeneous immunoassays make up the second
category, where physical separation of bound
from free labeled ligand is not required. A signal
that is related to bound and free labeled distribution
is obtained from the solution that contains all
of the participating analytical species. The signalproducing
species may be derived enzymatically
when the label is an enzyme whose substrate
turnover rate is reduced upon ligand–antibody
association, or the label can be a fluorophore
(fluoroimmunoassay) or a chemiluminogenic-active
entity (CL immunoassay). Separation-free,
homogeneous immunoassay protocols offer several
advantages over heterogeneous methods.
Since no separation is involved, the number of
procedural steps is reduced, decreasing the time
required per assay. Additionally, since the physical
transfer step is circumvented, potential sample
loss related to this step is eliminated.
CL immunoassay has proved to be a good
alternative to radioimmunoassay, especially in the
field of clinical chemistry, and a large number of
specific applications have been cited in the literature,
including veterinary and food analysis [26].
About five components are required for CL
reactions: the CL substrate which reacts to form
the light emitting species, oxidants, cofactor(s),
inorganic ions, and a catalyst. As a principle, any
of these components can be coupled to an antibody
or antigen. The labeled reagent can be used
in a competitive or non-competitive binding assay,
CL being initiated by the addition of the
remaining components of the reaction. For use as
a CL label, a compound must fulfil four requirements:
it must be capable of participation in a CL
reaction; it must be attachable it to the antigen or
antibody, to form a stable reagent until the reaction
is triggered; the label should retain a high
quantum yield and the necessary reaction kinetics
after coupling; it should not significantly alter the
physico-chemical properties of the molecule to
which it is attached, in particular its immunological
activity.
The following groups of compounds, amongst
many others, have been investigated extensively to
meet the cited requirements: synthetic organic
compounds (e.g. phthalazine diones, acridinium
esters); cofactors in bioluminescent reactions
(NAD and ATP); enzymes (peroxidase, oxidases,
kinases, luciferases). Covalent linking to either the
antigen or antibody is carried out by chemical
modification of the label (e.g. diazotisation, isothiocyanate,
N-hydroxysuccinimide, hemisuccinate,
imidoesters), by chemical modification of antigen
or antibody (e.g. hemisuccinate, glutaraldehyde),
or by conjugation using bifunctional reagents (e.g.
mixed anhydride, carbodiimide, bis (N-hydroxy
succinimides), azido-succinimides).
The growing success of CL in immunoassays is
based on the fact that CL labels (mostly aryl
hydrazides or acridinium derivatives) can replace
radioactive labels in almost all cases without less
performance, offering low detection limits and
good precision.
The instrumentation for CL light detection is
rapidly developing, as was demonstrated at the
1993 Banff (Canada) bioluminescence and chemiluminescence
meeting [41] (including the earlier
event [42]) and the Ghent (Belgium) biomedical
quantitative luminescence spectrometry [32] meetings.
Recent advances of biological imaging techniques
for example may provide important
aspects of signal transduction pathways within
living systems. Receptor–ligand and antigen–anW.
R.G. Baeyens et al. : J. Pharm. Biomed. Anal. 17 (1998) 941–953 951
tibody coupling may serve as striking examples of
these reactions. Although classical radioligandbinding
studies are still successful, nonisotopic
strategies are of increasing impact because of
several advantages. Besides the problems of radiolabeled
ligand handing, waste disposal and limited
shelf-life, some non-isotopic labels e.g. chemiluminogenic,
may provide dynamic signals that are
more jointly related to the target biological attribute.
For example, light-emitting sources that
decompose as conjugates of target molecules signal
the place and rate of degradation of the
substance being measured. Special enzyme-ligand
conjugates (e.g. alkaline phosphatases or
horseradish peroxidase) are frequently used to
induce an enzymatic decomposition of a luminogenic
substrate (e.g. 1,2-dioxetanes or luminol). If
the amount of available substrate does not limit
these reactions, the light emission intensity becomes
a direct measure of enzyme conjugates.
Imaging cameras for CL ELISA and for monitoring
bacterial bioluminescence, fast and nonpolluting
CL imaging and quantify systems as an
alternative to X-ray film approaches, quantitative
photon imaging techniques for CL measurements
of single cells and for simultaneously measuring
multiple samples in arrays (e.g. immunoassays
and screening of drugs and toxic agents), the use
of calibration standards for microtiter plate and
tube chemiluminometers employed in both research
and clinical laboratories have been described
extensively in the literature. The number
of chemiluminescent assays with applications in
human disease (e.g. tumor diagnostics, protein
and hormone measurements), DNA sequencing,
genetic and environmental research is rapidly
growing [11,26]. Moreover, taking into account
the increasing importance of CL in drug assay
procedures as surveyed in recent years [10,33],
many more applications are to be expected in the
coming years.
6. Conclusion
From the various detection techniques nowadays
available for detection in flowing streams
and in immunoassays, CL measuring systems and
devices are of growing importance with respect to
the low detection limits that may be reached using
selected techniques.
Reactor design for post-column derivatization
as taken from liquid chromatography and FIA is
clearly under development which certainly will be
of value for the development of CL detectors.
Many devices are still in the optimization phase
due to reproducibility problems when measuring
the CL light flash. Moreover, CL detectors often
produce poor separation efficiencies which limit
their usefulness in CE. It should be mentioned
that CL enhancement techniques, from the analytical
electrophoretic point of view, will likewise be
focused on in further research. Detection sensitivity
obviously may also be improved by techniques
of sample pre-concentration during injection,
apart from the development of suitable derivatizing
agents.
The importance of micromachining in CE instrumentation—
the technology used to produce
integrated circuits—is to be cited as originally
mentioned by Dovichi at the Orlando HPCE ’93
meeting [17] and ever since further developed, as
reported most recently by Ewing [57]. Not only
can separation channels be etched into a glass
substrate, but mixing and reaction chambers can
also be incorporated. Moreover, micromachining
allows mass production, and glass substrates with
a complex manifold of channels with cross-sectional
dimensions of 10 30 mm have been used.
CL-based immunoassay methods are in full development
mainly due to the various restrictions
encountered when using radioisotopes.
Finally, it should be mentioned that the rapid
evolution of immobilization techniques has improved
the application of CL-based measuring
systems, especially in FIA and liquid chromatographic
set-ups. Immobilization of enzymes can
be of use in CL detecting systems. For example,
immobilization of luciferase in a flow-cell in front
of the photomultiplier tube, so that light is emitted
as the substrates flow over the enzyme, allows
very selective and sensitive measurements. Immobilization
of the enzyme may occur within the
flowing stream in order to produce a product
which participates in a CL reaction, e.g. the action
of enzymes on various analytes to produce
W.R.952 G. Baeyens et al. : J. Pharm. Biomed. Anal. 17 (1998) 941–953
hydrogen peroxide which is determined by the
luminol reaction. Also, all applications of immobilized
reagents in CL can easily be adapted to
biosensing systems, an area which has tremendously
increased after the introduction of fiber
optics in analytical research. Immobilization of
enzymes on the fiber is focussed on since immobilization
of chemiluminogenic reagent(s) (e.g. luminol)
would cause problems due to fast reagent
consumption.